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1
RESEARCH ARTICLE 1 2
Thylakoid Membrane Architecture in Synechocystis Depends on CurT a 3 Homolog of the Granal CURVATURE THYLAKOID1 Proteins 4
5 Steffen Heinza Anna Rasta Lin Shaoa1 Andrian Gutub Irene L Guumlgelcd Eiri Heynoef 6 Mathias Labsg2 Birgit Rengstla Stefania Violag3 Marc M Nowaczyke Dario Leisterg 7 Joumlrg Nickelsena4 8
9 aMolekulare Pflanzenwissenschaften cBiochemie und Physiologie der Pflanzen and 10 gMolekularbiologie der Pflanzen Ludwig-Maximilians-Universitaumlt Muumlnchen Biozentrum 11 Groszlighaderner Str 2-4 82152 Planegg-Martinsried Germany 12 bDepartment of Molecular and Cellular Biology FAS Center for Systems Biology Harvard 13 University 52 Oxford Street Cambridge MA 02138 USA 14 dMunich Centre for Integrated Protein Science CiPSM Ludwig-Maximilians-Universitaumlt 15 Muumlnchen Department of Chemistry and Biochemistry Butenandtstr 5 ndash 13 81377 Munich 16 Germany 17 eBiochemie der Pflanzen Ruhr-Universitaumlt Bochum Universitaumltsstr 150 44801 Bochum 18 Germany 19 fMax-Planck-Institut fuumlr Chemische Energiekonversion Stiftstrasse 34-36 45470 Muumllheim an 20 der Ruhr Germany 21
22 Short title CurT shapes cyanobacterial thylakoids 23
24 One-sentence summary 25 CurT is required for shaping the thylakoid membrane architecture in Synechocystis and 26 mediates osmotic stress responses 27
28 29
The author responsible for distribution of materials integral to the findings presented in this 30 article in accordance with the policy described in the Instructions for Authors 31 (wwwplantcellorg) is Joumlrg Nickelsen (joergnickelsenlrzuni-muenchende) 32
33 34
1 Current address National Key Laboratory of Crop Genetic Improvement Huazhong Agricultural University Wuhan 430070 China 2 Current address KWS SAAT SE Gateway Research Center St Louis MO USA 3 Current address UMR7141 CNRSUniversiteacute Pierre et Marie Curie Institut de Biologie Physico-Chimique 13 Rue Pierre et Marie Curie 75005 Paris France 4 Address correspondence to Joumlrg Nickelsen Biozentrum LMU Muumlnchen Groszlighaderner Str 2-4 82152 Planegg-Martinsried Germany Tel +49 89 2180 74773 Fax +49 89 2180 9974773 e-mail joergnickelsenlrzuni-muenchende
Plant Cell Advance Publication Published on August 19 2016 doi101105tpc1600491
copy2016 American Society of Plant Biologists All Rights Reserved
2
Abstract 35 36
Photosynthesis occurs in thylakoids a highly specialized membrane system In the 37 cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis 6803) the thylakoids 38 are arranged parallel to the plasma membrane and occasionally converge towards it to form 39 biogenesis centers The initial steps in photosystem II (PSII) assembly are thought to take 40 place in these regions which contain a membrane subcompartment harboring the early 41 assembly factor PratA and are referred to as PratA-defined membranes (PDMs) Loss of 42 CurT the Synechocystis 6803 homolog of Arabidopsis thaliana grana-shaping proteins of the 43 CURVATURE THYLAKOID1 family results in disrupted thylakoid organization and the 44 absence of biogenesis centers As a consequence PSII is less efficiently assembled and 45 accumulates to only 50 of wild-type levels CurT induces membrane curvature in vitro and 46 is distributed all over the thylakoids with local concentrations at biogenesis centers There it 47 forms a sophisticated tubular network at the cell periphery as revealed by live-cell imaging 48 CurT is part of several high molecular-weight complexes and Blue NativeSDS-PAGE and 49 isoelectric focusing demonstrated that different isoforms associate with PDMs and thylakoids 50 Moreover CurT deficiency enhances sensitivity to osmotic stress adding a level of 51 complexity to CurT function We propose that CurT is crucial for the differentiation of 52 membrane architecture including the formation of PSII-related biogenesis centers in 53 Synechocystis 6803 54
55 Introduction 56 57
Oxygenic photosynthesis originated in cyanobacteria more than 24 billion years ago 58
and went on to transform Earthrsquos atmosphere and biosphere The underlying process of light-59
driven photosynthetic electron transport is mediated by multiproteinpigment complexes 60
which are located within a specialized system of membrane sheets termed thylakoids During 61
the evolutionary transition from cyanobacteria to present-day chloroplasts this system has 62
undergone substantial diversification (Mullineaux 2005 Allen et al 2011) Contemporary 63
forms range from undifferentiated thylakoids in cyanobacteria to elaborate systems that are 64
differentiated into grana and stroma regions in plant chloroplasts (Mullineaux 2005) 65
Despite this increase in complexity over the course of evolution even ldquosimplerdquo 66
cyanobacterial systems exhibit compositional and functional membrane heterogeneity 67
(Nickelsen and Rengstl 2013) Perhaps the most striking example is the cyanobacterium 68
Gloeobacter violaceus which lacks internal thylakoids and organizes its photosynthetic 69
complexes in distinct patches within the plasma membrane (Rexroth et al 2011) Moreover 70
spatial separation between developing and functional thylakoids has been observed in the 71
model cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis 6803) 72
Immunolocalization of the photosystem II (PSII) assembly factor PratA (for processing-73
associated TPR protein) in fractionated membranes and examination of ultrathin sections by 74
immunogold electron microscopy have revealed specialized PratA-defined membrane (PDM) 75
3
regions forming biogenic centers at peripheral sites in cells where thylakoids converge 76
(Schottkowski et al 2009b Stengel et al 2012) 77
Some details of the ultrastructure of these centers have begun to emerge (Stengel et al 78
2012) Some of the convergence areas are composed of a rod-like structure ndash previously 79
named the ldquothylakoid centerrdquo ndash which is in turn surrounded by membranous material within 80
which thylakoid lamellae appear to originate (van de Meene et al 2006 Stengel et al 2012 81
Nickelsen and Zerges 2013 Ruumltgers and Schroda 2013) A current working model for these 82
biogenesis centers postulates that the initial steps in the assembly of photosynthetic complexes 83
ndash and in particular photosystem II (PSII) ndash take place at the biogenic PDMs Subsequently 84
pre-complexes migrate laterally into thylakoid lamellae where their assembly is completed 85
(Nickelsen and Rengstl 2013) Recently evidence based on the subcellular distribution of the 86
D1 degradation-related FtsH protease and the PSII repair factor Slr0151 (Yang et al 2014 87
Sacharz et al 2015 Rast et al 2016) has been obtained that maintenance ie the repair of 88
PSII is also localized at or near these areas However whether or not plasma and thylakoid 89
membranes fuse at these sites has not yet been resolved (Liberton et al 2006 van de Meene 90
et al 2006 van de Meene et al 2012) 91
Only limited information is available on the spatial organization of thylakoid 92
membrane biogenesis in land plants Their chloroplasts harbor a dynamic thylakoid membrane 93
system which is comprised of non-appressed stromal thylakoids and appressed grana regions 94
Stromal thylakoids are likely to represent sites where membrane proteins are synthesized and 95
assembled within the membrane (Yamamoto et al 1981 Danielsson et al 2006) while the 96
physicochemical forces driving grana formation are still a matter of debate (Nevo et al 2012 97
Kirchhoff 2013 Pribil et al 2014) It has however been proposed that stromal moieties of 98
LHCII determine membrane stacking of adjacent thylakoid disks (Fristedt et al 2009 Daum 99
et al 2010 Anderson et al 2012) Moreover a family of thylakoid-shaping proteins with 100
four members named CURVATURE THYLAKOID1A-D (CURT1A-D) has been identified 101
in Arabidopsis thaliana (Armbruster et al 2013) CURT1 proteins localize to grana margins 102
where they induce membrane bending thereby determining the architecture of the thylakoid 103
network (Pribil et al 2014) Intriguingly cyanobacteria whose thylakoids do not differentiate 104
into grana regions also contain a single CURT1 homolog (Armbruster et al 2013) Here we 105
report on the characterization of this homolog CurT from Synechocystis 6803 Our data 106
reveal that the cyanobacterial protein is essential for the shaping of thylakoid membranes and 107
thereby promotes efficient assembly of PSII at the cell periphery Our data argue for an 108
ancient membrane-curving activity of CURT1-like proteins which are necessary to form an 109
4
efficient thylakoid system in cyanobacteria as well as having a critical role in the response to 110
osmotic stress 111
112
Results 113
Inactivation of curT affects membrane architecture 114
The open reading frame slr0483 encodes the only CURT1-like protein expressed in the 115
cyanobacterium Synechocystis 6803 The corresponding cyanobacterial gene was previously 116
named synCURT1 to emphasize its homology to CURT1 from Arabidopsis thaliana 117
(Armbruster et al 2013 Luque and Ochoa de Alda 2014) However in conformity with 118
conventional nomenclature for bacterial genes we adopt the name lsquocurTrsquo The CurT protein is 119
predicted to comprise 149 amino acids and contains within its C-terminal half two putative 120
transmembrane domains (TMDs) which exhibit a high degree of sequence similarity with the 121
Arabidopsis CURT1A-D proteins The N-terminal half shows less similarity to its 122
Arabidopsis counterparts but it harbors a predicted amphipathic α-helix (amino acids 46-62) 123
that has been implicated in membrane bending (Figure 1A and 1B Armbruster et al 2013) 124
Interestingly the TMDs of CURT homologs share sequence and structural features with a 125
domain that is found in some thylakoid-associated cyanobacterial aminoacyl-tRNA 126
synthetases and has been hypothesized to be involved in mediating the unusual membrane 127
attachment of these enzymes (Luque and Ochoa de Alda 2014) 128
To verify the predicted membrane association of CurT total membranes from wild-129
type cells were exposed to various agents and the solubility of CurT was assessed using an 130
antibody directed against a fragment comprising its first 58 N-terminal amino acids (Figure 131
1A) We first confirmed that CurT can be detected in the membrane fractions of cell lysates 132
and exposure to 01 M Na2CO3 4 M urea or 1 M NaCl failed to extract it as would be 133
expected for a membrane-bound protein (Figure 1C) Indeed treatment of the cell lysate with 134
the non-ionic detergent Triton X-100 rendered CurT soluble as was the case for the PSII 135
inner antenna protein CP47 ndash an integral membrane protein with six transmembrane α-helices 136
(Figure 1C) Thus CurT like its Arabidopsis counterparts is likely to be an integral 137
membrane protein 138
To test whether the cyanobacterial CurT has similar membrane-tubulating properties to 139
CURT1A of A thaliana we used the same liposome-based assay to probe its ability to form 140
tubules (Armbruster et al 2013) We expressed CurT in vitro using a cell-free extract 141
supplemented with liposomes similar in composition to the thylakoid membrane 142
Subsequently liposome topology was visualized by transmission electron microscopy (TEM 143
5
Figure 1D) Like the grana-forming CURT1A its Synechocystis 6803 homolog efficiently 144
induced localized ldquocompressionrdquo of liposomes into thin tubule-like segments revealing its 145
strong membrane-curving activity (Figure 1D) Hence the membrane-shaping function of 146
members of the CURT1 family is conserved from cyanobacteria to plants Nevertheless 147
liposome shapes caused by either CurT or CURT1A displayed some differences in the degree 148
of membrane tubulation These might be due to the variable N-termini of both proteins 149
(Figure 1D) 150
To dissect the function of CurT in vivo we generated a knock-out mutant by inserting 151
a kanamycin-resistance cassette into the unique AgeI site in the slr0483 reading frame 152
(Supplemental Figure 1A Methods) Complete segregation of the mutation was confirmed by 153
PCR and immunoblot analyses (Supplemental Figure 1B and 1C) Progressively higher levels 154
of antibiotic (up to 400 microg kanamycinml) were used for mutant selection as previous 155
attempts to select a fully segregated curT- mutant had been unsuccessful probably due to 156
application of insufficient selection pressure (Armbruster et al 2013) When growth rates of 157
wild-type and mutant cells were compared 20- and 15-fold increases in doubling time were 158
observed for curT- grown under photoauto- and photoheterotrophic conditions respectively 159
(Table 1 Supplemental Figure 2) 160
Strikingly curT inactivation also led to loss of competence for DNA uptake during 161
both transformation and conjugation-based experiments Hence attempts to complement the 162
curT- mutant were not applicable However in the course of the work four independent 163
rounds of transformation of wild-type cells were carried out to generate fresh curT- mutants 164
All mutant strains obtained the same phenotype strongly suggesting that no secondary sites 165
were involved in its establishment To rule out the possibility that read-through transcription 166
from the resistance marker gene affects the upstream reading frame slr0482 (of unknown 167
function) in the mutant RT-PCR with appropriate primers was performed (Supplemental 168
Figure 3) No change in slr0482 mRNA accumulation was observed confirming that the curT- 169
phenotype is caused by disruption of the curT gene In agreement with this finding recent 170
transcriptome analyses of Synechocystis 6803 have clearly shown that the curT mRNA is 171
monocistronic (Kopf et al 2014) 172
In view of CurTrsquos in vitro membrane-tubulating activity the ultrastructure of the curT- 173
mutant was examined by TEM As shown in Figure 2A the wild-type strain exhibits the 174
typical thylakoid organization with ordered thylakoid sheets at the cell periphery and 175
convergence zones next to the plasma membrane In contrast massive disorganization of the 176
thylakoid membrane system was observed in curT- In all cells analyzed the mutant 177
6
thylakoids appeared as disordered sheets that traversed the cytoplasm and completely lacked 178
the typical convergence zones with the corresponding biogenesis centers at the periphery 179
(Figures 2B-D) This indicates a strict requirement of CurT for the establishment of normal 180
thylakoid membrane architecture in Synechocystis 6803 181
182
Inactivation of curT affects photosynthetic performance 183
The severely altered morphology of the thylakoid membrane system in curT- cells and 184
in particular the lack of PSII-related biogenesis centers prompted us to investigate the 185
photosynthetic performance of the mutant (Stengel et al 2012) The chlorophyll content was 186
reduced by ~20 and absorption spectroscopy also revealed a reduction in phycobilisomes 187
as well as an increase in carotenoid levels (Table 1 Supplemental Figure 4) Despite changes 188
in ultrastructure and pigment levels size and number of curT- cells at OD750 were not 189
significantly different as judged by t-test-based statistical analysis (Table 1) 190
To explore the photosynthetic defects of the mutant in detail immunoblot analyses 191
were performed using antibodies raised against specific subunits of photosynthetic complexes 192
and some of their biogenesis factors In curT- PSII reaction-center subunits including D1 D2 193
CP47 and CP43 were found to be present at 44 42 56 and 45 of the wild-type level 194
respectively (Figure 3) Interestingly amounts of the PSII assemblyrepair factors PratA and 195
Slr0151 as well as the light-dependent protochlorophyllide oxidoreductase (POR) enzyme 196
were also reduced (Figure 3 Yang et al 2014 Rast et al 2016) Levels of the Pitt protein 197
which interacts with POR (Schottkowski et al 2009a) and the Oxa homolog YidC 198
(Ossenbuumlhl et al 2006) were slightly increased in curT- (Figure 3) On the other hand 199
Sll0933 the homolog of the PSII assembly factor PAM68 in A thaliana accumulated to 200
250 of the wild-type level (Armbruster et al 2010) In contrast amounts of both Cytf and 201
PsaA which serve as markers for the Cyt(b6f) and PSI complexes respectively the RbcL 202
subunit of the Rubisco enzyme the PSII assembly factor Ycf48 (Komenda et al 2008) and 203
VIPP1 remained unaltered in curT- (Figure 3) Overall the alterations in protein accumulation 204
indicate that loss of CurT preferentially affects the accumulation of PSII 205
This result was further corroborated by analysis of the photosynthetic performance of 206
curT- The rate of light-dependent oxygen evolution was reduced by approximately 50 in the 207
mutant (Table 1) and analysis of P700+ reduction kinetics revealed impaired electron donation 208
by PSII (Figure 4A) PSII-driven P700+ reduction is slowed down by a factor of three in curT-209
whereas the residual cyclic electron transfer activity in the presence of DCMU is less than 210
5 in both wild-type and curT- (Figure 4B) In contrast the light intensity-dependent electron 211
7
transfer capacity which was measured as relative electron transfer rate (rETR) did not differ 212
significantly between wild-type and curT- (Figure 4C) Both wild-type and curT- cells reach a 213
capacity limit at ~ 250 micromol photons m-2s-1 and the decay of rETR beyond the capacity limit 214
is very similar These results indicate that the point of onset of PSII-related photoinhibition is 215
the same in both strains which in turn implies that electron flow downstream of PSII is 216
unchanged in the mutant 217
Low PSII levels in curT- could be caused either by reduced synthesisbiogenesis or its 218
enhanced degradation To measure the rates of D1 repair the kinetics of D1 accumulation 219
upon induction of photoinhibition by high-light treatment (800 micromol photons m-2s-1) was 220
assayed by immunological means (Figure 4D) In wild-type cells the D1 level decreased to 221
60 (relative to the initial time point) after 90 min of treatment As previously shown the 222
drop is much more pronounced (~35 left) when repair synthesis of D1 is inhibited by the 223
addition of lincomycin (Figure 4D Komenda et al 2008) Surprisingly in curT- the level of 224
residual D1 is rather stable over the same time period and lincomycin treatment induces a 225
more modest decrease to 60 relative to the initial time point (Figure 4D) These data clearly 226
indicate that in absence of CurT residual D1 is less susceptible to degradation even though 227
its absolute amount is reduced In line with this no effect on growth rates was observed when 228
either wild-type or curT- cells were cultivated for two days at 200 micromol photons m-2s-1 229
(Supplemental Figure 5) 230
To assess the role of CurT in the assembly of PSII we compared the two-dimensional 231
profiles of thylakoid membrane proteins from wild-type and mutant cells by Blue-Native 232
(BN)SDS-PAGE PSII assembly intermediates were subsequently visualized via 233
immunodetection of PSII core subunits As shown in Figure 5A dimeric PSII core complexes 234
(RCCII) are drastically underrepresented in curT- whereas relative levels of earlier assembly 235
intermediates including the monomeric CP43-less RC47 complex and non-assembled CP43 236
increase Parallel detection of CurT itself revealed a ldquosmearedrdquo signal in the size range from 237
15 up to 500 kDa suggesting that CurT forms part of high-molecular-weight complexes 238
(Figure 5A) Finally in vivo 35S protein pulse-labeling experiments confirmed that RC and 239
RC47 complexes in particular are efficiently formed in curT- but the transition to RCCI 240
complexes is severely delayed (Figure 5B) As a consequence dimeric RCCII complexes do 241
not incorporate any radioactive label over the experimentrsquos time course of 25 min (Figure 5B) 242
Taken together these data show that curT inactivation has a severe impact on PSII biogenesis 243
but little or no effect on the degradation of the complex 244
8
The data presented so far suggest a PSII-related phenotype for curT- so we explored 245
the effects of curT disruption on photosynthesis further by comparing the low-temperature 246
(77K) fluorescence emission spectra in cell suspensions following excitation of chlorophyll at 247
440 nm (Figure 6A) The signals were normalized to the emission maximum at 514 nm of the 248
external standard fluorescein (Figure 6A) In curT- the PSI emission peak amplitude was 249
similar to that of the wild-type (Figure 6A) The spectra differed however around 685 nm 250
Here we observed an increase in fluorescence in the mutant strain an emission signature that 251
is probably related to the inner antenna protein CP43 (Figure 6A Nilsson et al 1992) 252
Increased chlorophyll fluorescence at 685 nm has previously been shown to be characteristic 253
for cells that are accumulating the stress-induced IsiA protein which shares structural 254
similarities with CP43 (Odom et al 1993 Yeremenko et al 2004 Wilson et al 2007) A 255
second indication for accumulation of IsiA is a diagnostic blue shift of the 725 nm 256
fluorescence peak by approximately 5 nm which is indeed observed in the curT- spectrum 257
(Figure 6A Sandstroumlm et al 2001) We therefore determined the level of IsiA in both wild-258
type and curT- by immunoblot analysis As expected IsiA was strongly induced in curT- 259
suggesting that the mutant suffers from severe stress (Figure 6C) When fluorescence at 77K 260
was recorded after excitation at 580 nm which mainly excites the phycobiliproteins the 261
signal at 685 nm (PSII related) was enhanced in the curT- mutant most likely reflecting IsiA 262
emission andor the presence of uncoupled phycobilisomes (Figure 6B Wilson et al 2007) 263
In contrast the 725 nm peak (PSI related) was reduced in curT- relative to the wild-type 264
suggesting less coupling of phycobilisomes to PSI (Figure 6B) The increase in uncoupled 265
phycobilisomes can be directly attributed to the reduction in RCCII levels found in the curT- 266
mutant (Figure 5A) since phycobilisomes are attached to PSII dimers for efficient light 267
harvesting (Watanabe and Ikeuchi 2013 Chang et al 2015) 268
Thus the curT- mutant exhibits a characteristic set of photosynthetic defects The 269
primary target of the CurT membrane-shaping function appears to be PSII in particular in its 270
biogenic phase However the reduction in PSII content cannot be responsible for the lack of 271
thylakoid convergence zones at the plasma membrane because these structures can still be 272
observed in the psbA- mutant TD41 which is lacking D1 and therefore unable to assemble any 273
PSII complexes (Supplemental Figure 6 Nilsson et al 1992) 274
275
Subcellular localization of CurT 276
We have previously proposed that the initial steps in PSII assembly take place in 277
biogenesis centers located at the interface between plasma and thylakoid membranes (Stengel 278
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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phylogenetic map for chloroplast photosynthesis Trends Plant Sci 16 645-655 868
Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
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Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
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Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
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thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
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complexes Plant Physiol Biochem 45 119-128 883
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Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
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Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
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28
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
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Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
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Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
2
Abstract 35 36
Photosynthesis occurs in thylakoids a highly specialized membrane system In the 37 cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis 6803) the thylakoids 38 are arranged parallel to the plasma membrane and occasionally converge towards it to form 39 biogenesis centers The initial steps in photosystem II (PSII) assembly are thought to take 40 place in these regions which contain a membrane subcompartment harboring the early 41 assembly factor PratA and are referred to as PratA-defined membranes (PDMs) Loss of 42 CurT the Synechocystis 6803 homolog of Arabidopsis thaliana grana-shaping proteins of the 43 CURVATURE THYLAKOID1 family results in disrupted thylakoid organization and the 44 absence of biogenesis centers As a consequence PSII is less efficiently assembled and 45 accumulates to only 50 of wild-type levels CurT induces membrane curvature in vitro and 46 is distributed all over the thylakoids with local concentrations at biogenesis centers There it 47 forms a sophisticated tubular network at the cell periphery as revealed by live-cell imaging 48 CurT is part of several high molecular-weight complexes and Blue NativeSDS-PAGE and 49 isoelectric focusing demonstrated that different isoforms associate with PDMs and thylakoids 50 Moreover CurT deficiency enhances sensitivity to osmotic stress adding a level of 51 complexity to CurT function We propose that CurT is crucial for the differentiation of 52 membrane architecture including the formation of PSII-related biogenesis centers in 53 Synechocystis 6803 54
55 Introduction 56 57
Oxygenic photosynthesis originated in cyanobacteria more than 24 billion years ago 58
and went on to transform Earthrsquos atmosphere and biosphere The underlying process of light-59
driven photosynthetic electron transport is mediated by multiproteinpigment complexes 60
which are located within a specialized system of membrane sheets termed thylakoids During 61
the evolutionary transition from cyanobacteria to present-day chloroplasts this system has 62
undergone substantial diversification (Mullineaux 2005 Allen et al 2011) Contemporary 63
forms range from undifferentiated thylakoids in cyanobacteria to elaborate systems that are 64
differentiated into grana and stroma regions in plant chloroplasts (Mullineaux 2005) 65
Despite this increase in complexity over the course of evolution even ldquosimplerdquo 66
cyanobacterial systems exhibit compositional and functional membrane heterogeneity 67
(Nickelsen and Rengstl 2013) Perhaps the most striking example is the cyanobacterium 68
Gloeobacter violaceus which lacks internal thylakoids and organizes its photosynthetic 69
complexes in distinct patches within the plasma membrane (Rexroth et al 2011) Moreover 70
spatial separation between developing and functional thylakoids has been observed in the 71
model cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis 6803) 72
Immunolocalization of the photosystem II (PSII) assembly factor PratA (for processing-73
associated TPR protein) in fractionated membranes and examination of ultrathin sections by 74
immunogold electron microscopy have revealed specialized PratA-defined membrane (PDM) 75
3
regions forming biogenic centers at peripheral sites in cells where thylakoids converge 76
(Schottkowski et al 2009b Stengel et al 2012) 77
Some details of the ultrastructure of these centers have begun to emerge (Stengel et al 78
2012) Some of the convergence areas are composed of a rod-like structure ndash previously 79
named the ldquothylakoid centerrdquo ndash which is in turn surrounded by membranous material within 80
which thylakoid lamellae appear to originate (van de Meene et al 2006 Stengel et al 2012 81
Nickelsen and Zerges 2013 Ruumltgers and Schroda 2013) A current working model for these 82
biogenesis centers postulates that the initial steps in the assembly of photosynthetic complexes 83
ndash and in particular photosystem II (PSII) ndash take place at the biogenic PDMs Subsequently 84
pre-complexes migrate laterally into thylakoid lamellae where their assembly is completed 85
(Nickelsen and Rengstl 2013) Recently evidence based on the subcellular distribution of the 86
D1 degradation-related FtsH protease and the PSII repair factor Slr0151 (Yang et al 2014 87
Sacharz et al 2015 Rast et al 2016) has been obtained that maintenance ie the repair of 88
PSII is also localized at or near these areas However whether or not plasma and thylakoid 89
membranes fuse at these sites has not yet been resolved (Liberton et al 2006 van de Meene 90
et al 2006 van de Meene et al 2012) 91
Only limited information is available on the spatial organization of thylakoid 92
membrane biogenesis in land plants Their chloroplasts harbor a dynamic thylakoid membrane 93
system which is comprised of non-appressed stromal thylakoids and appressed grana regions 94
Stromal thylakoids are likely to represent sites where membrane proteins are synthesized and 95
assembled within the membrane (Yamamoto et al 1981 Danielsson et al 2006) while the 96
physicochemical forces driving grana formation are still a matter of debate (Nevo et al 2012 97
Kirchhoff 2013 Pribil et al 2014) It has however been proposed that stromal moieties of 98
LHCII determine membrane stacking of adjacent thylakoid disks (Fristedt et al 2009 Daum 99
et al 2010 Anderson et al 2012) Moreover a family of thylakoid-shaping proteins with 100
four members named CURVATURE THYLAKOID1A-D (CURT1A-D) has been identified 101
in Arabidopsis thaliana (Armbruster et al 2013) CURT1 proteins localize to grana margins 102
where they induce membrane bending thereby determining the architecture of the thylakoid 103
network (Pribil et al 2014) Intriguingly cyanobacteria whose thylakoids do not differentiate 104
into grana regions also contain a single CURT1 homolog (Armbruster et al 2013) Here we 105
report on the characterization of this homolog CurT from Synechocystis 6803 Our data 106
reveal that the cyanobacterial protein is essential for the shaping of thylakoid membranes and 107
thereby promotes efficient assembly of PSII at the cell periphery Our data argue for an 108
ancient membrane-curving activity of CURT1-like proteins which are necessary to form an 109
4
efficient thylakoid system in cyanobacteria as well as having a critical role in the response to 110
osmotic stress 111
112
Results 113
Inactivation of curT affects membrane architecture 114
The open reading frame slr0483 encodes the only CURT1-like protein expressed in the 115
cyanobacterium Synechocystis 6803 The corresponding cyanobacterial gene was previously 116
named synCURT1 to emphasize its homology to CURT1 from Arabidopsis thaliana 117
(Armbruster et al 2013 Luque and Ochoa de Alda 2014) However in conformity with 118
conventional nomenclature for bacterial genes we adopt the name lsquocurTrsquo The CurT protein is 119
predicted to comprise 149 amino acids and contains within its C-terminal half two putative 120
transmembrane domains (TMDs) which exhibit a high degree of sequence similarity with the 121
Arabidopsis CURT1A-D proteins The N-terminal half shows less similarity to its 122
Arabidopsis counterparts but it harbors a predicted amphipathic α-helix (amino acids 46-62) 123
that has been implicated in membrane bending (Figure 1A and 1B Armbruster et al 2013) 124
Interestingly the TMDs of CURT homologs share sequence and structural features with a 125
domain that is found in some thylakoid-associated cyanobacterial aminoacyl-tRNA 126
synthetases and has been hypothesized to be involved in mediating the unusual membrane 127
attachment of these enzymes (Luque and Ochoa de Alda 2014) 128
To verify the predicted membrane association of CurT total membranes from wild-129
type cells were exposed to various agents and the solubility of CurT was assessed using an 130
antibody directed against a fragment comprising its first 58 N-terminal amino acids (Figure 131
1A) We first confirmed that CurT can be detected in the membrane fractions of cell lysates 132
and exposure to 01 M Na2CO3 4 M urea or 1 M NaCl failed to extract it as would be 133
expected for a membrane-bound protein (Figure 1C) Indeed treatment of the cell lysate with 134
the non-ionic detergent Triton X-100 rendered CurT soluble as was the case for the PSII 135
inner antenna protein CP47 ndash an integral membrane protein with six transmembrane α-helices 136
(Figure 1C) Thus CurT like its Arabidopsis counterparts is likely to be an integral 137
membrane protein 138
To test whether the cyanobacterial CurT has similar membrane-tubulating properties to 139
CURT1A of A thaliana we used the same liposome-based assay to probe its ability to form 140
tubules (Armbruster et al 2013) We expressed CurT in vitro using a cell-free extract 141
supplemented with liposomes similar in composition to the thylakoid membrane 142
Subsequently liposome topology was visualized by transmission electron microscopy (TEM 143
5
Figure 1D) Like the grana-forming CURT1A its Synechocystis 6803 homolog efficiently 144
induced localized ldquocompressionrdquo of liposomes into thin tubule-like segments revealing its 145
strong membrane-curving activity (Figure 1D) Hence the membrane-shaping function of 146
members of the CURT1 family is conserved from cyanobacteria to plants Nevertheless 147
liposome shapes caused by either CurT or CURT1A displayed some differences in the degree 148
of membrane tubulation These might be due to the variable N-termini of both proteins 149
(Figure 1D) 150
To dissect the function of CurT in vivo we generated a knock-out mutant by inserting 151
a kanamycin-resistance cassette into the unique AgeI site in the slr0483 reading frame 152
(Supplemental Figure 1A Methods) Complete segregation of the mutation was confirmed by 153
PCR and immunoblot analyses (Supplemental Figure 1B and 1C) Progressively higher levels 154
of antibiotic (up to 400 microg kanamycinml) were used for mutant selection as previous 155
attempts to select a fully segregated curT- mutant had been unsuccessful probably due to 156
application of insufficient selection pressure (Armbruster et al 2013) When growth rates of 157
wild-type and mutant cells were compared 20- and 15-fold increases in doubling time were 158
observed for curT- grown under photoauto- and photoheterotrophic conditions respectively 159
(Table 1 Supplemental Figure 2) 160
Strikingly curT inactivation also led to loss of competence for DNA uptake during 161
both transformation and conjugation-based experiments Hence attempts to complement the 162
curT- mutant were not applicable However in the course of the work four independent 163
rounds of transformation of wild-type cells were carried out to generate fresh curT- mutants 164
All mutant strains obtained the same phenotype strongly suggesting that no secondary sites 165
were involved in its establishment To rule out the possibility that read-through transcription 166
from the resistance marker gene affects the upstream reading frame slr0482 (of unknown 167
function) in the mutant RT-PCR with appropriate primers was performed (Supplemental 168
Figure 3) No change in slr0482 mRNA accumulation was observed confirming that the curT- 169
phenotype is caused by disruption of the curT gene In agreement with this finding recent 170
transcriptome analyses of Synechocystis 6803 have clearly shown that the curT mRNA is 171
monocistronic (Kopf et al 2014) 172
In view of CurTrsquos in vitro membrane-tubulating activity the ultrastructure of the curT- 173
mutant was examined by TEM As shown in Figure 2A the wild-type strain exhibits the 174
typical thylakoid organization with ordered thylakoid sheets at the cell periphery and 175
convergence zones next to the plasma membrane In contrast massive disorganization of the 176
thylakoid membrane system was observed in curT- In all cells analyzed the mutant 177
6
thylakoids appeared as disordered sheets that traversed the cytoplasm and completely lacked 178
the typical convergence zones with the corresponding biogenesis centers at the periphery 179
(Figures 2B-D) This indicates a strict requirement of CurT for the establishment of normal 180
thylakoid membrane architecture in Synechocystis 6803 181
182
Inactivation of curT affects photosynthetic performance 183
The severely altered morphology of the thylakoid membrane system in curT- cells and 184
in particular the lack of PSII-related biogenesis centers prompted us to investigate the 185
photosynthetic performance of the mutant (Stengel et al 2012) The chlorophyll content was 186
reduced by ~20 and absorption spectroscopy also revealed a reduction in phycobilisomes 187
as well as an increase in carotenoid levels (Table 1 Supplemental Figure 4) Despite changes 188
in ultrastructure and pigment levels size and number of curT- cells at OD750 were not 189
significantly different as judged by t-test-based statistical analysis (Table 1) 190
To explore the photosynthetic defects of the mutant in detail immunoblot analyses 191
were performed using antibodies raised against specific subunits of photosynthetic complexes 192
and some of their biogenesis factors In curT- PSII reaction-center subunits including D1 D2 193
CP47 and CP43 were found to be present at 44 42 56 and 45 of the wild-type level 194
respectively (Figure 3) Interestingly amounts of the PSII assemblyrepair factors PratA and 195
Slr0151 as well as the light-dependent protochlorophyllide oxidoreductase (POR) enzyme 196
were also reduced (Figure 3 Yang et al 2014 Rast et al 2016) Levels of the Pitt protein 197
which interacts with POR (Schottkowski et al 2009a) and the Oxa homolog YidC 198
(Ossenbuumlhl et al 2006) were slightly increased in curT- (Figure 3) On the other hand 199
Sll0933 the homolog of the PSII assembly factor PAM68 in A thaliana accumulated to 200
250 of the wild-type level (Armbruster et al 2010) In contrast amounts of both Cytf and 201
PsaA which serve as markers for the Cyt(b6f) and PSI complexes respectively the RbcL 202
subunit of the Rubisco enzyme the PSII assembly factor Ycf48 (Komenda et al 2008) and 203
VIPP1 remained unaltered in curT- (Figure 3) Overall the alterations in protein accumulation 204
indicate that loss of CurT preferentially affects the accumulation of PSII 205
This result was further corroborated by analysis of the photosynthetic performance of 206
curT- The rate of light-dependent oxygen evolution was reduced by approximately 50 in the 207
mutant (Table 1) and analysis of P700+ reduction kinetics revealed impaired electron donation 208
by PSII (Figure 4A) PSII-driven P700+ reduction is slowed down by a factor of three in curT-209
whereas the residual cyclic electron transfer activity in the presence of DCMU is less than 210
5 in both wild-type and curT- (Figure 4B) In contrast the light intensity-dependent electron 211
7
transfer capacity which was measured as relative electron transfer rate (rETR) did not differ 212
significantly between wild-type and curT- (Figure 4C) Both wild-type and curT- cells reach a 213
capacity limit at ~ 250 micromol photons m-2s-1 and the decay of rETR beyond the capacity limit 214
is very similar These results indicate that the point of onset of PSII-related photoinhibition is 215
the same in both strains which in turn implies that electron flow downstream of PSII is 216
unchanged in the mutant 217
Low PSII levels in curT- could be caused either by reduced synthesisbiogenesis or its 218
enhanced degradation To measure the rates of D1 repair the kinetics of D1 accumulation 219
upon induction of photoinhibition by high-light treatment (800 micromol photons m-2s-1) was 220
assayed by immunological means (Figure 4D) In wild-type cells the D1 level decreased to 221
60 (relative to the initial time point) after 90 min of treatment As previously shown the 222
drop is much more pronounced (~35 left) when repair synthesis of D1 is inhibited by the 223
addition of lincomycin (Figure 4D Komenda et al 2008) Surprisingly in curT- the level of 224
residual D1 is rather stable over the same time period and lincomycin treatment induces a 225
more modest decrease to 60 relative to the initial time point (Figure 4D) These data clearly 226
indicate that in absence of CurT residual D1 is less susceptible to degradation even though 227
its absolute amount is reduced In line with this no effect on growth rates was observed when 228
either wild-type or curT- cells were cultivated for two days at 200 micromol photons m-2s-1 229
(Supplemental Figure 5) 230
To assess the role of CurT in the assembly of PSII we compared the two-dimensional 231
profiles of thylakoid membrane proteins from wild-type and mutant cells by Blue-Native 232
(BN)SDS-PAGE PSII assembly intermediates were subsequently visualized via 233
immunodetection of PSII core subunits As shown in Figure 5A dimeric PSII core complexes 234
(RCCII) are drastically underrepresented in curT- whereas relative levels of earlier assembly 235
intermediates including the monomeric CP43-less RC47 complex and non-assembled CP43 236
increase Parallel detection of CurT itself revealed a ldquosmearedrdquo signal in the size range from 237
15 up to 500 kDa suggesting that CurT forms part of high-molecular-weight complexes 238
(Figure 5A) Finally in vivo 35S protein pulse-labeling experiments confirmed that RC and 239
RC47 complexes in particular are efficiently formed in curT- but the transition to RCCI 240
complexes is severely delayed (Figure 5B) As a consequence dimeric RCCII complexes do 241
not incorporate any radioactive label over the experimentrsquos time course of 25 min (Figure 5B) 242
Taken together these data show that curT inactivation has a severe impact on PSII biogenesis 243
but little or no effect on the degradation of the complex 244
8
The data presented so far suggest a PSII-related phenotype for curT- so we explored 245
the effects of curT disruption on photosynthesis further by comparing the low-temperature 246
(77K) fluorescence emission spectra in cell suspensions following excitation of chlorophyll at 247
440 nm (Figure 6A) The signals were normalized to the emission maximum at 514 nm of the 248
external standard fluorescein (Figure 6A) In curT- the PSI emission peak amplitude was 249
similar to that of the wild-type (Figure 6A) The spectra differed however around 685 nm 250
Here we observed an increase in fluorescence in the mutant strain an emission signature that 251
is probably related to the inner antenna protein CP43 (Figure 6A Nilsson et al 1992) 252
Increased chlorophyll fluorescence at 685 nm has previously been shown to be characteristic 253
for cells that are accumulating the stress-induced IsiA protein which shares structural 254
similarities with CP43 (Odom et al 1993 Yeremenko et al 2004 Wilson et al 2007) A 255
second indication for accumulation of IsiA is a diagnostic blue shift of the 725 nm 256
fluorescence peak by approximately 5 nm which is indeed observed in the curT- spectrum 257
(Figure 6A Sandstroumlm et al 2001) We therefore determined the level of IsiA in both wild-258
type and curT- by immunoblot analysis As expected IsiA was strongly induced in curT- 259
suggesting that the mutant suffers from severe stress (Figure 6C) When fluorescence at 77K 260
was recorded after excitation at 580 nm which mainly excites the phycobiliproteins the 261
signal at 685 nm (PSII related) was enhanced in the curT- mutant most likely reflecting IsiA 262
emission andor the presence of uncoupled phycobilisomes (Figure 6B Wilson et al 2007) 263
In contrast the 725 nm peak (PSI related) was reduced in curT- relative to the wild-type 264
suggesting less coupling of phycobilisomes to PSI (Figure 6B) The increase in uncoupled 265
phycobilisomes can be directly attributed to the reduction in RCCII levels found in the curT- 266
mutant (Figure 5A) since phycobilisomes are attached to PSII dimers for efficient light 267
harvesting (Watanabe and Ikeuchi 2013 Chang et al 2015) 268
Thus the curT- mutant exhibits a characteristic set of photosynthetic defects The 269
primary target of the CurT membrane-shaping function appears to be PSII in particular in its 270
biogenic phase However the reduction in PSII content cannot be responsible for the lack of 271
thylakoid convergence zones at the plasma membrane because these structures can still be 272
observed in the psbA- mutant TD41 which is lacking D1 and therefore unable to assemble any 273
PSII complexes (Supplemental Figure 6 Nilsson et al 1992) 274
275
Subcellular localization of CurT 276
We have previously proposed that the initial steps in PSII assembly take place in 277
biogenesis centers located at the interface between plasma and thylakoid membranes (Stengel 278
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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phylogenetic map for chloroplast photosynthesis Trends Plant Sci 16 645-655 868
Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
dynamic structural changes of plant thylakoid architecture Philos Trans R Soc 870
Lond B Biol Sci 367 3515-3524 871
Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
D Jahns P Weisshaar B Nickelsen J and Leister D (2010) The Arabidopsis 873
thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II 874
assembly Plant Cell 22 3439-3460 875
Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
Rojahn U Jensen PE Rappaport F Joliot P Doumlrmann P Wanner G and 877
Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify 878
thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
basic thylakoid membrane formation but not for the assembly of thylakoid protein 882
complexes Plant Physiol Biochem 45 119-128 883
Bartsevich VV and Pakrasi HB (1995) Molecular identification of an ABC transporter 884
complex for manganese analysis of a cyanobacterial mutant strain impaired in the 885
photosynthetic oxygen evolution process EMBO J 14 1845-1853 886
Bartsevich VV and Pakrasi HB (1996) Manganese transport in the cyanobacterium 887
Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
Synechocystis PCC 6803 Photosynth Res 99 205-216 891
Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
J (2012) Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH893
Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
248-254897
28
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
organization of an intact phycobilisome and its association with photosystem II Cell 899
Res 25 726-737 900
Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
Parsed CitationsAllen JF de Paula WBM Puthiyaveetil S and Nield J (2011) A structural phylogenetic map for chloroplast photosynthesisTrends Plant Sci 16 645-655
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Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of dynamic structural changes of plant thylakoidarchitecture Philos Trans R Soc Lond B Biol Sci 367 3515-3524
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
3
regions forming biogenic centers at peripheral sites in cells where thylakoids converge 76
(Schottkowski et al 2009b Stengel et al 2012) 77
Some details of the ultrastructure of these centers have begun to emerge (Stengel et al 78
2012) Some of the convergence areas are composed of a rod-like structure ndash previously 79
named the ldquothylakoid centerrdquo ndash which is in turn surrounded by membranous material within 80
which thylakoid lamellae appear to originate (van de Meene et al 2006 Stengel et al 2012 81
Nickelsen and Zerges 2013 Ruumltgers and Schroda 2013) A current working model for these 82
biogenesis centers postulates that the initial steps in the assembly of photosynthetic complexes 83
ndash and in particular photosystem II (PSII) ndash take place at the biogenic PDMs Subsequently 84
pre-complexes migrate laterally into thylakoid lamellae where their assembly is completed 85
(Nickelsen and Rengstl 2013) Recently evidence based on the subcellular distribution of the 86
D1 degradation-related FtsH protease and the PSII repair factor Slr0151 (Yang et al 2014 87
Sacharz et al 2015 Rast et al 2016) has been obtained that maintenance ie the repair of 88
PSII is also localized at or near these areas However whether or not plasma and thylakoid 89
membranes fuse at these sites has not yet been resolved (Liberton et al 2006 van de Meene 90
et al 2006 van de Meene et al 2012) 91
Only limited information is available on the spatial organization of thylakoid 92
membrane biogenesis in land plants Their chloroplasts harbor a dynamic thylakoid membrane 93
system which is comprised of non-appressed stromal thylakoids and appressed grana regions 94
Stromal thylakoids are likely to represent sites where membrane proteins are synthesized and 95
assembled within the membrane (Yamamoto et al 1981 Danielsson et al 2006) while the 96
physicochemical forces driving grana formation are still a matter of debate (Nevo et al 2012 97
Kirchhoff 2013 Pribil et al 2014) It has however been proposed that stromal moieties of 98
LHCII determine membrane stacking of adjacent thylakoid disks (Fristedt et al 2009 Daum 99
et al 2010 Anderson et al 2012) Moreover a family of thylakoid-shaping proteins with 100
four members named CURVATURE THYLAKOID1A-D (CURT1A-D) has been identified 101
in Arabidopsis thaliana (Armbruster et al 2013) CURT1 proteins localize to grana margins 102
where they induce membrane bending thereby determining the architecture of the thylakoid 103
network (Pribil et al 2014) Intriguingly cyanobacteria whose thylakoids do not differentiate 104
into grana regions also contain a single CURT1 homolog (Armbruster et al 2013) Here we 105
report on the characterization of this homolog CurT from Synechocystis 6803 Our data 106
reveal that the cyanobacterial protein is essential for the shaping of thylakoid membranes and 107
thereby promotes efficient assembly of PSII at the cell periphery Our data argue for an 108
ancient membrane-curving activity of CURT1-like proteins which are necessary to form an 109
4
efficient thylakoid system in cyanobacteria as well as having a critical role in the response to 110
osmotic stress 111
112
Results 113
Inactivation of curT affects membrane architecture 114
The open reading frame slr0483 encodes the only CURT1-like protein expressed in the 115
cyanobacterium Synechocystis 6803 The corresponding cyanobacterial gene was previously 116
named synCURT1 to emphasize its homology to CURT1 from Arabidopsis thaliana 117
(Armbruster et al 2013 Luque and Ochoa de Alda 2014) However in conformity with 118
conventional nomenclature for bacterial genes we adopt the name lsquocurTrsquo The CurT protein is 119
predicted to comprise 149 amino acids and contains within its C-terminal half two putative 120
transmembrane domains (TMDs) which exhibit a high degree of sequence similarity with the 121
Arabidopsis CURT1A-D proteins The N-terminal half shows less similarity to its 122
Arabidopsis counterparts but it harbors a predicted amphipathic α-helix (amino acids 46-62) 123
that has been implicated in membrane bending (Figure 1A and 1B Armbruster et al 2013) 124
Interestingly the TMDs of CURT homologs share sequence and structural features with a 125
domain that is found in some thylakoid-associated cyanobacterial aminoacyl-tRNA 126
synthetases and has been hypothesized to be involved in mediating the unusual membrane 127
attachment of these enzymes (Luque and Ochoa de Alda 2014) 128
To verify the predicted membrane association of CurT total membranes from wild-129
type cells were exposed to various agents and the solubility of CurT was assessed using an 130
antibody directed against a fragment comprising its first 58 N-terminal amino acids (Figure 131
1A) We first confirmed that CurT can be detected in the membrane fractions of cell lysates 132
and exposure to 01 M Na2CO3 4 M urea or 1 M NaCl failed to extract it as would be 133
expected for a membrane-bound protein (Figure 1C) Indeed treatment of the cell lysate with 134
the non-ionic detergent Triton X-100 rendered CurT soluble as was the case for the PSII 135
inner antenna protein CP47 ndash an integral membrane protein with six transmembrane α-helices 136
(Figure 1C) Thus CurT like its Arabidopsis counterparts is likely to be an integral 137
membrane protein 138
To test whether the cyanobacterial CurT has similar membrane-tubulating properties to 139
CURT1A of A thaliana we used the same liposome-based assay to probe its ability to form 140
tubules (Armbruster et al 2013) We expressed CurT in vitro using a cell-free extract 141
supplemented with liposomes similar in composition to the thylakoid membrane 142
Subsequently liposome topology was visualized by transmission electron microscopy (TEM 143
5
Figure 1D) Like the grana-forming CURT1A its Synechocystis 6803 homolog efficiently 144
induced localized ldquocompressionrdquo of liposomes into thin tubule-like segments revealing its 145
strong membrane-curving activity (Figure 1D) Hence the membrane-shaping function of 146
members of the CURT1 family is conserved from cyanobacteria to plants Nevertheless 147
liposome shapes caused by either CurT or CURT1A displayed some differences in the degree 148
of membrane tubulation These might be due to the variable N-termini of both proteins 149
(Figure 1D) 150
To dissect the function of CurT in vivo we generated a knock-out mutant by inserting 151
a kanamycin-resistance cassette into the unique AgeI site in the slr0483 reading frame 152
(Supplemental Figure 1A Methods) Complete segregation of the mutation was confirmed by 153
PCR and immunoblot analyses (Supplemental Figure 1B and 1C) Progressively higher levels 154
of antibiotic (up to 400 microg kanamycinml) were used for mutant selection as previous 155
attempts to select a fully segregated curT- mutant had been unsuccessful probably due to 156
application of insufficient selection pressure (Armbruster et al 2013) When growth rates of 157
wild-type and mutant cells were compared 20- and 15-fold increases in doubling time were 158
observed for curT- grown under photoauto- and photoheterotrophic conditions respectively 159
(Table 1 Supplemental Figure 2) 160
Strikingly curT inactivation also led to loss of competence for DNA uptake during 161
both transformation and conjugation-based experiments Hence attempts to complement the 162
curT- mutant were not applicable However in the course of the work four independent 163
rounds of transformation of wild-type cells were carried out to generate fresh curT- mutants 164
All mutant strains obtained the same phenotype strongly suggesting that no secondary sites 165
were involved in its establishment To rule out the possibility that read-through transcription 166
from the resistance marker gene affects the upstream reading frame slr0482 (of unknown 167
function) in the mutant RT-PCR with appropriate primers was performed (Supplemental 168
Figure 3) No change in slr0482 mRNA accumulation was observed confirming that the curT- 169
phenotype is caused by disruption of the curT gene In agreement with this finding recent 170
transcriptome analyses of Synechocystis 6803 have clearly shown that the curT mRNA is 171
monocistronic (Kopf et al 2014) 172
In view of CurTrsquos in vitro membrane-tubulating activity the ultrastructure of the curT- 173
mutant was examined by TEM As shown in Figure 2A the wild-type strain exhibits the 174
typical thylakoid organization with ordered thylakoid sheets at the cell periphery and 175
convergence zones next to the plasma membrane In contrast massive disorganization of the 176
thylakoid membrane system was observed in curT- In all cells analyzed the mutant 177
6
thylakoids appeared as disordered sheets that traversed the cytoplasm and completely lacked 178
the typical convergence zones with the corresponding biogenesis centers at the periphery 179
(Figures 2B-D) This indicates a strict requirement of CurT for the establishment of normal 180
thylakoid membrane architecture in Synechocystis 6803 181
182
Inactivation of curT affects photosynthetic performance 183
The severely altered morphology of the thylakoid membrane system in curT- cells and 184
in particular the lack of PSII-related biogenesis centers prompted us to investigate the 185
photosynthetic performance of the mutant (Stengel et al 2012) The chlorophyll content was 186
reduced by ~20 and absorption spectroscopy also revealed a reduction in phycobilisomes 187
as well as an increase in carotenoid levels (Table 1 Supplemental Figure 4) Despite changes 188
in ultrastructure and pigment levels size and number of curT- cells at OD750 were not 189
significantly different as judged by t-test-based statistical analysis (Table 1) 190
To explore the photosynthetic defects of the mutant in detail immunoblot analyses 191
were performed using antibodies raised against specific subunits of photosynthetic complexes 192
and some of their biogenesis factors In curT- PSII reaction-center subunits including D1 D2 193
CP47 and CP43 were found to be present at 44 42 56 and 45 of the wild-type level 194
respectively (Figure 3) Interestingly amounts of the PSII assemblyrepair factors PratA and 195
Slr0151 as well as the light-dependent protochlorophyllide oxidoreductase (POR) enzyme 196
were also reduced (Figure 3 Yang et al 2014 Rast et al 2016) Levels of the Pitt protein 197
which interacts with POR (Schottkowski et al 2009a) and the Oxa homolog YidC 198
(Ossenbuumlhl et al 2006) were slightly increased in curT- (Figure 3) On the other hand 199
Sll0933 the homolog of the PSII assembly factor PAM68 in A thaliana accumulated to 200
250 of the wild-type level (Armbruster et al 2010) In contrast amounts of both Cytf and 201
PsaA which serve as markers for the Cyt(b6f) and PSI complexes respectively the RbcL 202
subunit of the Rubisco enzyme the PSII assembly factor Ycf48 (Komenda et al 2008) and 203
VIPP1 remained unaltered in curT- (Figure 3) Overall the alterations in protein accumulation 204
indicate that loss of CurT preferentially affects the accumulation of PSII 205
This result was further corroborated by analysis of the photosynthetic performance of 206
curT- The rate of light-dependent oxygen evolution was reduced by approximately 50 in the 207
mutant (Table 1) and analysis of P700+ reduction kinetics revealed impaired electron donation 208
by PSII (Figure 4A) PSII-driven P700+ reduction is slowed down by a factor of three in curT-209
whereas the residual cyclic electron transfer activity in the presence of DCMU is less than 210
5 in both wild-type and curT- (Figure 4B) In contrast the light intensity-dependent electron 211
7
transfer capacity which was measured as relative electron transfer rate (rETR) did not differ 212
significantly between wild-type and curT- (Figure 4C) Both wild-type and curT- cells reach a 213
capacity limit at ~ 250 micromol photons m-2s-1 and the decay of rETR beyond the capacity limit 214
is very similar These results indicate that the point of onset of PSII-related photoinhibition is 215
the same in both strains which in turn implies that electron flow downstream of PSII is 216
unchanged in the mutant 217
Low PSII levels in curT- could be caused either by reduced synthesisbiogenesis or its 218
enhanced degradation To measure the rates of D1 repair the kinetics of D1 accumulation 219
upon induction of photoinhibition by high-light treatment (800 micromol photons m-2s-1) was 220
assayed by immunological means (Figure 4D) In wild-type cells the D1 level decreased to 221
60 (relative to the initial time point) after 90 min of treatment As previously shown the 222
drop is much more pronounced (~35 left) when repair synthesis of D1 is inhibited by the 223
addition of lincomycin (Figure 4D Komenda et al 2008) Surprisingly in curT- the level of 224
residual D1 is rather stable over the same time period and lincomycin treatment induces a 225
more modest decrease to 60 relative to the initial time point (Figure 4D) These data clearly 226
indicate that in absence of CurT residual D1 is less susceptible to degradation even though 227
its absolute amount is reduced In line with this no effect on growth rates was observed when 228
either wild-type or curT- cells were cultivated for two days at 200 micromol photons m-2s-1 229
(Supplemental Figure 5) 230
To assess the role of CurT in the assembly of PSII we compared the two-dimensional 231
profiles of thylakoid membrane proteins from wild-type and mutant cells by Blue-Native 232
(BN)SDS-PAGE PSII assembly intermediates were subsequently visualized via 233
immunodetection of PSII core subunits As shown in Figure 5A dimeric PSII core complexes 234
(RCCII) are drastically underrepresented in curT- whereas relative levels of earlier assembly 235
intermediates including the monomeric CP43-less RC47 complex and non-assembled CP43 236
increase Parallel detection of CurT itself revealed a ldquosmearedrdquo signal in the size range from 237
15 up to 500 kDa suggesting that CurT forms part of high-molecular-weight complexes 238
(Figure 5A) Finally in vivo 35S protein pulse-labeling experiments confirmed that RC and 239
RC47 complexes in particular are efficiently formed in curT- but the transition to RCCI 240
complexes is severely delayed (Figure 5B) As a consequence dimeric RCCII complexes do 241
not incorporate any radioactive label over the experimentrsquos time course of 25 min (Figure 5B) 242
Taken together these data show that curT inactivation has a severe impact on PSII biogenesis 243
but little or no effect on the degradation of the complex 244
8
The data presented so far suggest a PSII-related phenotype for curT- so we explored 245
the effects of curT disruption on photosynthesis further by comparing the low-temperature 246
(77K) fluorescence emission spectra in cell suspensions following excitation of chlorophyll at 247
440 nm (Figure 6A) The signals were normalized to the emission maximum at 514 nm of the 248
external standard fluorescein (Figure 6A) In curT- the PSI emission peak amplitude was 249
similar to that of the wild-type (Figure 6A) The spectra differed however around 685 nm 250
Here we observed an increase in fluorescence in the mutant strain an emission signature that 251
is probably related to the inner antenna protein CP43 (Figure 6A Nilsson et al 1992) 252
Increased chlorophyll fluorescence at 685 nm has previously been shown to be characteristic 253
for cells that are accumulating the stress-induced IsiA protein which shares structural 254
similarities with CP43 (Odom et al 1993 Yeremenko et al 2004 Wilson et al 2007) A 255
second indication for accumulation of IsiA is a diagnostic blue shift of the 725 nm 256
fluorescence peak by approximately 5 nm which is indeed observed in the curT- spectrum 257
(Figure 6A Sandstroumlm et al 2001) We therefore determined the level of IsiA in both wild-258
type and curT- by immunoblot analysis As expected IsiA was strongly induced in curT- 259
suggesting that the mutant suffers from severe stress (Figure 6C) When fluorescence at 77K 260
was recorded after excitation at 580 nm which mainly excites the phycobiliproteins the 261
signal at 685 nm (PSII related) was enhanced in the curT- mutant most likely reflecting IsiA 262
emission andor the presence of uncoupled phycobilisomes (Figure 6B Wilson et al 2007) 263
In contrast the 725 nm peak (PSI related) was reduced in curT- relative to the wild-type 264
suggesting less coupling of phycobilisomes to PSI (Figure 6B) The increase in uncoupled 265
phycobilisomes can be directly attributed to the reduction in RCCII levels found in the curT- 266
mutant (Figure 5A) since phycobilisomes are attached to PSII dimers for efficient light 267
harvesting (Watanabe and Ikeuchi 2013 Chang et al 2015) 268
Thus the curT- mutant exhibits a characteristic set of photosynthetic defects The 269
primary target of the CurT membrane-shaping function appears to be PSII in particular in its 270
biogenic phase However the reduction in PSII content cannot be responsible for the lack of 271
thylakoid convergence zones at the plasma membrane because these structures can still be 272
observed in the psbA- mutant TD41 which is lacking D1 and therefore unable to assemble any 273
PSII complexes (Supplemental Figure 6 Nilsson et al 1992) 274
275
Subcellular localization of CurT 276
We have previously proposed that the initial steps in PSII assembly take place in 277
biogenesis centers located at the interface between plasma and thylakoid membranes (Stengel 278
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
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Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
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Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
4
efficient thylakoid system in cyanobacteria as well as having a critical role in the response to 110
osmotic stress 111
112
Results 113
Inactivation of curT affects membrane architecture 114
The open reading frame slr0483 encodes the only CURT1-like protein expressed in the 115
cyanobacterium Synechocystis 6803 The corresponding cyanobacterial gene was previously 116
named synCURT1 to emphasize its homology to CURT1 from Arabidopsis thaliana 117
(Armbruster et al 2013 Luque and Ochoa de Alda 2014) However in conformity with 118
conventional nomenclature for bacterial genes we adopt the name lsquocurTrsquo The CurT protein is 119
predicted to comprise 149 amino acids and contains within its C-terminal half two putative 120
transmembrane domains (TMDs) which exhibit a high degree of sequence similarity with the 121
Arabidopsis CURT1A-D proteins The N-terminal half shows less similarity to its 122
Arabidopsis counterparts but it harbors a predicted amphipathic α-helix (amino acids 46-62) 123
that has been implicated in membrane bending (Figure 1A and 1B Armbruster et al 2013) 124
Interestingly the TMDs of CURT homologs share sequence and structural features with a 125
domain that is found in some thylakoid-associated cyanobacterial aminoacyl-tRNA 126
synthetases and has been hypothesized to be involved in mediating the unusual membrane 127
attachment of these enzymes (Luque and Ochoa de Alda 2014) 128
To verify the predicted membrane association of CurT total membranes from wild-129
type cells were exposed to various agents and the solubility of CurT was assessed using an 130
antibody directed against a fragment comprising its first 58 N-terminal amino acids (Figure 131
1A) We first confirmed that CurT can be detected in the membrane fractions of cell lysates 132
and exposure to 01 M Na2CO3 4 M urea or 1 M NaCl failed to extract it as would be 133
expected for a membrane-bound protein (Figure 1C) Indeed treatment of the cell lysate with 134
the non-ionic detergent Triton X-100 rendered CurT soluble as was the case for the PSII 135
inner antenna protein CP47 ndash an integral membrane protein with six transmembrane α-helices 136
(Figure 1C) Thus CurT like its Arabidopsis counterparts is likely to be an integral 137
membrane protein 138
To test whether the cyanobacterial CurT has similar membrane-tubulating properties to 139
CURT1A of A thaliana we used the same liposome-based assay to probe its ability to form 140
tubules (Armbruster et al 2013) We expressed CurT in vitro using a cell-free extract 141
supplemented with liposomes similar in composition to the thylakoid membrane 142
Subsequently liposome topology was visualized by transmission electron microscopy (TEM 143
5
Figure 1D) Like the grana-forming CURT1A its Synechocystis 6803 homolog efficiently 144
induced localized ldquocompressionrdquo of liposomes into thin tubule-like segments revealing its 145
strong membrane-curving activity (Figure 1D) Hence the membrane-shaping function of 146
members of the CURT1 family is conserved from cyanobacteria to plants Nevertheless 147
liposome shapes caused by either CurT or CURT1A displayed some differences in the degree 148
of membrane tubulation These might be due to the variable N-termini of both proteins 149
(Figure 1D) 150
To dissect the function of CurT in vivo we generated a knock-out mutant by inserting 151
a kanamycin-resistance cassette into the unique AgeI site in the slr0483 reading frame 152
(Supplemental Figure 1A Methods) Complete segregation of the mutation was confirmed by 153
PCR and immunoblot analyses (Supplemental Figure 1B and 1C) Progressively higher levels 154
of antibiotic (up to 400 microg kanamycinml) were used for mutant selection as previous 155
attempts to select a fully segregated curT- mutant had been unsuccessful probably due to 156
application of insufficient selection pressure (Armbruster et al 2013) When growth rates of 157
wild-type and mutant cells were compared 20- and 15-fold increases in doubling time were 158
observed for curT- grown under photoauto- and photoheterotrophic conditions respectively 159
(Table 1 Supplemental Figure 2) 160
Strikingly curT inactivation also led to loss of competence for DNA uptake during 161
both transformation and conjugation-based experiments Hence attempts to complement the 162
curT- mutant were not applicable However in the course of the work four independent 163
rounds of transformation of wild-type cells were carried out to generate fresh curT- mutants 164
All mutant strains obtained the same phenotype strongly suggesting that no secondary sites 165
were involved in its establishment To rule out the possibility that read-through transcription 166
from the resistance marker gene affects the upstream reading frame slr0482 (of unknown 167
function) in the mutant RT-PCR with appropriate primers was performed (Supplemental 168
Figure 3) No change in slr0482 mRNA accumulation was observed confirming that the curT- 169
phenotype is caused by disruption of the curT gene In agreement with this finding recent 170
transcriptome analyses of Synechocystis 6803 have clearly shown that the curT mRNA is 171
monocistronic (Kopf et al 2014) 172
In view of CurTrsquos in vitro membrane-tubulating activity the ultrastructure of the curT- 173
mutant was examined by TEM As shown in Figure 2A the wild-type strain exhibits the 174
typical thylakoid organization with ordered thylakoid sheets at the cell periphery and 175
convergence zones next to the plasma membrane In contrast massive disorganization of the 176
thylakoid membrane system was observed in curT- In all cells analyzed the mutant 177
6
thylakoids appeared as disordered sheets that traversed the cytoplasm and completely lacked 178
the typical convergence zones with the corresponding biogenesis centers at the periphery 179
(Figures 2B-D) This indicates a strict requirement of CurT for the establishment of normal 180
thylakoid membrane architecture in Synechocystis 6803 181
182
Inactivation of curT affects photosynthetic performance 183
The severely altered morphology of the thylakoid membrane system in curT- cells and 184
in particular the lack of PSII-related biogenesis centers prompted us to investigate the 185
photosynthetic performance of the mutant (Stengel et al 2012) The chlorophyll content was 186
reduced by ~20 and absorption spectroscopy also revealed a reduction in phycobilisomes 187
as well as an increase in carotenoid levels (Table 1 Supplemental Figure 4) Despite changes 188
in ultrastructure and pigment levels size and number of curT- cells at OD750 were not 189
significantly different as judged by t-test-based statistical analysis (Table 1) 190
To explore the photosynthetic defects of the mutant in detail immunoblot analyses 191
were performed using antibodies raised against specific subunits of photosynthetic complexes 192
and some of their biogenesis factors In curT- PSII reaction-center subunits including D1 D2 193
CP47 and CP43 were found to be present at 44 42 56 and 45 of the wild-type level 194
respectively (Figure 3) Interestingly amounts of the PSII assemblyrepair factors PratA and 195
Slr0151 as well as the light-dependent protochlorophyllide oxidoreductase (POR) enzyme 196
were also reduced (Figure 3 Yang et al 2014 Rast et al 2016) Levels of the Pitt protein 197
which interacts with POR (Schottkowski et al 2009a) and the Oxa homolog YidC 198
(Ossenbuumlhl et al 2006) were slightly increased in curT- (Figure 3) On the other hand 199
Sll0933 the homolog of the PSII assembly factor PAM68 in A thaliana accumulated to 200
250 of the wild-type level (Armbruster et al 2010) In contrast amounts of both Cytf and 201
PsaA which serve as markers for the Cyt(b6f) and PSI complexes respectively the RbcL 202
subunit of the Rubisco enzyme the PSII assembly factor Ycf48 (Komenda et al 2008) and 203
VIPP1 remained unaltered in curT- (Figure 3) Overall the alterations in protein accumulation 204
indicate that loss of CurT preferentially affects the accumulation of PSII 205
This result was further corroborated by analysis of the photosynthetic performance of 206
curT- The rate of light-dependent oxygen evolution was reduced by approximately 50 in the 207
mutant (Table 1) and analysis of P700+ reduction kinetics revealed impaired electron donation 208
by PSII (Figure 4A) PSII-driven P700+ reduction is slowed down by a factor of three in curT-209
whereas the residual cyclic electron transfer activity in the presence of DCMU is less than 210
5 in both wild-type and curT- (Figure 4B) In contrast the light intensity-dependent electron 211
7
transfer capacity which was measured as relative electron transfer rate (rETR) did not differ 212
significantly between wild-type and curT- (Figure 4C) Both wild-type and curT- cells reach a 213
capacity limit at ~ 250 micromol photons m-2s-1 and the decay of rETR beyond the capacity limit 214
is very similar These results indicate that the point of onset of PSII-related photoinhibition is 215
the same in both strains which in turn implies that electron flow downstream of PSII is 216
unchanged in the mutant 217
Low PSII levels in curT- could be caused either by reduced synthesisbiogenesis or its 218
enhanced degradation To measure the rates of D1 repair the kinetics of D1 accumulation 219
upon induction of photoinhibition by high-light treatment (800 micromol photons m-2s-1) was 220
assayed by immunological means (Figure 4D) In wild-type cells the D1 level decreased to 221
60 (relative to the initial time point) after 90 min of treatment As previously shown the 222
drop is much more pronounced (~35 left) when repair synthesis of D1 is inhibited by the 223
addition of lincomycin (Figure 4D Komenda et al 2008) Surprisingly in curT- the level of 224
residual D1 is rather stable over the same time period and lincomycin treatment induces a 225
more modest decrease to 60 relative to the initial time point (Figure 4D) These data clearly 226
indicate that in absence of CurT residual D1 is less susceptible to degradation even though 227
its absolute amount is reduced In line with this no effect on growth rates was observed when 228
either wild-type or curT- cells were cultivated for two days at 200 micromol photons m-2s-1 229
(Supplemental Figure 5) 230
To assess the role of CurT in the assembly of PSII we compared the two-dimensional 231
profiles of thylakoid membrane proteins from wild-type and mutant cells by Blue-Native 232
(BN)SDS-PAGE PSII assembly intermediates were subsequently visualized via 233
immunodetection of PSII core subunits As shown in Figure 5A dimeric PSII core complexes 234
(RCCII) are drastically underrepresented in curT- whereas relative levels of earlier assembly 235
intermediates including the monomeric CP43-less RC47 complex and non-assembled CP43 236
increase Parallel detection of CurT itself revealed a ldquosmearedrdquo signal in the size range from 237
15 up to 500 kDa suggesting that CurT forms part of high-molecular-weight complexes 238
(Figure 5A) Finally in vivo 35S protein pulse-labeling experiments confirmed that RC and 239
RC47 complexes in particular are efficiently formed in curT- but the transition to RCCI 240
complexes is severely delayed (Figure 5B) As a consequence dimeric RCCII complexes do 241
not incorporate any radioactive label over the experimentrsquos time course of 25 min (Figure 5B) 242
Taken together these data show that curT inactivation has a severe impact on PSII biogenesis 243
but little or no effect on the degradation of the complex 244
8
The data presented so far suggest a PSII-related phenotype for curT- so we explored 245
the effects of curT disruption on photosynthesis further by comparing the low-temperature 246
(77K) fluorescence emission spectra in cell suspensions following excitation of chlorophyll at 247
440 nm (Figure 6A) The signals were normalized to the emission maximum at 514 nm of the 248
external standard fluorescein (Figure 6A) In curT- the PSI emission peak amplitude was 249
similar to that of the wild-type (Figure 6A) The spectra differed however around 685 nm 250
Here we observed an increase in fluorescence in the mutant strain an emission signature that 251
is probably related to the inner antenna protein CP43 (Figure 6A Nilsson et al 1992) 252
Increased chlorophyll fluorescence at 685 nm has previously been shown to be characteristic 253
for cells that are accumulating the stress-induced IsiA protein which shares structural 254
similarities with CP43 (Odom et al 1993 Yeremenko et al 2004 Wilson et al 2007) A 255
second indication for accumulation of IsiA is a diagnostic blue shift of the 725 nm 256
fluorescence peak by approximately 5 nm which is indeed observed in the curT- spectrum 257
(Figure 6A Sandstroumlm et al 2001) We therefore determined the level of IsiA in both wild-258
type and curT- by immunoblot analysis As expected IsiA was strongly induced in curT- 259
suggesting that the mutant suffers from severe stress (Figure 6C) When fluorescence at 77K 260
was recorded after excitation at 580 nm which mainly excites the phycobiliproteins the 261
signal at 685 nm (PSII related) was enhanced in the curT- mutant most likely reflecting IsiA 262
emission andor the presence of uncoupled phycobilisomes (Figure 6B Wilson et al 2007) 263
In contrast the 725 nm peak (PSI related) was reduced in curT- relative to the wild-type 264
suggesting less coupling of phycobilisomes to PSI (Figure 6B) The increase in uncoupled 265
phycobilisomes can be directly attributed to the reduction in RCCII levels found in the curT- 266
mutant (Figure 5A) since phycobilisomes are attached to PSII dimers for efficient light 267
harvesting (Watanabe and Ikeuchi 2013 Chang et al 2015) 268
Thus the curT- mutant exhibits a characteristic set of photosynthetic defects The 269
primary target of the CurT membrane-shaping function appears to be PSII in particular in its 270
biogenic phase However the reduction in PSII content cannot be responsible for the lack of 271
thylakoid convergence zones at the plasma membrane because these structures can still be 272
observed in the psbA- mutant TD41 which is lacking D1 and therefore unable to assemble any 273
PSII complexes (Supplemental Figure 6 Nilsson et al 1992) 274
275
Subcellular localization of CurT 276
We have previously proposed that the initial steps in PSII assembly take place in 277
biogenesis centers located at the interface between plasma and thylakoid membranes (Stengel 278
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
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Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
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Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
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1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
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Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
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localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
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supercomplex Photosynth Res 116 265-276 1048
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Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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5
Figure 1D) Like the grana-forming CURT1A its Synechocystis 6803 homolog efficiently 144
induced localized ldquocompressionrdquo of liposomes into thin tubule-like segments revealing its 145
strong membrane-curving activity (Figure 1D) Hence the membrane-shaping function of 146
members of the CURT1 family is conserved from cyanobacteria to plants Nevertheless 147
liposome shapes caused by either CurT or CURT1A displayed some differences in the degree 148
of membrane tubulation These might be due to the variable N-termini of both proteins 149
(Figure 1D) 150
To dissect the function of CurT in vivo we generated a knock-out mutant by inserting 151
a kanamycin-resistance cassette into the unique AgeI site in the slr0483 reading frame 152
(Supplemental Figure 1A Methods) Complete segregation of the mutation was confirmed by 153
PCR and immunoblot analyses (Supplemental Figure 1B and 1C) Progressively higher levels 154
of antibiotic (up to 400 microg kanamycinml) were used for mutant selection as previous 155
attempts to select a fully segregated curT- mutant had been unsuccessful probably due to 156
application of insufficient selection pressure (Armbruster et al 2013) When growth rates of 157
wild-type and mutant cells were compared 20- and 15-fold increases in doubling time were 158
observed for curT- grown under photoauto- and photoheterotrophic conditions respectively 159
(Table 1 Supplemental Figure 2) 160
Strikingly curT inactivation also led to loss of competence for DNA uptake during 161
both transformation and conjugation-based experiments Hence attempts to complement the 162
curT- mutant were not applicable However in the course of the work four independent 163
rounds of transformation of wild-type cells were carried out to generate fresh curT- mutants 164
All mutant strains obtained the same phenotype strongly suggesting that no secondary sites 165
were involved in its establishment To rule out the possibility that read-through transcription 166
from the resistance marker gene affects the upstream reading frame slr0482 (of unknown 167
function) in the mutant RT-PCR with appropriate primers was performed (Supplemental 168
Figure 3) No change in slr0482 mRNA accumulation was observed confirming that the curT- 169
phenotype is caused by disruption of the curT gene In agreement with this finding recent 170
transcriptome analyses of Synechocystis 6803 have clearly shown that the curT mRNA is 171
monocistronic (Kopf et al 2014) 172
In view of CurTrsquos in vitro membrane-tubulating activity the ultrastructure of the curT- 173
mutant was examined by TEM As shown in Figure 2A the wild-type strain exhibits the 174
typical thylakoid organization with ordered thylakoid sheets at the cell periphery and 175
convergence zones next to the plasma membrane In contrast massive disorganization of the 176
thylakoid membrane system was observed in curT- In all cells analyzed the mutant 177
6
thylakoids appeared as disordered sheets that traversed the cytoplasm and completely lacked 178
the typical convergence zones with the corresponding biogenesis centers at the periphery 179
(Figures 2B-D) This indicates a strict requirement of CurT for the establishment of normal 180
thylakoid membrane architecture in Synechocystis 6803 181
182
Inactivation of curT affects photosynthetic performance 183
The severely altered morphology of the thylakoid membrane system in curT- cells and 184
in particular the lack of PSII-related biogenesis centers prompted us to investigate the 185
photosynthetic performance of the mutant (Stengel et al 2012) The chlorophyll content was 186
reduced by ~20 and absorption spectroscopy also revealed a reduction in phycobilisomes 187
as well as an increase in carotenoid levels (Table 1 Supplemental Figure 4) Despite changes 188
in ultrastructure and pigment levels size and number of curT- cells at OD750 were not 189
significantly different as judged by t-test-based statistical analysis (Table 1) 190
To explore the photosynthetic defects of the mutant in detail immunoblot analyses 191
were performed using antibodies raised against specific subunits of photosynthetic complexes 192
and some of their biogenesis factors In curT- PSII reaction-center subunits including D1 D2 193
CP47 and CP43 were found to be present at 44 42 56 and 45 of the wild-type level 194
respectively (Figure 3) Interestingly amounts of the PSII assemblyrepair factors PratA and 195
Slr0151 as well as the light-dependent protochlorophyllide oxidoreductase (POR) enzyme 196
were also reduced (Figure 3 Yang et al 2014 Rast et al 2016) Levels of the Pitt protein 197
which interacts with POR (Schottkowski et al 2009a) and the Oxa homolog YidC 198
(Ossenbuumlhl et al 2006) were slightly increased in curT- (Figure 3) On the other hand 199
Sll0933 the homolog of the PSII assembly factor PAM68 in A thaliana accumulated to 200
250 of the wild-type level (Armbruster et al 2010) In contrast amounts of both Cytf and 201
PsaA which serve as markers for the Cyt(b6f) and PSI complexes respectively the RbcL 202
subunit of the Rubisco enzyme the PSII assembly factor Ycf48 (Komenda et al 2008) and 203
VIPP1 remained unaltered in curT- (Figure 3) Overall the alterations in protein accumulation 204
indicate that loss of CurT preferentially affects the accumulation of PSII 205
This result was further corroborated by analysis of the photosynthetic performance of 206
curT- The rate of light-dependent oxygen evolution was reduced by approximately 50 in the 207
mutant (Table 1) and analysis of P700+ reduction kinetics revealed impaired electron donation 208
by PSII (Figure 4A) PSII-driven P700+ reduction is slowed down by a factor of three in curT-209
whereas the residual cyclic electron transfer activity in the presence of DCMU is less than 210
5 in both wild-type and curT- (Figure 4B) In contrast the light intensity-dependent electron 211
7
transfer capacity which was measured as relative electron transfer rate (rETR) did not differ 212
significantly between wild-type and curT- (Figure 4C) Both wild-type and curT- cells reach a 213
capacity limit at ~ 250 micromol photons m-2s-1 and the decay of rETR beyond the capacity limit 214
is very similar These results indicate that the point of onset of PSII-related photoinhibition is 215
the same in both strains which in turn implies that electron flow downstream of PSII is 216
unchanged in the mutant 217
Low PSII levels in curT- could be caused either by reduced synthesisbiogenesis or its 218
enhanced degradation To measure the rates of D1 repair the kinetics of D1 accumulation 219
upon induction of photoinhibition by high-light treatment (800 micromol photons m-2s-1) was 220
assayed by immunological means (Figure 4D) In wild-type cells the D1 level decreased to 221
60 (relative to the initial time point) after 90 min of treatment As previously shown the 222
drop is much more pronounced (~35 left) when repair synthesis of D1 is inhibited by the 223
addition of lincomycin (Figure 4D Komenda et al 2008) Surprisingly in curT- the level of 224
residual D1 is rather stable over the same time period and lincomycin treatment induces a 225
more modest decrease to 60 relative to the initial time point (Figure 4D) These data clearly 226
indicate that in absence of CurT residual D1 is less susceptible to degradation even though 227
its absolute amount is reduced In line with this no effect on growth rates was observed when 228
either wild-type or curT- cells were cultivated for two days at 200 micromol photons m-2s-1 229
(Supplemental Figure 5) 230
To assess the role of CurT in the assembly of PSII we compared the two-dimensional 231
profiles of thylakoid membrane proteins from wild-type and mutant cells by Blue-Native 232
(BN)SDS-PAGE PSII assembly intermediates were subsequently visualized via 233
immunodetection of PSII core subunits As shown in Figure 5A dimeric PSII core complexes 234
(RCCII) are drastically underrepresented in curT- whereas relative levels of earlier assembly 235
intermediates including the monomeric CP43-less RC47 complex and non-assembled CP43 236
increase Parallel detection of CurT itself revealed a ldquosmearedrdquo signal in the size range from 237
15 up to 500 kDa suggesting that CurT forms part of high-molecular-weight complexes 238
(Figure 5A) Finally in vivo 35S protein pulse-labeling experiments confirmed that RC and 239
RC47 complexes in particular are efficiently formed in curT- but the transition to RCCI 240
complexes is severely delayed (Figure 5B) As a consequence dimeric RCCII complexes do 241
not incorporate any radioactive label over the experimentrsquos time course of 25 min (Figure 5B) 242
Taken together these data show that curT inactivation has a severe impact on PSII biogenesis 243
but little or no effect on the degradation of the complex 244
8
The data presented so far suggest a PSII-related phenotype for curT- so we explored 245
the effects of curT disruption on photosynthesis further by comparing the low-temperature 246
(77K) fluorescence emission spectra in cell suspensions following excitation of chlorophyll at 247
440 nm (Figure 6A) The signals were normalized to the emission maximum at 514 nm of the 248
external standard fluorescein (Figure 6A) In curT- the PSI emission peak amplitude was 249
similar to that of the wild-type (Figure 6A) The spectra differed however around 685 nm 250
Here we observed an increase in fluorescence in the mutant strain an emission signature that 251
is probably related to the inner antenna protein CP43 (Figure 6A Nilsson et al 1992) 252
Increased chlorophyll fluorescence at 685 nm has previously been shown to be characteristic 253
for cells that are accumulating the stress-induced IsiA protein which shares structural 254
similarities with CP43 (Odom et al 1993 Yeremenko et al 2004 Wilson et al 2007) A 255
second indication for accumulation of IsiA is a diagnostic blue shift of the 725 nm 256
fluorescence peak by approximately 5 nm which is indeed observed in the curT- spectrum 257
(Figure 6A Sandstroumlm et al 2001) We therefore determined the level of IsiA in both wild-258
type and curT- by immunoblot analysis As expected IsiA was strongly induced in curT- 259
suggesting that the mutant suffers from severe stress (Figure 6C) When fluorescence at 77K 260
was recorded after excitation at 580 nm which mainly excites the phycobiliproteins the 261
signal at 685 nm (PSII related) was enhanced in the curT- mutant most likely reflecting IsiA 262
emission andor the presence of uncoupled phycobilisomes (Figure 6B Wilson et al 2007) 263
In contrast the 725 nm peak (PSI related) was reduced in curT- relative to the wild-type 264
suggesting less coupling of phycobilisomes to PSI (Figure 6B) The increase in uncoupled 265
phycobilisomes can be directly attributed to the reduction in RCCII levels found in the curT- 266
mutant (Figure 5A) since phycobilisomes are attached to PSII dimers for efficient light 267
harvesting (Watanabe and Ikeuchi 2013 Chang et al 2015) 268
Thus the curT- mutant exhibits a characteristic set of photosynthetic defects The 269
primary target of the CurT membrane-shaping function appears to be PSII in particular in its 270
biogenic phase However the reduction in PSII content cannot be responsible for the lack of 271
thylakoid convergence zones at the plasma membrane because these structures can still be 272
observed in the psbA- mutant TD41 which is lacking D1 and therefore unable to assemble any 273
PSII complexes (Supplemental Figure 6 Nilsson et al 1992) 274
275
Subcellular localization of CurT 276
We have previously proposed that the initial steps in PSII assembly take place in 277
biogenesis centers located at the interface between plasma and thylakoid membranes (Stengel 278
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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phylogenetic map for chloroplast photosynthesis Trends Plant Sci 16 645-655 868
Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
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Lond B Biol Sci 367 3515-3524 871
Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
D Jahns P Weisshaar B Nickelsen J and Leister D (2010) The Arabidopsis 873
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Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
Rojahn U Jensen PE Rappaport F Joliot P Doumlrmann P Wanner G and 877
Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify 878
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Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
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Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
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Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
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Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
organization of an intact phycobilisome and its association with photosystem II Cell 899
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Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
6
thylakoids appeared as disordered sheets that traversed the cytoplasm and completely lacked 178
the typical convergence zones with the corresponding biogenesis centers at the periphery 179
(Figures 2B-D) This indicates a strict requirement of CurT for the establishment of normal 180
thylakoid membrane architecture in Synechocystis 6803 181
182
Inactivation of curT affects photosynthetic performance 183
The severely altered morphology of the thylakoid membrane system in curT- cells and 184
in particular the lack of PSII-related biogenesis centers prompted us to investigate the 185
photosynthetic performance of the mutant (Stengel et al 2012) The chlorophyll content was 186
reduced by ~20 and absorption spectroscopy also revealed a reduction in phycobilisomes 187
as well as an increase in carotenoid levels (Table 1 Supplemental Figure 4) Despite changes 188
in ultrastructure and pigment levels size and number of curT- cells at OD750 were not 189
significantly different as judged by t-test-based statistical analysis (Table 1) 190
To explore the photosynthetic defects of the mutant in detail immunoblot analyses 191
were performed using antibodies raised against specific subunits of photosynthetic complexes 192
and some of their biogenesis factors In curT- PSII reaction-center subunits including D1 D2 193
CP47 and CP43 were found to be present at 44 42 56 and 45 of the wild-type level 194
respectively (Figure 3) Interestingly amounts of the PSII assemblyrepair factors PratA and 195
Slr0151 as well as the light-dependent protochlorophyllide oxidoreductase (POR) enzyme 196
were also reduced (Figure 3 Yang et al 2014 Rast et al 2016) Levels of the Pitt protein 197
which interacts with POR (Schottkowski et al 2009a) and the Oxa homolog YidC 198
(Ossenbuumlhl et al 2006) were slightly increased in curT- (Figure 3) On the other hand 199
Sll0933 the homolog of the PSII assembly factor PAM68 in A thaliana accumulated to 200
250 of the wild-type level (Armbruster et al 2010) In contrast amounts of both Cytf and 201
PsaA which serve as markers for the Cyt(b6f) and PSI complexes respectively the RbcL 202
subunit of the Rubisco enzyme the PSII assembly factor Ycf48 (Komenda et al 2008) and 203
VIPP1 remained unaltered in curT- (Figure 3) Overall the alterations in protein accumulation 204
indicate that loss of CurT preferentially affects the accumulation of PSII 205
This result was further corroborated by analysis of the photosynthetic performance of 206
curT- The rate of light-dependent oxygen evolution was reduced by approximately 50 in the 207
mutant (Table 1) and analysis of P700+ reduction kinetics revealed impaired electron donation 208
by PSII (Figure 4A) PSII-driven P700+ reduction is slowed down by a factor of three in curT-209
whereas the residual cyclic electron transfer activity in the presence of DCMU is less than 210
5 in both wild-type and curT- (Figure 4B) In contrast the light intensity-dependent electron 211
7
transfer capacity which was measured as relative electron transfer rate (rETR) did not differ 212
significantly between wild-type and curT- (Figure 4C) Both wild-type and curT- cells reach a 213
capacity limit at ~ 250 micromol photons m-2s-1 and the decay of rETR beyond the capacity limit 214
is very similar These results indicate that the point of onset of PSII-related photoinhibition is 215
the same in both strains which in turn implies that electron flow downstream of PSII is 216
unchanged in the mutant 217
Low PSII levels in curT- could be caused either by reduced synthesisbiogenesis or its 218
enhanced degradation To measure the rates of D1 repair the kinetics of D1 accumulation 219
upon induction of photoinhibition by high-light treatment (800 micromol photons m-2s-1) was 220
assayed by immunological means (Figure 4D) In wild-type cells the D1 level decreased to 221
60 (relative to the initial time point) after 90 min of treatment As previously shown the 222
drop is much more pronounced (~35 left) when repair synthesis of D1 is inhibited by the 223
addition of lincomycin (Figure 4D Komenda et al 2008) Surprisingly in curT- the level of 224
residual D1 is rather stable over the same time period and lincomycin treatment induces a 225
more modest decrease to 60 relative to the initial time point (Figure 4D) These data clearly 226
indicate that in absence of CurT residual D1 is less susceptible to degradation even though 227
its absolute amount is reduced In line with this no effect on growth rates was observed when 228
either wild-type or curT- cells were cultivated for two days at 200 micromol photons m-2s-1 229
(Supplemental Figure 5) 230
To assess the role of CurT in the assembly of PSII we compared the two-dimensional 231
profiles of thylakoid membrane proteins from wild-type and mutant cells by Blue-Native 232
(BN)SDS-PAGE PSII assembly intermediates were subsequently visualized via 233
immunodetection of PSII core subunits As shown in Figure 5A dimeric PSII core complexes 234
(RCCII) are drastically underrepresented in curT- whereas relative levels of earlier assembly 235
intermediates including the monomeric CP43-less RC47 complex and non-assembled CP43 236
increase Parallel detection of CurT itself revealed a ldquosmearedrdquo signal in the size range from 237
15 up to 500 kDa suggesting that CurT forms part of high-molecular-weight complexes 238
(Figure 5A) Finally in vivo 35S protein pulse-labeling experiments confirmed that RC and 239
RC47 complexes in particular are efficiently formed in curT- but the transition to RCCI 240
complexes is severely delayed (Figure 5B) As a consequence dimeric RCCII complexes do 241
not incorporate any radioactive label over the experimentrsquos time course of 25 min (Figure 5B) 242
Taken together these data show that curT inactivation has a severe impact on PSII biogenesis 243
but little or no effect on the degradation of the complex 244
8
The data presented so far suggest a PSII-related phenotype for curT- so we explored 245
the effects of curT disruption on photosynthesis further by comparing the low-temperature 246
(77K) fluorescence emission spectra in cell suspensions following excitation of chlorophyll at 247
440 nm (Figure 6A) The signals were normalized to the emission maximum at 514 nm of the 248
external standard fluorescein (Figure 6A) In curT- the PSI emission peak amplitude was 249
similar to that of the wild-type (Figure 6A) The spectra differed however around 685 nm 250
Here we observed an increase in fluorescence in the mutant strain an emission signature that 251
is probably related to the inner antenna protein CP43 (Figure 6A Nilsson et al 1992) 252
Increased chlorophyll fluorescence at 685 nm has previously been shown to be characteristic 253
for cells that are accumulating the stress-induced IsiA protein which shares structural 254
similarities with CP43 (Odom et al 1993 Yeremenko et al 2004 Wilson et al 2007) A 255
second indication for accumulation of IsiA is a diagnostic blue shift of the 725 nm 256
fluorescence peak by approximately 5 nm which is indeed observed in the curT- spectrum 257
(Figure 6A Sandstroumlm et al 2001) We therefore determined the level of IsiA in both wild-258
type and curT- by immunoblot analysis As expected IsiA was strongly induced in curT- 259
suggesting that the mutant suffers from severe stress (Figure 6C) When fluorescence at 77K 260
was recorded after excitation at 580 nm which mainly excites the phycobiliproteins the 261
signal at 685 nm (PSII related) was enhanced in the curT- mutant most likely reflecting IsiA 262
emission andor the presence of uncoupled phycobilisomes (Figure 6B Wilson et al 2007) 263
In contrast the 725 nm peak (PSI related) was reduced in curT- relative to the wild-type 264
suggesting less coupling of phycobilisomes to PSI (Figure 6B) The increase in uncoupled 265
phycobilisomes can be directly attributed to the reduction in RCCII levels found in the curT- 266
mutant (Figure 5A) since phycobilisomes are attached to PSII dimers for efficient light 267
harvesting (Watanabe and Ikeuchi 2013 Chang et al 2015) 268
Thus the curT- mutant exhibits a characteristic set of photosynthetic defects The 269
primary target of the CurT membrane-shaping function appears to be PSII in particular in its 270
biogenic phase However the reduction in PSII content cannot be responsible for the lack of 271
thylakoid convergence zones at the plasma membrane because these structures can still be 272
observed in the psbA- mutant TD41 which is lacking D1 and therefore unable to assemble any 273
PSII complexes (Supplemental Figure 6 Nilsson et al 1992) 274
275
Subcellular localization of CurT 276
We have previously proposed that the initial steps in PSII assembly take place in 277
biogenesis centers located at the interface between plasma and thylakoid membranes (Stengel 278
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
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Microbiology 111 1-61 1005
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thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
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Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
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Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
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tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
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1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
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Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
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supercomplex Photosynth Res 116 265-276 1048
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Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
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Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
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Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
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Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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7
transfer capacity which was measured as relative electron transfer rate (rETR) did not differ 212
significantly between wild-type and curT- (Figure 4C) Both wild-type and curT- cells reach a 213
capacity limit at ~ 250 micromol photons m-2s-1 and the decay of rETR beyond the capacity limit 214
is very similar These results indicate that the point of onset of PSII-related photoinhibition is 215
the same in both strains which in turn implies that electron flow downstream of PSII is 216
unchanged in the mutant 217
Low PSII levels in curT- could be caused either by reduced synthesisbiogenesis or its 218
enhanced degradation To measure the rates of D1 repair the kinetics of D1 accumulation 219
upon induction of photoinhibition by high-light treatment (800 micromol photons m-2s-1) was 220
assayed by immunological means (Figure 4D) In wild-type cells the D1 level decreased to 221
60 (relative to the initial time point) after 90 min of treatment As previously shown the 222
drop is much more pronounced (~35 left) when repair synthesis of D1 is inhibited by the 223
addition of lincomycin (Figure 4D Komenda et al 2008) Surprisingly in curT- the level of 224
residual D1 is rather stable over the same time period and lincomycin treatment induces a 225
more modest decrease to 60 relative to the initial time point (Figure 4D) These data clearly 226
indicate that in absence of CurT residual D1 is less susceptible to degradation even though 227
its absolute amount is reduced In line with this no effect on growth rates was observed when 228
either wild-type or curT- cells were cultivated for two days at 200 micromol photons m-2s-1 229
(Supplemental Figure 5) 230
To assess the role of CurT in the assembly of PSII we compared the two-dimensional 231
profiles of thylakoid membrane proteins from wild-type and mutant cells by Blue-Native 232
(BN)SDS-PAGE PSII assembly intermediates were subsequently visualized via 233
immunodetection of PSII core subunits As shown in Figure 5A dimeric PSII core complexes 234
(RCCII) are drastically underrepresented in curT- whereas relative levels of earlier assembly 235
intermediates including the monomeric CP43-less RC47 complex and non-assembled CP43 236
increase Parallel detection of CurT itself revealed a ldquosmearedrdquo signal in the size range from 237
15 up to 500 kDa suggesting that CurT forms part of high-molecular-weight complexes 238
(Figure 5A) Finally in vivo 35S protein pulse-labeling experiments confirmed that RC and 239
RC47 complexes in particular are efficiently formed in curT- but the transition to RCCI 240
complexes is severely delayed (Figure 5B) As a consequence dimeric RCCII complexes do 241
not incorporate any radioactive label over the experimentrsquos time course of 25 min (Figure 5B) 242
Taken together these data show that curT inactivation has a severe impact on PSII biogenesis 243
but little or no effect on the degradation of the complex 244
8
The data presented so far suggest a PSII-related phenotype for curT- so we explored 245
the effects of curT disruption on photosynthesis further by comparing the low-temperature 246
(77K) fluorescence emission spectra in cell suspensions following excitation of chlorophyll at 247
440 nm (Figure 6A) The signals were normalized to the emission maximum at 514 nm of the 248
external standard fluorescein (Figure 6A) In curT- the PSI emission peak amplitude was 249
similar to that of the wild-type (Figure 6A) The spectra differed however around 685 nm 250
Here we observed an increase in fluorescence in the mutant strain an emission signature that 251
is probably related to the inner antenna protein CP43 (Figure 6A Nilsson et al 1992) 252
Increased chlorophyll fluorescence at 685 nm has previously been shown to be characteristic 253
for cells that are accumulating the stress-induced IsiA protein which shares structural 254
similarities with CP43 (Odom et al 1993 Yeremenko et al 2004 Wilson et al 2007) A 255
second indication for accumulation of IsiA is a diagnostic blue shift of the 725 nm 256
fluorescence peak by approximately 5 nm which is indeed observed in the curT- spectrum 257
(Figure 6A Sandstroumlm et al 2001) We therefore determined the level of IsiA in both wild-258
type and curT- by immunoblot analysis As expected IsiA was strongly induced in curT- 259
suggesting that the mutant suffers from severe stress (Figure 6C) When fluorescence at 77K 260
was recorded after excitation at 580 nm which mainly excites the phycobiliproteins the 261
signal at 685 nm (PSII related) was enhanced in the curT- mutant most likely reflecting IsiA 262
emission andor the presence of uncoupled phycobilisomes (Figure 6B Wilson et al 2007) 263
In contrast the 725 nm peak (PSI related) was reduced in curT- relative to the wild-type 264
suggesting less coupling of phycobilisomes to PSI (Figure 6B) The increase in uncoupled 265
phycobilisomes can be directly attributed to the reduction in RCCII levels found in the curT- 266
mutant (Figure 5A) since phycobilisomes are attached to PSII dimers for efficient light 267
harvesting (Watanabe and Ikeuchi 2013 Chang et al 2015) 268
Thus the curT- mutant exhibits a characteristic set of photosynthetic defects The 269
primary target of the CurT membrane-shaping function appears to be PSII in particular in its 270
biogenic phase However the reduction in PSII content cannot be responsible for the lack of 271
thylakoid convergence zones at the plasma membrane because these structures can still be 272
observed in the psbA- mutant TD41 which is lacking D1 and therefore unable to assemble any 273
PSII complexes (Supplemental Figure 6 Nilsson et al 1992) 274
275
Subcellular localization of CurT 276
We have previously proposed that the initial steps in PSII assembly take place in 277
biogenesis centers located at the interface between plasma and thylakoid membranes (Stengel 278
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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8
The data presented so far suggest a PSII-related phenotype for curT- so we explored 245
the effects of curT disruption on photosynthesis further by comparing the low-temperature 246
(77K) fluorescence emission spectra in cell suspensions following excitation of chlorophyll at 247
440 nm (Figure 6A) The signals were normalized to the emission maximum at 514 nm of the 248
external standard fluorescein (Figure 6A) In curT- the PSI emission peak amplitude was 249
similar to that of the wild-type (Figure 6A) The spectra differed however around 685 nm 250
Here we observed an increase in fluorescence in the mutant strain an emission signature that 251
is probably related to the inner antenna protein CP43 (Figure 6A Nilsson et al 1992) 252
Increased chlorophyll fluorescence at 685 nm has previously been shown to be characteristic 253
for cells that are accumulating the stress-induced IsiA protein which shares structural 254
similarities with CP43 (Odom et al 1993 Yeremenko et al 2004 Wilson et al 2007) A 255
second indication for accumulation of IsiA is a diagnostic blue shift of the 725 nm 256
fluorescence peak by approximately 5 nm which is indeed observed in the curT- spectrum 257
(Figure 6A Sandstroumlm et al 2001) We therefore determined the level of IsiA in both wild-258
type and curT- by immunoblot analysis As expected IsiA was strongly induced in curT- 259
suggesting that the mutant suffers from severe stress (Figure 6C) When fluorescence at 77K 260
was recorded after excitation at 580 nm which mainly excites the phycobiliproteins the 261
signal at 685 nm (PSII related) was enhanced in the curT- mutant most likely reflecting IsiA 262
emission andor the presence of uncoupled phycobilisomes (Figure 6B Wilson et al 2007) 263
In contrast the 725 nm peak (PSI related) was reduced in curT- relative to the wild-type 264
suggesting less coupling of phycobilisomes to PSI (Figure 6B) The increase in uncoupled 265
phycobilisomes can be directly attributed to the reduction in RCCII levels found in the curT- 266
mutant (Figure 5A) since phycobilisomes are attached to PSII dimers for efficient light 267
harvesting (Watanabe and Ikeuchi 2013 Chang et al 2015) 268
Thus the curT- mutant exhibits a characteristic set of photosynthetic defects The 269
primary target of the CurT membrane-shaping function appears to be PSII in particular in its 270
biogenic phase However the reduction in PSII content cannot be responsible for the lack of 271
thylakoid convergence zones at the plasma membrane because these structures can still be 272
observed in the psbA- mutant TD41 which is lacking D1 and therefore unable to assemble any 273
PSII complexes (Supplemental Figure 6 Nilsson et al 1992) 274
275
Subcellular localization of CurT 276
We have previously proposed that the initial steps in PSII assembly take place in 277
biogenesis centers located at the interface between plasma and thylakoid membranes (Stengel 278
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
References 866
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phylogenetic map for chloroplast photosynthesis Trends Plant Sci 16 645-655 868
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assembly Plant Cell 22 3439-3460 875
Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
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thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
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Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
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the impact of antenna size and external factors on electron transport dynamics in 890
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Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
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Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
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Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
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of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
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cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
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your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
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Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
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Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
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Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
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PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
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Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
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Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
9
et al 2012) These biogenic membranes (PDMs) are marked by the PSII Mn2+ delivery factor 279
PratA and can be separated from thylakoids by a two-step sucrose-gradient centrifugation 280
procedure (Schottkowski et al 2009b Rengstl et al 2011) When the distributions of various 281
proteins within such membrane fractions from the wild-type and curT- were compared two 282
striking changes were detected in the mutant First the precursor of the D1 subunit of PSII 283
(pD1) is absent from PDMs in the mutant and secondly the inner antenna protein CP47 ndash but 284
not CP43 ndash of PSII shifts towards fractions of lower density (Figure 7) Hence the 285
organization of PDMs appears to be perturbed in curT- In wild-type cells most of the CurT 286
protein was found in thylakoid membranes only a minor fraction similar in amount to that of 287
the PSII assembly factors Ycf48 Pitt and YidC as well as the VIPP1 protein comigrated with 288
PDMs (Figure 7) According to rough estimates based on densitometric signal analysis 25 289
of total cellular CurT is normally found in the PDMs and 75 in the thylakoids (Figure 7) 290
To further analyze the localization of CurT in its cellular context in vivo we 291
constructed a translational fusion in which the monomeric enhanced cyan fluorescent protein 292
(CFP) mTurquoise2 (Goedhart et al 2012) is attached to the C-terminus of CurT and is 293
expressed under the control of the native curT promoter The resulting strain curT-CFP 294
showed a fully restored wild-type growth phenotype indicating that the CFP tag does not 295
affect CurTrsquos function (Supplemental Figure 7A) In agreement with this the fusion protein 296
accumulated to wild-type levels (96 plusmn 16 Supplemental Figure 7B) and localized to the 297
same membrane sub-fractions as does native CurT (Supplemental Figure 7C) The CurT-CFP 298
fluorescence signal was distinctly discernible above the wild-type autofluorescence 299
background (Supplemental Figure 8A) and was distributed in a network-like pattern with 300
concentrated areas at the cell periphery at mid-plane Analysis of CurT-CFP fluorescence in 301
Z-axis montages revealed that these signals often appeared as rod-like structures which 302
seemed to follow the spherical inner surface of the cell (Figure 8A B Supplemental Movie 1 303
and 2) In some cases these structures also appear to extend through the cytoplasm (Figure 304
8A and 8B Supplemental Movie 3) 305
When chlorophyll autofluorescence indicative of thylakoid membranes was visualized 306
in the same cell only a partial overlap with the CurT-CFP signal was observed (Figure 8 307
Supplemental Movie 1-3) Strikingly the peripheral CurT-CFP signals frequently reached 308
their maximal intensity in those areas where chlorophyll fluorescence was low ie where 309
thylakoid convergence zones are expected to form (Sacharz et al 2015) This becomes even 310
clearer when the intensity of a circumferential profile that follows the thylakoidrsquos 311
10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
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Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
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Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
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Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
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Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
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Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
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In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
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Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
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Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
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Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
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Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
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Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
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Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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10
fluorescence signal is quantified separately for each of the two fluorescent channels (Figure 312
8C) 313
In contrast the chlorophyll autofluorescence in the curT- mutant seems to be evenly 314
distributed (Supplemental Figure 9) Following the fluorescence in an intensity profile as in 315
curT-CFP no regions with a similar decline in fluorescence were found most likely due to 316
the absence of biogenesis centers In addition some circular structures presenting chlorophyll 317
autofluorescence were detected in the interior of the cells Hence the disturbed curT- 318
ultrastructure is also reflected in the distribution of chlorophyll pigments (Supplemental 319
Figure 9) 320
To confirm the network-like distribution of CurT-CFP in the cell we used 321
immunofluorescence to detect CurT in the wild-type in situ Synechocystis 6803 cells were 322
fixed and treated with the αCurT antibody and an Oregon Green-conjugated secondary 323
antibody As shown in Figure 9A and B both techniques revealed similar network-like 324
patterns of the CurT signal which coalesced into rod-like structures at the cell surface and 325
essentially filled up the gaps between the autofluorescent thylakoid regions (negative controls 326
shown in Supplemental Figure 8B and 8C) Again regions of low chlorophyll fluorescence 327
typical of biogenesis centers generally exhibited stronger CurT-related fluorescence (Figure 328
8C) Occasionally CurT-CFP signals were also observed near traversing thylakoid lamellae 329
(when these were present in the cell analyzed) but the autofluorescence intensity of such 330
thylakoids is much weaker (see Figure 8B cell b in slice 6 and Supplemental Movie 3) 331
Finally CurT was localized by immunogold labeling experiments on ultrathin sections 332
of wild-type Synechocystis 6803 cells Almost no signals were detected when wild-type and 333
curT- sections were processed in the absence of the primary antibody as a negative control 334
(Figure 10A and Supplemental Figure 10A respectively) When mutant curT- cells were 335
processed in the presence of the αCurT antiserum a low level of randomly distributed 336
background signals was observed (Supplemental Figure 10B Supplemental Table 1) 337
However the number of signals located at the thylakoid membrane was clearly reduced 338
relative to the wild-type Therefore these signals were treated as non-specific background 339
Upon incubation of wild-type cell sections with antibodies directed against the N-terminus of 340
CurT (Figure 1A) the antigen was detected on the cytoplasmic surface of thylakoid 341
membranes (Figure 10B and 10C) This strongly suggests that its N-terminus is oriented 342
towards the cytoplasm Overall CurT signals appeared to be distributed along both thylakoids 343
and PDMs This agrees with the broad distribution of CurT across various fractions in the 344
membrane fractionation experiments (Figure 7) In only a few cases (12 ) however was 345
11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
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Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
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1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
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Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
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Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
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localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
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supercomplex Photosynth Res 116 265-276 1048
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Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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11
some clustering of immunogold signals at thylakoid convergence regions found (Figure 10D 346
and 10E) Thus the high local concentration of CurT at biogenic centers seen in the 347
fluorescence-based approaches (Figures 8 and 9) was not quantitatively reflected in the 348
immunogold labeling data This is likely due to the fact that immunogold electron microscopy 349
can only detect antigens on the surface of the section This issue is further complicated by the 350
heterogeneity of the CurT assemblies (see below and Discussion section) 351
Nevertheless closer inspection of the immunogold signals revealed an asymmetric 352
distribution of CurT signals with regard to the two faces of thylakoid sheets As illustrated in 353
Figure 10G curved thylakoid sheets possess a longer convex and a shorter concave face 354
When we analyzed a total of approximately 1500 CurT immunogold signals (from a total of 355
95 cells) which were unambiguously located to one side of the thylakoid membrane (for a 356
representative example see Supplemental Figure 11) 561 were found to be located at the 357
convex face while the other 439 localized to the concave side (Figure 10G Supplemental 358
Table 2) When differently curved regions (Figure 10F) including thylakoid lamellae (i) that 359
follow the overall shape of the cell (green) (ii) bend away from the plasma membrane (red) or 360
(iii) towards it ie where thylakoids converge to form biogenesis centers (yellow) were 361
examined separately red and green regions showed a CurT distribution in the range of 55 362
convex to 45 concave (Figure 10G Supplemental Table 2) However at the biogenesis 363
centers (the regions highlighted in yellow in Figure 10F) the uneven distribution of the 364
immunogold signals was more pronounced 61 of signals were found on the convex side 365
and 39 on the concave face of thylakoid lamellae (Figure 10G Supplemental Table 2) 366
Statistical analyses confirmed the significance of these differences in signal distributions 367
along the thylakoid membrane types (Supplemental Table 3) 368
Taken together with the finding that CurT is required for the formation of the 369
convergence sites forming biogenesis centers these data are consistent with the idea that the 370
thylakoid sheets are shaped by CurT which induces membrane curvature via asymmetric 371
intercalation on the two sides of thylakoid sheet 372
373
Different forms of CurT are found in PDMs and thylakoids 374
CurT was found to localize to both highly curved PDMs and less bent thylakoids Its 375
high local concentration at biogenic centers as seen in the fluorescence-based approaches 376
suggests a dosage-dependent effect of CurT on membrane shaping An alternative possibility 377
is that different CurT variants might exist in the two membrane types which could potentially 378
serve different functions To explore this possibility we asked whether CurT is found in 379
12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
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Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
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Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
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Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
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1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
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Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
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localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
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supercomplex Photosynth Res 116 265-276 1048
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Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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12
different complexes by using 2D BNSDS-PAGE to characterize membrane material isolated 380
via two-step sucrose gradient centrifugation (see Figure 7) In accordance with the data in 381
Figure 5A we find several low-molecular-weight complexes containing CurT (Figure 11A) 382
Smaller complexes of ~80 (complex I) and ~100 kDa (complex II) accumulate in membrane 383
fraction 5 (representing PDMs) but these are far less prevalent in fraction 9 representing 384
thylakoids (Figure 11A) In addition CurT-containing sub-complexes of ~140 (complex III) 385
and ~200 kDa (complex IV) are more prominently represented in thylakoids together with 386
even larger complexes ranging up to 670 kDa (Figure 11A) 387
Even more strikingly PDMs and thylakoids also differ with respect to the forms of 388
CurT they contain Thus isoelectric focusing (IEF) analysis of both membrane fractions 389
revealed at least four different CurT isoforms (a-d Figure 11B) PDMs contain mainly forms 390
b and d as well as trace amounts of a while thylakoids apparently possess very low levels of 391
form d and accumulate forms a b and c (Figure 11B) The nature of the underlying CurT 392
modifications still has to be determined Interestingly however CurT has recently been 393
identified as a phosphoprotein in a proteomic study (Spaumlt et al 2015) and the CurT variants 394
in Figure 11B may differ in their phosphorylation states At all events PDMs and thylakoids 395
can be distinguished by their complement of CurT isoforms which potentially play a role in 396
formation of the CurT sub-complexes that define the two membrane fractions 397
398
CurT is involved in the osmotic stress response 399
To further explore the stress-related role of CurT (Figure 6) and its link to IsiA 400
induction the effect of other potential stressors on curT- cells was tested High light levels had 401
a minor effect on curT- growth and its photosynthetic performance (Figure 4 Supplemental 402
Figure 5 and section above) suggesting that other environmental factors induced isiA 403
expression Our search was also motivated by a previous proteomic survey of plasma 404
membrane proteins of Synechocystis 6803 in high salt conditions in which CurT and VIPP1 405
were found among the most highly induced targets (Huang et al 2006) First we cultured 406
wild-type and curT- cells in different salt concentrations and found that growth of curT- was 407
severely affected in high salt (Figure 12A) Wild-type cells were only slightly affected by the 408
addition of salt to the medium The deleterious effect of the loss of curT on growth was even 409
more pronounced when maltose an osmotically active compound was present in the medium 410
(Figure 12B) Indeed curT- cells failed to survive exposure to 150 mM maltose whereas 411
growth of the wild-type was only slightly compromised These findings assign an additional 412
function to CurT ie a protective role during osmotic stress 413
13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
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van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
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Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
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Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
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Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
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Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
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Proteomics 6 910-920 933
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Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
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DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
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Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
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PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
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Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
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Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
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Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
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plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
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Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
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Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
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Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
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Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
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supercomplex Photosynth Res 116 265-276 1048
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Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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13
When the curT-CFP strain was analyzed under these stress conditions enhanced 414
accumulation of CurT at the cell periphery was found mostly outside the autofluorescent 415
thylakoids and consistent with accumulation at the plasma membrane (Figure 12C) This was 416
further substantiated by membrane fractionation experiments which also revealed an 417
increased relative abundance of CurT in the plasma membrane fraction in the first sucrose 418
gradient (Figure 12E) Determination of total cellular CurT levels demonstrated that its 419
absolute amount did not increase under stress (Figure 12D) Therefore its higher abundance 420
in the plasma membrane under osmotic stress is likely to be due to CurT trafficking towards 421
the plasma membrane instead of increased synthesis 422
The same appears to hold true for VIPP1 which has been implicated in membrane 423
maintenance in both cyanobacteria and chloroplasts (Zhang and Sakamoto 2015) At stress 424
conditions an increase of the VIPP1 signal in the plasma membrane can be monitored in 425
wild-type (Figure 12E) However enhanced accumulation of VIPP1 in plasma membranes is 426
unaffected in the curT- mutant suggesting that thylakoid convergence zones are not strictly 427
required for the localization of VIPP1 428
429
Discussion 430
CurT determines thylakoid architecture 431
This study demonstrates that the protein CurT a homolog of the grana-forming 432
CURT1 proteins in plants is essential for establishing the proper thylakoid membrane 433
architecture in the cyanobacterium Synechocystis 6803 In particular CurTrsquos membrane-434
bending activity is likely to be required for the formation of thylakoid biogenesis centers at 435
points on the cell periphery where thylakoids converge towards the plasma membrane Here 436
PratA-mediated preloading of early PSII assembly intermediates with Mn2+ ions has been 437
proposed to take place together with PSII repair (Stengel et al 2012 Sacharz et al 2015) 438
This idea is supported by the following lines of evidence (i) The curT- mutant is devoid of 439
any thylakoid sectors that have convergence zones at their distal ends instead thylakoid 440
membranes are displaced and disposed as disordered or continuous rings (ii) CurT similarly 441
to CURT1A possesses a membrane-curving activity in vitro and intercalates asymmetrically 442
into thylakoids (iii) A high local concentration of CurT is observed at regions of high 443
curvature where biogenesis centers are found (iv) Lack of CurT affects the formation and 444
accumulation of PSII complexes as well as the abundance of some of its assembly factors 445
The drastic effects of CurT inactivation indicate an important role for this factor in membrane 446
shaping In contrast to the situation for CURT1A from Arabidopsis attempts to stably 447
14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
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Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
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Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
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Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify 878
thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
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complexes Plant Physiol Biochem 45 119-128 883
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Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
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Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
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28
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
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Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
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Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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14
overexpress CurT in Synechocystis 6803 failed Although short-lived transient increases in 448
CurT levels were observed when curT was expressed via strong heterologous promoters 449
wild-type levels of the protein were restored during subsequent rounds of cultivation of 450
transgenic lines Apparently Synechocystis 6803 cells do not tolerate substantial alterations in 451
CurT dosage and counter-select against these 452
This is in agreement with a dosage-dependent membrane-curving activity of CurT 453
which is suggested by its high local concentration at the edges of thylakoid autofluorescent 454
regions which mark the sites of the highly curved thylakoid convergence zones (Figures 8 9 455
and 13 Supplemental Movie 1 2 and 3) In addition some CurT was detected in structures 456
extending through the cytoplasm which most likely represent thylakoid sheets traversing the 457
cell from one thylakoid convergence zone to another (Figures 2A and 8) CurT might be 458
involved in the formation and stabilization of these structures 459
Members of the CURT1 family contain an N-terminal amphipathic α-helix that may 460
be involved in the membrane-bending activity of CurT (Armbruster et al 2013) Several 461
eukaryotic membrane-shaping proteins possess amphipathic helices as part of either an ENTH 462
(epsin N-terminal homology) or an N-BAR (Bin amphiphysin Rvs with N-terminal 463
amphipathic helix) domain (Ford et al 2002 Gallop and McMahon 2005 Zimmerberg and 464
Kozlov 2006) ENTH and N-BAR domains are rather large (~200 amino acids) and it has 465
been shown that amphipatic helices present in those domains insert into one leaflet of the lipid 466
bilayer and thereby induce curvature of the membrane (reviewed by Gallop and McMahon 467
2005 Zimmerberg and Kozlov 2006) However CurT itself is a small protein (149 amino 468
acids) that contains two transmembrane domains which insert into both leaflets of the lipid 469
bilayer (Figure 1A) as already suggested by Armbruster et al (2013) The amphipathic helix 470
faces the cytoplasm in Synechocystis 6803 or the stroma in A thaliana and most likely it 471
fine-tunes the degree of membrane bending mediated by CurT (Armbruster et al 2013) 472
In addition the increase in curvature correlated with CurT accumulation at convex 473
sides of thylakoid lamella (Figure 10 and 13) Hence curving might be mediated by 474
asymmetric integration of CurT into the lipid bilayers forming opposite faces of thylakoid 475
membrane sheets (Figure 13) However the mechanism causing this asymmetry remains 476
elusive 477
The presence of different CurT isoforms and complexes in PDMs and thylakoids adds 478
a further level of complexity and most likely reflects the dynamic regulation of this system 479
(Figure 11) The formation of different complexes might be primed by the phosphorylation of 480
CurT within its N-terminal cytoplasmic part (Spaumlt et al 2015) It seems likely that these 481
15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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phylogenetic map for chloroplast photosynthesis Trends Plant Sci 16 645-655 868
Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
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Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
D Jahns P Weisshaar B Nickelsen J and Leister D (2010) The Arabidopsis 873
thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II 874
assembly Plant Cell 22 3439-3460 875
Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
Rojahn U Jensen PE Rappaport F Joliot P Doumlrmann P Wanner G and 877
Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify 878
thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
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Bartsevich VV and Pakrasi HB (1995) Molecular identification of an ABC transporter 884
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Bartsevich VV and Pakrasi HB (1996) Manganese transport in the cyanobacterium 887
Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
Synechocystis PCC 6803 Photosynth Res 99 205-216 891
Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
248-254897
28
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
organization of an intact phycobilisome and its association with photosystem II Cell 899
Res 25 726-737 900
Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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15
complexes play distinct roles in mediating the different CurT functions ie membrane 482
bending and maintenance 483
However it remains to be seen how exactly curvature is established maintained and 484
fine-tuned In addition other determinants of membrane topology and their putative interplay 485
with CurT have to be considered eg lipid and protein (complex) composition of 486
membranes cellular turgor pressure and other factors required for membrane 487
biogenesismaintenance eg the VIPP1 protein before precise molecular models of CurTrsquos 488
mode of action can be constructed (Rast et al 2015 and see below) That these other factors 489
can be crucial for membrane architecture is documented by the fact that some marine 490
cyanobacteria such as Prochlorococcus and Synechococcus contain curved thylakoids (but 491
no thylakoid convergence zones at the plasma membrane) although they lack a curT gene 492
493
The role of biogenesis centers 494
The absence of biogenesis centers in the curT- mutant now enables one to answer some 495
questions relating to the role of these thylakoid sub-structures which form in some but not all 496
cyanobacteria (Kunkel 1982 van de Meene et al 2006) First they are not essential since 497
even the curT- mutant still accumulates PSII to ~50 of wild-type levels On the other hand 498
in the mutant assembly of PSII is impaired as revealed by the increased accumulation of early 499
assembly intermediates In addition PSII dimers are almost completely absent in curT- 500
Whether the reduction in PSII dimers is attributable to an assembly problem linked to the 501
absence of biogenesis centers or to a secondary defect in dimer stabilization caused by 502
changes in overall thylakoid membrane ultrastructure remains to be determined Thus 503
biogenesis centers are more likely to represent evolutionary ldquoadd-onsrdquo that facilitated efficient 504
thylakoid biogenesis This is compatible with the fact that some cyanobacteria are devoid of 505
any biogenesis center-related substructures Second their absence compromises PSII but the 506
accumulation of other photosynthetic complexes ie PSI and the Cyt(b6f) complex is not 507
affected by CurT deficiency (Figure 3) This observation agrees with previous findings that 508
neither the PsaA subunit of PSI nor its assembly factor Ycf37 is detectable in isolated PDM 509
fractions (Rengstl et al 2011) 510
In addition to reduced de novo PSII assembly a higher relative stability of D1 in high-511
light experiments has been detected in curT- which suggests that D1 degradation during PSII 512
repair is less efficient in the mutant Photo-damaged D1 is degraded by the FtsH2FtsH3 513
complex which has previously been shown to partly localize to thylakoid convergence zones 514
at the cell periphery ie biogenesis centers (Boehm et al 2012 Sacharz et al 2015) It 515
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
16
therefore seems possible that the absence of biogenesis centers in curT- leads to 516
mislocalization of the FtsH2FtsH3 complex and less effective D1 degradation 517
Moreover a PSII related-function for CurT is supported by a meta-analysis of the 518
Synechocystis 6803 transcriptome When the CyanoEXpress 22 database (Hernaacutendez-Prieto 519
and Futschik 2012 Hernaacutendez-Prieto et al 2016) was queried for genes co-expressed with 520
curT (slr0483) genes for five PSII subunits ndash psbE psbO psbF psbL and psb30 ndash were 521
found among the first ten hits (Supplemental Table 4) From this we infer that biogenic 522
centers do not play an essential role in the assembly of all photosynthetic complexes but 523
serve to enhance PSII assemblyrepair possibly through the efficient delivery of Mn2+ 524
Residual PSII in curT- cells is likely to be supplied with cytoplasmic Mn2+ via a second 525
independently operating ABC transporter-based system in the plasma membrane named the 526
Mnt pathway (Bartsevich and Pakrasi 1995 Bartsevich and Pakrasi 1996) 527
Formally we cannot exclude that CurT is directly involved in the PSII assembly 528
process However considering CurTrsquos membrane bending capacity and the mutant phenotype 529
(Figure 1D and Figure 2) it appears more likely that CurT forms cellular substructures for 530
efficient PSII biogenesis ie biogenesis centers 531
As mentioned above some ultrastructural features of biogenesis centers have emerged 532
from studies employing EM-based tomography (van de Meene et al 2006) To date however 533
a high-resolution picture of the precise membrane architecture within these centers remains 534
elusive In particular whether or not a direct connection between plasma membrane and 535
thylakoids exists continues to be debated As recently discussed and in line with this gap in 536
knowledge different membrane fractionation techniques have revealed different distributions 537
of PSII-related subunits and assembly factors between plasma and thylakoid membranes 538
(Heinz et al 2016 Liberton et al 2016 Selatildeo et al 2016) The overall picture that emerges 539
is that the initial hypothesis that PSII biogenesis is initiated at the plasma membrane is no 540
longer tenable whereas a specialized thylakoid sub-fraction like the PDMs that make contact 541
with the plasma membrane could explain many of the available data and thus clarify some 542
aspects of the debate (Zak et al 2001 Pisareva et al 2011 Rast et al 2015) Moreover it 543
has previously been hypothesized that ldquoribosome-decorated membrane-like complexesrdquo 544
forming at the innermost thylakoid sheet might represent biogenic compartments (van de 545
Meene et al 2006 Mullineaux 2008) Whether these structures are affected in curT- cannot 546
be judged based on our TEM analysis but requires ultrastructural data of higher resolution 547
However the accessibility of thylakoids for ribosomes should be increased in the less 548
compressed membrane system of curT- (Figure 2B-D) 549
17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
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Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
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Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
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Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
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Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
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Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
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DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
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PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
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Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
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Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
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plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
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Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
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Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
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Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
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Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
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supercomplex Photosynth Res 116 265-276 1048
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Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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17
A scenario similar to that of Synechocystis 6803 holds for the situation in chloroplasts 550
of the green alga Chlamydomonas reinhardtii in which de novo PSII biogenesis has been 551
shown to be initiated in punctate regions named T-zones located close to the pyrenoid 552
(Uniacke and Zerges 2007) Unlike the mRNAs for the PSII components neither RNAs for 553
PSI subunits nor PSI assembly factors are localized to T-zones indicating that PSI and PSII 554
assembly processes are spatially separated (Uniacke and Zerges 2009 Nickelsen and Zerges 555
2013 Rast et al 2015) Interestingly a recent cryoelectron tomography study demonstrated 556
that next to T-zones at the chloroplast base-lobe junction the inner chloroplast envelope 557
exhibits invaginationsconnections to thylakoids that structurally resemble the organization of 558
cyanobacterial biogenesis centers (Engel et al 2015) For future investigations it will be 559
interesting to see if a homolog of the CURT1 family is involved in the formation of these 560
structures in C reinhardtii 561
562
Evolution of CURT1 function 563
Homologs of the CURT1 protein family can be found throughout cyanobacteria green 564
algae and plants (Armbruster et al 2013) The Arabidopsis CURT1A-D proteins have 565
recently been shown to induce bending of thylakoid membranes at grana margin regions 566
(Armbruster et al 2013) Interestingly and in contrast to the situation in Synechocystis 6803 567
their inactivation did not result in severe photosynthetic deficiencies although the thylakoids 568
formed lacked defined grana stacks Nevertheless the data available support the idea that the 569
function of thylakoid membrane structures that were regarded as being independent ie 570
cyanobacterial biogenesis centers and grana are closely related Intriguingly both of these 571
thylakoid membrane structures are dedicated to aspects of PSII function but do not involve 572
PSI (Pribil et al 2014) In both the prokaryotic and eukaryotic systems CURT1 homologs 573
appear to bend thylakoid membranes as indicated by (i) the distortion of membrane 574
ultrastructure seen in mutants devoid of curved thylakoids (ii) the localization of CURT1 575
homologs at grana margins and their local concentration at biogenesis centers (iii) the in vitro 576
membrane-curving activity of both CURT forms The phenotypic differences between 577
A thaliana and Synechocystis 6803 mutants may however be attributable to the different 578
functions of the membrane sub-compartments affected in the two model organisms Whereas 579
the role of grana is still under debate one function of the cyanobacterial biogenesis centers is 580
the above-mentioned high-throughput uptake of Mn2+ ions for efficient assembly of PSII 581
(Stengel et al 2012 Nickelsen and Rengstl 2013 Pribil et al 2014) However it appears 582
that in both cases the primary function of CURT1 proteins is to generate curved membrane 583
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
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Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
18
regions These discoveries suggest that until now two independently observed structures ie 584
cyanobacterial thylakoid biogenesis centers and chloroplast grana regions are closely related 585
The degree of curvature is much less pronounced in the cyanobacterial system As previously 586
discussed it was probably the subsequent evolution of a membrane-localized light-harvesting 587
system in chloroplasts that made the formation of tightly curved grana thylakoids possible 588
(Mullineaux 2005 Nevo et al 2012 Pribil et al 2014) 589
590
CurT is involved in osmotic stress response 591
One unexpected phenotype of the curT- mutant is its sensitivity to osmotic stress 592
These data revealed that in addition to shaping membranes CurT also influences their 593
functional integrity This latter function might be intrinsic to CurT or could be mediated by 594
the VIPP1 protein which has been shown to be required for membrane maintenance in 595
bacteria and chloroplasts probably by acting as a supplier of lipids (for an overview see 596
Zhang and Sakamoto 2015) Both factors accumulate in the plasma membrane upon osmotic 597
stress but VIPP1 localization is not severely affected by CurT inactivation which suggests 598
that VIPP1 operates independently of CurT In agreement with this repeated co-599
immunoprecipitations revealed no interaction between both proteins Therefore it remains to 600
be established whether a direct functional relationship between them exists 601
Previous investigations of osmotic stress in Synechocystis 6803 showed a kidney-602
shaped form of wild-type cells under very high concentrations of osmotically active 603
compounds (Marin et al 2006) The cells were severely deformed indicating changes in the 604
structure of the cellular envelope Since CurT shifts towards the plasma membrane already 605
under lower osmolyte concentrations we suggest a stabilizing function of CurT in the plasma 606
membrane CurT might be necessary to tolerate the forces trying to deform the cells under 607
osmotic stress 608
In conclusion this analysis has identified a crucial determinant for shaping and 609
maintaining the cyanobacterial thylakoid membrane system ie CurT a homolog of the 610
grana-forming CURT1 protein family from A thaliana In particular CurT is involved in the 611
formation of specific thylakoid substructures close to the plasma membrane at which PSII is 612
assembledrepaired Future work will dissect the precise ultrastructure of these centers and 613
enable us to elaborate a molecular model for the spatiotemporal organization of PSII assembly 614
in cyanobacteria 615
616
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
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Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
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thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II 874
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Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
Rojahn U Jensen PE Rappaport F Joliot P Doumlrmann P Wanner G and 877
Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify 878
thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
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complexes Plant Physiol Biochem 45 119-128 883
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Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
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Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
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28
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
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Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
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Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
19
Methods 617
Strains and growth conditions 618
Wild-type and mutant Synechocystis 6803 cells were grown on solid or in liquid BG 619
11 medium (Rippka et al 1979) supplemented with 5 mM glucose (unless indicated 620
otherwise) at 30degC under continuous illumination at a photon irradiance of 30 μmol m-2 s-1 of 621
white light For growth during high-light experiments for investigation of D1 repair cells 622
were cultivated in a FMT150 photobioreactor (Photon Systems Instruments Draacutesov Czech 623
Republic) at 800 micromol photons m-2 s-1 in the presence or absence of lincomycin (100 microgml) 624
Cell density was monitored photometrically at 750 nm Doubling times were determined after 625
two days of growth To generate the insertion mutant curT- the curT gene (slr0483) was first 626
amplified from wild-type genomic DNA by PCR using the primer pair 04835 627
(GAAGCCTATTTAGCTAAGGCCGAAAG) and 04833 628
(TAGTACCTGGTCTTCCATGGCGT) and the resulting fragment was cloned into the 629
Bluescript pKS vector (Stratagene Waldbronn Germany) A kanamycin resistance cassette 630
was then inserted into the single AgeI restriction site (159 bp downstream of the start codon) 631
and this disruption construct was used to replace the wild-type gene as described (Wilde et al 632
2001) 633
634
Bioinformatic and computational analysis 635
CURT1 sequences of Synechocystis 6803 were obtained from CyanoBase 636
(httpgenomekazusaorjpcyanobaseSynechocystis) and CURT1 homologs in other 637
organisms were retrieved from NCBIBLAST (httpblastncbinlmnihgovBlastcgi) 638
Numbers and positions of putative transmembrane domains in the predicted proteins were 639
determined with TMHMM20 (httpwwwcbsdtudkservicesTMHMM) The sequence for 640
the N-terminal amphipathic helix was predicted by Jpred 3 641
(httpwwwcompbiodundeeacukwww-jpred) and plotted using a helical wheel projection 642
script (httprzlabucreduscriptswheelwheelcgi) The programs ClustalW2 643
(httpwwwebiacuktoolsclustalw2) and GeneDoc (httpwwwpscedubiomedgenedoc) 644
were used to generate multiple alignments of amino acid sequences Quantitative analysis of 645
immunoblots was performed with the AIDA Image Analyzer V325 (Raytest 646
Isotopenmessgeraumlte GmbH Straubenhardt Germany) 647
Co-expression patterns of curT were analyzed using the CyanoEXpress 22 database 648
(httpcyanoexpresssysbiolabeu Hernaacutendez-Prieto and Futschik 2012 Hernaacutendez-Prieto et 649
al 2016) 650
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
20
651
Measurement of chlorophyll concentration and oxygen production 652
Determination of chlorophyll content was carried out according to Wellburn and 653
Lichtenthaler (1984) For oxygen evolution measurements cells were grown in BG-11 654
without glucose harvested in mid-log phase and diluted to an OD750 nm = 05 Oxygen 655
evolution rates were measured with a Clark-type oxygen electrode (Hansatech Instruments 656
Ltd Kingrsquos Lynn Norfolk UK) at 1000 micromol photons m-2 s-1 (cool white light) after 5 min of 657
dark acclimation Oxygen evolution under saturating light conditions was measured without 658
any additives 659
660
Cell size and cell number estimation 661
Synechocystis 6803 strains were grown in liquid BG11 media containing 5 mM 662
glucose For cell size estimation the cells were analyzed with a confocal laser scanning 663
microscope TCS SP5 (Leica Wetzlar Germany) The diameter of the cells was measured 664
with the LAS AF Lite software (Leica Wetzlar Germany) Cells that were about to divide 665
were not taken into account For the determination of cell number cultures were set to an 666
OD750 = 1 and analyzed by using a Neubauer counting chamber (Paul Marienfeld GmbH amp 667
Co KG Lauda-Koumlnigshofen Germany) Statistical significance was determined by Studentrsquos 668
t-test 669
670
Measurements of P700+ reduction kinetics and relative electron transport rates 671
P700+ reduction kinetics and changes in chlorophyll fluorescence yield at different 672
light intensities were measured at a chlorophyll concentration of 5 microgml with a Dual-PAM-673
100 instrument (Heinz Walz GmbH Effeltrich Germany) Complete P700 oxidation was 674
achieved by a 50-ms multiple turnover pulse (10000 micromol photons m-2 s-1) after 2 min of dark 675
incubation P700+ reduction kinetics were recorded without any additions as well as in the 676
presence of 10 microM DCMU Ten technical replicates were averaged and fitted with single 677
exponential functions Data interpretation was done according to Bernaacutet et al (2009) 678
Light saturation measurements were performed according to Xu et al (2008) The 679
actinic light intensity was increased stepwise from 0 to 665 micromol photons m-2 s-1 with 30-s 680
adaptation periods Maximal fluorescence yields were obtained by applying saturating light 681
pulses (duration 600 ms intensity 10000 micromol photons m-2 s-1) 682
683
Low-temperature fluorescence measurements 684
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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phylogenetic map for chloroplast photosynthesis Trends Plant Sci 16 645-655 868
Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
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Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
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of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
21
Fluorescence emission spectra were measured with an AMINCO Bowman Series 2 685
Luminescence Spectrometer Wild-type and curT- cells were grown in the presence of 5 mM 686
glucose to mid log-phase and concentrated to 25 microgml chlorophyll adding 2 microM fluorescein 687
as an internal standard Samples (1 ml) were transferred to thin glass tubes dark-adapted for 688
30 min at 30 degC and shock frozen in liquid N2 To quantify chlorophyllfluorescein and 689
phycobilisome-mediated fluorescence samples were excited and emission recorded at 440 690
nm510 nm (chlorophyll and fluorescein excitationmax fluorescein emission) and 580 691
nm685 nm respectively Emission was scanned at 490-800 nm or at 640-800 nm 692
respectively 693
694
Antibody production and protein analysis 695
For generation of an αCurT antibody the N-terminal coding region of curT (amino 696
acid positions 1-58) was amplified by PCR using primers N04835 697
(AAGGATCCGTGGGCCGTAAACATTCAA) and N04833 698
(AAGTCGACGCACTTCCCACACCGGTTGTA) and cloned into the BamHI and XhoI sites 699
of the pGEX-4T-1 vector (GE Healthcare Freiburg Germany) Expression of the GST fusion 700
protein in Escherichia coli BL21(DE3) and subsequent affinity purification on Glutathione-701
Sepharose 4B (GE Healthcare) were carried out as described (Schottkowski et al 2009b) A 702
polyclonal antiserum was raised against this protein fragment in rabbits (Pineda Berlin 703
Germany) Other primary antibodies used in this study have been described previously ie 704
αpD1 (Schottkowski et al 2009b) αD1 (Schottkowski et al 2009b) αD2 (Klinkert et al 705
2006) αPratA (Klinkert et al 2004) αYcf48 (Rengstl et al 2011) αPitt (Schottkowski et 706
al 2009a) αPOR (Schottkowski et al 2009a) αYidC (Ossenbuumlhl et al 2006) αVIPP1 707
(Aseeva et al 2007) αSll0933 (Armbruster et al 2010) and αSlr0151 (Rast et al 2016) or 708
were purchased from Agrisera (Vaumlnnaumls Sweden) ie αCP43 αCP47 αPsaA αCyt f 709
αRbcL αIsiA The αGFP-HRP antibody was acquired from Miltenyi Biotech (Bergisch 710
Gladbach Germany) 711
Protein concentrations were determined according to Bradford (1976) using RotiQuant 712
(Carl Roth GmbH amp Co KG Karlsruhe Germany) Protein extraction membrane 713
fractionation on sucrose-density gradients and quantification of Western signal intensities was 714
performed as described previously (Rengstl et al 2011) Solubility properties of CurT were 715
determined according to Schottkowski et al (2009a) 716
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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complex for manganese analysis of a cyanobacterial mutant strain impaired in the 885
photosynthetic oxygen evolution process EMBO J 14 1845-1853 886
Bartsevich VV and Pakrasi HB (1996) Manganese transport in the cyanobacterium 887
Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
Synechocystis PCC 6803 Photosynth Res 99 205-216 891
Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
J (2012) Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH893
Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
248-254897
28
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
organization of an intact phycobilisome and its association with photosystem II Cell 899
Res 25 726-737 900
Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann D Jahns P Weisshaar B NickelsenJ and Leister D (2010) The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem IIassembly Plant Cell 22 3439-3460
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
22
Sucrose-density centrifugation separating PDM and thylakoid membrane fractions by 717
two consecutive gradients was carried out as described previously (Schottkowski et al 718
2009b Rengstl et al 2011) 719
Two-dimensional fractionations of cyanobacterial membrane proteins via BNSDS-720
PAGE and of pulse-labeled proteins by BNSDS-PAGE were performed as previously 721
reported (Klinkert et al 2004 Schottkowski et al 2009b Rengstl et al 2013) For the 722
analysis of native complexes in fractions obtained by sucrose-density centrifugation the 723
volume corresponding to 350 microg of protein in fraction 7 was applied to BNSDS-PAGE 724
For isoelectric focusing (IEF) the volume corresponding to 50 microg of protein from 725
fraction 7 in 200 microl of IEF buffer (8 M urea 4 CHAPS 1 IPG buffer pH 4-7 (GE 726
Healthcare)) was applied to an 11-cm Immobiline DryStrip pH 4-7 (GE Healthcare) in a 727
Protean IEF Cell (Bio-Rad Munich Germany) Strips were rehydrated for 12 h at 20 degC and 728
50V Focusing was carried out in the following steps 500 V rapid 1500 Vh 1000 V linear 729
880 Vh 6000 V linear 9680 Vh 6000 V rapid 5390 Vh 50 microA per gel focus temperature 730
20degC Subsequently the strips were incubated in equilibration buffer I (6 M urea 0375 M 731
Tris pH 88 20 glycerol 2 SDS 2 DTT) and equilibration buffer II (6 M urea 0375 732
M Tris pH 88 20 glycerol 2 SDS 25 iodoacetamide) for 10 min in each buffer 733
Then the IEF strips were applied to second-dimension SDS-PAGE 734
735
Transmission electron microscopy and immunogold labeling 736
Sample preparation for transmission electron microscopy including freeze substitution 737
and immunogold labeling of CurT was performed as described previously (Stengel et al 738
2012) Ultrathin sections were cut to thicknesses of 35 - 45 nm and 45 - 65 nm for 739
ultrastructural analyses and immunogold labeling respectively For immunogold labeling 740
sections collected on collodion-coated Ni grids were incubated for 18 h at 4degC with (rabbit) 741
αCurT (diluted 1100 1500 11000 or 14000 in blocking buffer (5 fetal calf serum) with 742
005 Tween 20) and further processed as described previously (Stengel et al 2012) As a 743
negative control wild-type Synechocystis 6803 cells were incubated without the primary 744
antibody and exposed to the gold-labeled anti-rabbit IgG To image the ultrastructure and the 745
immunogold-treated samples a Fei Morgagni 268 transmission electron microscope was used 746
and micrographs were taken at 80 kV 747
To avoid errors in the interpretation of the immunogold labeling experiment only 748
signals which were unambiguously localized to distinctly visible thylakoid membranes were 749
taken into account For each cell different regions (see Figure 10F) were defined and in each 750
23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
References 866
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Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
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Lond B Biol Sci 367 3515-3524 871
Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
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thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II 874
assembly Plant Cell 22 3439-3460 875
Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
Rojahn U Jensen PE Rappaport F Joliot P Doumlrmann P Wanner G and 877
Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify 878
thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
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complexes Plant Physiol Biochem 45 119-128 883
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photosynthetic oxygen evolution process EMBO J 14 1845-1853 886
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Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
Synechocystis PCC 6803 Photosynth Res 99 205-216 891
Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
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Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
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Res 25 726-737 900
Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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23
region the signals on the convex and concave sides of the thylakoid membrane were analyzed 751
separately (see Supplemental Figure 11 for representative counts) Statistical significance was 752
assessed by χ2 test (Zoumlfel 1988) 753
For the analysis of clusters of CurT signals in biogenesis centers a cluster was defined 754
as a minimum of four signals in close proximity Overall 219 biogenesis centers were 755
examined with regard to CurT cluster formation 756
757
Preparation of proteoliposomes 758
Liposomes with a lipid mixture of 25 monogalactosyl diacylglycerol 42 759
digalactosyl diacylglycerol 16 sulfoquinovosyl diacylglycerol and 17 760
phosphatidylglycerol (Lipid Products South Nutfield UK) were prepared as described 761
previously (Armbruster et al 2013) To generate proteoliposomes CurT and CURT1A 762
proteins were expressed in the presence of liposomes for 3 h at 37degC using a PURExpress In 763
Vitro Protein Synthesis Kit (New England Biolabs Ipswich MA USA) The liposomes were 764
separated from the mix by centrifugation in a discontinuous sucrose gradient (100000 g 18 765
h) Subsequently the fraction containing the liposomes was extracted and dialyzed against 20 766
mM HEPES (pH 76) For transmission electron microscopy the proteoliposomes were 767
negatively stained with 1 uranyl acetate on a carbon-coated copper grid Micrographs were 768
taken on a Fei Morgagni 268 TEM at 80 kV 769
770
curT-CFP strain generation 771
Gibson assembly was used for single-step cloning (Gibson 2011) of the slr0483 gene 772
(together with its ~08 kb upstream region) the mTurquoise2 gene fragment the gentamycin-773
resistance cassette (aacC1) and an ~17-kb downstream region of slr0483 into pBR322 774
cleaved with EcoRV and NheI The slr0483 gene was fused in frame to the codon-optimized 775
mTurquoise2 (DNA20) preceded by a linker sequence encoding GSGSG The resulting 776
plasmid was used to transform Synechocystis 6803 as described previously (Zang et al 2007) 777
After ~ 10 days of incubation on BG11-GelRite (15 ) medium in the presence of 2 microgml 778
gentamycin several antibiotic resistant colonies were obtained and streaked out on fresh 779
medium These transformants were then tested for full segregation of the tagged allele by 780
colony PCR with primers flanking the slr0483 gene 781
782
Fluorescence microscopy 783
24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
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Lond B Biol Sci 367 3515-3524 871
Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
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thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II 874
assembly Plant Cell 22 3439-3460 875
Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
Rojahn U Jensen PE Rappaport F Joliot P Doumlrmann P Wanner G and 877
Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify 878
thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
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complexes Plant Physiol Biochem 45 119-128 883
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Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
Synechocystis PCC 6803 Photosynth Res 99 205-216 891
Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
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Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
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Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
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Res 25 726-737 900
Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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24
An aliquot of a cell suspension in mid-log phase was placed under a BG11-agarose 784
pad on a glass coverslip (15) and imaged on an inverted microscope (Zeiss 785
AxioObserverZ1) equipped with an ORCA Flash40 V2 (Hamamatsu) camera a Lumenecor 786
SOLA II Light Engine and a Plan-Apochromat 63x140 Oil DIC M27 (NA 14) objective 787
(Zeiss) Image acquisition was done with Zen 21 software in cyan and far-red fluorescence 788
channels (Zeiss Filter Sets 47 HE and 50) as Z-series with a step size of 250 nm and effective 789
pixel size of 103 nm Acquired image files were subsequently deconvolved (Constrained 790
Iterative Method) using a default theoretical Point Spread Function in Zen 20 (Zeiss) 791
Extraction of intensities for line profiles was performed by image thresholding the thylakoid 792
autofluorescence channel and eroding the resulting binary mask in Fiji (Schindelin et al 793
2012) 794
For immunofluorescence analysis cells were grown to mid-log phase as in the case of 795
the cells prepared for live-cell microscopy A 10-ml culture was fixed for 20 min in 37 796
formaldehyde and following extensive washing with phosphate-buffered saline (PBS) cells 797
were first permeabilized in 005 Triton-X for 20 min and then in lysis buffer (50 mM Tris-798
HCl 100 mM NaCl 5 mgml lysozyme) at 37degC for 2 h with gentle shaking To block 799
unspecific binding sites the cell pellet was incubated in 5 bovine serum albumin in PBS at 800
30degC for 1 h The αCurT serum (1500 dilution) was added to cells in fresh blocking buffer 801
for 1 h at 30degC with gentle shaking Cells were then washed three times with blocking buffer 802
and incubated with goat anti-rabbit IgG secondary antibodies conjugated to Oregon Green 488 803
(Invitrogen) (11000 dilution) under the conditions as for the primary antibodies Cells were 804
washed three times with PBS and prepared for microscopy as above Imaging was done as 805
described for live-cell microscopy except that a Colibri2 LED light source was employed for 806
illumination and a CoolSnap HQ camera (Photometrics) was used for image acquisition Cells 807
were imaged as Z-series (step size 280 nm) in the yellow and far-red fluorescent channels 808
(Zeiss Filter Sets 46 HE and 50) using the 505 nm and 625 nm LED modules Effective pixel 809
size in the final image was 102 nm Deconvolution and image analysis was done as described 810
above As judged by the staining intensity of the Oregon Green-conjugated antibodies 811
approximately half of the cells in any given field of view appeared to have been successfully 812
permeabilised a rate that is common in our experience No significant signal was detected in 813
the control sample that had not been exposed to the primary antibodies 814
815
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
References 866
Allen JF de Paula WBM Puthiyaveetil S and Nield J (2011) A structural 867
phylogenetic map for chloroplast photosynthesis Trends Plant Sci 16 645-655 868
Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of 869
dynamic structural changes of plant thylakoid architecture Philos Trans R Soc 870
Lond B Biol Sci 367 3515-3524 871
Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann 872
D Jahns P Weisshaar B Nickelsen J and Leister D (2010) The Arabidopsis 873
thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II 874
assembly Plant Cell 22 3439-3460 875
Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP 876
Rojahn U Jensen PE Rappaport F Joliot P Doumlrmann P Wanner G and 877
Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify 878
thylakoid architecture by inducing membrane curvature Plant Cell 25 2661-2678 879
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J 880
Wanner G Westhoff P Soll J and Vothknecht UC (2007) Vipp1 is required for 881
basic thylakoid membrane formation but not for the assembly of thylakoid protein 882
complexes Plant Physiol Biochem 45 119-128 883
Bartsevich VV and Pakrasi HB (1995) Molecular identification of an ABC transporter 884
complex for manganese analysis of a cyanobacterial mutant strain impaired in the 885
photosynthetic oxygen evolution process EMBO J 14 1845-1853 886
Bartsevich VV and Pakrasi HB (1996) Manganese transport in the cyanobacterium 887
Synechocystis sp PCC 6803 J Biol Chem 271 26057-26061 888
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production 889
the impact of antenna size and external factors on electron transport dynamics in 890
Synechocystis PCC 6803 Photosynth Res 99 205-216 891
Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield 892
J (2012) Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH893
Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683894
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram 895
quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72 896
248-254897
28
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural 898
organization of an intact phycobilisome and its association with photosystem II Cell 899
Res 25 726-737 900
Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
Mamedov F (2006) Dimeric and monomeric organization of photosystem II 902
Distribution of five distinct complexes in the different domains of the thylakoid 903
membrane J Biol Chem 281 14241-14249 904
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement 905
of photosystem II and ATP synthase in chloroplast membranes of Spinach and Pea 906
Plant Cell 22 1299-1312 907
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
(2015) Native architecture of the Chlamydomonas chloroplast revealed by in situ 909
cryo-electron tomography eLife 4 e04889 910
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon 911
HT (2002) Curvature of clathrin-coated pits driven by epsin Nature 419 361-366 912
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) 913
Phosphorylation of photosystem II controls functional macroscopic folding of 914
photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964 915
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing 916
your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
Parsed CitationsAllen JF de Paula WBM Puthiyaveetil S and Nield J (2011) A structural phylogenetic map for chloroplast photosynthesisTrends Plant Sci 16 645-655
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
25
Accession numbers 816
Sequence data from this article can be found in the CyanoBase and EMBLGenBank 817
data libraries under accession number slr0483 818
819
Supplemental Data 820
Supplemental Figure 1 Construction of the curT- mutant 821
Supplemental Figure 2 Growth phenotype of curT- 822
Supplemental Figure 3 Expression analysis of the open reading frame slr0482 (located 823
upstream of curT) in the curT- strain 824
Supplemental Figure 4 Absorbance spectra of curT- and wild-type cells 825
Supplemental Figure 5 High-light growth phenotypes of wild-type Synechocystis 6803 and 826
the curT- strain 827
Supplemental Figure 6 Ultrastructure of the TD41 mutant which lacks the psbA transcript 828
and the D1 protein 829
Supplemental Figure 7 Expression and localization of CurT-CFP 830
Supplemental Figure 8 Localization of CurT and CurT-CFP by fluorescence microscopy 831
Supplemental Figure 9 Chlorophyll autofluorescence of curT- 832
Supplemental Figure 10 The curT- mutant as a negative control for immunogold labeling 833
Supplemental Figure 11 Representative counts of immunogold signals 834
Supplemental Table 1 Overview of immunogold signals in curT- cells 835
Supplemental Table 2 Overview of immunogold signals in specific regions of Synechocystis 836
6803 wild-type cells 837
Supplemental Table 3 Statistical analysis of immunogold signals for wild-type 838
Synechocystis 6803 cells 839
Supplemental Table 4 Co-expression of curT 840
Supplemental Movie 1 Scan through the Z-axis of a curT-CFP cell 841
Supplemental Movie 2 Rendered 3D-model of a CurT-CFP detection 842
Supplemental Movie 3 Scan through the Z-axis of a curT-CFP cell with a traversing 843
thylakoid lamella 844
845
Acknowledgements 846
We thank Y Nishiyama for providing αpD1 and J Soll for αVIPP1 and αYidC antibodies 847
respectively and Nina Dyczmons-Nowaczyk for support with the fluorescence measurements 848
This work was supported by grants awarded to JN (Ni3909) MMN (NO 8363) and DL 849
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments In Methods in Enzymology VChristopher ed (Academic Press) pp 349-361
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Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA van Weeren L Gadella TWJ andRoyant A (2012) Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751
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Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II biogenesis in cyanobacteria BiochimBiophys Acta 1857 274-287
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for exploration and visualisation of the integratedtranscriptome of cyanobacterium Synechocystis sp PCC6803 Bioinformation 8 634-638
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The Transcriptional Landscape of thePhotosynthetic Model Cyanobacterium Synechocystis sp PCC6803 Sci Rep 6 22168
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-stress-induced changes in plasmamembranes of Synechocystis sp strain PCC 6803 Proteomics 6 910-920
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 116 481-487Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by asecondary RNA structure blocking the AUG start codon Nucleic Acids Res 34 386-394
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) PratA a periplasmic tetratricopeptiderepeat protein involved in biogenesis of photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644
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Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The cyanobacterial homologue ofHCF136YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly andrepair of photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399
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Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) Comparative analysis of the primarytranscriptome of Synechocystis sp PCC 6803 DNA Res 21 527-539
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial photosynthetic membrane system ArchMicrobiol 133 97-99
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Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure of the membrane systems in theunicellular cyanobacterium Synechocystis sp strain PCC 6803 Protoplasma 227 129-138
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Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD Koppenaal DW and Pakrasi HB (2016) GlobalProteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium Mol CellProteomics 15 2021-2032
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Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid curvature and gene translation TrendsPlant Sci 19 63-66
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Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress in Synechocystis sp PCC 6803 lowtolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation Microbiology 1522023-2030
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Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the photosynthetic apparatus In TheCyanobacteria A Herrero and E Flores eds (Norfolk UK Caister Academic Press) pp 289-304
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Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants Annu Rev Plant Biol 64 609-635Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial cell biology Front Plant Sci 4 458Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
26
(Le126529) by the Deutsche Forschungsgemeinschaft in the context of Research Unit 850
FOR2092 and the Cluster of Excellence RESOLV (EXC 1069) AG is a Howard Hughes 851
Medical Institute postdoctoral fellow in the laboratory of Erin OrsquoShea at Harvard University 852
LS was supported by a PhD fellowship from the Chinese Scholarship Council 853
854
Author Contributions 855
SH AR LS MMN DL and JN designed the research SH AR LS AG ILG EH ML BR 856
SV performed the research all authors contributed to data analysis SH AR and JN wrote the 857
paper 858
859
Competing Financial Interest statement 860
The authors declare no competing financial interests 861
862
We wish to dedicate this paper to Fabrice Rappaport an outstanding colleague and friend who 863
passed away so early 864
865
27
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your curves to the BAR Biochem Soc Symp 72 223-231 917
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
In Methods in Enzymology V Christopher ed (Academic Press) pp 349-361 919
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA 920
van Weeren L Gadella TWJ and Royant A (2012) Structure-guided evolution of 921
cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 922
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B (2011) Model for Membrane Organizationand Protein Sorting in the Cyanobacterium Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate SequenceAnalyses J Proteome Res 10 3617-3631
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Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land plants J Exp Bot 65 1955-1972Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim Biophys Acta 1847 821-830Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a tetratricopeptide repeat protein fromSynechocystis sp PCC 6803 during Photosystem II assembly and repair Front Plant Sci 7 605
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane Subfraction in Cyanobacteria Is Involved inan Assembly Network for Photosystem II Biogenesis J Biol Chem 286 21944-21951
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a Synechocystis double mutant lacking thephotosystem II assembly factors YCF48 and Sll0933 Planta 237 471-480
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) The Plasma Membrane of theCyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains Plant Cell 23 2379-2390
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic Assignments Strain Histories andProperties of Pure Cultures of Cyanobacteria Microbiology 111 1-61
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystembiogenesis Plant Signal Behav 8 e27037
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux CW (2015) Sub-cellular location ofFtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoidmembranes Mol Microbiol 96 448-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43 the isiA Gene Product Functions as an Excitation EnergyDissipator in the Cyanobacterium Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S SchmidB Tinevez J-Y White DJ Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-source platform forbiological-image analysis Nat Methods 9 676-682
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel tetratricopeptide repeat proteininvolved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 MolPlant 2 1289-1297
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E and Nickelsen J (2009b) Interaction ofthe periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J BiolChem 284 1813-1819
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II Assembly Steps Take Place in theThylakoid Membrane of the Cyanobacterium Synechocystis sp PCC6803 Plant Cell Physiol 57 878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spaumlt P Macek B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium Synechocystis sp PCC 6803 and itsdynamics during nitrogen starvation Front Microbiol 6 248
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial steps of photosystem II de novo assemblyand preloading with manganese take place in biogenesis centers in Synechocystis Plant Cell 24 660-675
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially localized in Chlamydomonas Plant Cell 193640-3654
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized translation in Chlamydomonas Proc Natl AcadSci USA 106 1439-1444
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The three-dimensional structure of thecyanobacterium Synechocystis sp PCC 6803 Arch Microbiol 184 259-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and Roberson RW (2012) Gross morphologicalchanges in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 BiochimBiophys Acta 1818 1427-1434
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting supercomplex Photosynth Res 116 265-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B ofLeaf Extracts in Different Solvents In Advances in Photosynthesis Research Proceedings of the VIth International Congress onPhotosynthesis Brussels Belgium August 1-6 1983 C Sybesma ed (Dordrecht Springer Netherlands) pp 9-12
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of the cyanobacterial ycf37 mutationdecreases the photosystem I content Biochem J 357 211-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced Energy Dissipation in Iron-StarvedCyanobacteria Roles of OCP and IsiA Proteins Plant Cell 19 656-672
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) Properties of Mutants of Synechocystis spStrain PCC 6803 Lacking Inorganic Carbon Sequestration Systems Plant Cell Physiol 49 1672-1677
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea chloroplast thylakoid membranes locationand release in vitro by high salt puromycin and RNase Plant Physiol 67 940-949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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the impact of antenna size and external factors on electron transport dynamics in 890
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Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and 901
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Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W 908
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Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments 918
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Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II 923
biogenesis in cyanobacteria Biochim Biophys Acta 1857 274-287 924
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for 925
exploration and visualisation of the integrated transcriptome of cyanobacterium 926
Synechocystis sp PCC6803 Bioinformation 8 634-638 927
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The 928
Transcriptional Landscape of the Photosynthetic Model Cyanobacterium 929
Synechocystis sp PCC6803 Sci Rep 6 22168 930
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Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
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Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
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Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
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2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
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Annu Rev Plant Biol 64 609-635 971
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Biol 23 1255-1264 979
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tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
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Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
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Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
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Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
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1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
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(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
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Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
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Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
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structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
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Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
29
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-931
stress-induced changes in plasma membranes of Synechocystis sp strain PCC 6803 932
Proteomics 6 910-920 933
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 934
116 481-487 935
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in 936
Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start 937
codon Nucleic Acids Res 34 386-394 938
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) 939
PratA a periplasmic tetratricopeptide repeat protein involved in biogenesis of 940
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644 941
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The 942
cyanobacterial homologue of HCF136YCF48 is a component of an early photosystem 943
II assembly complex and is important for both the efficient assembly and repair of 944
photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399 945
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) 946
Comparative analysis of the primary transcriptome of Synechocystis sp PCC 6803 947
DNA Res 21 527-539 948
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial 949
photosynthetic membrane system Arch Microbiol 133 97-99 950
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure 951
of the membrane systems in the unicellular cyanobacterium Synechocystis sp strain 952
PCC 6803 Protoplasma 227 129-138 953
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD 954
Koppenaal DW and Pakrasi HB (2016) Global Proteomic Analysis Reveals an 955
Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model 956
Cyanobacterium Mol Cell Proteomics 15 2021-2032 957
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid 958
curvature and gene translation Trends Plant Sci 19 63-66 959
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress 960
in Synechocystis sp PCC 6803 low tolerance towards nonionic osmotic stress results 961
from lacking activation of glucosylglycerol accumulation Microbiology 152 2023-962
2030 963
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525 964
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim Biophys Acta 1847 821-830Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane Subfraction in Cyanobacteria Is Involved inan Assembly Network for Photosystem II Biogenesis J Biol Chem 286 21944-21951
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a Synechocystis double mutant lacking thephotosystem II assembly factors YCF48 and Sll0933 Planta 237 471-480
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) The Plasma Membrane of theCyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains Plant Cell 23 2379-2390
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic Assignments Strain Histories andProperties of Pure Cultures of Cyanobacteria Microbiology 111 1-61
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystembiogenesis Plant Signal Behav 8 e27037
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux CW (2015) Sub-cellular location ofFtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoidmembranes Mol Microbiol 96 448-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43 the isiA Gene Product Functions as an Excitation EnergyDissipator in the Cyanobacterium Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S SchmidB Tinevez J-Y White DJ Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-source platform forbiological-image analysis Nat Methods 9 676-682
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel tetratricopeptide repeat proteininvolved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 MolPlant 2 1289-1297
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E and Nickelsen J (2009b) Interaction ofthe periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J BiolChem 284 1813-1819
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II Assembly Steps Take Place in theThylakoid Membrane of the Cyanobacterium Synechocystis sp PCC6803 Plant Cell Physiol 57 878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spaumlt P Macek B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium Synechocystis sp PCC 6803 and itsdynamics during nitrogen starvation Front Microbiol 6 248
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial steps of photosystem II de novo assemblyand preloading with manganese take place in biogenesis centers in Synechocystis Plant Cell 24 660-675
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially localized in Chlamydomonas Plant Cell 193640-3654
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized translation in Chlamydomonas Proc Natl AcadSci USA 106 1439-1444
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The three-dimensional structure of thecyanobacterium Synechocystis sp PCC 6803 Arch Microbiol 184 259-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and Roberson RW (2012) Gross morphologicalchanges in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 BiochimBiophys Acta 1818 1427-1434
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting supercomplex Photosynth Res 116 265-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B ofLeaf Extracts in Different Solvents In Advances in Photosynthesis Research Proceedings of the VIth International Congress onPhotosynthesis Brussels Belgium August 1-6 1983 C Sybesma ed (Dordrecht Springer Netherlands) pp 9-12
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of the cyanobacterial ycf37 mutationdecreases the photosystem I content Biochem J 357 211-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced Energy Dissipation in Iron-StarvedCyanobacteria Roles of OCP and IsiA Proteins Plant Cell 19 656-672
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) Properties of Mutants of Synechocystis spStrain PCC 6803 Lacking Inorganic Carbon Sequestration Systems Plant Cell Physiol 49 1672-1677
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea chloroplast thylakoid membranes locationand release in vitro by high salt puromycin and RNase Plant Physiol 67 940-949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
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is available atPlant Physiology and The Plant CellSubscription Information for
ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
30
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the 965
photosynthetic apparatus In The Cyanobacteria A Herrero and E Flores eds 966
(Norfolk UK Caister Academic Press) pp 289ndash304 967
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and 968
dynamics of the photosynthetic apparatus in higher plants Plant J 70 157-176 969
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants 970
Annu Rev Plant Biol 64 609-635 971
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial 972
cell biology Front Plant Sci 4 458 973
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and 974
biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA 975
genes Arch Biochem Biophys 295 340-347 976
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of 977
Synechocystis sp PCC 6803 in iron-supplied and iron-deficient media Plant Mol 978
Biol 23 1255-1264 979
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The 980
Synechocystis sp PCC 6803 Oxa1 homolog is essential for membrane integration of 981
reaction center precursor protein pD1 Plant Cell 18 2236-2246 982
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B 983
(2011) Model for Membrane Organization and Protein Sorting in the Cyanobacterium 984
Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate Sequence 985
Analyses J Proteome Res 10 3617-3631 986
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land 987
plants J Exp Bot 65 1955-1972 988
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim 989
Biophys Acta 1847 821-830 990
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a 991
tetratricopeptide repeat protein from Synechocystis sp PCC 6803 during Photosystem 992
II assembly and repair Front Plant Sci 7 605 993
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane 994
Subfraction in Cyanobacteria Is Involved in an Assembly Network for Photosystem II 995
Biogenesis J Biol Chem 286 21944-21951 996
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
31
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a 997
Synechocystis double mutant lacking the photosystem II assembly factors YCF48 and 998
Sll0933 Planta 237 471-480 999
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) 1000
The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains 1001
Segregated Bioenergetic Domains Plant Cell 23 2379-2390 1002
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic 1003
Assignments Strain Histories and Properties of Pure Cultures of Cyanobacteria 1004
Microbiology 111 1-61 1005
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within 1006
thylakoid centers as sites of photosystem biogenesis Plant Signal Behav 8 e27037 1007
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux 1008
CW (2015) Sub-cellular location of FtsH proteases in the cyanobacterium1009
Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid1010
membranes Mol Microbiol 96 448-4621011
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43prime the isiA Gene 1012
Product Functions as an Excitation Energy Dissipator in the Cyanobacterium 1013
Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437 1014
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T 1015
Preibisch S Rueden C Saalfeld S Schmid B Tinevez J-Y White DJ 1016
Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-1017
source platform for biological-image analysis Nat Methods 9 676-682 1018
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel 1019
tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis 1020
and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 Mol Plant 2 1021
1289-1297 1022
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E 1023
and Nickelsen J (2009b) Interaction of the periplasmic PratA factor and the PsbA 1024
(D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J 1025
Biol Chem 284 1813-1819 1026
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II 1027
Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium 1028
Synechocystis sp PCC6803 Plant Cell Physiol 57 878 1029
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially localized in Chlamydomonas Plant Cell 193640-3654
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized translation in Chlamydomonas Proc Natl AcadSci USA 106 1439-1444
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The three-dimensional structure of thecyanobacterium Synechocystis sp PCC 6803 Arch Microbiol 184 259-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and Roberson RW (2012) Gross morphologicalchanges in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 BiochimBiophys Acta 1818 1427-1434
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting supercomplex Photosynth Res 116 265-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B ofLeaf Extracts in Different Solvents In Advances in Photosynthesis Research Proceedings of the VIth International Congress onPhotosynthesis Brussels Belgium August 1-6 1983 C Sybesma ed (Dordrecht Springer Netherlands) pp 9-12
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of the cyanobacterial ycf37 mutationdecreases the photosystem I content Biochem J 357 211-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced Energy Dissipation in Iron-StarvedCyanobacteria Roles of OCP and IsiA Proteins Plant Cell 19 656-672
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) Properties of Mutants of Synechocystis spStrain PCC 6803 Lacking Inorganic Carbon Sequestration Systems Plant Cell Physiol 49 1672-1677
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea chloroplast thylakoid membranes locationand release in vitro by high salt puromycin and RNase Plant Physiol 67 940-949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
32
Spaumlt P Maček B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium 1030
Synechocystis sp PCC 6803 and its dynamics during nitrogen starvation Front 1031
Microbiol 6 248 1032
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial 1033
steps of photosystem II de novo assembly and preloading with manganese take place 1034
in biogenesis centers in Synechocystis Plant Cell 24 660-675 1035
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially 1036
localized in Chlamydomonas Plant Cell 19 3640-3654 1037
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized 1038
translation in Chlamydomonas Proc Natl Acad Sci USA 106 1439-1444 1039
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The 1040
three-dimensional structure of the cyanobacterium Synechocystis sp PCC 6803 Arch 1041
Microbiol 184 259-270 1042
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and 1043
Roberson RW (2012) Gross morphological changes in thylakoid membrane 1044
structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 1045
Biochim Biophys Acta 1818 1427-1434 1046
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting 1047
supercomplex Photosynth Res 116 265-276 1048
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total 1049
Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents In 1050
Advances in Photosynthesis Research Proceedings of the VIth International Congress 1051
on Photosynthesis Brussels Belgium August 1ndash6 1983 C Sybesma ed (Dordrecht 1052
Springer Netherlands) pp 9-12 1053
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of 1054
the cyanobacterial ycf37 mutation decreases the photosystem I content Biochem J 1055
357 211-216 1056
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced 1057
Energy Dissipation in Iron-Starved Cyanobacteria Roles of OCP and IsiA Proteins 1058
Plant Cell 19 656-672 1059
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) 1060
Properties of Mutants of Synechocystis sp Strain PCC 6803 Lacking Inorganic Carbon 1061
Sequestration Systems Plant Cell Physiol 49 1672-1677 1062
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
33
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea 1063
chloroplast thylakoid membranes location and release in vitro by high salt 1064
puromycin and RNase Plant Physiol 67 940-949 1065
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) 1066
Slr0151 in Synechocystis sp PCC 6803 is required for efficient repair of photosystem 1067
II under high-light condition J Integr Plant Biol 56 1136-1150 1068
Yeremenko N Kouřil R Ihalainen JA DHaene S van Oosterwijk N 1069
Andrizhiyevskaya EG Keegstra W Dekker HL Hagemann M Boekema EJ 1070
Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual 1071
Function of the IsiA Chlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 1072
10308-10313 1073
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The 1074
initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes 1075
Proc Natl Acad Sci USA 98 13443-13448 1076
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum 1077
Conditions for Transformation of Synechocystis sp PCC 6803 J Microbiol 45 241-1078
245 1079
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast 1080
membranes Biochim Biophys Acta 1847 831-837 1081
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane 1082
curvature Nat Rev Mol Cell Biol 7 9-19 1083
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag) 1084
1085
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
Parsed CitationsAllen JF de Paula WBM Puthiyaveetil S and Nield J (2011) A structural phylogenetic map for chloroplast photosynthesisTrends Plant Sci 16 645-655
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Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of dynamic structural changes of plant thylakoidarchitecture Philos Trans R Soc Lond B Biol Sci 367 3515-3524
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
34
Figure Legends 1086
Figure 1 General characteristics of CurT A) Sequence alignment of CurT and CURT1A-D 1087
from A thaliana Predicted chloroplast transit-peptide sequences of CURT1A-D are omitted 1088
(Armbruster et al 2013) Positions of predicted C-terminal transmembrane domains (TDM) 1089
are marked (black bar) as is the position of the predicted amphipathic N-terminal helix in 1090
CurT (red bar) The green bar denotes the peptide sequence used for antibody production 1091
Identical and related amino acids that are conserved in 100 80 and 60 of the sequences 1092
are highlighted in black dark grey and light grey respectively B) Helical-wheel 1093
representation of the N-terminal amphipathic helix of CurT (red bar in A) The color code 1094
reflects the physicochemical properties of amino-acid side-chains green polar and 1095
uncharged blue polar and positively charged red polar and negatively charged yellow 1096
hydrophobic C) Solubility of CurT Membrane-associated proteins (50 microg) from wild-type 1097
cells were extracted with 5 mM HEPES (pH 76) containing either 1 M NaCl 01 M Na2CO3 1098
4 M urea 01 Triton X-100 or no additional solute After separation of membrane-bound 1099
(M) and solubilized (S) proteins by centrifugation proteins were fractionated by SDS-PAGE1100
and CurT was immunodetected on immunoblots As a control the integral membrane protein 1101
CP47 from PSII was analyzed in parallel D) Transmission electron micrographs of negatively 1102
stained liposomes Cell-free expression (CFE) of CurT and A thaliana CURT1A was 1103
performed in the presence of liposomes similar in composition to the lipids of the thylakoid 1104
membrane Liposomes before and after cell-free protein expression in the absence of DNA 1105
served as negative controls Black arrowheads indicate tubular connections between 1106
liposomes Scale bars 250 nm 1107
1108
Figure 2 Ultrastructural analysis of curT- cells Transmission electron micrographs of typical 1109
wild-type (A) and curT- (B-D) Synechocystis 6803 cells taken at 44000times (A B) and 28000times 1110
(C D) magnification respectively Ultrathin sections (35 to 45 nm) of cryofixed samples 1111
were stained with osmium tetroxide and post-stained using lead citrate Arrowheads indicate 1112
biogenesis centers Scale bars 200 nm (A B) and 500 nm (C D) respectively 1113
1114
Figure 3 Accumulation of components of photosynthetic complexes in the curT- mutant A) 1115
Whole cell extracts (30 microg protein) from wild-type and curT- strains were fractionated by 1116
SDS-PAGE and analyzed on immunoblots using the indicated antibodies B) The histogram 1117
shows levels of the indicated proteins in curT- extracts relative to wild-type samples (dashed 1118
red line) Data are means plusmn SD of three independent experiments Significant differences from 1119
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
35
wild-type protein levels according to Studentrsquos t-test with error probabilities of 5 and 1 1120
are indicated by and respectively 1121
1122
Figure 4 P700+ reduction kinetics and high-light effects in curT- cells A) Averaged traces 1123
(10 single measurements) of P700+ decay after complete oxidation by a 50-ms multiple 1124
turnover pulse (10000 micromol photons m-2 s-1) in the absence and presence of 10 microM DCMU 1125
B) Rate constants for P700+ reduction were obtained by fitting of the data with single1126
exponential functions Error bars indicate the SD of three independent measurements C) 1127
Impact of light intensity on the (relative) electron transport rate (rETR) Cells were exposed to 1128
gradually increasing light intensities which resulted in increasing electron transport until the 1129
capacity limit was reached PAR light intensity (micromol photons m-2 s-1) Error bars indicate the 1130
SD of three independent measurements D) D1 protein level in wild-type and curT- after high-1131
light treatment (800 micromol photons m-2s-1) in the presence and absence of lincomycin Samples 1132
were taken every 30 min and processed as in Figure 3 to determine the level of D1 present 1133
100 refers to wild-type D1 level at the beginning of the experiment 1134
1135
Figure 5 PSII assembly in curT- cells A) BNSDS-PAGE of membrane proteins Membrane 1136
fractions from wild-type and curT- cells were solubilized with 13 n-dodecyl-β-maltoside 1137
and proteins were fractionated by two-dimensional BNSDS-PAGE blotted and probed with 1138
antibodies against the PSII subunits D1 D2 CP47 and CP43 Wild-type blots were also 1139
probed with the αCurT antibody The sizes of marker proteins and the positions of PSII 1140
assembly intermediates (RCCII RCCI RC47) are shown above and below each image B) In 1141
vivo pulse labeling of membrane proteins with 35S Pulse-labeled PSII core proteins were 1142
separated via BNSDS-PAGE and visualized by autoradiography 1143
1144
Figure 6 Loss of CurT induces stress A) Low-temperature (77 K) fluorescence emission of 1145
Synechocystis 6803 wild-type (blue) and curT- (red) cells excited at 440 nm The spectra were 1146
normalized to the added fluorescein standard B) Low-temperature (77 K) fluorescence 1147
emission of Synechocystis 6803 wild-type (blue) and curT- (red) excited at 580 nm The 1148
spectra were normalized to 780 nm C) Level of IsiA in the curT- mutant relative to the wild-1149
type A mean value of 368 plusmn 96 (SD) was determined from three independent experiments 1150
1151
Figure 7 Membrane sublocalization of PSII-related factors Cell extracts from wild-type and 1152
curT- cells were fractionated by two consecutive rounds of sucrose-density gradient 1153
36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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36
centrifugation (Schottkowski et al 2009b) The second linear gradient (20-60 sucrose) was 1154
apportioned into 14 fractions which were analyzed by immunoblotting using the indicated 1155
antibodies Fractions 1ndash6 represent PDMs and fractions 7ndash14 thylakoid membranes (TMs) 1156
To facilitate comparison between gradients sample volumes were normalized to the volume 1157
of fraction 7 that contained 40 microg of protein 1158
1159
Figure 8 Live-cell imaging of the curT-CFP strain by fluorescence microscopy In the curT-1160
CFP strain the native curT gene has been replaced by a CFP-tagged version resulting in the 1161
expression of the CurT-CFP fusion protein under the control of the native promotor A) 1162
Close-up views of the mid-cell plane in the CFP and far-red autofluorescence channels shown 1163
in slice 7 in panel B B) Synechocystis 6803 cells expressing CurT C-terminally fused to the 1164
mTurquoise2 variant of CFP from its native chromosomal promoter The two imaging 1165
channels are displayed as Z-axis montages (auto-scaled contrast step size 250 nm) in 1166
grayscale and color composite configuration The thylakoid autofluorescent channel as 1167
imaged in far-red fluorescence is depicted in magenta and CurT signal as imaged in cyan 1168
fluorescence is depicted in cyan C) Fluorescence intensity profiles of the two channels along 1169
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 7 The maximum projection of 1170
the entire Z montage is also shown Scale bars (light grey) 2 microm 1171
1172
Figure 9 Immunofluorescence detection of CurT A) Close-ups of slice 5 in panel B B) 1173
Wild-type Synechocystis 6803 cells labeled with αCurT antibodies and detected with Oregon 1174
Green-conjugated secondary antibodies The Z-axis montages are shown as in Figure 8B 1175
except that the step size is 280 nm (1 to 10) C) Intensity profiles of the two channels along 1176
the autofluorescent peripheral cell ldquoringrdquo are shown for slice 5 The maximum projection of 1177
the entire Z montage is also shown Scale bars (light grey) 2 microm 1178
1179
Figure 10 Subcellular localization of CurT Ultrathin sections of wild-type Synechocystis 1180
6803 cells were incubated with rabbit αCurT prior to incubation with gold-conjugated goat 1181
anti-rabbit IgG A) Negative control (wild-type processed without addition of primary 1182
antibody) B C) Immunogold-labeled sections showing CurT localization at the cytoplasmic 1183
side of the thylakoid membrane D) Cluster formation of CurT signals at a putative biogenesis 1184
center from C E) Biogenesis center from the same cell (C) without any obvious CurT 1185
clustering of gold particles F) Schematic depiction of a wild-type cell (from Figure 2A) with 1186
color-coded curved membranes green = thylakoids that follow the curvature of the cell red = 1187
37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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37
thylakoids that bend away from the plasma membrane yellow = thylakoids that bend toward 1188
the plasma membrane to converge on sites of biogenesis centers G) Relative distribution of 1189
gold signals between convex (dark color) and concave (light color) sides of membranes (n = 1190
1584 signals from a total of 95 cells) The undifferentiated overall signal distribution is given 1191
in blue Bars = 200 nm (overview) and 100 nm (details) 1192
1193
Figure 11 Differentially modified forms of CurT are found in different complexes in PDMs 1194
and thylakoid membranes Samples were isolated by fractionation of wild-type membranes 1195
via two consecutive sucrose density gradients according to Schottkowski et al (2009b) After 1196
density centrifugation samples were applied to BNSDS-PAGE analysis (A) or isoelectric 1197
focusing (B) Numbers on the right indicate the respective fraction from the second gradient 1198
(see Figure 7) Fraction 5 represents the PDMs and fraction 9 the thylakoid membrane 1199
1200
Figure 12 Loss of CurT increases sensitivity to abiotic stress A B) Doubling times of wild-1201
type and curT- under salt stress (A) or osmotic stress (B) Doubling times are means plusmn SD of 1202
three independent cultures C) Localization of CurT-CFP under salt and osmotic stress 1203
Samples were examined as described in Figure 8 Scale bars 1 microm D) Protein levels of wild-1204
type cells after growth in BG11 medium containing 500 mM NaCl Samples were analyzed on 1205
the same gel but unrelated samples between the presented signals were omitted E) 1206
Membrane preparations obtained from wild-type cells wild-type cells grown in the presence 1207
of 500 mM NaCl and curT- cells were subjected to the first gradient step in the membrane 1208
fractionation scheme The gradient was divided into five fractions and 10 of fractions I-IV 1209
and 02 of fraction V were analyzed by SDS-PAGE Fraction II includes the plasma 1210
membrane and fraction V consists of PratA-defined membrane and thylakoid membrane 1211
1212
Figure 13 Model of CurT distribution at convergence zones in Synechocystis 6803 The 1213
distribution of CurT in thylakoid (green) and plasma membranes (brown) is shown The 1214
pattern of localization of CurT in the thylakoid membrane is derived from membrane 1215
fractionation via sucrose density centrifugation fluorescence-based detection of the CurT-1216
CFP fusion and endogenous CurT and immunogold labeling experiments (Figures 7-10) The 1217
different CurT variants a b c and d identified in the IEF experiments (Figure 11) are depicted 1218
in yellow pink blue and black respectively CurT present in the plasma membrane is 1219
highlighted in gray as its precise nature and role remain to be defined (Figure 12) 1220
Membranes are represented as lipid bilayers Convex and concave faces of bent thylakoid 1221
38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
Parsed CitationsAllen JF de Paula WBM Puthiyaveetil S and Nield J (2011) A structural phylogenetic map for chloroplast photosynthesisTrends Plant Sci 16 645-655
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of dynamic structural changes of plant thylakoidarchitecture Philos Trans R Soc Lond B Biol Sci 367 3515-3524
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann D Jahns P Weisshaar B NickelsenJ and Leister D (2010) The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem IIassembly Plant Cell 22 3439-3460
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP Rojahn U Jensen PE Rappaport F JoliotP Doumlrmann P Wanner G and Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify thylakoid architectureby inducing membrane curvature Plant Cell 25 2661-2678
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J Wanner G Westhoff P Soll J andVothknecht UC (2007) Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid proteincomplexes Plant Physiol Biochem 45 119-128
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bartsevich VV and Pakrasi HB (1995) Molecular identification of an ABC transporter complex for manganese analysis of acyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process EMBO J 14 1845-1853
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bartsevich VV and Pakrasi HB (1996) Manganese transport in the cyanobacterium Synechocystis sp PCC 6803 J Biol Chem271 26057-26061
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production the impact of antenna size andexternal factors on electron transport dynamics in Synechocystis PCC 6803 Photosynth Res 99 205-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield J (2012) Subunit Organization of aSynechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle ofprotein-dye binding Anal Biochem 72 248-254
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural organization of an intact phycobilisome and itsassociation with photosystem II Cell Res 25 726-737
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and Mamedov F (2006) Dimeric andmonomeric organization of photosystem II Distribution of five distinct complexes in the different domains of the thylakoidmembrane J Biol Chem 281 14241-14249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement of photosystem II and ATP synthase in
chloroplast membranes of Spinach and Pea Plant Cell 22 1299-1312Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W (2015) Native architecture of theChlamydomonas chloroplast revealed by in situ cryo-electron tomography eLife 4 e04889
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Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon HT (2002) Curvature of clathrin-coatedpits driven by epsin Nature 419 361-366
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Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) Phosphorylation of photosystem IIcontrols functional macroscopic folding of photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964
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Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing your curves to the BAR Biochem SocSymp 72 223-231
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Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments In Methods in Enzymology VChristopher ed (Academic Press) pp 349-361
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Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA van Weeren L Gadella TWJ andRoyant A (2012) Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751
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Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II biogenesis in cyanobacteria BiochimBiophys Acta 1857 274-287
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Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for exploration and visualisation of the integratedtranscriptome of cyanobacterium Synechocystis sp PCC6803 Bioinformation 8 634-638
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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38
sheets are indicated by black arrows The PDM which is dedicated to PSII assembly and 1222
repair is depicted in blue The dotted blue line emphasizes that the ultrastructure in this region 1223
has not yet been fully resolved PP periplasm PM plasma membrane TM thylakoid 1224
membrane L lumen PDM PratA-defined membrane 1225
1226
Tables 1227
Table 1 Physiological characteristics of the curT- mutant 1228
Strain Doubling time [h]
Oxygen Evolution[nmol h-1 OD750
-1]c
Chlorophyll content
[microg OD750-1]
d
Cell size [microm]e
Cell number [ml-1 OD750
-1]f
Wild type 817plusmn017a 1600plusmn042
b 1933plusmn187 251plusmn014 264plusmn020 390plusmn006 x 107
curT- 1259plusmn020a 3226plusmn056
b 1074plusmn20 195plusmn029 274plusmn044 367plusmn016 x 107
1229 abDoubling times in the presence (a) or absence (b) of 5 mM glucose measured under 1230
continuous illumination at 30 micromol photons m-2 s-1 and CO2-limiting conditions cOxygen 1231
evolution is expressed in nmol O2 produced per hour per OD750 unit dChlorophyll content is 1232
expressed in microg OD750-1 eThe diameter of non-dividing cells is presented in microm fCell number 1233
was determined per ml per OD750 Data are means plusmn SD of at least three independent 1234
experiments 1235
1236
Parsed CitationsAllen JF de Paula WBM Puthiyaveetil S and Nield J (2011) A structural phylogenetic map for chloroplast photosynthesisTrends Plant Sci 16 645-655
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Anderson JM Horton P Kim E-H and Chow WS (2012) Towards elucidation of dynamic structural changes of plant thylakoidarchitecture Philos Trans R Soc Lond B Biol Sci 367 3515-3524
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Armbruster U Zuumlhlke J Rengstl B Kreller R Makarenko E Ruumlhle T Schuumlnemann D Jahns P Weisshaar B NickelsenJ and Leister D (2010) The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem IIassembly Plant Cell 22 3439-3460
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Armbruster U Labs M Pribil M Viola S Xu W Scharfenberg M Hertle AP Rojahn U Jensen PE Rappaport F JoliotP Doumlrmann P Wanner G and Leister D (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify thylakoid architectureby inducing membrane curvature Plant Cell 25 2661-2678
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Aseeva E Ossenbuumlhl F Sippel C Cho WK Stein B Eichacker LA Meurer J Wanner G Westhoff P Soll J andVothknecht UC (2007) Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid proteincomplexes Plant Physiol Biochem 45 119-128
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bartsevich VV and Pakrasi HB (1995) Molecular identification of an ABC transporter complex for manganese analysis of acyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process EMBO J 14 1845-1853
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bartsevich VV and Pakrasi HB (1996) Manganese transport in the cyanobacterium Synechocystis sp PCC 6803 J Biol Chem271 26057-26061
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bernaacutet G Waschewski N and Roumlgner M (2009) Towards efficient hydrogen production the impact of antenna size andexternal factors on electron transport dynamics in Synechocystis PCC 6803 Photosynth Res 99 205-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boehm M Yu J Krynicka V Barker M Tichy M Komenda J Nixon PJ and Nield J (2012) Subunit Organization of aSynechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair Plant Cell 24 3669-3683
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle ofprotein-dye binding Anal Biochem 72 248-254
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chang L Liu X Li Y Liu C-C Yang F Zhao J and Sui S-F (2015) Structural organization of an intact phycobilisome and itsassociation with photosystem II Cell Res 25 726-737
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Danielsson R Suorsa M Paakkarinen V Albertsson P-Aring Styring S Aro E-M and Mamedov F (2006) Dimeric andmonomeric organization of photosystem II Distribution of five distinct complexes in the different domains of the thylakoidmembrane J Biol Chem 281 14241-14249
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Daum B Nicastro D Austin J McIntosh JR and Kuumlhlbrandt W (2010) Arrangement of photosystem II and ATP synthase in
chloroplast membranes of Spinach and Pea Plant Cell 22 1299-1312Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Engel BD Schaffer M Kuhn Cuellar L Villa E Plitzko JM and Baumeister W (2015) Native architecture of theChlamydomonas chloroplast revealed by in situ cryo-electron tomography eLife 4 e04889
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ford MGJ Mills IG Peter BJ Vallis Y Praefcke GJK Evans PR and McMahon HT (2002) Curvature of clathrin-coatedpits driven by epsin Nature 419 361-366
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Fristedt R Willig A Granath P Cregravevecoeur M Rochaix J-D and Vener AV (2009) Phosphorylation of photosystem IIcontrols functional macroscopic folding of photosynthetic membranes in Arabidopsis Plant Cell 21 3950-3964
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gallop JL and McMahon HT (2005) BAR domains and membrane curvature bringing your curves to the BAR Biochem SocSymp 72 223-231
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gibson DG (2011) Chapter fifteen - Enzymatic Assembly of Overlapping DNA Fragments In Methods in Enzymology VChristopher ed (Academic Press) pp 349-361
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Goedhart J von Stetten D Noirclerc-Savoye M Lelimousin M Joosen L Hink MA van Weeren L Gadella TWJ andRoyant A (2012) Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Heinz S Liauw P Nickelsen J and Nowaczyk M (2016) Analysis of photosystem II biogenesis in cyanobacteria BiochimBiophys Acta 1857 274-287
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for exploration and visualisation of the integratedtranscriptome of cyanobacterium Synechocystis sp PCC6803 Bioinformation 8 634-638
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The Transcriptional Landscape of thePhotosynthetic Model Cyanobacterium Synechocystis sp PCC6803 Sci Rep 6 22168
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-stress-induced changes in plasmamembranes of Synechocystis sp strain PCC 6803 Proteomics 6 910-920
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 116 481-487Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by asecondary RNA structure blocking the AUG start codon Nucleic Acids Res 34 386-394
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) PratA a periplasmic tetratricopeptiderepeat protein involved in biogenesis of photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The cyanobacterial homologue ofHCF136YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly andrepair of photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) Comparative analysis of the primarytranscriptome of Synechocystis sp PCC 6803 DNA Res 21 527-539
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial photosynthetic membrane system ArchMicrobiol 133 97-99
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure of the membrane systems in theunicellular cyanobacterium Synechocystis sp strain PCC 6803 Protoplasma 227 129-138
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD Koppenaal DW and Pakrasi HB (2016) GlobalProteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium Mol CellProteomics 15 2021-2032
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid curvature and gene translation TrendsPlant Sci 19 63-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress in Synechocystis sp PCC 6803 lowtolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation Microbiology 1522023-2030
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the photosynthetic apparatus In TheCyanobacteria A Herrero and E Flores eds (Norfolk UK Caister Academic Press) pp 289-304
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and dynamics of the photosynthetic apparatus inhigher plants Plant J 70 157-176
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants Annu Rev Plant Biol 64 609-635Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial cell biology Front Plant Sci 4 458Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and biochemical characterization of aSynechocystis 6803 mutant with inactivated psbA genes Arch Biochem Biophys 295 340-347
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of Synechocystis sp PCC 6803 in iron-supplied
and iron-deficient media Plant Mol Biol 23 1255-1264Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The Synechocystis sp PCC 6803 Oxa1 homolog isessential for membrane integration of reaction center precursor protein pD1 Plant Cell 18 2236-2246
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B (2011) Model for Membrane Organizationand Protein Sorting in the Cyanobacterium Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate SequenceAnalyses J Proteome Res 10 3617-3631
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Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land plants J Exp Bot 65 1955-1972Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim Biophys Acta 1847 821-830Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a tetratricopeptide repeat protein fromSynechocystis sp PCC 6803 during Photosystem II assembly and repair Front Plant Sci 7 605
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Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane Subfraction in Cyanobacteria Is Involved inan Assembly Network for Photosystem II Biogenesis J Biol Chem 286 21944-21951
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Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a Synechocystis double mutant lacking thephotosystem II assembly factors YCF48 and Sll0933 Planta 237 471-480
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) The Plasma Membrane of theCyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains Plant Cell 23 2379-2390
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Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystembiogenesis Plant Signal Behav 8 e27037
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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Hernaacutendez-Prieto M and Futschik M (2012) CyanoEXpress A web database for exploration and visualisation of the integratedtranscriptome of cyanobacterium Synechocystis sp PCC6803 Bioinformation 8 634-638
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hernaacutendez-Prieto MA Semeniuk TA Giner-Lamia J and Futschik ME (2016) The Transcriptional Landscape of thePhotosynthetic Model Cyanobacterium Synechocystis sp PCC6803 Sci Rep 6 22168
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang F Fulda S Hagemann M and Norling B (2006) Proteomic screening of salt-stress-induced changes in plasmamembranes of Synechocystis sp strain PCC 6803 Proteomics 6 910-920
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kirchhoff H (2013) Architectural switches in plant thylakoid membranes Photosynth Res 116 481-487Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Klinkert B Elles I and Nickelsen J (2006) Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by asecondary RNA structure blocking the AUG start codon Nucleic Acids Res 34 386-394
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Klinkert B Ossenbuumlhl F Sikorski M Berry S Eichacker L and Nickelsen J (2004) PratA a periplasmic tetratricopeptiderepeat protein involved in biogenesis of photosystem II in Synechocystis sp PCC 6803 J Biol Chem 279 44639-44644
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The cyanobacterial homologue ofHCF136YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly andrepair of photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) Comparative analysis of the primarytranscriptome of Synechocystis sp PCC 6803 DNA Res 21 527-539
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial photosynthetic membrane system ArchMicrobiol 133 97-99
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure of the membrane systems in theunicellular cyanobacterium Synechocystis sp strain PCC 6803 Protoplasma 227 129-138
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD Koppenaal DW and Pakrasi HB (2016) GlobalProteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium Mol CellProteomics 15 2021-2032
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid curvature and gene translation TrendsPlant Sci 19 63-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress in Synechocystis sp PCC 6803 lowtolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation Microbiology 1522023-2030
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the photosynthetic apparatus In TheCyanobacteria A Herrero and E Flores eds (Norfolk UK Caister Academic Press) pp 289-304
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and dynamics of the photosynthetic apparatus inhigher plants Plant J 70 157-176
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants Annu Rev Plant Biol 64 609-635Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial cell biology Front Plant Sci 4 458Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and biochemical characterization of aSynechocystis 6803 mutant with inactivated psbA genes Arch Biochem Biophys 295 340-347
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of Synechocystis sp PCC 6803 in iron-supplied
and iron-deficient media Plant Mol Biol 23 1255-1264Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The Synechocystis sp PCC 6803 Oxa1 homolog isessential for membrane integration of reaction center precursor protein pD1 Plant Cell 18 2236-2246
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B (2011) Model for Membrane Organizationand Protein Sorting in the Cyanobacterium Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate SequenceAnalyses J Proteome Res 10 3617-3631
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land plants J Exp Bot 65 1955-1972Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim Biophys Acta 1847 821-830Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a tetratricopeptide repeat protein fromSynechocystis sp PCC 6803 during Photosystem II assembly and repair Front Plant Sci 7 605
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane Subfraction in Cyanobacteria Is Involved inan Assembly Network for Photosystem II Biogenesis J Biol Chem 286 21944-21951
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a Synechocystis double mutant lacking thephotosystem II assembly factors YCF48 and Sll0933 Planta 237 471-480
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) The Plasma Membrane of theCyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains Plant Cell 23 2379-2390
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic Assignments Strain Histories andProperties of Pure Cultures of Cyanobacteria Microbiology 111 1-61
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystembiogenesis Plant Signal Behav 8 e27037
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux CW (2015) Sub-cellular location ofFtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoidmembranes Mol Microbiol 96 448-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43 the isiA Gene Product Functions as an Excitation EnergyDissipator in the Cyanobacterium Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S SchmidB Tinevez J-Y White DJ Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-source platform forbiological-image analysis Nat Methods 9 676-682
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel tetratricopeptide repeat proteininvolved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 MolPlant 2 1289-1297
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E and Nickelsen J (2009b) Interaction ofthe periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J BiolChem 284 1813-1819
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II Assembly Steps Take Place in theThylakoid Membrane of the Cyanobacterium Synechocystis sp PCC6803 Plant Cell Physiol 57 878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spaumlt P Macek B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium Synechocystis sp PCC 6803 and itsdynamics during nitrogen starvation Front Microbiol 6 248
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial steps of photosystem II de novo assemblyand preloading with manganese take place in biogenesis centers in Synechocystis Plant Cell 24 660-675
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially localized in Chlamydomonas Plant Cell 193640-3654
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized translation in Chlamydomonas Proc Natl AcadSci USA 106 1439-1444
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The three-dimensional structure of thecyanobacterium Synechocystis sp PCC 6803 Arch Microbiol 184 259-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and Roberson RW (2012) Gross morphologicalchanges in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 BiochimBiophys Acta 1818 1427-1434
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting supercomplex Photosynth Res 116 265-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B ofLeaf Extracts in Different Solvents In Advances in Photosynthesis Research Proceedings of the VIth International Congress onPhotosynthesis Brussels Belgium August 1-6 1983 C Sybesma ed (Dordrecht Springer Netherlands) pp 9-12
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of the cyanobacterial ycf37 mutationdecreases the photosystem I content Biochem J 357 211-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced Energy Dissipation in Iron-StarvedCyanobacteria Roles of OCP and IsiA Proteins Plant Cell 19 656-672
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) Properties of Mutants of Synechocystis spStrain PCC 6803 Lacking Inorganic Carbon Sequestration Systems Plant Cell Physiol 49 1672-1677
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea chloroplast thylakoid membranes locationand release in vitro by high salt puromycin and RNase Plant Physiol 67 940-949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
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chloroplast membranes of Spinach and Pea Plant Cell 22 1299-1312Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The cyanobacterial homologue ofHCF136YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly andrepair of photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) Comparative analysis of the primarytranscriptome of Synechocystis sp PCC 6803 DNA Res 21 527-539
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial photosynthetic membrane system ArchMicrobiol 133 97-99
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure of the membrane systems in theunicellular cyanobacterium Synechocystis sp strain PCC 6803 Protoplasma 227 129-138
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD Koppenaal DW and Pakrasi HB (2016) GlobalProteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium Mol CellProteomics 15 2021-2032
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid curvature and gene translation TrendsPlant Sci 19 63-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress in Synechocystis sp PCC 6803 lowtolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation Microbiology 1522023-2030
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the photosynthetic apparatus In TheCyanobacteria A Herrero and E Flores eds (Norfolk UK Caister Academic Press) pp 289-304
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and dynamics of the photosynthetic apparatus inhigher plants Plant J 70 157-176
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Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants Annu Rev Plant Biol 64 609-635Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial cell biology Front Plant Sci 4 458Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystembiogenesis Plant Signal Behav 8 e27037
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux CW (2015) Sub-cellular location ofFtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoidmembranes Mol Microbiol 96 448-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43 the isiA Gene Product Functions as an Excitation EnergyDissipator in the Cyanobacterium Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S SchmidB Tinevez J-Y White DJ Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-source platform forbiological-image analysis Nat Methods 9 676-682
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel tetratricopeptide repeat proteininvolved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 MolPlant 2 1289-1297
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E and Nickelsen J (2009b) Interaction ofthe periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J BiolChem 284 1813-1819
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II Assembly Steps Take Place in theThylakoid Membrane of the Cyanobacterium Synechocystis sp PCC6803 Plant Cell Physiol 57 878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spaumlt P Macek B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium Synechocystis sp PCC 6803 and itsdynamics during nitrogen starvation Front Microbiol 6 248
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial steps of photosystem II de novo assemblyand preloading with manganese take place in biogenesis centers in Synechocystis Plant Cell 24 660-675
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially localized in Chlamydomonas Plant Cell 193640-3654
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized translation in Chlamydomonas Proc Natl AcadSci USA 106 1439-1444
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The three-dimensional structure of thecyanobacterium Synechocystis sp PCC 6803 Arch Microbiol 184 259-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and Roberson RW (2012) Gross morphologicalchanges in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 BiochimBiophys Acta 1818 1427-1434
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting supercomplex Photosynth Res 116 265-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B ofLeaf Extracts in Different Solvents In Advances in Photosynthesis Research Proceedings of the VIth International Congress onPhotosynthesis Brussels Belgium August 1-6 1983 C Sybesma ed (Dordrecht Springer Netherlands) pp 9-12
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of the cyanobacterial ycf37 mutationdecreases the photosystem I content Biochem J 357 211-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced Energy Dissipation in Iron-StarvedCyanobacteria Roles of OCP and IsiA Proteins Plant Cell 19 656-672
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) Properties of Mutants of Synechocystis spStrain PCC 6803 Lacking Inorganic Carbon Sequestration Systems Plant Cell Physiol 49 1672-1677
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea chloroplast thylakoid membranes locationand release in vitro by high salt puromycin and RNase Plant Physiol 67 940-949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
Permissions httpswwwcopyrightcomcccopenurldosid=pd_hw1532298Xampissn=1532298XampWTmc_id=pd_hw1532298X
eTOCs httpwwwplantcellorgcgialertsctmain
Sign up for eTOCs at
CiteTrack Alerts httpwwwplantcellorgcgialertsctmain
Sign up for CiteTrack Alerts at
Subscription Information httpwwwaspborgpublicationssubscriptionscfm
is available atPlant Physiology and The Plant CellSubscription Information for
ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
Komenda J Nickelsen J Tichyacute M Praacutešil O Eichacker LA and Nixon PJ (2008) The cyanobacterial homologue ofHCF136YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly andrepair of photosystem II in Synechocystis sp PCC 6803 J Biol Chem 283 22390-22399
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kopf M Klaumlhn S Scholz I Matthiessen JKF Hess WR and Voszlig B (2014) Comparative analysis of the primarytranscriptome of Synechocystis sp PCC 6803 DNA Res 21 527-539
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kunkel DD (1982) Thylakoid centers Structures associated with the cyanobacterial photosynthetic membrane system ArchMicrobiol 133 97-99
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liberton M Howard Berg R Heuser J Roth R and Pakrasi BH (2006) Ultrastructure of the membrane systems in theunicellular cyanobacterium Synechocystis sp strain PCC 6803 Protoplasma 227 129-138
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Liberton M Saha R Jacobs JM Nguyen AY Gritsenko MA Smith RD Koppenaal DW and Pakrasi HB (2016) GlobalProteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium Mol CellProteomics 15 2021-2032
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Luque I and Ochoa de Alda JAG (2014) CURT1CAAD-containing aaRSs thylakoid curvature and gene translation TrendsPlant Sci 19 63-66
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Marin K Stirnberg M Eisenhut M Kraumlmer R and Hagemann M (2006) Osmotic stress in Synechocystis sp PCC 6803 lowtolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation Microbiology 1522023-2030
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mullineaux CW (2005) Function and evolution of grana Trends Plant Sci 10 521-525Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mullineaux CW (2008) Biogenesis and dynamics of thylakoid membranes and the photosynthetic apparatus In TheCyanobacteria A Herrero and E Flores eds (Norfolk UK Caister Academic Press) pp 289-304
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nevo R Charuvi D Tsabari O and Reich Z (2012) Composition architecture and dynamics of the photosynthetic apparatus inhigher plants Plant J 70 157-176
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nickelsen J and Rengstl B (2013) Photosystem II assembly From cyanobacteria to plants Annu Rev Plant Biol 64 609-635Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nickelsen J and Zerges W (2013) Thylakoid biogenesis has joined the new era of bacterial cell biology Front Plant Sci 4 458Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nilsson F Simpson DJ Jansson C and Andersson B (1992) Ultrastructural and biochemical characterization of aSynechocystis 6803 mutant with inactivated psbA genes Arch Biochem Biophys 295 340-347
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Odom WR Hodges R Chitnis PR and Guikema JA (1993) Characterization of Synechocystis sp PCC 6803 in iron-supplied
and iron-deficient media Plant Mol Biol 23 1255-1264Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The Synechocystis sp PCC 6803 Oxa1 homolog isessential for membrane integration of reaction center precursor protein pD1 Plant Cell 18 2236-2246
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B (2011) Model for Membrane Organizationand Protein Sorting in the Cyanobacterium Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate SequenceAnalyses J Proteome Res 10 3617-3631
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land plants J Exp Bot 65 1955-1972Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim Biophys Acta 1847 821-830Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a tetratricopeptide repeat protein fromSynechocystis sp PCC 6803 during Photosystem II assembly and repair Front Plant Sci 7 605
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane Subfraction in Cyanobacteria Is Involved inan Assembly Network for Photosystem II Biogenesis J Biol Chem 286 21944-21951
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a Synechocystis double mutant lacking thephotosystem II assembly factors YCF48 and Sll0933 Planta 237 471-480
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) The Plasma Membrane of theCyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains Plant Cell 23 2379-2390
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic Assignments Strain Histories andProperties of Pure Cultures of Cyanobacteria Microbiology 111 1-61
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystembiogenesis Plant Signal Behav 8 e27037
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux CW (2015) Sub-cellular location ofFtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoidmembranes Mol Microbiol 96 448-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43 the isiA Gene Product Functions as an Excitation EnergyDissipator in the Cyanobacterium Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S SchmidB Tinevez J-Y White DJ Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-source platform forbiological-image analysis Nat Methods 9 676-682
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel tetratricopeptide repeat proteininvolved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 MolPlant 2 1289-1297
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E and Nickelsen J (2009b) Interaction ofthe periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J BiolChem 284 1813-1819
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II Assembly Steps Take Place in theThylakoid Membrane of the Cyanobacterium Synechocystis sp PCC6803 Plant Cell Physiol 57 878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spaumlt P Macek B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium Synechocystis sp PCC 6803 and itsdynamics during nitrogen starvation Front Microbiol 6 248
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial steps of photosystem II de novo assemblyand preloading with manganese take place in biogenesis centers in Synechocystis Plant Cell 24 660-675
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially localized in Chlamydomonas Plant Cell 193640-3654
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized translation in Chlamydomonas Proc Natl AcadSci USA 106 1439-1444
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The three-dimensional structure of thecyanobacterium Synechocystis sp PCC 6803 Arch Microbiol 184 259-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and Roberson RW (2012) Gross morphologicalchanges in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 BiochimBiophys Acta 1818 1427-1434
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting supercomplex Photosynth Res 116 265-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B ofLeaf Extracts in Different Solvents In Advances in Photosynthesis Research Proceedings of the VIth International Congress onPhotosynthesis Brussels Belgium August 1-6 1983 C Sybesma ed (Dordrecht Springer Netherlands) pp 9-12
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of the cyanobacterial ycf37 mutationdecreases the photosystem I content Biochem J 357 211-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced Energy Dissipation in Iron-StarvedCyanobacteria Roles of OCP and IsiA Proteins Plant Cell 19 656-672
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) Properties of Mutants of Synechocystis spStrain PCC 6803 Lacking Inorganic Carbon Sequestration Systems Plant Cell Physiol 49 1672-1677
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea chloroplast thylakoid membranes locationand release in vitro by high salt puromycin and RNase Plant Physiol 67 940-949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
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Subscription Information httpwwwaspborgpublicationssubscriptionscfm
is available atPlant Physiology and The Plant CellSubscription Information for
ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
and iron-deficient media Plant Mol Biol 23 1255-1264Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ossenbuumlhl F Inaba-Sulpice M Meurer J Soll J and Eichacker LA (2006) The Synechocystis sp PCC 6803 Oxa1 homolog isessential for membrane integration of reaction center precursor protein pD1 Plant Cell 18 2236-2246
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pisareva T Kwon J Oh J Kim S Ge C Wieslander Aring Choi J-S and Norling B (2011) Model for Membrane Organizationand Protein Sorting in the Cyanobacterium Synechocystis sp PCC 6803 Inferred from Proteomics and Multivariate SequenceAnalyses J Proteome Res 10 3617-3631
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Pribil M Labs M and Leister D (2014) Structure and dynamics of thylakoids in land plants J Exp Bot 65 1955-1972Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Heinz S and Nickelsen J (2015) Biogenesis of thylakoid membranes Biochim Biophys Acta 1847 821-830Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rast A Rengstl B Heinz S Klingl A and Nickelsen J (2016) The role of Slr0151 a tetratricopeptide repeat protein fromSynechocystis sp PCC 6803 during Photosystem II assembly and repair Front Plant Sci 7 605
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Oster U Stengel A and Nickelsen J (2011) An Intermediate Membrane Subfraction in Cyanobacteria Is Involved inan Assembly Network for Photosystem II Biogenesis J Biol Chem 286 21944-21951
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rengstl B Knoppovaacute J Komenda J and Nickelsen J (2013) Characterization of a Synechocystis double mutant lacking thephotosystem II assembly factors YCF48 and Sll0933 Planta 237 471-480
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rexroth S Mullineaux CW Ellinger D Sendtko E Roumlgner M and Koenig F (2011) The Plasma Membrane of theCyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains Plant Cell 23 2379-2390
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rippka R Deruelles J Waterbury JB Herdman M and Stanier RY (1979) Generic Assignments Strain Histories andProperties of Pure Cultures of Cyanobacteria Microbiology 111 1-61
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ruumltgers M and Schroda M (2013) A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystembiogenesis Plant Signal Behav 8 e27037
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sacharz J Bryan SJ Yu J Burroughs NJ Spence EM Nixon PJ and Mullineaux CW (2015) Sub-cellular location ofFtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoidmembranes Mol Microbiol 96 448-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sandstroumlm S Park Y-I Oumlquist G and Gustafsson P (2001) CP43 the isiA Gene Product Functions as an Excitation EnergyDissipator in the Cyanobacterium Synechococcus sp PCC 7942 Photochem Photobiol 74 431-437
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schindelin J Arganda-Carreras I Frise E Kaynig V Longair M Pietzsch T Preibisch S Rueden C Saalfeld S SchmidB Tinevez J-Y White DJ Hartenstein V Eliceiri K Tomancak P and Cardona A (2012) Fiji an open-source platform forbiological-image analysis Nat Methods 9 676-682
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel tetratricopeptide repeat proteininvolved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 MolPlant 2 1289-1297
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E and Nickelsen J (2009b) Interaction ofthe periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J BiolChem 284 1813-1819
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II Assembly Steps Take Place in theThylakoid Membrane of the Cyanobacterium Synechocystis sp PCC6803 Plant Cell Physiol 57 878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spaumlt P Macek B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium Synechocystis sp PCC 6803 and itsdynamics during nitrogen starvation Front Microbiol 6 248
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial steps of photosystem II de novo assemblyand preloading with manganese take place in biogenesis centers in Synechocystis Plant Cell 24 660-675
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially localized in Chlamydomonas Plant Cell 193640-3654
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized translation in Chlamydomonas Proc Natl AcadSci USA 106 1439-1444
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The three-dimensional structure of thecyanobacterium Synechocystis sp PCC 6803 Arch Microbiol 184 259-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and Roberson RW (2012) Gross morphologicalchanges in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 BiochimBiophys Acta 1818 1427-1434
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting supercomplex Photosynth Res 116 265-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B ofLeaf Extracts in Different Solvents In Advances in Photosynthesis Research Proceedings of the VIth International Congress onPhotosynthesis Brussels Belgium August 1-6 1983 C Sybesma ed (Dordrecht Springer Netherlands) pp 9-12
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of the cyanobacterial ycf37 mutationdecreases the photosystem I content Biochem J 357 211-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced Energy Dissipation in Iron-StarvedCyanobacteria Roles of OCP and IsiA Proteins Plant Cell 19 656-672
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) Properties of Mutants of Synechocystis spStrain PCC 6803 Lacking Inorganic Carbon Sequestration Systems Plant Cell Physiol 49 1672-1677
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea chloroplast thylakoid membranes locationand release in vitro by high salt puromycin and RNase Plant Physiol 67 940-949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
Permissions httpswwwcopyrightcomcccopenurldosid=pd_hw1532298Xampissn=1532298XampWTmc_id=pd_hw1532298X
eTOCs httpwwwplantcellorgcgialertsctmain
Sign up for eTOCs at
CiteTrack Alerts httpwwwplantcellorgcgialertsctmain
Sign up for CiteTrack Alerts at
Subscription Information httpwwwaspborgpublicationssubscriptionscfm
is available atPlant Physiology and The Plant CellSubscription Information for
ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Ratke J Oster U Nowaczyk M and Nickelsen J (2009a) Pitt a novel tetratricopeptide repeat proteininvolved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp PCC 6803 MolPlant 2 1289-1297
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schottkowski M Gkalympoudis S Tzekova N Stelljes C Schuumlnemann D Ankele E and Nickelsen J (2009b) Interaction ofthe periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp PCC 6803 J BiolChem 284 1813-1819
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Selatildeo TT Zhang L Knoppovaaacute J Komenda J and Norling B (2016) Photosystem II Assembly Steps Take Place in theThylakoid Membrane of the Cyanobacterium Synechocystis sp PCC6803 Plant Cell Physiol 57 878
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Spaumlt P Macek B and Forchhammer K (2015) Phosphoproteome of the cyanobacterium Synechocystis sp PCC 6803 and itsdynamics during nitrogen starvation Front Microbiol 6 248
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Stengel A Guumlgel IL Hilger D Rengstl B Jung H and Nickelsen J (2012) Initial steps of photosystem II de novo assemblyand preloading with manganese take place in biogenesis centers in Synechocystis Plant Cell 24 660-675
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2007) Photosystem II assembly and repair are differentially localized in Chlamydomonas Plant Cell 193640-3654
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Uniacke J and Zerges W (2009) Chloroplast protein targeting involves localized translation in Chlamydomonas Proc Natl AcadSci USA 106 1439-1444
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AL Hohmann-Marriott M Vermaas WJ and Roberson R (2006) The three-dimensional structure of thecyanobacterium Synechocystis sp PCC 6803 Arch Microbiol 184 259-270
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
van de Meene AML Sharp WP McDaniel JH Friedrich H Vermaas WFJ and Roberson RW (2012) Gross morphologicalchanges in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp PCC 6803 BiochimBiophys Acta 1818 1427-1434
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Watanabe M and Ikeuchi M (2013) Phycobilisome architecture of a light-harvesting supercomplex Photosynth Res 116 265-276
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wellburn AR and Lichtenthaler H (1984) Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B ofLeaf Extracts in Different Solvents In Advances in Photosynthesis Research Proceedings of the VIth International Congress onPhotosynthesis Brussels Belgium August 1-6 1983 C Sybesma ed (Dordrecht Springer Netherlands) pp 9-12
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilde A Lunser K Ossenbuumlhl F Nickelsen J and Borner T (2001) Characterization of the cyanobacterial ycf37 mutationdecreases the photosystem I content Biochem J 357 211-216
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wilson A Boulay C Wilde A Kerfeld CA and Kirilovsky D (2007) Light-Induced Energy Dissipation in Iron-StarvedCyanobacteria Roles of OCP and IsiA Proteins Plant Cell 19 656-672
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Xu M Bernaacutet G Singh A Mi H Roumlgner M Pakrasi HB and Ogawa T (2008) Properties of Mutants of Synechocystis spStrain PCC 6803 Lacking Inorganic Carbon Sequestration Systems Plant Cell Physiol 49 1672-1677
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yamamoto T Burke J Autz G and Jagendorf AT (1981) Bound ribosomes of Pea chloroplast thylakoid membranes locationand release in vitro by high salt puromycin and RNase Plant Physiol 67 940-949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang H Liao L Bo T Zhao L Sun X Lu X Norling B and Huang F (2014) Slr0151 in Synechocystis sp PCC 6803 isrequired for efficient repair of photosystem II under high-light condition J Integr Plant Biol 56 1136-1150
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yeremenko N Kouril R Ihalainen JA DHaene S van Oosterwijk N Andrizhiyevskaya EG Keegstra W Dekker HLHagemann M Boekema EJ Matthijs HCP and Dekker JP (2004) Supramolecular Organization and Dual Function of the IsiAChlorophyll-Binding Protein in Cyanobacteria Biochemistry 43 10308-10313
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zak E Norling B Maitra R Huang F Andersson B and Pakrasi HB (2001) The initial steps of biogenesis of cyanobacterialphotosystems occur in plasma membranes Proc Natl Acad Sci USA 98 13443-13448
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zang X Liu B Liu S Arunakumara KKIU and Zhang X (2007) Optimum Conditions for Transformation of Synechocystis spPCC 6803 J Microbiol 45 241-245
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang L and Sakamoto W (2015) Possible function of VIPP1 in maintaining chloroplast membranes Biochim Biophys Acta 1847831-837
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zimmerberg J and Kozlov MM (2006) How proteins produce cellular membrane curvature Nat Rev Mol Cell Biol 7 9-19Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zoumlfel P (1988) Statistik in der Praxis (Stuttgart Germany Gustav Fischer Verlag)Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
This information is current as of May 25 2020
Supplemental Data contentsuppl20160819tpc1600491DC1html
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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DOI 101105tpc1600491 originally published online August 19 2016Plant Cell
Rengstl Stefania Viola Marc M Nowaczyk Dario Leister and Joumlrg NickelsenSteffen Heinz Anna Rast Lin Shao Andrian Gutu Irene L Guumlgel Eiri Heyno Mathias Labs Birgit
CURVATURE THYLAKOID1 ProteinsThylakoid Membrane Architecture in Synechocystis Depends on CurT a Homolog of the Granal
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