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Short title: Plant GnTI localization 1
2
Author for contact: Richard Strasser, Department of Applied Genetics and Cell Biology, University 3
of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria, 4
phone: ++43-1-47654-94145, fax: ++43-1-47654-94009 6
7
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The Golgi localization of GnTI requires a polar amino acid residue within its 9
transmembrane domain 10
11
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Jennifer Schoberer1, Eva Liebminger1, Ulrike Vavra1, Christiane Veit1, Clemens Grünwald-Gruber2 13
Friedrich Altmann2, Stanley W. Botchway3, Richard Strasser1 14
15
1Department of Applied Genetics and Cell Biology, University of Natural Resources and Life 16
Sciences, Vienna, Austria 17
2Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria 18
3Research Complex at Harwell, Central Laser Facility, Science and Technology Facilities Council, 19
Rutherford Appleton Laboratory, Harwell-Oxford, Didcot OX11 0QX, United Kingdom 20
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Author for correspondence: [email protected] 22
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Author contributions: J.S. and R.S. conceived the original research plan, designed the experiments 24
and analysed the data; J.S. and E.L. performed most of the experiments; U.V. and C.V. provided 25
technical assistance. C.G-G. and F.A. performed MS-based analysis and interpretation of the data. 26
S.W.B. assisted in design and interpretation of fluorescence imaging data. R.S. wrote the manuscript 27
with contributions from all the authors. 28
29
This work was supported by grants from the Austrian Science Fund (FWF): P23906-B20 (to R.S.), 30
P28218-B22 (to R.S.), T655-B20 (to J.S.) and the Mizutani Foundation for Glycosciences (to R.S.). 31
We thank the STFC for funding access to the Central Laser Facility (grant no. 12130006 to Chris 32
Hawes, Oxford Brookes University). 33
34
One-sentence summary 35
An amino acid sequence motif in the N-acetylglucosaminyltransferase I transmembrane domain 36
governs its Golgi localization and glycan processing function in plants. 37
Plant Physiology Preview. Published on April 10, 2019, as DOI:10.1104/pp.19.00310
Copyright 2019 by the American Society of Plant Biologists
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ABSTRACT 39
The Golgi apparatus consists of stacked cisternae filled with enzymes that facilitate the sequential and 40
highly controlled modification of glycans from proteins that transit through the organelle. Although 41
the glycan processing pathways have been extensively studied, the underlying mechanisms that 42
concentrate Golgi-resident glycosyltransferases and glycosidases in distinct Golgi compartments are 43
poorly understood. The single-pass transmembrane domain of N-acetylglucosaminyltransferase I 44
(GnTI) accounts for its steady-state distribution in the cis/medial-Golgi. Here, we investigated the 45
contribution of individual amino acid residues within the transmembrane domain of Arabidopsis 46
thaliana and Nicotiana tabacum GnTI towards Golgi localization and N-glycan processing. Conserved 47
sequence motifs within the transmembrane domain were replaced with those from the established 48
trans-Golgi enzyme 2,6-sialyltransferase (ST) and site-directed mutagenesis was used to exchange 49
individual amino acid residues. Subsequent subcellular localization of fluorescent fusion proteins and 50
N-glycan profiling revealed that a conserved glutamine residue in the GnTI transmembrane domain is 51
essential for its cis/medial-Golgi localization. Substitution of the crucial glutamine residue with other 52
amino acids resulted in mislocalization to the vacuole and impaired N-glycan processing in vivo. Our 53
results suggest that sequence-specific features of the GnTI transmembrane domain are required for its 54
interaction with a Golgi-resident adaptor protein or a specific lipid environment that likely promotes 55
COPI-mediated retrograde transport, thus maintaining the steady-state distribution of GnTI in the 56
cis/medial-Golgi of plants. 57
58
INTRODUCTION 59
The Golgi apparatus has a central role in protein processing and transport along the secretory pathway. 60
In the Golgi apparatus, secreted glycoproteins from animals and plants acquire complex glycan 61
structures which furnish them with distinct functions (Helenius and Aebi, 2001; Strasser, 2016). The 62
formation of complex N-glycans in the Golgi is carried out by specific glycosyltransferases and 63
glycosidases that are asymmetrically distributed throughout the different Golgi cisternae (Dunphy and 64
Rothman, 1983; Rabouille et al., 1995). The distinct, yet overlapping, distribution is a prerequisite for 65
controlled glycosylation by spatial separation of glycan processing enzymes that compete for the same 66
acceptor substrates. In mammals, diseases like congenital disorders of glycosylation are caused by 67
missense mutations that affect trafficking and organization of Golgi-resident glycosylation enzymes 68
(Fisher and Ungar, 2016). Despite their important role in maintenance of Golgi organization and 69
function, the mechanisms for steady-state distribution of Golgi-resident glycosyltransferases and 70
glycosidases are still largely unknown (Tu and Banfield, 2010). A better understanding of the Golgi 71
organization is further complicated by the fact that the mode of cargo transport through the Golgi is 72
still controversial in most organisms and different cell types (Glick and Luini, 2011; Ito et al., 2014). 73
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Sequence motifs that govern the intra-Golgi distribution can either function as a retention signal for a 74
distinct Golgi sub-compartment, a retrieval signal for retrograde transport, or a combination of both. 75
A common feature of Golgi-resident N-glycan processing enzymes is their type II membrane protein 76
topology with a short cytoplasmic tail, a single transmembrane domain (TMD) and a stem or linker 77
region that orients the large catalytic domain into the Golgi lumen. Proposed models for Golgi 78
retention and retrieval include oligomerization of glycosyltransferases and glycosidases with 79
subsequent exclusion of large protein complexes from intra-Golgi transport (Machamer, 1991; Nilsson 80
et al., 1993), TMD length-dependent sorting (Munro, 1995), lipid-based partitioning (Patterson et al., 81
2008) and cytoplasmic tail-dependent processes (Uliana et al., 2006; Petrosyan et al., 2015). An 82
adaptor-mediated sorting mechanism involving binding to a specific amino acid motif within the 83
cytoplasmic tail of glycosyltransferases and interaction with the coat protein complex I (COPI) 84
retrograde transport system has been described in yeast and mammals (Schmitz et al., 2008; Tu et al., 85
2008; Eckert et al., 2014). Yeast VPS74 acts as an adaptor protein that binds to the sequence motif in 86
the cytoplasmic tail of late-Golgi glycosyltransferases and to COPI subunits to mediate the retrograde 87
transport in the Golgi. Apart from VPS74 and its mammalian homolog GOLPH3, which may have a 88
similar function, no adaptor proteins for binding to cytoplasmic tails have been identified so far and 89
for many Golgi-resident enzymes, like those involved in complex N-glycan processing in mammals or 90
plants, the signals and mechanisms for their steady-state distribution are not well known. Noteworthy, 91
an adapter-mediated mechanism has not been described in plants and VPS74/GOLPH3-like proteins 92
appear absent. Interestingly, a recent study showed a direct binding of COPI subunits to an amino acid 93
motif present in the cytoplasmic tail of several mammalian glycosyltransferases (Liu et al., 2018) 94
indicating an adaptor-protein independent sorting. Golgi localization of plant N-glycan processing 95
enzymes is typically mediated by sequence features within the N-terminal region comprising the 96
cytoplasmic tail, the TMD and the stem (CTS region) without any contribution from the catalytic 97
domain (Saint-Jore-Dupas et al., 2006; Schoberer et al., 2010; Schoberer et al., 2013). However, 98
within this N-terminal targeting region, a conserved sequence motif contributing to Golgi 99
compartment-specific localization has yet to be revealed. 100
N-glycosylation is an essential posttranslational modification that is initiated in the endoplasmic 101
reticulum (ER) by transfer of a preassembled oligosaccharide to asparagine residues within the 102
consensus sequence Asn-X-Ser/Thr (X can be any amino acid except proline) on nascent proteins 103
(Strasser, 2016). Upon transfer, the N-glycans are processed by specific -glucosidases and -104
mannosidases, and glycoproteins with oligomannosidic N-glycans exit the ER towards the Golgi 105
complex. In the Golgi, additional mannose residues are removed by Golgi -mannosidase I 106
(Liebminger et al., 2009) and complex N-glycan formation is initiated by 1,2-N-107
acetylglucosaminyltransferase I (GnTI, called MGAT1 in mammals), which transfers a single GlcNAc 108
residue to Man5GlcNAc2. This is a critical step in the pathway because all further N-glycan processing 109
reactions in the Golgi depend on the presence of the single GlcNAc added by GnTI (von Schaewen et 110
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al., 1993; Strasser et al., 1999). Importantly, the central function of GnTI in complex N-glycan 111
formation is conserved in higher eukaryotes and GnTI deficiency is embryo-lethal in mammals (Ioffe 112
and Stanley, 1994). GnTI-deficient Lotus japonicus display severe growth defects and Oryza sativa 113
(rice) lacking GnTI show developmental phenotypes leading to early lethality (Fanata et al., 2013; 114
Pedersen et al., 2017). Arabidopsis thaliana lacking GnTI are viable, but less tolerant to abiotic stress 115
and display severe growth defects when combined with mutations that affect in plant cell wall 116
formation (von Schaewen et al., 1993; Kang et al., 2008). In summary, these data highlight the 117
importance of complex N-glycan initiation by GnTI for different biological processes across 118
kingdoms. 119
In accordance with its N-glycan processing function, GnTI is mainly located in the cis/medial-Golgi in 120
plants (Reichardt et al., 2007; Schoberer et al., 2010). In a previous study, we generated chimeric 121
GnTI variants and analyzed their Golgi localization and functionality to identify regions for Golgi 122
targeting and retrieval/retention (Schoberer et al., 2014). The results for Nicotiana tabacum (tobacco) 123
GnTI demonstrated that the cytoplasmic tail, the stem region and the catalytic domain are not involved 124
the intra-Golgi distribution of GnTI. By contrast, the TMD plays a pivotal role for its cis/medial-Golgi 125
distribution. Here, we investigated the contribution of individual sequence motifs and amino acid 126
residues in the TMD to Golgi localization and GnTI function. Tobacco and Arabidopsis GnTI (herein 127
NtGnTI and AtGnTI, respectively) were analyzed to identify a common mechanism for their Golgi 128
distribution. We identified a single amino acid residue conserved in the TMD of plant GnTI enzymes 129
that plays an essential role in GnTI cis/medial-Golgi localization and N-glycan processing. 130
Mutagenesis of this conserved residue altered the subcellular localization of GnTI and impaired its 131
function when expressed under native-like conditions. In summary, our study reveals a motif that 132
governs Golgi-resident enzyme distribution and provides insights into the subcellular localization 133
mechanisms of Golgi-located type II membrane proteins. 134
135
RESULTS 136
The exoplasmic half of the GnTI TMD is important for the cis/medial-Golgi localization 137
We compared the GnTI TMD sequences from different plant species to identify amino acid motifs that 138
are involved in the cis/medial-Golgi distribution. The sequence alignment revealed the presence of 139
several highly conserved amino acid residues, especially in the exoplasmic half of the TMD (Fig. 1A 140
and Suppl. Fig. S1). Consequently, we exchanged the exoplasmic half of the TMD from the well-141
characterized trans-Golgi marker rat 2,6-sialyltransferase (ST) (Boevink et al., 1998) with 9 142
exoplasmic amino acids from the 18-residue TMD of NtGnTI. The resulting chimeric CTS regions, 143
ST/GnTI and GnTI/ST (Fig. 1B and Suppl. Fig. S2), were C-terminally fused to the NtGnTI catalytic 144
domain and GFP, transiently expressed in Nicotiana benthamiana leaves and examined using a 145
confocal microscope. Co-localization was carried out with the 1,2-galactosyltransferase MUR3, a 146
xyloglucan synthesizing enzyme that has been associated with medial-Golgi cisternae (Chevalier et al., 147
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2010). As expected, both constructs harbouring the chimeric CTS regions displayed Golgi localization 148
(Suppl. Fig. S3). To assess the degree of co-localization in the Golgi, the Pearson’s correlation co-149
efficient was determined for co-localization with the cis-Golgi protein AtCASP (Osterrieder et al., 150
2010; Schoberer et al., 2010) fused to mRFP and the trans-Golgi marker ST-CTS-mRFP (Fig. 1C and 151
Suppl. Table S1). The experiment was carried with the CTS regions lacking the catalytic domains to 152
enhance their expression. The co-localization analysis revealed a similar correlation for ST/GnTI-153
CTS-GFP and NtGnTI-CTS-GFP as well as for GnTI/ST-CTS-GFP and ST-CTS-GFP, indicating that 154
the 9 exoplasmic amino acids from the GnTI TMD contain a sequence motif for the concentration in a 155
distinct Golgi sub-compartment. 156
To obtain further support for our finding, we fused the ST/GnTI-CTS and GnTI/ST-CTS regions to the 157
catalytic domain of human 1,4-galactosyltransferase (GALT) and performed an N-glycan processing 158
assay. In this assay, the co-expression of GALT with a secreted glycoprotein reporter results in the 159
transfer of 1,4-galactose to the non-reducing end of N-glycans on the reporter (Schoberer et al., 160
2014). Dependent on the Golgi compartmental localization of the chimeric GALT, a different N-161
glycan pattern is obtained due to competition of GALT with endogenous N-glycan processing 162
enzymes (Suppl. Fig. S4). Thus, the N-glycan processing assay allows cis/medial-Golgi-localized 163
GALT (leading to incompletely processed N-glycans) to be distinguished from medial/trans-Golgi-164
localized GALT (higher amount of completely processed N-glycans). Mass spectrometry-based 165
analysis of a glycopeptide derived from the reporter revealed incompletely processed N-glycans for 166
ST/GnTI-CTS and a substantial amount of completely processed N-glycans for GnTI/ST-CTS (Suppl. 167
Fig. S5), providing additional evidence for a role of the exoplasmic TMD half in intra-Golgi 168
localization. 169
We have previously shown that a trans-Golgi targeted GnTI carrying the ST-CTS region fails to 170
complement the complex N-glycan processing defect of GnTI-deficient Arabidopsis plants (Schoberer 171
et al., 2014). Here, we fused the chimeric CTS regions with the exchanged exoplasmic TMD domains 172
to the AtGnTI catalytic domain. GFP-fused AtGnTI variants were expressed in Arabidopsis gntI T-173
DNA insertional mutants under the control of a weak endogenous AtGnTI promoter and the processing 174
to complex N-glycans was analyzed by immunoblotting. Consistent with an effect on cis/medial-Golgi 175
localization, GnTI/ST-AtGnTI-GFP carrying the exoplasmic TMD region from ST did not restore 176
complex N-glycan formation (Suppl. Fig. S6). However, 8 out of 12 tested transgenic ST/GnTI-177
AtGnTI-GFP lines displayed considerable amounts of complex N-glycans indicating relocation of the 178
protein to an earlier Golgi compartment where it initiated complex N-glycan processing. Taken 179
together, our results show that an amino acid motif in the exoplasmic half of the NtGnTI TMD 180
contains a signal for efficient steady-state cis/medial-Golgi localization. 181
182
A single amino acid residue in the NtGnTI TMD is crucial for cis/medial-Golgi localization 183
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To further characterize the sequence motif in the exoplasmic half of the NtGnTI TMD, we generated 184
another chimeric GALT variant where the LFAV tetrapeptide sequence from ST was replaced by the 185
FIYIQ amino acid motif from NtGnTI (STFIYIQ-GALT) and carried out the N-glycan processing assay. 186
Co-expression of STFIYIQ-GALT resulted in the generation of incompletely processed N-glycans 187
(Suppl. Fig. S5), indicating that the FIYIQ-motif plays a role in NtGnTI cis/medial-Golgi localization. 188
One of the two fully conserved residues in this motif is the glutamine at position 25 (Fig. 1A). We 189
replaced this glutamine (Q25) with alanine and analyzed the localization of NtGnTI-Q25A fused to 190
GFP or mRFP in N. benthamiana leaf epidermal cells. NtGnTI-Q25A was found in Golgi bodies as 191
well as other intracellular compartments indicating that the subcellular localization was changed 192
(Suppl. Fig. S3). Co-localization of NtGnTI-Q25A-GFP with mRFP-AtCASP and ST-CTS-mRFP 193
indicated a shift in the Golgi localization towards the trans-Golgi (Fig. 1D). The N-glycan processing 194
assay revealed the presence of processed N-glycans when NtGnTI-Q25A-GALT was co-expressed 195
with the glycoprotein reporter and the N-glycan profile was comparable to that derived from ST-196
GALT (Fig. 1E). 197
In addition to the accumulation in the trans-Golgi, GFP- or mRFP-tagged NtGnTI-Q25A was 198
observed in the vacuole (Fig. 2A and Suppl. Fig. S3), which was clearly visible three to four days after 199
infiltration. Co-localization with aleu-GFP (Humair et al., 2001) confirmed targeting to the lytic 200
vacuole (Fig. 2B). No ER localization was observed for NtGnTI-Q25A fusion proteins, but 201
accumulation of fluorescence in the apoplast was occasionally observed indicating the secretion of 202
full-length NtGnTI-Q25A or a degradation product. In addition to vacuolar and trans-Golgi 203
localization, we observed smaller puncta in NtGnTI-Q25A expressing cells that did not represent 204
typical Golgi bodies (Fig. 2C). Additional co-localization experiments with different subcellular 205
markers were carried out to identify these small puncta. However, the cellular structures overlapped 206
with neither the TGN marker mRFP-SYP61 (Sanderfoot et al., 2001) (Fig. 2D), the early endosome 207
marker GFP-RABF2b (ARA7) (Kotzer et al., 2004) (Fig. 2E), the autophagosome marker GFP-208
ATG8e (Contento et al., 2005) (Fig. 2F), nor the late endosome/multivesicular body marker GFP-209
RABF1 (ARA6) (Ueda et al., 2001) (Fig. 2G). These data show that the glutamine residue confers 210
efficient cis/medial-Golgi localization of NtGnTI. The mutated NtGnTI-Q25A, on the other hand, 211
accumulates in the trans-Golgi and substantial amounts are released to post-Golgi compartments 212
including the vacuole by an unknown trafficking route. 213
214
Arabidopsis GnTI-Q23A fails to complement GnTI-deficient plants 215
To investigate the functional relevance of the identified glutamine residue within the TMD of GnTI, 216
we expressed GFP- and mRFP-tagged AtGnTI and the AtGnTI-Q23A variant with the conserved 217
glutamine (at position 23 in AtGnTI) changed to an alanine in the gntI mutant. A functional AtGnTI in 218
the cis/medial-Golgi will restore the N-glycan processing which can be analyzed by immunoblotting 219
using antibodies directed against Golgi-processed N-glycans carrying 1,2-xylose and core 1,3-220
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fucose (Strasser et al., 2004). When expressed under the control of the weak AtGnTI promoter, 221
complex N-glycans were detected in gntI plants expressing AtGnTI-GFP, but not in AtGnTI-Q23A-222
GFP expressing plants (Fig. 3A). AtGnTI-GFP could be found on immunoblots, whereas AtGnTI-223
Q23A-GFP was hardly detectable. Comparison of the transcript levels between the two AtGnTI 224
variants by reverse transcription quantitative PCR (RT-qPCR) did not reveal considerable variation in 225
mRNA levels (Fig. 3B), suggesting that the difference in protein abundance is caused by a decreased 226
stability of the AtGnTI-Q23A-GFP protein. The same result was obtained for complementation of 227
Arabidopsis cgl1 plants that carry a missense mutation in the AtGnTI gene (von Schaewen et al., 1993; 228
Strasser et al., 2005) or when mRFP-tagged AtGnTI variants were used for complementation of gntI 229
(Suppl. Fig. S7). MALDI-TOF MS analysis of N-glycans from gntI expressing AtGnTI-GFP under the 230
control of the weak endogenous AtGnTI promoter showed partial formation of complex N-glycans. By 231
contrast, no peaks corresponding to complex N-glycans were detectable for AtGnTI-Q23A-GFP 232
(Suppl. Fig. S8). Expression of AtGnTI-mRFP or AtGnTI-Q23A-mRFP under the control of the 233
constitutive ubiquitin 10 (UBQ10) promoter restored the complex N-glycan formation in gntI plants 234
(Fig. 3C). However, in gntI expressing the mutant AtGnTI-Q23A variant, MALDI-TOF MS analysis 235
revealed the presence of considerable amounts of unprocessed Man5GlcNAc2 (Man5), which is the 236
major N-glycan in AtGnTI-deficient plants (Fig. 3D and Suppl. Fig. S8) (von Schaewen et al., 1993; 237
Strasser et al., 2005). Thus, even the overexpression of the AtGnTI-Q23A variant could not fully 238
complement the N-glycan processing defect of gntI which would result in reduced levels of Man5 and 239
more of the truncated N-glycan carrying 1,2-xylose and core 1,3-fucose (MMXF). This finding is 240
fully consistent with the observed mistargeting due to the disturbance of the conserved sequence motif 241
within the GnTI TMD. 242
Immunoblot analysis of AtGnTI-mRFP stably expressed under the control of the UBQ10 promoter in 243
gntI revealed a single band of the expected size of approximately 77 kDa for the fusion protein. By 244
contrast, the AtGnTI-Q23A variant displayed 3 to 4 bands (Fig. 3C). The upper one corresponds to the 245
size of the full-length AtGnTI-mRFP protein, whereas the other 2–3 bands represent faster migrating 246
variants of different sizes suggesting the presence of degradation products. In a previous study, we 247
observed that GnTI forms a homodimer which requires a sequence domain from the stem region 248
(Schoberer et al., 2013; Schoberer et al., 2014). Here, we tested whether mistargeted AtGnTI-Q23A 249
can still interact with the non-mutated AtGnTI. Both proteins were co-expressed in N. benthamiana 250
leaf epidermal cells and subjected to co-immunoprecipitation analysis. In contrast to wild-type 251
AtGnTI, a substantially decreased amount of AtGnTI-Q23A was co-purified (Fig. 3E). Notably, only 252
the band representing full-length AtGnTI interacted, whereas the faster migrating bands from AtGnTI-253
Q23A were not co-purified providing potential evidence that they represent spatially separated 254
variants from post-Golgi compartments. FRET-FLIM analysis of transiently expressed NtGnTI and 255
NtGnTI-Q25A fusion proteins showed that the FRET efficiency determined by FLIM was also 256
reduced for the wild-type/mutant NtGnTI combination compared to the homodimer formation of wild-257
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type NtGnTI (Table 1). Confocal microscopy of transgenic seedlings confirmed the Golgi localization 258
of AtGnTI-mRFP. AtGnTI-Q23A-mRFP-expressing seedlings hardly displayed any Golgi 259
localization, but showed a fluorescence signal in the apoplast which may be derived from the cleavage 260
and secretion of mRFP. A similar secretion has been described for the trans-Golgi marker ST-mRFP 261
(Runions et al., 2006), but in the case of AtGnTI-Q23A-mRFP, the effect is less pronounced (Fig. 3F). 262
To investigate the fate of the mutant variant in more detail, 10-day-old seedlings were treated with 263
cycloheximide and the AtGnTI-mRFP protein level was analyzed at different time points by 264
immunoblotting. AtGnTI-mRFP levels were stable during the chase period. The AtGnTI-Q23A-mRFP 265
levels, on the other hand, decreased over time indicating a faster clearance (Fig. 4A). Moreover, we 266
treated Arabidopsis seedlings with the proteasomal inhibitor MG132 (Fig. 4B) as well as with the 267
vacuolar H+-ATPase inhibitor concanamycin A (Dettmer et al., 2006) to see whether the discrete 268
degradation products were generated in an acidic compartment like the vacuole (Fig. 4C). By 269
increasing the pH in the organelle, the catalytic activity of proteases is diminished which may result in 270
the accumulation of proteins targeted for vacuolar degradation (Chamberlain et al., 2008). 271
Pharmacological inhibition with MG132 or concanamycin A did not result in substantial changes in 272
the pattern of AtGnTI-Q23A-mRFP degradation products. However, incubation of Arabidopsis 273
seedlings in a combination of MG132 and concanamycin A increased the amounts of the intact 274
AtGnTI-Q23A-mRFP protein (Fig. 4D). By contrast, the ER-associated degradation inhibitor 275
kifunensine did not stabilize AtGnTI-Q23A-RFP (Fig. 4E). 276
277
A polar or positively charged amino acid is required for proper Golgi targeting and in vivo N-278
glycan processing 279
Different features of the TMD have been suggested to play a role for the subcellular localization of 280
type II membrane proteins including the TMD length, the presence of polar residues in the exoplasmic 281
region of the leaflet or changes in the average amino acid volume in the exoplasmic half (Munro, 282
1995; Sharpe et al., 2010; Quiroga et al., 2013). To define the signal for proper AtGnTI localization in 283
more detail, we analyzed the contribution of different amino acid residues at this position to GnTI 284
localization and processing of complex N-glycans. We performed site-directed mutagenesis and 285
generated mutants where Q23 from AtGnTI was replaced by His, Leu, Glu, Tyr, Val and Ser. 286
Confocal microscopy of mRFP-fusion proteins expressed under the control of the ubiquitin 10 287
promoter showed that AtGnTI-Q23V, AtGnTI-Q23L and AtGnTI-Q23S behaved like AtGnTI-Q23A 288
and displayed mistargeting to post-Golgi compartments. AtGnTI-Q23H showed mainly Golgi 289
localization similar to that of AtGnTI. AtGnTI-Q23E and AtGnTI-Q23Y displayed Golgi localization 290
as well as post-Golgi distribution (Fig. 5A and Suppl. Fig. S9). In agreement with altered localization, 291
an increase of the faster migrating degradation product was observed for various AtGnTI mutants (Fig. 292
5B). Together, these data suggest that a polar amino acid residue with a long side chain (glutamine) or 293
a positively charged residue like histidine is tolerated at this particular position within the GnTI TMD, 294
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whereas hydrophobic residues with small (e.g. alanine) or large volumes (e.g. leucine) are not 295
tolerated (Suppl. Fig. S10). To determine the effect of the different residues on in vivo N-glycan 296
processing, transgenic Arabidopsis gntI expressing the different mutant variants under the control of 297
the AtGnTI promoter were generated. In agreement with our localization data, only AtGnTI-Q23H-298
RFP displayed a similar degree of complex N-glycan formation like AtGnTI-RFP. Other mutants did 299
not complement the defect at all or resulted only in partial complementation (e.g. AtGnTI-Q23E) (Fig. 300
5C). Likewise, substitution of adjacent highly conserved amino acids (F19A, M24I and R25A) did not 301
obviously affect the sub-Golgi distribution of AtGnTI and resulted in gntI complementation (Suppl. 302
Fig. S11). 303
304
The Arabidopsis Golgi -mannosidase MNS1 is mistargeted and non-functional when fused to 305
the AtGnTI-CTS-Q23A domain 306
The conserved glutamine in the TMD is important for the cis/medial-Golgi localization of GnTI. To 307
see whether other N-glycan processing enzymes are mistargeted when their CTS region is exchanged 308
with the mutated GnTI domain, we fused the AtGnTI CTS region to the catalytic domain of the 309
Arabidopsis Golgi -mannosidase MNS1, which trims mannose residues from N-glycans in the Golgi 310
and acts upstream of GnTI by converting Man8GlcNAc2 to Man5GlcNAc2 (Liebminger et al., 2009). 311
We generated chimeric AtGnTI-CTS-MNS1-GFP and AtGnTI-CTS-Q23A-MNS1-GFP constructs, 312
where the CTS region from GnTI is fused to the catalytic domain of MNS1 and GFP. The chimeric 313
proteins were expressed under the control of the endogenous MNS1 promoter in the mns1 mns2 mns3 314
triple mutant that generates mainly Man9GlcNAc2 structures. Restoration of MNS1 activity in the 315
cis/medial-Golgi of mns1 mns2 mns3 leads to the formation of complex N-glycans (Liebminger et al., 316
2009). In contrast to AtGnTI-CTS-MNS1-GFP, the MNS1 variant with the Q23A substitution in the 317
AtGnTI TMD failed to restore complex N-glycan formation (Fig. 6A) and no Golgi localization was 318
detected in root cells (Fig. 6B). We previously showed that the mns1 mns2 mns3 mutant displays a 319
severe root-growth phenotype due to the block in N-glycan processing (Liebminger et al., 2009). 320
Expression of MNS1:AtGnTI-CTS-MNS1-GFP in mns1 mns2 mns3 led to an almost wild type-like 321
root growth of seedlings whereas the MNS1 variant harbouring the GnTI targeting region with the 322
mutated glutamine displayed aberrantly swollen and truncated roots (Fig. 6C). The difference was 323
even more pronounced when seeds were grown on 4% sucrose which enhances the phenotype (Fig. 6D 324
and Suppl. Fig. S12). These results highlight the importance of proper Golgi localization mediated by 325
the glutamine-containing sequence motif in the GnTI TMD. 326
327
Knockdown of COPI subunits leads to GnTI mislocalization to the vacuole 328
To investigate whether COPI subunits are involved in Golgi localization of GnTI, we carried out 329
immunoprecipitation of AtGnTI-mRFP and AtGnTI-Q23A-mRFP and monitored if endogenous 330
Arabidopsis COP is co-purified. Immunoblot analysis did not reveal any signal corresponding to 331
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10
COP suggesting that there is no or only a very weak interaction with AtGnTI (Suppl. Fig. S13). Next, 332
we generated RNAi constructs to silence endogenous COP and COP in N. benthamiana leaves. 333
When the RNAi constructs were transiently co-expressed with the Golgi protein RFP-EMP12 that 334
carries a COPI binding site, mistargeting to the vacuole was observed which is in agreement with 335
earlier studies (Gao et al., 2014; Woo et al., 2015). Likewise, NtGnTI-mRFP was mislocalized to the 336
vacuole in cells co-infiltrated with COP or COP RNAi constructs (Fig. 7A and Suppl. Fig. S13) and 337
the trans-Golgi-resident ST-NtGnTI-mRFP was found extracellularly, as described previously (Ahn et 338
al., 2015), and in the vacuole (Fig. 7A). Silencing of these COPI subunits targeted AtGnTI-mRFP (or 339
AtGnTI-GFP) to the vacuole, whereas for AtGnTI-mRFP-Q23A, the vacuolar localization was not 340
changed. AtGnTI-mRFP-Q23A was also localized predominately to the vacuole when incubated for 1 341
or 2 h in brefeldin A (BFA) (Fig. 7B). Immunoblot analysis revealed an accumulation of the intact 342
mRFP fusion protein upon co-expression of the COPI silencing constructs accompanied by an increase 343
of cleaved mRFP (Fig. 7C). This finding indicates that the Golgi-resident GnTI is not efficiently 344
recycled in the Golgi complex when COPI formation is impaired leading to vacuolar targeting. 345
Moreover, transient overexpression of AtGnTI-mRFP in N. benthamiana caused an increase in 346
truncated fragments (Suppl. Fig. S13), suggesting a saturation of the GnTI localization mechanism. 347
348
DISCUSSION 349
How can a distinct amino acid motif within the transmembrane domain mediate specific 350
subcellular localization of a Golgi-resident protein? 351
A large number of secretory proteins are glycosylated and undergo glycan processing reactions in the 352
Golgi. The concentration of processing enzymes in distinct Golgi cisternae is a prerequisite for the 353
sequential and orderly modification of glycans. However, the organization of Golgi-resident enzymes 354
and the mechanism of cargo transport through the Golgi are under debate. In the stable 355
compartment/vesicular transport model, the cargo processing enzymes remain in their cisternae and 356
cargo is transported from the cis-Golgi to the trans-Golgi by vesicular transport and/or transient 357
tubular connections (Rabouille and Klumperman, 2005; Glick and Luini, 2011). An important feature 358
of the model is the absence of Golgi-resident enzymes in anterograde trafficking carriers and the 359
differential localization of Golgi-resident enzymes is achieved by retention in specific Golgi cisternae. 360
According to this model, the motif in the GnTI TMD constitutes a cis/medial-Golgi retention signal 361
that prevents anterograde transport. 362
In the cisternal maturation model, cargo stays in the cisternae, which matures from cis-to-trans 363
together with Golgi-resident enzymes. To maintain their gradient-like distribution across the Golgi 364
stack, the cargo processing enzymes are cycled back from late to earlier Golgi cisternae by COPI 365
vesicles (Villeneuve et al., 2017). The recycling of Golgi processing enzymes transforms 366
biosynthetically inactive cisternae into biosynthetically active cisternae (Donohoe et al., 2013). In the 367
cisternal maturation model, the GnTI TMD motif acts as a signal for incorporation into retrograde 368
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11
transport carriers that mediate recycling of the glycosyltransferase. Even though an interaction with 369
COPI subunits was not found so far, the mislocalization of GnTI to post Golgi compartments after 370
COP and COP knockdown can be explained by impaired recycling from the trans-Golgi to an 371
earlier cisternae as proposed by the cisternal maturation model (Fig. 8). The existence of two types of 372
COPI vesicles in plants has been shown by electron tomography (Donohoe et al., 2007). COPIa-type 373
vesicles are located in the vicinity of cis cisternae and likely contribute to Golgi-to-ER transport. 374
COPIb-type vesicles have been found next to medial- and trans-Golgi cisternae. Immunolabelling 375
showed that the cis/medial-Golgi N-glycan processing enzyme MNS1 that generates the N-glycan 376
substrate for GnTI is present in COPIb-type vesicles (Donohoe et al., 2007). Likewise, GnTI may also 377
be incorporated into COPIb-type carriers that cycle from the trans-Golgi to the cis/medial-Golgi. In 378
the absence of recycling, GnTI accumulates in the trans-Golgi from where it is transported to post-379
Golgi compartments and the vacuole. A comparable recycling mechanism has been proposed for the 380
cis/medial-Golgi-located multiple transmembrane protein AtEMP12 (Gao et al., 2012; Woo et al., 381
2015). AtEMP12 has a conserved COPI-interacting KXD/E motif in its C-terminal cytoplasmic 382
region. This amino acid sequence is not present in the short cytoplasmic tail of GnTI and the 383
conserved glutamine residue in the exoplasmic half of the transmembrane protein is not accessible for 384
a direct interaction with COPI subunits. Therefore, GnTI may bind via its transmembrane domain to 385
an integral membrane protein which serves as an adaptor for incorporation into COPI carriers. 386
The polytopic yeast membrane proteins ERV14 and RER1 are cargo sorting receptors that recognize 387
transmembrane domain features and sort cargo into COPII or COPI carriers (Sato et al., 2001; Herzig 388
et al., 2012; Gomez-Navarro and Miller, 2016). Interestingly, RER1 recognizes polar residues in the 389
transmembrane domain of single TMD proteins such as SEC12 or SEC71 (Sato et al., 2001, 2003) and 390
retrieves them from the Golgi to the ER. Moreover, proper localization of the yeast type II membrane 391
protein ER 1,2-mannosidase I depends on the presence of RER1 (Massaad et al., 1999). The TMD of 392
ER 1,2-mannosidase I interacts with RER1 and ER 1,2-mannosidase I is missorted to the vacuole in 393
the absence of a functional RER1. The Arabidopsis RER1 family consists of three members that can 394
complement the mislocalization of cargo proteins in Saccharomyces cerevisiae rer1, thus suggesting 395
a similar function in plants. However, RER1-interacting proteins and a role of RER1 in intra-Golgi 396
transport have not been described in plants. In addition to ERV14 and RER1, EMP family proteins are 397
another group of Golgi-resident integral membrane proteins that may directly interact with GnTI and 398
play a role in its subcellular localization. 399
Apart from a direct protein-protein interaction and the involvement of an adapter protein, the motif in 400
the GnTI TMD may be crucial for a specific protein-lipid interaction to partition GnTI into a 401
membrane domain competent for recycling to earlier Golgi cisternae (Fig. 8). In mammalian cells, the 402
interaction of a distinct sequence motif in the transmembrane domain of p24 with an individual 403
sphingolipid affects the COPI-dependent retrograde transport (Contreras et al., 2012). Moreover, 404
disruption of sphingomyelin homeostasis has led to changes in glycan processing by disturbance of 405
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12
glycosyltransferase organization in distinct Golgi membranes (van Galen et al., 2014). In plants, 406
alterations in the Golgi lipid composition affect protein secretion and Golgi morphology, but the Golgi 407
localization of the trans-Golgi marker ST appears unaltered (Melser et al., 2010). 408
The function of GnTI in N-glycan processing as well as its subcellular localization are conserved in 409
mammals and plants (von Schaewen et al., 1993; Gomez and Chrispeels, 1994; Bakker et al., 1999; 410
Strasser et al., 1999) and there is evidence for cycling of mammalian GnTI (Hoe et al., 1995). A 411
recently identified COPI-binding motif in mammalian glycosyltransferases is not present in the 412
cytoplasmic tail of human GnTI and, in contrast to other cis/medial-Golgi enzymes, no direct binding 413
of GnTI to COPI subunits was found (Liu et al., 2018). GOLPH3, the mammalian VPS74 homolog, is 414
involved in the incorporation of certain glycosyltransferases into COPI vesicles (Eckert et al., 2014) 415
but its absence had no effect on GnTI localization in human cells (Liu et al., 2018). A role for the 416
TMD of mammalian GnTI in Golgi localization has also been described in other studies (Burke et al., 417
1992; Tang et al., 1992; Burke et al.; Nilsson et al., 1996) and the TMD from mammalian GnTIs share 418
a sequence motif with a highly conserved asparagine residue at a similar position. Whether this residue 419
or other features of the TMD are involved in intra-Golgi localization of mammalian GnTI remains to 420
be shown. A general role of the TMD in Golgi retention of type II membrane proteins has been 421
proposed for yeast and mammals (Quiroga et al., 2013) and individual amino acids including polar 422
residues within the TMD contribute to Golgi localization of mammalian proteins (Aoki et al., 1992; 423
Machamer et al., 1993), but a specific receptor or lipid interaction has not been described. 424
In summary, our data show that a transmembrane motif-based sorting mechanism mediates the steady-425
state localization of GnTI in plants. Our findings are fully consistent with the fact that GnTI homomer 426
formation plays no or only a minor role in cis/medial Golgi localization (Schoberer et al., 2013; 427
Schoberer et al., 2014). The mechanism which is functional in Arabidopsis as well as in N. 428
benthamiana is distinct from the earlier proposed bilayer thickness-mediated retention, the kin 429
recognition and the more recently recognized cytoplasmic tail-dependent sorting (Tu and Banfield, 430
2010). Our model proposes that a conserved polar amino acid in the TMD promotes the interaction 431
with a yet unknown adaptor protein/complex or the segregation into a distinct lipid domain. Impaired 432
recycling in the absence of the specific interaction results in GnTI exit from the Golgi and trafficking 433
to post-Golgi compartments including the vacuole where GnTI is eventually degraded. This trafficking 434
route may highlight the default degradation pathway for glycosyltransferases that are not needed 435
anymore in a plant cell. 436
437
MATERIALS AND METHODS 438
Plant material and growth conditions 439
Arabidopsis thaliana (ecotype Col-0) and mutant plants were grown at 22°C under long-day 440
conditions (16-h-light/8-h-dark photoperiod) on soil or on 0.5 × Murashige and Skoog (MS) medium 441
containing 1% (w/v) sucrose and 0.8% (w/v) agar. The mns1 mns2 mns3 (Liebminger et al., 2009), 442
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13
gntI (Schoberer et al., 2014) and cgl1 (von Schaewen et al., 1993; Strasser et al. 2005) mutants were 443
available from previous studies. For the measurement of the root length, Arabidopsis seedlings were 444
grown for 10 days on 0.5 x MS medium supplemented with 4% sucrose. For the inhibitor treatments, 445
Arabidopsis seedlings were incubated for 17 h in liquid MS medium supplemented with 1 µM 446
concanamycin A (Sigma Aldrich), 20 µM MG132 (Sigma Aldrich), 100 µg/ml cycloheximide (Sigma 447
Aldrich) or 20 µM kifunensine (Santa Cruz Biotechnology). Nicotiana benthamiana were grown 448
under long-day conditions at 24°C. 449
450
Cloning of constructs 451
DNA fragments coding for the chimeric GnTI/ST-, ST/GnTI- and STFIYIQ-CTS regions were obtained 452
by custom DNA synthesis (GeneArt, Thermo Fisher Scientific) and ligated into XbaI/BamHI sites of 453
p20-Fc (CaMV35S promoter, used for co-localization analysis) (Suppl. Fig. S2) (Schoberer et al., 454
2009), pF (CaMV35S promoter, GALT fusions containing the catalytic domain of human 1,4-455
galactosyltransferase, used for the N-glycan processing assay) (Schoberer et al., 2014), p46 (UBQ10 456
promoter, expression of proteins containing a CTS region fused to the catalytic domain of NtGnTI and 457
GFP) (Schoberer et al., 2014), p49 (like p46 but with a C-terminal mRFP tag), p57 (386-bp weak 458
AtGnTI promoter, complementation of gntI plants with AtGnTI catalytic domain fused to GFP) 459
(Schoberer et al., 2014), p66 (UBQ10 promoter, expression of proteins containing a CTS region fused 460
to the catalytic domain of AtGnTI and GFP), p67 (1000-bp AtGnTI promoter, complementation of gntI 461
plants with AtGnTI catalytic domain fused to GFP), p68 (like p67 but with a C-terminal mRFP tag) 462
and p69 (like p66 but with a C-terminal mRFP tag). Plant expression vector p67 was generated by 463
insertion of the 1000-bp AtGnTI promoter region into the HindIII/XbaI site of p57. The 1000-bp 464
AtGnTI promoter region was amplified from Arabidopsis genomic DNA by PCR using 465
AthGnT33/AthGnTI_13R (Suppl. Table S2). p68 was generated by replacing GFP in vector p67 with 466
mRFP. p66 and p69 were generated by replacement of the AtGnTI promoter with the UBQ10 467
promoter. 468
The constructs for expression of the glycoprotein reporter (monoclonal antibody) (Strasser et al., 469
2009), ST-mRFP, ST-GFP, NtGnTI-CTS-GFP (Boevink et al., 1998; Schoberer et al., 2009), mRFP-470
AtCASP (Schoberer et al., 2010), NtGnTI-GFP, NtGnTI-RFP, ST-NtGnTI-GFP, ST-NtGnTI-mRFP 471
(Schoberer et al., 2014), aleu-GFP (Shin et al. 2017), mRFP-EMP12 (Shin et al., 2018), mRFP-SYP61 472
(Sanderfoot et al., 2001), GFP-RABF2b (Kotzer et al., 2004) and GFP-RABF1 (Ueda et al., 2001) 473
were all available from previous studies. p48-MUR3 was generated by PCR amplification from Col-0 474
cDNA of the MUR3 (AT2G20370) coding region using primers KAM1_1F/KAM1_3R. The PCR 475
product was XbaI/BglII digested and cloned into p48 (Hüttner et al., 2014). For GFP-ATG8e 476
expression, the coding region for Arabidopsis ATG8e (AT2G45170) was amplified by PCR from Col-477
0 cDNA using primers ATG8e_1F/ATG8e_2R. The PCR product was XbaI/BamHI digested and 478
cloned into XbaI/BamHI digested vector p37 (CaMV35S promoter). For the generation of p37, the 479
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14
GFP coding region lacking the stop codon was PCR-amplified from the vector p20F using primers 480
GFP-14F/GFP-15R. The PCR product was SpeI/XbaI-digested and ligated into XbaI-linearized pPT2m 481
(Strasser et al., 2005) resulting in the N-terminal GFP expression vector p37. 482
NtGnTI and AtGnTI CTS regions with mutated single amino acids were generated by site-directed 483
mutagenesis using the Quikchange site directed mutagenesis protocol (Stratagene) and cloned into the 484
XbaI/BamHI sites of the used expression vectors. The expression construct p87 (MNS1 promoter, 485
MNS1 catalytic domain fused to GFP) for the chimeric MNS1 fusion proteins was described recently 486
(Veit et al., 2018). For expression of AtGnTI and AtGnTI-Q23A CTS regions, the corresponding 487
DNA was amplified with primers AthGnTI_14F/AthGnTI_16R and ligated into XbaI/BamHI digested 488
p87. 489
490
LC-ESI-MS analysis 491
Five-week-old N. benthamiana plants were used for Agrobacterium tumefaciens-mediated transient 492
expression of indicated constructs using the agroinfiltration technique as described previously 493
(Schoberer et al., 2009). The glycoprotein reporter was purified by affinity chromatography, subjected 494
to SDS-PAGE and Coomassie Brilliant Blue staining (Schoberer et al., 2014). The purified protein 495
band was excised from the gel, destained, carbamidomethylated, in-gel trypsin digested and analyzed 496
by liquid-chromatography electrospray ionization-mass spectrometry (LC-ESI-MS) (Stadlmann et al., 497
2008; Schoberer et al., 2009). A detailed explanation of N-glycan abbreviations and nomenclature can 498
be found at http://www.proglycan.com. 499
500
Complementation of Arabidopsis mutants 501
Arabidopsis gntI, cgl1 and mns1 mns2 mns3 mutants were transformed with different constructs by 502
floral dipping and selected on hygromycin (Strasser et al., 2004). The presence of the construct was 503
confirmed by PCR with specific primers. Proteins extracted from leaves of five-week-old plants were 504
subjected to SDS-PAGE under reducing conditions. Glycoproteins with processed N-glycans were 505
analyzed by immunoblotting with anti-horseradish peroxidase antibodies (anti-HRP, Sigma) that bind 506
to complex, truncated and hybrid N-glycans carrying 1,2-xylose and/or core 1,3-fucose residues 507
(Strasser et al., 2004; Jin et al., 2008; Kaulfürst-Soboll et al., 2011). MALDI-MS analysis was carried 508
out from 500 mg of leaves as described (Strasser et al., 2004). 509
510
Confocal imaging of fluorescent protein fusions 511
Leaves of five-week-old N. benthamiana plants were infiltrated with agrobacterium suspensions 512
carrying the protein(s) of interest with optical densities (OD600) from 0.05 to 0.2. Confocal images 513
were acquired 2–4 days post infiltration (dpi) on an upright Leica SP5 II confocal microscope using 514
the Leica LAS AF software system. GFP and mRFP were excited with the 488-nm and 561-nm laser 515
line, respectively, and detected at 500–530 nm and 600–630 nm, respectively. Dual-color image 516
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15
acquisition of cells expressing both GFP and mRFP was performed simultaneously. Post-acquisition 517
image processing was performed in Adobe Photoshop CS5. 518
For co-localization analyses of co-expressed fluorescent protein fusions, images of latrunculin B-519
treated cells expressing the indicated NtGnTI and ST-CTS constructs together with the cis/medial-520
Golgi protein mRFP-AtCASP (Schoberer et al., 2010) or the trans-Golgi marker ST-mRFP (Boevink 521
et al., 1998) were acquired 2 dpi under non-saturating conditions using a 512 x 512 image format, 522
zoom factor 3 and a ×100/1.40 NA oil immersion objective of the Leica SP5. The pinhole was set to 1 523
airy unit, and background noise was reduced by 4–8 times line averaging. Only cells with comparable 524
GFP and mRFP fluorescence levels were considered for analysis. The images obtained were used for 525
co-localization analysis using the Pearson’s correlation coefficient. Calculations were made on 9–15 526
confocal images per co-expressed combination using the IMAGEJ 1.46 m plug-in JACOP (Bolte and 527
Cordelières, 2006). Statistical analyses were performed using a two-tailed Student’s t-test for the 528
comparison of two samples, assuming equal variances. FRET-FLIM data acquisition and analysis was 529
carried out as described previously (Schoberer et al., 2014). 530
531
Co-immunoprecipitation and immunoblotting 532
For analysis of AtGnTI-Q23A and AtGnTI interaction, AtGnTI-Q23A-mRFP (p69 vector) or AtGnTI-533
mRFP (p69 vector) was co-expressed with AtGnTI-GFP (p66 vector) by agroinfiltration of N. 534
benthamiana leaves. Two days after infiltration, the GFP-tagged protein was purified using GFP-Trap-535
A beads (ChromoTek) and co-purified protein was analyzed by immunoblotting with anti-mRFP 536
antibodies. NBR1 was detected with anti-NBR1 (Agrisera) and antibodies against actin (Agrisera) and 537
-tubulin (Sigma) were used as loading controls. 538
539
RT-qPCR analysis 540
RNA was extracted from 18-day-old Arabidopsis seedlings using the SV Total RNA Isolation System 541
(Promega) and the iScript cDNA Synthesis Kit (Bio-Rad, Vienna Austria). An aliquot of the cDNA 542
was used for the PCR amplification using iQ SYBR Green Supermix (Bio-Rad) with an iCycler (Bio-543
Rad) as described in detail previously (Hüttner et al., 2014). Expression of GFP-fusion proteins was 544
monitored using GFP-specific primers GFP-Q1F and GFP-Q2R and normalized to the UBQ5 545
transcript expression monitored with primers UBQ5_Q1F/UBQ5_Q2R. 546
547
Knockdown of N. benthamiana coatomer subunits 548
To knockdown the expression of the N. benthamiana COP and COP subunits, RNAi constructs were 549
generated as described (Shin et al., 2017). A synthetic DNA fragment consisting of intron 2 from 550
Arabidopsis 1,2-xylosyltransferase and an antisense DNA fragment corresponding to the coding 551
sequence for amino acids 35–186 of COP or 306–473 of COP were obtained by GeneArt gene 552
synthesis. The vector with the synthetic DNA was used as a template for PCR with primers Nb-eCOP-553
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16
1F and Nb-eCOP-2R for COP and Nb-dCOP-1F and Nb-dCOP-2R for COP. The ‘sense’ PCR 554
product was digested with XbaI/KpnI and cloned into the synthetic DNA containing vector to generate 555
a sense–intron–antisense hairpin construct. The sense–intron–antisense sequence was subsequently 556
excised by XbaI/BamHI digestion and ligated into XbaI/BamHI digested plant expression vector 557
pPT2m. The pPT2-NbCOP-RNAi and pPT2-NbCOP-RNAi vectors were transformed into 558
agrobacteria and transiently expressed by agroinfiltration in N. benthamiana leaves together with the 559
fluorescent-tagged proteins. 560
561
Accession Numbers 562
Sequence data from this article can be found in the GenBank database under the following accession 563
numbers: M18769 (ST), AJ295993 (NtGnTI), in the Arabidopsis Information Resource under the 564
following accession numbers: AT4G38240 (AtGnTI), AT1G51590 (MNS1) and in the Nicotiana 565
benthamiana draft genome sequence v1.0.1 from the Sol Genomics Network: 566
Niben101Scf03249g01008 (COP) and Niben101Scf09552g01014 (COP). 567
568
Supplemental Data 569
Supplemental Figure S1. Amino acid sequence alignment of the N-terminal sequence (including the 570
TMD) from 74 putative plant GnTI sequences. 571
Supplemental Figure S2. CTS regions and used expression vectors. 572
Supplemental Figure S3. Subcellular localization of fusion proteins containing a chimeric or mutated 573
CTS region. 574
Supplemental Figure S4. Schematic illustration of the N-glycan processing assay. 575
Supplemental Figure S5. LC-ESI-MS analysis of the glycopeptide derived from a secreted 576
glycoreporter (IgG antibody) co-expressed with ST/GnTI-GALT, GnTI/ST-GALT or STFIYIQ-GALT. 577
Supplemental Figure S6. Complementation of the Arabidopsis gntI N-glycan processing defect. 578
Supplemental Figure S7. Complementation of Arabidopsis gntI and cgl1 plants. 579
Supplemental Figure S8. MALDI-TOF spectra of gntI expressing the indicated constructs under the 580
control of a weak AtGnTI promoter (386 bp). 581
Supplemental Figure S9. Co-localization of mutated AtGnTI with the trans-Golgi marker ST-582
GFPglyc. 583
Supplemental Figure S10. Comparison of the TMD sequence composition. 584
Supplemental Figure S11. AtGnTI variants with other conserved amino acids mutated reside in the 585
Golgi and are functional. 586
Supplemental Figure S12. Phenotypic comparison of complemented mns1 mns2 mns3 seedlings. 587
Supplemental Figure S13. Knockdown of the coatomer subunits COP or COP results in 588
mislocalization of Golgi-resident GnTI. 589
Supplemental Table S1. Statistical analyses of co-localization data. 590
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17
Supplemental Table S2. List of used primers. 591
592
ACKNOWLEDGEMENTS 593
We thank Martina Dicker, Josephine Grass, Julia König, Daniel Maresch, Karin Polacsek and Yun-ji 594
Shin (all from BOKU) for technical assistance and help with N-glycan and glycopeptide analysis. 595
Verena Kriechbaumer (Oxford Brookes University) is acknowledged for supplying plants used at the 596
Central Laser Facility. This work was supported by grants from the Austrian Science Fund (FWF): 597
P23906-B20 (to R.S.), P28218-B22 (to R.S.), T655-B20 (to J.S.) and the Mizutani Foundation for 598
Glycosciences (to R.S.). We thank the STFC for funding access to the Central Laser Facility (grant no. 599
12130006 to Chris Hawes, Oxford Brookes University). We thank BOKU-VIBT Imaging Center for 600
access and expertise. 601
602
603
Table 1. FRET efficiency determined by FLIM 604
Donor Acceptor 𝝉D ± SD (ns) 𝝉DA ± SD (ns) Δ𝝉 (ns) E (%)
NtGnTI-CTS-GFP NtGnTI-CTS-mRFP 2.44 ± 0.06 2.08 ± 0.09 0.36 14.56
(n = 412) (n = 204)
NtGnTI-CTS-GFP ST-CTS-mRFP 2.44 ± 0.06 2.36 ± 0.06 0.08 3.35
(n = 412) (n = 238)
NtGnTI-CTS-GFP NtGnTI-CTS-Q25A-mRFP 2.44 ± 0.06 2.37 ± 0.06 0.07 2.89
(n = 412) (n = 273)
𝜏D, lifetime of the donor in the absence of the acceptor; 𝜏DA, lifetime of the donor in the presence of the 605 acceptor; Δ𝜏, lifetime contrast (𝜏D- 𝜏DA); E, FRET efficiency calculated according to (1-[𝜏D- 𝜏DA]) × 606 100; ns, nanosecond; SD, standard deviation; n, number of Golgi bodies analyzed. A minimum 607 decrease of the average excited-state fluorescence lifetime of the donor molecule by 0.20 ns or 8% in 608 the presence of the acceptor molecule was considered relevant to indicate interaction. 609 610
FIGURE LEGENDS 611
612
Figure 1. The exoplasmic half of the NtGnTI TMD contains a signal for cis/medial-Golgi 613
localization. (A) Sequence logo illustration of the degree of amino acid conservation within the TMD 614
of plant GnTI sequences (74 sequences in total). (B) Schematic illustration of the NtGnTI, ST, 615
ST/GnTI and GnTI/ST CTS regions. The amino acid sequence of the TMD is shown for each CTS 616
region. C: cytoplasmic tail, S: stem region. Sequences corresponding to the NtGnTI TMD are 617
highlighted in bold. (C) Co-localization analysis. NtGnTI-CTS-GFP, ST/GnTI-CTS-GFP and ST-618
CTS-GFP and GnTI/ST-GFP were co-expressed with mRFP-AtCASP (CASP) or ST-CTS-mRFP (ST) 619
(data are the mean ± SD, n > 10 confocal images). Asterisks indicate a significant difference between 620
CASP and ST localization (p ≤ 0.001, two tailed t-Test: two-sample assuming equal variances), ns: not 621
significant (p > 0.05). (D) Co-localization of NtGnTI-Q25A-GFP with mRFP-AtCASP or with ST-622
CTS-mRFP. Left images: scale bars = 5 µm, right images: scale bars = 1 µm. Magnified images of the 623
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18
boxed region from the left images are shown in the right images. Graphs show relative fluorescence 624
intensities (NtGnTI-Q25A-GFP in green and mRFP-AtCASP in magenta) along the white lines (from 625
the right images). (E) Glycan processing assay. NtGnTI-Q25A-GALT (NtGnTI-Q25A) or the 626
indicated controls were co-expressed with a glycoprotein reporter. LC-ESI-MS analysis of the 627
glycopeptide “EEQYNSTYR” derived from the glycoprotein is shown. Reporter without GALT 628
(control), reporter co-expressed with NtGnTI-GALT (NtGnTI), ST-GALT (ST) or NtGnTI-Q25A-629
GALT. The schematic presentation of the major N-glycan structure is given. The symbols are drawn 630
according to the nomenclature from the Consortium for Functional Glycomics. For further details on 631
the abbreviation of the N-glycan structures see Supplemental Figure S4. 632
633
Figure 2. NtGnTI-Q25A locates in the Golgi, small puncta and the vacuole. Confocal microscopy 634
analysis was performed on N. benthamiana leaf epidermal cells expressing NtGnTI-Q25A fused to 635
mRFP or GFP and different subcellular markers. (A) Localization of NtGnTI-Q25A-mRFP was 636
monitored at different time points post-infiltration (2, 3 and 4 days post infiltration – dpi). (B) Co-637
localization with aleu-GFP (vacuolar marker). (C) Co-localization of NtGnTI-Q25A-GFP with 638
NtGnTI-mRFP reveals the labelling of small punctate structures (white arrows) that are distinct from 639
NtGnTI-labelled Golgi stacks. (D) Co-localization with mRFP-SYP61 (TGN-marker). (E) Co-640
localization with GFP-RABF2b (early endosome marker). (F) Co-localization with GFP-ATG8e 641
(autophagosome marker). (G) Co-localization with GFP-RABF1 (late endosome/multivesicular body 642
marker). Scale bars = 25 µm (A and B) and 10 µm (C to G). 643
644
Figure 3. AtGnTI-Q23A fusion protein expression does not restore the N-glycan processing 645
defect of gntI. (A) Immunoblot analysis of gntI AtGnTI:AtGnTI-GFP (WT) and gntI 646
AtGnTI:AtGnTI-Q23A-GFP (Q23A) seedlings with anti-HRP to detect plant complex N-glycans, with 647
GFP antibodies (anti-GFP) to detect the AtGnTI-GFP fusion protein and with antibodies against 648
ARGONAUTE 1 (anti-AGO) used as a loading control. (B) RT-pPCR analysis of transcripts from gntI 649
AtGnTI:AtGnTI-GFP (WT) and gntI AtGnTI:AtGnTI-Q23A-GFP (Q23A) seedlings. PCR for the 650
transgene expression was carried out with GFP-specific primers and normalized to UBQ5 transcript 651
expression. Data represent mean values ± SE, n = 3. (C) Immunoblot analysis of protein extracts from 652
gntI UBQ10:AtGnTI-mRFP (WT) or gntI UBQ10:AtGnTI-Q23A-mRFP (Q23A) with anti-HRP and 653
anti-RFP. (D) N-glycan analysis of complemented gntI expressing UBQ10:AtGnTI-mRFP (WT) or 654
UBQ10:AtGnTI-Q23A-mRFP (Q23A) by MALDI-MS. (E) Co-IP of AtGnTI (WT) and AtGnTI-655
Q23A (Q23A). UBQ10:AtGnTI-GFP co-expressed with UBQ10:AtGnTI-mRFP (WT) or 656
UBQ10:AtGnTI-Q23A-mRFP (Q23A) was purified with GFP-trap beads from N. benthamiana and 657
the co-purification of mRFP-fusion proteins was monitored by immunoblotting with mRFP antibodies. 658
(F) Localization of AtGnTI-mRFP, AtGnTI-Q23A-mRFP and ST-mRFP expressed in A. thaliana 659
seedlings under the control of the ubiquitin 10 promoter. Scale bars = 10 µm. 660
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661
Figure 4. AtGnTI-Q23A-mRFP degradation is blocked in the presence of both MG132 and 662
concanamycin A. (A) CHX-degradation assay. 9-day-old Arabidopsis gntI UBQ10:AtGnTI-mRFP 663
(WT) and gntI UBQ10:AtGnTI-Q23A-mRFP (Q23A) seedlings were incubated for the indicated time 664
in 100 µg/ml cycloheximide (CHX) and subjected to SDS-PAGE and immunoblotting. The intact 665
fusion protein is marked by an arrowhead. Detection of actin was used as a control. (B) gntI 666
UBQ10:AtGnTI-mRFP (WT) and gntI UBQ10:AtGnTI-Q23A-mRFP (Q23A) seedlings were 667
incubated for 17 h with 20 µM MG132, (C) with 1 µM concanamycin A (ConcA) or (D) with 1 µM 668
ConcA and 20 µM MG132. AtGnTI expression was monitored with anti-RFP. NBR1 detection served 669
as a control for the inhibitor treatment (increased NBR1 signal) and tubulin as a loading control. The 670
quantitation shows the relative GnTI-mRFP amount normalized to tubulin expression. Values are 671
means ± SEM (n = 4 independent experiments). (E) gntI UBQ10:AtGnTI-mRFP (WT) and gntI 672
UBQ10:AtGnTI-Q23A-mRFP (Q23A) seedlings were incubated for 24 h with 20 µM of the class I -673
mannosidase inhibitor kifunensine (kif) and proteins were subjected to SDS-PAGE and 674
immunoblotting with anti-RFP antibodies. 675
676
Figure 5. AtGnTI variants with an altered amino acid at position 23 are mistargeted in plants 677
and do not complement the gntI N-glycan processing defect. (A) Confocal microscopy analysis in 678
N. benthamiana leaf epidermal cells of transiently expressed AtGnTI-Q23-mRFP mutants. Confocal 679
images were acquired 48 h after infiltration. (B) Transiently expressed AtGnTI-Q23-mRFP mutants 680
analyzed by immunoblotting using an anti-mRFP antibody. The quantitation shows the relative 681
amount of the upper (intact AtGnTI-mRFP) and the lower (AtGnTI-mRFP degradation product) 682
bands. Values are means ± SD (n = 4 independent experiments. The single amino acid labelling 683
indicates the substituted amino acid at position 23. (C) Immunoblot analysis of transgenic gntI plants 684
expressing the different AtGnTI-Q23-mRFP variants under the control of the endogenous AtGnTI 685
promoter. Protein extracts from leaves were analyzed with antibodies against complex plant N-glycans 686
(anti-HRP). Two independent lines are shown for each AtGnTI-Q23-mRFP variant, Col-0 (wt) and 687
gntI protein extracts were included as controls. Ponceau S staining of the membrane was used as a 688
loading control. 689
690
igure 6. Mistargeted MNS1-GFP does not complement the N-glycan processing defect and root 691
growth phenotype of mns1 mns2 mns3. MNS1:AtGnTI-MNS1-GFP and MNS1:AtGnTI-Q23A-692
MNS1-GFP were expressed in the Arabidopsis mns1 mns2 mns3 mutant. (A) Protein extracts were 693
analyzed from wild-type (Col-0), mns1 mns2 mns3 (123), mns1 mns2 mns3 complemented with 694
MNS1:AtGnTI-MNS1-GFP (WT) or MNS1:AtGnTI-Q23A-MNS1-GFP (Q23A) by SDS-PAGE and 695
immunoblotting using antibodies against complex plant N-glycans (anti-HRP) and GFP. Ponceau S 696
(Ponc.) staining of the membrane was used as a loading control. (B) Confocal microscopy of 697
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20
Arabidopsis roots expressing the indicated constructs. Scale bars = 10 µm. (C) Phenotypic comparison 698
of 12-day-old Col-0, mns1 mns2 mns3 (123), mns1 mns2 mns3 complemented with MNS1:AtGnTI-699
MNS1-GFP (WT) or MNS1:AtGnTI-Q23A-MNS1-GFP (Q23A) seedlings grown on 0.5 x MS 700
supplemented with 1% sucrose. Scale bar = 1 cm. (D) Quantification of the root length from 8-day-old 701
seedlings grown on 0.5 x MS supplemented with 4% sucrose. For WT and Q23A the data from two 702
independent lines are shown. The mns3 single and the mns1 mns2 mns3 triple mutant are included as 703
controls. Data represent means ± SE (n > 50). 704
705
Figure 7. Knockdown of the coatomer subunits COP or COP results in mislocalization of 706
GnTI. (A) The left panel of images shows expression in the absence of any RNAi construct. “+COP” 707
indicates co-infiltration with the N. benthamiana COP RNAi construct. ”+COP” indicates co-708
infiltration with the N. benthamiana COP RNAi construct. mRFP-EMP12 (mRFP-tagged 709
Arabidopsis EMP12 protein lacking the N-terminal luminal domain), NtGnTI-mRFP, ST-NtGnTI-710
mRFP, AtGnTI-mRFP and AtGnTI-Q23A-mRFP were observed 48 h after infiltration using confocal 711
microscopy. Scale bars = 25 µm. (B) AtGnTI-Q23A-mRFP was treated with BFA (100 µg/ml) for the 712
indicated time. Scale bars = 25 µm. (C) Immunoblot analysis of NtGnTI-mRFP, ST-NtGnTI-mRFP, 713
AtGnTI-mRFP and AtGnTI-Q23A-mRFP. Proteins were co-expressed with the indicated COP RNAi 714
constructs, extracted 72 h after infiltration of N. benthamiana leaves and analyzed with anti-RFP 715
antibodies. An arrowhead marks the intact fusion protein. 716
717
Figure 8. Proposed working model for GnTI retrieval and its steady-state localization in the 718
cis/medial-Golgi. The model proposes a TMD-motif based sorting including the retrograde transport 719
from the trans-Golgi with COPI variants (e.g. COPIb) (Donohoe et al., 2007). The TMD sequence 720
motif with the conserved glutamine is either required for a specific protein-protein interaction with an 721
adaptor protein (indicated on the right side) or for interaction with a specific lipid class or Golgi 722
membrane environment (indicated on the left side) that partitions GnTI into retrograde transport 723
carriers to concentrate the glycosyltransferase in cis/medial-Golgi membranes. The large black arrows 724
indicate cargo that is transported from the ER trough the Golgi apparatus. 725
726
727
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728
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