Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120

J. Mol. Biol. (1998) 278, 369±388

Reconstitution, Characterisation and Mass Analysis ofthe Pentacylindrical Allophycocyanin Core Complexfrom the Cyanobacterium Anabaena sp. PCC 7120

Axel Ducret1, Shirley A. MuÈ ller2, Kenneth N. Goldie2, Andreas Hefti2

Walter A. Sidler1, Herbert Zuber1 and Andreas Engel2*

1Institute for Molecular Biologyand BiophysicsFederal Institute of TechnologyCH-8093 ZuÈ rich, Switzerland2Maurice E. MuÈ ller Institutefor Microscopy at theBiozentrum, University ofBasel, CH-4056 BaselSwitzerland

This paper is dedicated to Walte({ 19.1.1996).

Present address: A. Ducret, MercBiochemistry and Molecular BiologPointe-Claire-Dorval, QueÂbec, H9R

Abbreviations used: A, absorbanAP, allophycocyanin; Lc

8.9, AP-assopolypeptide; LCM, core-membraneLR, rod linker polypeptide; LRC, ropolypeptide; PBS, phycobilisome; Pphycoerythrin; PEC, phycoerythrocrod complex plus linker polypeptidtransmission electron microscopy/

0022±2836/98/170369±20 $25.00/0/mb

The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed todissociate into its constituents and the resulting allophycocyanin (AP)fraction was puri®ed. Its reconstitution yielded a complex which accord-ing to negative stain electron microscopy and spectral analysis was iden-tical to the native pentacylindrical PBS core domain. Each cylinder of thecentral tricylindric unit was comprised of four AP (ab)3 disks. Mass anal-ysis using the scanning transmission electron microscope (STEM) showedthe presence of 16 AP trimers in the intact reconstitute, which had a totalmass of 1966(�66) kDa. Composition analysis indicated an AP trimer dis-tribution of (AP-II):(AP-LCM):(AP-B):(AP-I) � 6:2:2:6, i.e. an addition oftwo AP-I and two AP-II complexes compared to a tricylindrical PBS coredomain. Therefore, we suggest that each supplementary half-core cylin-der found in pentacylindrical AP core domains is comprised of one AP-Iand one AP-II trimer, in agreement with the current model.

The structural signi®cance of the 127 kDa core membrane linker polypep-tide was further investigated by subjecting the AP core reconstitute tomild chymotryptic degradation. After isolation, the digested complexexhibited a tricylindrical appearance while STEM mass analysis con-®rmed the presence of only 12 AP complexes. Polypeptide analysis bySDS-PAGE and Edman degradation related the half-cylinder loss tocleavage of the Rep4 domain of the core membrane linker polypeptide.On the basis of these data, a general model for the assembly of the threehemidiscoidal PBS types known to date is discussed.

# 1998 Academic Press Limited

Keywords: phycobilisome; allophycocyanin; scanning transmissionelectron microscope; mass analysis; photosynthesis
*Corresponding author
Introduction

Cyanobacteria and red algae are characterisedby the presence of phycobilisomes (PBSs), giant

r A. Sidler

k-Frosst Canada Inc.,y, P.O. Box 1005,4P8, Canada.

ce; aa, amino acid;ciated linkerlinker polypeptide;d-core linkerC, phycocyanin; PE,yanin; R, hexamerice; STEM, scanning

microscope.

981678

extramembranous light-harvesting antennae ontheir thylakoid membranes (for reviews, see Baldet al., 1996; Bryant, 1991; Gantt, 1988; Glazer, 1987;Sidler, 1994; Zuber, 1987). PBSs are the main light-harvesting antenna of these organisms and replacethe intramembranous antennae generally found inmost eukaryotic algae and higher plants. They areprimarily (80 to 85%) composed of brilliantlycoloured, water soluble polypeptides known asphycobiliproteins (Glazer, 1987, 1989). The pre-sence of different chromophores among themallows their classi®cation into three groups(Glazer, 1987; Ducret et al., 1994; Sidler, 1994): (1)the phycoerythrins (PE; lmax � 490 to 570 nm); (2)the phycocyanins (PC; lmax � 490 to 625 nm) andthe phycoerythrocyanins (PEC; lmax � 560 to600 nm); and (3) the allophycocyanins (AP;

# 1998 Academic Press Limited

370 The Allophycocyanin Core Complex of Anabaena

lmax � 650 to 665 nm). The fundamental unit ofeach is a trimeric aggregate of two dissimilar poly-peptides, a and b, present in equal stoichiometry.Several PC (ab) trimeric units have been analysedby X-ray crystallography revealing ring-shapedcomplexes of 11 nm diameter and 3 nm thicknesswith a central hole of 3 nm diameter (Schirmeret al., 1985, 1987; Duerring et al., 1990, 1991; Ficneret al., 1992), while analogous AP (ab) trimers havea maximum diameter of 9.5 nm (Brejce et al., 1995).The production of higher aggregates requires thepresence of another protein family, the linker poly-peptides. These polypeptides control the assemblyof the phycobiliproteins into the precisely organ-ised PBS required for high ef®ciency light harvest-ing (Bryant, 1991; Sidler, 1994).

Five distinct morphological PBS families havebeen described (for reviews, see Wehrmeyer, 1983,1990): (1) hemidiscoidal; (2) hemiellipsoidal; (3)bundle-shaped; (4) block-shaped; and (5) ``hemielli-discoidal''. While some families have only a fewmembers, most rhodophytan and cyanobacterial,including Anabaena sp. PCC 7120, PBS belong tothe hemidiscoidal genus. In electron micrographs,hemidiscoidal PBS show two discrete subdomains:the ``core'' and the 11 nm diameter (Lundell &Glazer, 1983a; Williams et al., 1980) ``peripheralrods''. The core domain is generally observed infront-projection as several circular objects arrangedside-by-side. These correspond to cylinders consist-ing of concentrically stacked, trimeric disks. Intypical hemidiscoidal PBS, the peripheral roddomain is formed by six to eight cylindrical rodsthat radiate from the core. Each rod is generallycomprised of two to four, 6 nm � 11 nm disk-shaped elements (Glick & Zilinskas, 1982). Theseare PC, PE and PEC hexameric disks and theirlinker polypeptides. A PC hexamer is always clo-sest to the core (Sidler, 1994).

The hemidiscoidal family has been divided inturn into three subgroups according to structuraldifferences of the core domains (for a review, seeSidler, 1994): (1) bicylindrical PBS, as exempli®edby Synechococcus sp. PCC 6301 (Yamanaka et al.,1980). PBS of this subgroup have only been iso-lated in two cyanobacteria to date. (2) Tricylindri-cal PBS, as observed in Synechococcus sp. PCC 7002(Bryant et al., 1990; De Lorimier et al., 1990) and inSynechocystis sp. PCC 6701 (Williams et al., 1980).They are found in numerous cyanobacteria andred algae. (3) Pentacylindrical PBS, as exempli®edby Mastigocladus laminosus (Glauser et al., 1992;Sidler, 1994), Anabaena variabilis (Isono & Katoh1983, 1987) and Anabaena sp. PCC 7120 (Ducretet al., 1996). This subfamily was only addedrecently, after re-interpretation of the electronmicrographs prompted by studies made on thevarious linker polypeptides found in the sub-groups (see Sidler, 1994).

All hemidiscoidal PBS core domains analysedconsist of a single phycobiliprotein family, the allo-phycocyanin (AP), and two linker polypeptides,the allophycocyanin-associated small linker poly-

peptide (Lc8.9, 68 amino acids for Anabaena sp. PCC

7120; Cai, 1997) and the core-membrane linkerpolypeptide (LCM, 1132 amino acids for Anabaenasp. PCC 7120; Cai, 1997). Four AP trimeric com-plexes can be differentiated on the basis of theirpolypeptide composition (for reviews, see Bryant,1991; Sidler, 1994). AP-II, of composition (aAPbAP)3,represents the basic AP trimeric complex. AP-I, ofcomposition (aAPbAP)3Lc

8.9, consists of an AP-IIcomplex associated with one Lc

8.9 linker polypep-tide. AP-B, of composition (aAP-BaAP

2 bAP3 )Lc

8.9, is anAP-I complex in which one aAP chain has beenreplaced by an aAP-B subunit. The aAP-B polypep-tide confers a red-shifted ¯uorescence maximum tothe complex. As a result, the AP-B complex is con-sidered to be one of the two terminal emittersof the PBS. Finally AP-LCM, of composition(aAP

2 bAP2 b16)LCM, represents a heterogeneous com-

plex in which one LCM domain replaces one aAP

subunit. The bAP subunit believed to associate withthis unusual aAP chain is the so-called b16 polypep-tide. The AP-LCM complex exhibits the most red-shifted ¯uorescence maximum and is considered tobe the second terminal emitter of the PBS.

The functional importance of the LCM in deter-mining the ultrastructure of PBS core domains wasrecently unveiled by the systematic cloning of thegenes encoding the polypeptides present in thePBS of several cyanobacteria (as reviewed byEsteban, 1993). The number and type of linkerpolypeptide-like domains, termed Rep domains,present in this highly conserved (75 to 95%sequence identity) polypeptide is key to the archi-tecture displayed. Analogous to the situation forPC and PEC assembly into hexameric complexes(Bryant, 1991; Sidler, 1994; Gottschalk et al., 1994),homologous LCM Rep domains are considered toful®l identical functions in AP core complexes,always assembling the same AP trimers in thesame way. Thus, the two 72 kDa LCMs of bicylind-rical PBS both possess the Rep1 and Rep2 domains(Capuano et al., 1991), which organise a total ofeight AP trimeric complexes in the core. The result-ing core domain (Figure 1a) consists of two parallelfour-disk cylinders lying side-by-side. Six rodsradiate from it in a hemidiscoidal manner. Further,the 95 kDa LCMs of tricylindrical PBS not only pos-sess Rep1 and Rep2, but also an additional Rep3domain (Bryant, 1991). Together these organise 12AP trimeric complexes. In this case, the coredomain consists of three parallel closely packedcylinders. Tricylindric cores are surrounded by sixrods, four are bound to the ``upper'' core cylinderwhile one is coupled to each basal cylinder(Figure 1b). Finally, the recent cloning and charac-terisation of the 127 kDa LCM from the pentacy-lindrical core of Mastigocladus laminosus (Esteban,1993) revealed the additional presence of a fourthRep domain, Rep4, predicting the incorporation of16 AP trimeric complexes. The core domain ofthese grape-like PBS has not been fully analysed.In projection, it is similar to the tricylindrical corewith two supplementary elements ¯anking the

Figure 1. Schematic representationof the three types of hemidiscoidalPBS known to date. a, Bicylindrical;b, tricylindrical; c, pentacylindrical.The core and rod domains (top)and the current AP core models(bottom) are shown. The structureand phycobiliprotein compositionof the bi- and tricylindrical coredomains have been investigated inprevious studies whereas the struc-tural details of the pentacylindricalcore domain are the subject of thepresent work. Core cylinders Aand B are presumed to have anidentical composition in all models.Core cylinder C, which is onlypresent in pentacylindrical coredomains, is probably half of a Bcylinder.

The Allophycocyanin Core Complex of Anabaena 371

upper cylinder (Figure 1c). Up to eight relativelyshort rods have been observed to radiate from it.

The ultrastructural organisation of the PBS coredomain and its phycobiliprotein composition hasonly been studied in detail for two cyanobacteria,Synechococcus sp. PCC 6301 (Yamanaka et al., 1982;Lundell & Glazer, 1983a,b,c) and Synechocystis sp.PCC 6701 (Gingrich et al., 1983; Anderson &Eiserling, 1986). The bicylindrical PBS core domainof Synechococcus sp. PCC 6301 contains equimolaramounts of the four different AP trimer complexes,one copy being present in each core cylinder(Figure 1a). Yamanaka et al. (1982) and Lundell &Glazer (1983c) were able to characterise the com-plex (AP-II)(AP-LCM)(AP-B) by partial dissociationand digestion experiments, whereas the position ofthe AP-I complex remained uncertain. The tricy-lindrical PBS core domain of Synechocystis sp. PCC6701 contains the AP complex distribution(AP-II):(AP-LCM):(AP-B):(AP-I) � 4:2:2:4. Assumingan organisation of the two basal core cylindersidentical with that in Synechococcus sp. PCC 6301,Gingrich et al. (1983) proposed the upper corecylinder to consist of two copies of the AP-I andAP-II complexes. Finally, Anderson & Eiserling(1986) repeated some dissociation experiments per-formed by Gingrich et al. (1983) and deduced thatcomplexes AP-B and AP-I were located on distalends of the core cylinders. Accordingly, theypostulated that the basal core cylinders of bothcyanobacteria exhibit the arrangement (AP-I)(AP-

II)(AP-LCM)(AP-B), termed cylinder type A, andsuggested the organisation (AP-I)(AP-II)(AP-II)(AP-I), termed cylinder type B, for the top cylinderof the PBS core domain of Synechocystis sp. PCC6701 (Figure 1a and b).

Based on sequence homologies, structural con-siderations and general appearance, pentacylindri-cal PBS core domains are expected to resembletheir tricylindrical counterparts with a small differ-ence caused by the two supplementary elements(Glauser et al. 1992; Isono & Katoh 1983, 1987).Initially, these additional AP trimers were pro-posed to ful®l a rod-like function beingimplemented at the base of two rods, and attachedto a tricylindrical AP core complex (Glauser et al.,1992). This model demands an orientation perpen-dicular to the core cylinders, as observed for rodhexamers. More recently, and in better agreementwith their appearance on electron micrographs andthe now known function of the LCM, they havebeen proposed as core constituents in the form oftwo AP half-cylinders ¯anking the other core cylin-ders (Sidler, 1994; Ducret et al., 1996). This currentmodel is schematically represented in Figure 1c.Cylinders A and B are considered to have thecomposition and orientation of a tricylindrical core.The two half-cylinders C are thought to consist ofeither AP-I, AP-II trimeric disks or a mixturethereof.

Here we present the reconstitution and charac-terisation of the pentacylindrical PBS core domain


of the cyanobacterium Anabaena sp. PCC 7120. TheAP fraction of the PBS isolated from Anabaena sp.PCC 7120 was puri®ed using a modi®ed protocolfrom Isono & Katoh (1983) and reconstituted. Thecomposition, the ultrastructure and the spectralproperties of the reconstitute were analysed indetail and con®rm the presence of intact pentacy-lindrical PBS core complexes. Our results elucidatethe structure of the pentacylindrical PBS coredomain and con®rm the current model for thistype of hemidiscoidal PBS. The general structuralimplications are discussed.

Results

Reconstitution, isolation, and preliminarycharacterisation of the AP core complex fromAnabaena sp. PCC 7120

All attempts to isolate the native AP core com-plex of Anabaena sp. PCC 7120 by partial dis-sociation experiments failed. The products wereseparated by sucrose density gradient ultracentri-fugation and analysed by SDS-PAGE and spec-troscopy. These results indicated that AP-I and AP-B complexes dissociated from the core before allrod elements could be removed. Moreover, only

Figure 2. Isolation and characterisation of the AP core recentrifugation. Intact AP core reconstitutes were present inby SDS-PAGE. P, PBS; AP, pool puri®ed by hydroxyapatiS, molecular mass standard (kDa). The bands labelled PBanalysis see Ducret et al., 1996). The gel was stained withAP fraction 1 (upper spectra), and AP fraction 2 (lowerabsorption spectrum, (- - - -) second derivative; right, (- - -escence spectrum.

modest amounts of the rod components PEC andPC could be isolated as trimeric and hexamericcomplexes in the lighter fractions of the sucrosegradients, while most of the PC rod-core com-plexes remained associated to the core under theconditions examined. Harsher isolation conditionsresulted either in the precipitation of the complexes(more acidic or basic pH) or in the full dissociationof the PBS (lower phosphate buffer concentrations).

As direct isolation did not succeed, a similar pro-tocol to that used by Isono & Katoh (1983) toobtain the core complex of the related cyanobacter-ium Anabaena variabilis was employed. Dialysis ofthe isolated PBS of Anabaena sp. PCC 7120 against5 mM KH2PO4 buffer (pH 7.0) caused completedissociation. Subsequent hydroxyapatite chroma-tography allowed most rod components to beremoved. The use of a high molar concentration ofphosphate buffer in this step initiated reconstitu-tion of the PBS core complex. This ``crude'' APfraction yielded two AP-containing fractions andone PC-containing fraction on sucrose gradientcentrifugation (Figure 2a) which were characterisedby SDS-PAGE (Figure 2b) and by absorption and¯uorescence spectroscopy (Figure 2c). The lighterfraction, fraction 1, contained the AP a and b sub-

constitutes. a, Separation by sucrose density gradient ultra-the lower band, labelled fraction 2. b, Polypeptide analysiste chromatography; I-II, AP fraction 1 and 2, respectively;P arise from AP and PC a and b subunits (for a detailedCoomassie blue. c, Absorption and ¯uorescence spectra ofspectra) in 900 mM KH2PO4 buffer (pH 7.0). Left, (ÐÐ)-) excitation ¯uorescence spectrum, (ÐÐ) emission ¯uor-


units contaminated with PC and the LR and LC8.9

linker polypeptides (Figure 2b), while the highmolecular mass LCM

127 was absent. Its absorptionspectrum, with a maximum at 653 nm, was verysimilar to that of the pure AP-I complex (Ducretet al., 1996). The second derivative of the absorp-tion spectrum indicated that this fraction also con-tained a small amount of AP-B complex (Lundell& Glazer, 1981). The heavier fraction, fraction 2,contained in addition to the AP polypeptides pre-sent in fraction 1, the LCM

127 and a small amount ofPC with its associated linker polypeptides. It exhib-ited an absorption maximum at 653 nm and a¯uorescence excitation maximum at 650 nm. Thesecond derivative of the absorption spectrumrevealed the presence of a supplementary absorb-ing element in the 630 nm spectral region probablyrelated to the presence of the LCM

127 (Figure 2c, asindicated by the arrow; see also Ducret et al., 1996).The ¯uorescence emission maxima of fraction 2were situated at 665 nm and 681 nm (Figure 2c),similar to PBS emission maxima (Ducret et al.,1996) con®rming the presence of complexes withan integral and functional energy pathway. Unlessotherwise stated, fraction 2 was used in all sub-sequent analyses and will be termed AP corereconstitute in the following. The occurrence of thelighter AP fraction indicates that reconstitutionwas in fact not always complete. The degree ofhomogeneity, which could not be assessed spectro-scopically or by composition analysis, was laterdetermined by STEM mass measurement (seebelow).

The most successful reconstitutions wereobtained with freshly puri®ed AP solutions (prefer-entially, reconstitutions were initiated during thesecond hydroxyapatite chromatography step, theAP fraction being eluted with high concentrationphosphate buffer). Yields were markedly lower

Table 1. Reconstitution experiments

Experiment Componentsa Ratio o

1 Crude AP fraction2 AP-I, AP-B, AP-II3 APLcm, AP-II4 APLcm, AP-I5 APLcm, AP-I, AP-II6 APLcm, AP-B, AP-II7 APLcm, AP-I, AP-II, AP-B

n.d., not determined.a Crude AP-fraction in 100 mM KH2PO4 buffer (p

fraction was directly obtained from the hydroxyapameric complexes.

APLcm in 100 mM KH2PO4 buffer (pH 7.0), A � 0.9AP-I in 25 mM KH2PO4 buffer (pH 7.0), A � 3.84 aAP-II in 70 mM KH2PO4 buffer (pH 7.0), A � 0.3 aAP-B in 10 mM KH2PO4 buffer (pH 7.0), A � 0.24APLcm denotes the APLcm complex comprised of

could not be separated by ion-exchange chromatograb A positive result corresponds to the formation

(experiment 1).c A faint band was nevertheless visible under UV

taminating the AP-B preparation.

when older AP solutions were used or when pro-tease inhibitors were omitted (data not shown).

Distribution and quantification of the APtrimeric complexes present in the APcore reconstitute

The reconstitution behaviour of various AP tri-meric complex mixtures puri®ed from the crudeextract (see Materials and Methods) was examinedto determine the composition of the AP complexesin the AP core. As indicated in Table 1, all combi-nations containing the APLcm and AP-I complexesformed aggregates with identical sedimentationvelocities to the AP core reconstitute. However,emission ¯uorescence analysis showed that onlywhen all four AP trimeric complexes, i.e. AP-I, AP-II, AP-B and AP-LCM, were present, was the com-plex formed also spectroscopically similar to theAP core reconstitute (Figure 3). As expected, thecombinations AP-I/APLcm and AP-I/APLcm/AP-IIdid not exhibit the 680 nm emission band typicalof associated AP-B. The combination containingAP-B, APLcm and AP-II apparently failed to pro-duce a high molecular mass complex. Due to theirhigh dilutions, none of these high mass reconstitu-tion products were characterised further.

To determine the relative proportions of theAP-I, AP-II, AP-B and AP-LCM trimeric complexespresent in AP core reconstitutes, two independentsamples (fraction 2, Figure 2a) were dissociated,fractionated by sucrose gradient density ultracen-trifugation (Table 2A) and their AP trimericcomplex content assessed spectroscopically asdescribed in Materials and Methods. Both prep-arations contained approximately the same relativeamounts of AP trimeric complexes (Table 2B). Themeasured amount of AP-B was lower thanexpected in comparison to structurally de®ned bi-and tricylindrical core domains (Gingrich et al.,

f compounds (by vol.) Reconstitution resultb

n.d. �2:0.5:1 ÿ

2:1 ÿ1:1 �

2:1:1 �1:0.5:1 (ÿ)c

1:0.5:0.5:0.5 �

H 7.0), A � 9.02 at 653.0 nm (positive control). Thistite chromatography step and contained all AP tri-

2 at 652.6 nm.t 653.0 nm.t 651.4 nm.at 654.0 nm.AP-LCM � AP-II and small quantities of AP-I whichphy.of a complex similar in size to an AP core complex

illumination, probably arising from AP-I trimers con-

Figure 3. Fluorescence emission spectroscopy analysis ofthe products reconstituted from experiments 1, 4, 5, 6and 7 (Table 1). The spectra were normalised at theiremission maxima. ( � � � ), reconstitutions 4, 5 and 6; (- - -),reconstitution 7; (Ð), reconstitution 1 (positive control).


1983; Lundell & Glazer, 1983c; Yamanaka et al.,1982). This was especially the case for the oldersample, preparation 1, Table 2B.

Electron microscopy and mass analysis of theAP core reconstitute

Images of negatively stained freshly isolated APcore reconstitutes recorded with the STEM at500,000� , are presented in Figure 4a. When

B. Quantification of the AP trimeric complexes contained in the reconstituIEX analysis of band 1

AP core complexpreparation

AP trimericcomplex

Peak area(mV �min)

% Connormali

AP-B 23.78 6.51 AP-I 214.73 53.0

AP-II 113.33 40.3APLcm ± ±

AP-B 29.03 10.22 AP-I 180.00 57.6

AP-II 69.87 32.1APLcm ± ±

a The results are the average of two runs of ion-exchange chromab Calculated as described in Materials and Methods.c The total concentration of AP-II complexes contained in the AP

tions of the AP-II and the APLcm complexes (according to assumptio

Table 2. Quanti®cation of the AP complexes

A. Quantification of the AP complexes separated by sucrose gradient ultra

AP core complex preparationa Sucrose gradient band

1 Band 1Band 2

2 Band 1Band 2

a AP core reconstitute, preparation 1: A � 1.79 at 653 nm in 900 mAP reconstitute, preparation 2: A � 3.98 at 653 nm in 900 mM KH

preparation 2.

oriented end-on the complexes exhibit ®ve circularelements. Three of these are homogeneous inappearance and form the triangular centre of thecomplex. Two supplementary disk-shaped units¯ank this triangular unit to produce the typical®ve-disk pattern. Cylinders and single disks cannotbe visually distinguished in end-on projection. Evi-dence for the cylindrical nature of the various com-ponents was obtained from the few side-onprojections observed (data not shown), and fromSTEM mass analysis of unstained samples (seebelow). Although the dimensions indicate a paral-lel cylinder orientation of the supplementary cylin-ders, their end-on projection was not alwaysstrictly circular (Figure 4a), implying that theymight be rather loosely attached to the more rigidcentral unit. The average obtained after angularand translational alignment of the 64 projectionspresented in Figure 4a, according to their distincttriangular centres, is shown in Figure 4b. Multi-variate statistical analysis (Frank et al., 1987)allowed 51 of the 64 projections to be classi®edinto three major classes to yield the averagesshown in Figures. 4c to e. All four results revealedspokes linking the subunits. While the average inFigure 4c arose from apparently intact core com-plexes, those of Figures 4d and e correspond tocores that are missing some of the supplementarydisks. Measurements on the average displayed inFigure 4b showed the cylinders of the triangularcore to be essentially circular in projection, havinga diameter of 8.4(�0.3) nm (n � 5). In contrast, thesupplementary disks exhibit an oval projectionwith a long axis of 10.0(�0.5) nm (n � 4) and a

ted AP core complexa Quantification of the AP trimeric complexestent,sedb Amount of AP trimersb

% Content,relativeb

7 98 nM 4.989 715 nM 36.384 791 nM 1152 nMc 58.63

361 nM ± ±

1 0.45 mM 7.190 2.30 mM 36.749 1.86 mM 3.51 mMc 56.07

1.65 mM ± ±

tography (IEX) per preparation.

core reconstitute was obtained by adding the partial concentra-ns 2 and 3 described in Materials and Methods).

centrifugationSucrose gradient quantification Absorbance (A)

Volume (ml) at absorption maximum

9.4 0.1557.2 0.0549.4 0.4587.8 0.227

M KH2PO4 buffer (pH 7.0).

2PO4 buffer (pH 7.0). Preparation 1 was three weeks older than

Figure 4. STEM images of a negatively stained AP core reconstitute preparation. a, AP core reconstitute complexes inend-on projection. The ®ve circular elements typical of pentacylindrical cores are generally distinguishable. Additionalend-on oriented structures, characterised by their diameter as rod components can also be seen (arrows), as can APcore reconstitutes bearing one or more speci®cally associated rod hexamers (arrowheads). b, Average calculated fromthe core regions of the 64 complexes shown. The resolution of this image determined by the radial correlation func-tion (Saxton & Baumeister, 1982), the phase residual (Frank et al., 1981) and the spectral signal to noise ratio (Unseret al., 1987) is 1.8 nm, 2.6 nm and 2.2 nm, respectively. c to e, Averages resulting after classi®cation of the particlesinto three classes. While c represents intact core complexes, d and e arise from complexes which are missing part ofthe supplementary disks. The box width represents 43 nm.



short axis of 6.0(�1.1) nm (n � 4). The centraltricylindric unit could be inscribed in a circle of20.4 nm diameter. The whole complex was foundto have a maximum width of 25.3(�0.4) nm(n � 12) and a maximum height of 18.6(�0.9) nm(n � 12). As illustrated by Figure 4a (arrows), insome cases there was the random association ofone or more additional structures with the com-plexes. Their diameter, 10.5(�1.4) nm (n � 6), istypical of PC complexes oriented end-on (i.e.10.8 nm; Schirmer et al., 1986). In addition, a rela-tively large proportion of the AP core reconstitutesbore speci®cally associated rods (arrowheads).These were formed from one to four disk-shapedcomplexes oriented side-on whose striped stainingpattern, height, 5.5(�0.2) nm (n � 22) and diam-eter, 10.8(�0.4) nm (n � 5), are typical of PC hex-amers. Interestingly, the rods were most oftenobserved bound to one of the supplementary APcore half-cylinders.

The different elements of the AP core reconsti-tute could only occasionally be clearly distin-guished on the low dose digital images ofunstained preparations recorded at 200,000� formass determination (Figure 5a). However, differ-ences in the size and shape of the complexes, indi-cating the attachment of additional cylinders orrods, could be discerned. The presence of differentspecies was re¯ected in the mass histogram shownin Figure 5b. The mass data from 275 unstainedAP core reconstitutes revealed the existence of sixdifferent populations present in the proportions1:4:3:4:2:1. The sequences of Anabaena sp. PCC 7120AP polypeptides available to date, supplementedwhere necessary by the known polypeptide masses

Figure 5. Mass analysis of AP core reconstitutes. a, STEMaration. The scalebar represents 100 nm. b, Mass histogramThe data could be deconvoluted into six populations. TheParticles from the third peak which corresponds to the intac

of the pentacylindrical Mastigocladus laminosus corecomplex (detailed in Table 3), were used to makethe assignments listed in Table 4. The presence oftwo AP-LCM, two AP-B, six AP-I and 6 AP-II in theintact core complex was assumed, in accordancewith the pentacylindrical core model (Figure 1c).Data collected from the third population exhibiteda Gauss peak at 1966(�66) kDa, in good agree-ment with the theoretical mass of 2000 kDa calcu-lated for an intact AP core complex. Size andshape of the particles present in this population(Figure 5b, gallery) roughly corresponded to thatof the negatively stained AP core reconstitutes pre-sented in Figure 4a. The lower mass particles form-ing populations 1 and 2 probably represent eitherincomplete reconstitution products or partially dis-sociated AP core reconstitutes missing two to fourdistal AP trimers. Loss may have occurred at thetrimeric central unit (loss of one AP-B or one AP-Icomplex) and/or at the half-cylinders (loss of oneAP-I or one AP-II complex). In negatively stainedsamples, such complexes were observed as APcore reconstitutes partially missing their sup-plementary AP half-cylinders (Figure 4d to e). Theheavier particles constituting population 4 prob-ably correspond to AP cores reconstitutes, withone associated hexameric PC rod complex (averagemass 255 kDa for Anabaena sp. PCC7120, Table 3)but missing one distal AP trimer. The presence ofsome core reconstitutes missing two trimeric com-plexes but with an associated rod hexamer inpopulations 3 and 4 cannot be excluded. Variouscombinations of populations 2, 3 and 4 withadditional rods and/or an AP half-cylinder areprobably also included in population 5. Finally,

micrograph of an unstained AP core reconstitute prep-from the analysis of 275 AP core reconstitute particles.

assignments and Gauss peak values are given in Table 4.t complex are shown in the gallery, box width is 52 nm.

Table 3. Molecular mass of selected complexes present in the PBS of Anabaena sp. PCC 7120

Complex Polypeptide composition Molecular mass (Da)a

AP-II (aAPbAP)3 107,091AP-I (aAPbAP)3Lc

8.9 114,931AP-B (aAP-Ba2

APb3AP)Lc

8.9 115,761AP-LCM (a2

APb2APb16.2)LCM

127 218,287

PC hexameric complex (aPCbPC)6LRCPC, (aPCbPC)6LR

PC 254,976b

Core cylinder A 1 AP-I, 1 AP-II, 1 AP-B, 1 AP-LCM 556,070Core cylinder B 2 AP-I, 2 AP-II 444,044Core cylinder C 1 AP-I, 1 AP-II 222,022

AP core complex, intact 2 cylinders A, 1 cylinder B, 2 cylinders C 2,000,228AP core complex, digested AP core intactÿ2 cylinders Cÿ2 LCM

F c 1,460,184

a Except for aAP-B and b16.2, the mass of the polypeptides belonging to the AP family are from Anabaena sp. PCC 7120, mutant SB12(Cai, 1997). Failing sequence data, the mass of aAP-B and b16.2 from Mastigocladus laminosus (Esteban, 1993) have been used. The massof the polypeptides belonging to the PC family are from Anabaena sp. PCC 7120 (Belknap & Haselkorn, 1987; Bryant et al., 1991).The additional mass arising from the six phycobilin pigments associated with each AP-complex and from the 18 associated witheach PC hexamer has been included (257 Da per pigment).

b This mass represents the average of four possible hexameric PC complexes as the precise identity of the PC complexes presentduring the reconstitution procedure was not determined.

c LCMF symbolises the four segments lost from the core-membrane linker proteins on digestion (a total of 96 kDa).


particles contained in population 6 represent high-er aggregates bearing several additional rods orassociated cylinders as shown in Figure 4a (arrowsand arrowheads). The width of the Gauss curves,standard deviation �66 kDa, makes the occurrenceof various closely related dissociation/reconstitu-tion products in the populations possible.

Table 4. STEM mass analysis of the AP core reconstitute

Peak Measured (kDa) (�n) Estimate

1 1514 � 66 (10) 155

2 1764 � 66 (89) 177

3 1966 � 66 (49) 200

4 2136 � 66 (75) 213

5 2348 � 66 (36) Mixtu

225

227

239

6 2611 � 66 (10)

a Core cylinder nomenclature as presented in Figure 1c. R represb The average number of AP trimers missing overall, calculated

was 1.3. The value was unchanged when only particles of peaks 1sidered.

Digestion of the reconstituted AP corecomplex with chymotrypsin;biochemical characterisation

The structural function of the LCM in the PBSwas further substantiated. AP core reconstitutesfrom Anabaena sp. PCC 7120 were digested withchymotrypsin and the resulting complexes were

d (kDa) Assignmenta,b

6

0 ÿ(AP-B),ÿ (AP-I)

0 (i.e. intact)

9 ÿ(AP-B), �R

re of:

5 � R

9 ÿ(AP-B), ÿ (AP-I) � 2R

4 ÿ(AP-B), �2R

Higher aggregates:�cylinders, �R

ents a rod hexamer plus its linker protein; mass 255 kDa (Table 3).using the listed assignments and the value of n for peaks 1 to 5,to 4 for which a more exact assignment was possible, were con-

Figure 6. Digestion of AP core reconstitutes with chymotrypsin and subsequent isolation and analysis of the digestionproducts. a, SDS-PAGE analysis of the digestion procedure. P, PBS; 1, untreated AP core reconstitute; 2, digested APcore reconstitute; S, molecular mass standard (kDa). The bands labelled PBP arise from AP and PC (lane 1 only) aand the AP b subunits. The bPC band is labelled separately. The gel was stained with Coomassie blue. b, Absorptionand ¯uorescence spectra of digested AP core reconstitute in 900 mM KH2PO4 buffer (pH 7.0). Left, (Ð) absorptionspectrum, (- - - -) second derivative; right, (- - - -) excitation ¯uorescence spectrum, (Ð) emission ¯uorescencespectrum.


isolated by sucrose gradient density ultracentrifu-gation. The low molecular mass band contained amixture of chymotrypsin and trimeric PC and APcomplexes and was not analysed further. The highmolecular mass band, which contained the par-tially digested cores, was characterised by SDS-PAGE (Figure 6a) and by absorption and ¯uor-escence spectrometry (Figure 6b). The absorptionmaximum of the digested AP core reconstitutewas situated at 654.7 nm, slightly red-shiftedcompared to the intact moiety. Interestingly, theA654/A600 absorption ratio of the digested coreswas signi®cantly increased compared to theuntreated core (Figure 2c). The excitation ¯uor-escence spectrum showed that the energy trans-fer resulting from complexes absorbing at 620and 640 nm was greatly reduced while theabsorption second derivative con®rmed that thechromophores absorbing in the 630 nm spectralband in the intact AP core reconstitute prep-aration were absent from the digested complex.Finally, the emission ¯uorescence maximum at669 nm was also signi®cantly red-shifted com-pared to the untreated AP core reconstitutepreparation, con®rming that short-wavelength

Houmard et al., 1990) are shown. /, Indicates the end of thing to the LCM

127 amino acid sequence of Mastigocladus laminos

AP-elements were absent from the digested APcore preparation.

SDS-PAGE of the digested AP core reconstituterevealed two major bands, the aAP and bAP sub-units, a distinct low molecular mass band, the LC

8.9,and several bands at higher molecular mass, themost prominent being the LCM, and a completeabsence of the LR, LRC and rod protein (Figure 6a,lane 2). The aAP-B and the b16 subunits, only pre-sent in a very low amount, could not be detectedby this method. The molecular mass of the LCM

was greatly reduced, being estimated as 79 kDafrom its mobility, rather than the 127 kDa expectedfrom sequence data (Cai, 1997). This degradationproduct was further analysed by N-terminalEdman sequencing to determine the site of clea-vage (Figure 7). Comparison with the knownsequence, allowed the resulting 27 amino acidsequence to be unambiguously assigned to position151 to 177. Thus, 150 N-terminal amino acids hadbeen lost, which only accounts for 16 kDa. Conse-quently, a C-terminal fragment of approximately32 kDa, which would correspond to 283 aminoacids of the sequence (Cai, 1997), must also havebeen cleaved off during the chymotryptic diges-tion.

Figure7.Amino-terminalsequenceof the digested LCM. Homologouspositions of the LCM sequenceof Mastigocladus laminosus (M. lami-nosus, Esteban, 1993) and Calothrixsp. PCC 7601 (Calothrix 7601,

e primary structure determination. Numeration is accord-us.

Figure 8. STEM micrographs of a digested, negatively stained AP core reconstitute preparation. The box width rep-resents 33 nm. Rarely, the association of rod-like elements could be observed (arrowheads). The average shown inthe bottom right corner was calculated from 54 well preserved particles. The resolution of this image determined bythe radial correlation function (Saxton & Baumeister, 1982), the phase residual (Frank et al., 1981) and the spectral sig-nal to noise ratio (Unser et al., 1987) is 2.2 nm, 2.8 nm and 2.5 nm, respectively.


Electron microscopy and mass analysis of theAP core reconstitute afterchymotrypsin digestion

Images of negatively stained reconstituted APcore complexes after digestion, recorded with the

Figure 9. Mass of digested AP core reconstitutes. a, STEMpreparation. The scalebar represents 100 nm. b, Mass histoAP core reconstitute particles. The data were deconvolutedvalues are given in Table 5. Typical particles are shown in

STEM at 500,000� are presented in Figure 8. Whenoriented end-on as shown, they exhibit threeelements packed in homogeneous triangular struc-tures. As expected, the digested cores look verysimilar to intact AP core complexes which havelost the two supplementary disk-shaped units.

micrograph of a digested, unstained AP core reconstitutegram generated by the analysis by STEM of 379 digestedinto three populations. The assignments and Gauss peak

the gallery, box width is 51 nm.

Table 5. STEM mass analysis of the AP core reconstitute after chymotrypsin digestion

Peak Measured (kDa) (�n) Estimated (kDa) Assignmenta

1 1181 � 69 (54) 1229 ÿ LCMF , ÿ(AP-B), ÿ (AP-I)

2 1352 � 69 (262) 1344 ÿ LCMF , ÿ(AP-B)

3 1487 � 69 (47) 1460 ÿ LCMF (i.e., digested)

a Core cylinder nomenclature as presented in Figure 1c. LCMF symbolises the total of four segments lost from the core-membrane

linker proteins on digestion, estimated mass 96 kDa (based on SDS-PAGE and N-terminal Edman sequencing).


Only rarely were the digested complexes associ-ated with rod-like elements (Figure 8, arrowheads),con®rming the analysis by SDS-PAGE (Figure 6a).Many of the cylinders exhibited an almost circular,stained indentation surrounding a central protru-sion (Figure 8). In a few cases spokes linking thecylinders could be detected. An average of 54aligned complexes gives a clearer picture of thisarchitecture (Figure 8, insert). Measurements onthis average showed the subunits to be more ovalin projection than the triangular core cylinders ofthe untreated reconstitutes (Figure 4). Correspond-ingly, their long axis was found to be 9.1(�0.3) nm(n � 3) and their short axis 7.9(�0.2) nm (n � 3).The whole complex could be inscribed in a circle of21.2 nm diameter. Occasional side views exhibitedthe AP disks (data not shown) whose height was3.1(�0.2) nm (n � 18).

Low dose digital images were recorded at200,000� from unstained preparations for massdetermination. In this case, it was quite often poss-ible to distinguish the three elements of thedigested AP core reconstitute. A large proportionof the particles exhibited the typical triangularshape (Figure 9a and b, gallery). Mass data from379 unstained digested AP core reconstitutes(Figure 9b, Table 5) document the existence ofthree populations, present in the proportions 1:5:1.The assignments listed in Table 5 were made usingthe AP polypeptide masses from Anabaena sp. PCC7120 and Mastigocladus laminosus detailed in Table 3taking into account the lower mass of the LCM

caused by digestion, and assuming two AP-LCM,two AP-B, four AP-I and 4 AP-II in the digestedcore complex (see Figures 1b and c). Data collectedfrom the heaviest species (population 3; about 15%of the total) exhibited a Gauss peak at1487(�69) kDa, very close to the theoretical valueof 1460 kDa. However, about 70% of the particlesmeasured (population 2) correspond to complexeswhich have lost one distal AP trimer. Finally, aminor portion of the analysed particles displayedmasses which roughly correlate to the loss of twoor three AP trimers. The absence of high masspeaks con®rmed the ef®ciency of digestion, mostof the subsidiary cylinders and hexameric PC rodimpurities having been removed.

Discussion

Partial dissociation of the intact rod-containingPBS of Anabaena sp. PCC 7120 to yield bare corecomplexes proved impossible since distal AP tri-meric disks always dissociated from the core. Inaddition, the rod-core junction, consisting of a PChexamer, a rod-core linker polypeptide, and an APtrimer or hexamer, was found to be extremelystable and dif®cult to break under all of the con-ditions examined. Thus, since it is well establishedthat functionally intact PBS can be reconstitutedfrom their constitutive parts (Canaani et al., 1980;Isono & Katoh, 1983; Kume et al., 1982; Lu & Yu,1986), the PBS were dissociated, separated frommost of the rod components, and reconstituted.Complete removal of all PC hexamers was neverachieved. Characterisation of the AP core reconsti-tute (Figure 2a, fraction 2) by absorption and ¯uor-escence spectroscopy indicated the presence ofcomplexes with an integral and functional energypathway, suggesting that structurally complete APcores had been formed. The lower yields obtainedwhen older AP solutions were used or when pro-tease inhibitors were omitted, were mainly due todegradation of the LCM with time (data notshown).

The reconstituted Anabaena sp. PCC 7120 corecomplexes contained all four AP trimeric com-plexes, AP-I, AP-II, AP-B and AP-LCM, identi®ed inother PBS cores. The relative amounts of these ischaracteristic of core ultrastructure (Bryant, 1991;Sidler, 1994; see Figure 1). Thus, for a bicylindriccore there are equal amounts of all four complexes(Lundell & Glazer, 1983b) and for a tricylindriccore 16.7% AP-B:33.3% AP-I:33.3% AP-II:16.7%AP-LCM is indicated (Gingrich et al., 1983;Anderson & Eiserling, 1986), while the pentacy-lindrical core model presented in Figure 1c predictsthe proportions, 12.5% AP-B:37.5% AP-I:37.5% AP-II:12.5% AP-LCM (Sidler, 1994; Ducret et al., 1996).Quantitative analysis of the Anabaena sp. PCC 7120AP core reconstitute by anion-exchange chroma-tography and absorption spectroscopy yielded theaverage ratio, 6% AP-B:37% AP-I:57% (AP-II � AP-LCM), the AP-II and AP-LCM trimeric complexesbeing assessed together due to the formation of the


stable APLcm complex (average of % content, rela-tive, Table 2b). While the AP-I and (AP-II � AP-LCM) amounts approximately agree with the pre-dicted values, the AP-B content was 50% lowerthan expected. This may be explained by an incom-plete reconstitution and/or the average loss of oneAP-B complex per reconstituted core prior to theanalysis. In agreement with this, the STEM massmeasurement indicated the frequent absence of atleast one AP trimeric complex from the reconsti-tutes (see below).

The fact that the AP-LCM complex was alwaysassociated with two or three AP-II trimericcomplexes (Table 2B) indicates the close proximityof these disks in the core. The reconstitution beha-viour of various AP trimeric complex mixturesyielded further information. Consistent with theproposed organising function of the LCM, high mol-ecular mass products were only formed when theAP-LCM trimeric complex was present in the mix-ture (Table 1). The presence of additional AP-Balone did not result in a high mass complex.Instead, incorporation of AP-B depended on thepresence of AP-I (Table 1). Thus, AP-B could notsubstitute for AP-I although these two complexesdiffer by only one a subunit. This con®rms that theLCM Rep domains stack speci®c AP trimericcomplexes, and suggests that AP-B cannot be inte-grated into the core domain before AP-I has beencorrectly assembled. All four trimeric complexeswere required for full activity of the core reconsti-tutes, according to spectroscopic criteria (Figure 3).Low reconstitution yields were always obtainedwhen these highly puri®ed, almost PC-free AP-I,AP-II, AP-B trimers and APLcm complexes wereemployed (Table 1), prohibiting their furtheranalysis. Although proteolytic degradation or inhi-bition of reconstitution by the low starting materialconcentrations (Kume et al., 1982) cannot beexcluded, low yields may be explained by theabsence of a stabilising effect of the rod-core junc-tion. Indeed, survey of the literature suggests thatentirely rod-less cores exhibit only limited stability(Bryant et al., 1990).

On chymotrypsin digestion, a 16 kDa N-terminaland an approximately 32 kDa C-terminal fragmentwere removed from the LCM

127 . In negative stain elec-tron microscopy, the AP core reconstitutes took onthe symmetric triangular appearance typical oftricylindric cores viewed end-on (Figure 8), andcould be inscribed in a circle of 21.2 nm diameter.The end-on projection of the individual cylinderswas not strictly circular, the long axis being9.1(�0.3) nm and the short axis 7.9(�0.2) nm. Sideviews showed the core cylinders to be assembledfrom four disks of thickness 3.1(�0.2) nm. Thespokes clearly seen between the three cylinders(Figure 8, inset), may be assigned to the residualLCM domain. As con®rmed by mass analysis(Table 5, Figure 9b), most reconstitutes had lostboth supplementary structures and the rod con-taminants. However, only 15% of the particles hadmasses corresponding to the expected full quota of

12 AP complexes (tricylindric core model,Figure 1b) taking into account the 48 kDa lost fromeach LCM on digestion. Instead, most particles hadgenerally lost one AP trimer. This is proposed tobe an AP-B, corroborating the spectroscopic anal-ysis of the intact core reconstitutes (Table 2B). Onlyvery few particles lacked a second trimeric com-plex. According to the tricylindric core model, thismust have been the second AP-B or an AP-I trimer.The absence of essentially all rod constituentsaccording to SDS-PAGE (Figure 6a, lane 2) resultedfrom the digestion of rod-core linkers. In contrast,only terminal regions of the LCM were cleaved.Sequence comparisons reveal that the C-terminalLCM region lost on digestion of the AP core recon-stitutes contained the Rep4 domain (see Esteban,1993 and compare Cai, 1997). The loss of thisdomain together with the two additional structuresof pentacylindrical cores, con®rms its proposedimportance for their assembly (Bryant, 1991; Isono& Katoh, 1987; Sidler, 1994).

In negative stain electron microscopy, theappearance of the untreated AP core reconstitutesvaried considerably. While in some cases thiswas typical of pentacylindrical cores viewedend-on (Figure 4c), in others the supplementarycylinders were partially missing (Figure 4dand e). The maximum core dimensions were25.3(�0.4) nm � 18.6(�0.9) nm. In contrast to theirdigested counterparts, exhibiting elliptical cylin-ders (7.9 nm � 9.1 nm) assembled from 3.1 nmdisks, cylinders of the tricylindric unit were circu-lar in end-on projection with a diameter of8.4(�0.3) nm. This is considerably smaller than the11 nm diameter measured by electron microscopyfor the two core cylinders of Synechococcus 6301(Yamanaka et al., 1980), but in reasonable agree-ment with X-ray data. The latter, obtained for APtrimeric disks of Spirulina platensis, yield minimumand maximum diameters of 8.5 nm and 9.5 nm,respectively, and a height of 3.4 nm (Brejce et al.,1995). The supplementary disks displayed thesame staining pattern as the tricylindric unit cylin-ders (Figure 4c), but had a distinctly oval end-onprojection with a long axis of 10.0(�0.5) nm and ashort axis of 6.0(�1.1) nm. This may be explainedby slight tilting on sample preparation, whichwould be compatible with the presence of two APtrimeric disks, rather than the four found in theother cylinders (pentacylindrical core model,Figure 1c). As after digestion, spokes were clearlyseen between the three central cylinders (Figure 4bto e). In addition, connections to the subsidiarycylinders could be discerned (Figure 4c). All ofthese regions may be assigned to LCM domains. Asexpected from the heterogeneous appearance ofthe untreated AP core reconstitutes, the mass histo-gram from these particles was complex (Table 4,Figure 5b). Only about 20% of the selected reconsti-tute particles had a mass corresponding to the pre-sence of the full complement of 16 AP complexes,predicted by their LCM length and proposed by thepentacylindrical model. The mass difference


between these and the corresponding digested corereconstitutes con®rms the two supplementary coreelements as half-cylinders, each consisting of twoAP trimeric complexes. The main lower mass peakwas not compatible with the loss of a single AP tri-meric complex, as expected from the results for thedigested sample. Instead, the loss of two trimerswas indicated. While according to the compositionanalysis, one of these missing disks was an AP-B,the other, considering the relative stability of thedigested cores, probably correlates to incompletesupplementary half-cylinders. Very few cores weremissing four trimeric complexes, as indicated bythe minor peak at even lower mass. The highermass, 2136 kDa, shown by about 25% of the APcore reconstitutes, correlates to the absence ofonly one AP trimer, most probably an AP-B, andthe presence of one PC hexamer. In this case, theattachment of rod hexamers apparently stabilisedone trimeric complex against loss. According tothe results from the digested sample, this wasprobably an AP-I trimer present in the sup-plementary half-cylinders. In support of this,images from negatively stained samples fre-quently showed rod impurities attached in thisarea (Figure 4, arrowheads), indicating a particu-larly stable association. Interaction of the C-term-inal region of the LCM, with a hexameric PC rod-core complex linking the latter to the AP coredomain has been proposed (Sidler, 1994). Thehigher masses measured re¯ect the association offurther rod hexamers.

The precision of the mass measurement (1.7%deviation from the calculated value for the com-plete core complex) is excellent considering thehigh heterogeneity present in the sample. The rela-tively small differences between the expected andmeasured STEM mass values might relate to minorsequence variations between the polypeptidesaAP-B and b16 of Anabaena sp. PCC 7120 and thoseof Mastigocladus laminosus, whose masses wereused in the calculations. In addition, it should benoted that while the untreated reconstitute massestend to be lower than those predicted from sequen-cing data, those from the digested reconstitute aremarginally higher. Thus, the amount of LCM

cleaved from the C terminus may have been over-estimated by SDS-PAGE.

The presence of AP-I trimers and a small amountof AP-B trimers in the lighter AP fraction, fraction1 (Figure 2a), obtained on sucrose gradients afterreconstitution, is compatible with the above assign-ments. Although spectroscopy indicated the pre-sence of intact core complex reconstitutes in theheavier AP fraction (fraction 2) a quantitative anal-ysis of the reconstitution was only possible bySTEM mass measurement. According to the popu-lation numbers (Table 4), an average of 1.3 trimericdisks were missing per complex, in the reconsti-tute. When based on the average presence of 14.7trimeric complexes as indicated by STEM, theaverage of the composition analyses reported inTable 2B gives an overall ratio of 0.9 AP-B disks:

5.4 AP-I disks: 8.4 (AP-II � AP-LCM) disks in thereconstitute. Assuming the loss of an AP-B andan AP-I trimeric complex, the proposed pentacy-lindrical core model (Figure 1c) predicts arelative content of 1 AP-B disk:5 AP-I disks:8(AP-II � AP-LCM) disks, in excellent agreementwith the measured values.

Combined, the results con®rm the pentacylindri-cal core model, i.e. the presence of a tricylindricelement, as found in tricylindric cores, plus twoadditional half-cylinders formed from AP-I andAP-II trimers in equal ratio. Thus, the principle ofa common architecture for the hemidiscoidal PBSis supported. Accordingly, the results indicate thesame trimer organisation in the four-disk cylindersas observed for bi- and tricylindric cores (Bryantet al., 1990; Bryant, 1991; Yamanaka et al., 1980)and show the extended size of the LCM to beresponsible for binding the two supplementarycylinders, as has been previously suggested for thepentacylindrical core of Anabaena variabilis (Isono &Katoh, 1987). A more important function of therod-core junction in maintaining the AP core struc-tural integrity is also implied, as contaminating PCrod-core complexes were invariably observed toincrease the stability of the reconstituting AP corecomplex and to facilitate the incorporation of itsAP trimers.

In the LCM, the conserved Rep domains are sep-arated by amino acid stretches of various lengthand composition, the Arms, which show no signi®-cant sequence homology with each other or withother polypeptides contained in the PBS. The Repdomains usually span approximately 120 aminoacids, being shorter than the corresponding iso-residue domain of rod or rod-core linker polypep-tides which assemble phycobiliprotein hexamers(Sidler, 1994). The morphological differencesbetween the three hemidiscoidal PBS types high-light the structural functions of the Rep domains(Figure 10). Rep1 and Rep2, being present in allcharacterised LCMs, organise the two basal cylin-ders. Due to the shortness of Arm1, Rep1 is prob-ably involved in binding an AP complex located inthe same core cylinder as the LCM, while the lengthof Arm2 would allow Rep2 to be located in theadjacent basal core cylinder (Capuano et al., 1991).Rep3, which is found in the 96 kDa and 127 kDaLCMs of tri- and pentacylindrical AP cores, respect-ively, is implemented in building the upper four-disk cylinder whereas Rep4, occurring only in the127 kDa LCM, promotes the assembly of the twohalf-cylinders observed in pentacylindrical PBScore domains (Sidler, 1994). The relationshipbetween PBS type and LCM length could be furthersubstantiated at the experimental level. Recently,Capuano et al. (1993) introduced the gene of anLCM known to promote the building of tricylindri-cal PBS to a cyanobacterium assembling bicylindri-cal PBS. The authors could demonstrate that asmall number of PBS isolated from the transformedcyanobacterium exhibited the expected tricylindri-cal morphology. In addition, the deduced amino

Figure 10. Schematic representation of the LCMs structure based on amino acid (aa) sequence comparisons of theapcE gene products of Synechococcus sp. PCC 6301 (Syn. 6301; Capuano et al., 1991), Synechococcus sp. PCC 7002(Syn. 7002; Bryant, 1991), Calothrix sp. PCC 6701 (Cal. 6701; Houmard et al., 1990) and Anabaena sp. PCC 7120 (Cai,1997). The degradation of Arm4 and Rep4 domains of Calothrix sp. PCC 7601 in vivo is indicated by the shadow-ing. The arrows show the possible interaction of the Rep domains with speci®c AP trimers. PBPD, phycobiliproteindomain.


acid sequence of the core-membrane linker ofCalothrix sp. PCC 7601 predicts a 120 kDa poly-peptide exhibiting four Rep domains (Houmardet al., 1990) although PBS isolated from this cya-nobacterium were clearly tricylindrical in mor-phology (Glauser et al., 1992). At the proteinlevel, however, the LCM molecular mass was esti-mated as only 94 kDa by SDS-PAGE (Glauseret al., 1992). Its amino-terminal sequence wasidentical to the sequence predicted by its genesequence, except that the initiator methionine wasmissing. The core-membrane linker had probablybeen processed at its carboxy terminus by anunknown proteolysis mechanism and so con-tained only three Rep domains. Therefore, thispolypeptide promoted the assembly of the sametricylindrical PBS core domain as isolated fromSynechocystis sp. PCC 6701 and Synechococcus sp.PCC 7002 (Sidler, 1994). Reconstitution exper-iments (Table 1) indicate that each Rep domainspeci®cally interacts with two AP trimers, oneAP-II or AP-LCM, and one distal complex, AP-Ior AP-B (see Figure 10). The strong associationof AP-II trimeric complexes with the LCM foundin the present work and reported previously(Reuter & Wehrmeyer, 1990), indicates that theinteracting Rep domains might be buried insideof these trimers, similar to the way in whichother linker polypeptides associate with theirphycobiliproteins (Bryant, 1991; Sidler, 1994;Gottschalk et al., 1994). A different interaction isthought to assemble the distal AP trimers ofthe PBS core domain since these are alreadyassociated via a linker polypeptide, the Lc

8.9.Nevertheless, the presence of distal complexspeci®city was illustrated by the inability of AP-

B to substitute for AP-I during Anabaena sp.PCC 7120 core reconstitution. Thus, the incor-poration of AP-B might require the presence ofthe AP-LCM complex, which is considered to belocated in an adjacent position.

Both a parallel (Anderson & Eiserling, 1986) andan antiparallel (Lundell & Glazer, 1983c & Bryantet al., 1979) organisation of the AP core domainhave been proposed. The knowledge gained fromthe structural analysis of the LCM makes it possibleto discriminate between these two hypotheses.A parallel arrangement of the basal core cylinderswould imply a parallel orientation of the LCMs.Consequently, the two AP hexamers organised bythe Rep3 s of these parallel LCMs, must also exhibita parallel orientation, thus forming two adjacenthalf-cylinders. Only an antiparallel arrangement ofthe basal core cylinders would provide an antipar-allel disposition of the Reps, as required to buildthe apparently coherent top core cylinder observedby electron microscopy. Similarly, the pentacylind-rical PBS core domains of Mastigocladus laminosus,Anabaena variabilis and Anabaena sp. PCC 7120,would probably exhibit a tetracylindrical core mor-phology if their 127 kDa LCMs were oriented inparallel. However, if Reps 1, 2, and 3 ful®l thesame structural functions as in a tricylindricalPBS core domain, an antiparallel arrangement ofthe AP complexes must be assumed, in whichcase the Rep4 domain of the 127 kDa LCM cannotbuild a fourth whole-cylinder. The latter has tobe split into two half-cylinders which will belocated on opposite sides of the tricylindrical unitto build the typical pentacylindrical core domainobserved on electron micrographs (Figure 4;model in Figure 1c).


Materials and Methods

Strain, chemicals and enzymes

The cyanobacterium Anabaena sp. PCC 7120 (alsoknown as Nostoc muscorum ISU and Anabaena sp. ATCC270893) was kindly supplied by Professor D. A.Bryant, of the Pennsylvania State University, Univer-sity Park, USA. Growth conditions were as describedby Ducret et al. (1996). Acrylamide (2 � crystallised)and bis-acrylamide (2 � crystallised) were purchasedfrom Serva. Pefabloc1, (4-(2-aminoethyl)benzenesul-phonyl ¯uoride hydrochloride) was purchased fromPentapharm Ltd. whereas the dialysis bags were fromUnion Carbide. Molecular mass standards for SDS-PAGEwere obtained from BioRad. All other chemicals were ofanalytical grade and were purchased from Fluka, Sigmaor Merck. Chymotrypsin A4 from bovine pancreas (90units/mg) was obtained from Boehringer-Mannheim,and Benzon nuclease2 (250,000 units/ml) was purchasedfrom Merck.

Phycobilisome isolation and partialdissociation experiments

The isolation of Anabaena PBS was carried out follow-ing a modi®ed protocol from Gantt et al. (1979) asdescribed elsewhere (Ducret et al., 1996).

Partial dissociation experiments were performed in anattempt to isolate the AP core complex. PBS were dia-lysed for six hours at either 0, 20 or 37�C against from500 to 800 mM KH2PO4 solutions buffered betweenpH 4.0 and pH 9.5. The dissociation products were sub-sequently separated by sucrose density gradient ultra-centrifugation and analysed by SDS-PAGE andabsorption spectrometry.

Isolation of AP from the Anabaena PBS andreconstitution of a complete AP core complex

The procedure outlined by Ducret et al. (1996) for theisolation and puri®cation of AP subcomplexes wasemployed except that 900 mM, rather than 100 mM,KH2PO4 buffer (pH 7.0, complemented with 2 mMK2EDTA and 1 mM NaN3) and a different sucrose gradi-ent were employed in the later stages. Thus, the secondhydroxyapatite column was eluted with 900 mMKH2PO4 buffer (pH 7.0). Up to 3 ml of the resultingcrude AP solution (with an A of 0.5 to 3.0 at the absorp-tion maximum; the exact composition was never deter-mined) was then directly applied to a 32 ml linearsucrose gradient (0.2 to 0.8 M sucrose) containing900 mM KH2PO4 buffer (pH 7.0). Ultracentrifugation at97,000 to 114,000 g (TST28.38 rotor, 23,000 to 25,000 rpm)at 20 �C for 12 to 14 hours yielded two AP containingfractions. The lower fraction contained the AP core com-plexes. Unless otherwise stated, this fraction, termed theAP core reconstitute, was used in all further analyses.The samples were either stored in the presence ofsucrose at room temperature or were snap-frozen inliquid nitrogen and stored at ÿ20 �C.

Isolation and purification of the APtrimeric complexes

AP trimeric complexes were isolated from AP corereconstitute solutions or from crude AP solutions puri-®ed by hydroxyapatite chromatography as described by

Ducret et al. (1996). The AP solutions were dialysedagainst 5 mM KH2PO4 buffer (pH 7.0; 2 � 100 samplevolumes) for six hours at room temperature and up to3 ml were applied onto a 32 ml linear sucrose gradient(0.1 to 0.6 M sucrose) containing the same buffer. Gradi-ents were ultracentrifuged for 12 to 14 hours at 97,000 to114,000 g (TST28.38, 23,000 to 25,000 rpm) at 20 �C. Theupper band, band 1, which contained the trimeric APcomplexes AP-I, AP-II and AP-B, was concentratedwith an Amicon ultra®ltration membrane (type PM10) and up to 3 ml of this solution (with an A of 0.5to 3.0 at the absorption maximum) were applied ontoa Pharmacia MonoQ HR 5/5. The AP complexes wereeluted from the ion-exchange column according to theprogram described by Ducret et al. (1996). The lowerhigh molecular mass band, band 2 (the APLcm com-plex), contained the AP-LCM trimeric complex associ-ated with several AP-II trimeric complexes and asmall amount of contaminating AP-I trimeric complex(Ducret et al., 1996). This fraction could not be puri-®ed further as the LCM irreversibly bound to the ion-exchange column.

Composition analysis of the AP core reconstitute

AP core reconstitutes were dissociated by dialysisagainst 5 mM KH2PO4 buffer (pH 7.0) and the resultingAP trimeric complexes fractionated by sucrose gradientdensity ultracentrifugation as described above. The rela-tive amounts of bands 1 and 2 were determined bymeasuring the absorbance at their absorption maxima.This immediately gave the amount of the APLcm complexpresent (band 2). Band 1 was subjected to ion-exchangechromatography which separated the three trimeric com-plexes it contained and allowed quantitative analysis.The concentration of each complex was calculatedaccording to the following assumptions: (1) all distalcomplexes (i.e. the AP-I and AP-B complexes) are quanti-tatively recovered in their trimeric aggregation state(Reuter & Wehrmeyer, 1990). Their overall populationsare isolated in the low molecular mass band of thesucrose gradient; (2) the AP-II population is accountedfor either as trimeric AP-II complex in the low molecularmass band of the sucrose gradient or as an LCM-associ-ated AP complex in the high molecular mass band of thesucrose gradient. These two populations differ by theirmolar extinction coef®cients. (3) All LCM-associated APcomplexes (including the APLcm complex) are assumedto exhibit a molar extinction coef®cient similar to that ofAP-I (Gottschalk et al., 1994). The normalised portion ofa particular AP trimer complex (APx) was calculated asfollows:

% APx ��APx

� eAp-I

eAPx

�AP-I ��AP-II � eAP-I

eAP-II��AP-B � eAP-I

eAP-B

� 100

where � denotes the peak area of each AP complex, andthe molar extinction coef®cients were:

eAP-B � 9:77� 105 Mÿ1 cmÿ1

eAP-I � 1:076� 106 Mÿ1 cmÿ1

eAP-II � 7:47� 105 Mÿ1 cmÿ1

(FuÈ glistaller et al., 1987).The amount of a particular AP complex (APx � AP-I,

AP-II or AP-B) in the band 1 or the amount of the APLcm


complex in the band 2 isolated from the sucrose densitygradient centrifugation (Table 2A) was calculated as fol-lows:

Amount APx (in M) � (volume of band 1 � A of band1 � normalised content APx)/eAPx

Amount APLcm and associated AP-II (in M) � (volumeof band 2 � A of band 2)/eAP-I (according to assumption3 above).

The relative portion of a particular AP trimer (APx)was calculated as follows:

% content APx � amount APx

overall AP trimer amount� 100

Proteolytic digestion of the AP core reconstitutewith chymotrypsin

The digestion procedure was adapted from a protocolestablished by Isono & Katoh (1987). The AP core recon-stitute was dialysed against 900 mM KH2PO4 buffer(pH 7.0) to remove the sucrose and the pH of the sol-ution was adjusted to 8.3 to 8.4 with 6 M KOH. Diges-tion was initiated by adding 900 units chymotrypsin permg AP core complex and the sample was incubated forthree hours at room temperature. Up to 3.5 ml of thesolution was then applied onto a 32 ml linear sucrosegradient (0.15 to 0.6 M sucrose) in 900 mM KH2PO4 buf-fer (pH 7.0) and the digested cores were puri®ed fromchymotrypsin and degraded components by ultracentri-fugation at 97,000 g (TST28.38 rotor, 23,000 rpm) for 14hours at 20�C.

Reconstitution behaviour of the APtrimeric complexes

Various mixtures of the puri®ed AP-I, AP-II and AP-Btrimeric complexes and the inseparable APLcm mixturewere dialysed for six hours at room temperature against900 mM KH2PO4 buffer (pH 7.0), 0.1 M sucrose, andthen applied to a 32 ml linear sucrose gradient (0.2 to0.8 M sucrose) containing the same buffer. The gradientswere ultracentrifuged at 97,000 to 114,000 g (TST28.38rotor, 23,000 to 25,000 rpm) at 20�C for 12-14 h andexamined for high molecular mass products. The latterwere characterised spectroscopically but otherwise notused further.

Spectroscopic measurements

Absorption spectra and their second derivatives weremeasured on a Perkin-Elmer Lambda 5 UV/VIS spectro-photometer at room temperature in 1 cm � 0.4 cm(path � 1 cm) cuvettes. The sample concentration wasadjusted to an A of 0.1 to 1.2 at the absorption maxi-mum. Spectra were recorded with a scan speed of120 nm/minute with a time constant adjusted to 0.2seconds. The second derivative spectra were calculatedover a delta wavelength of 6 nm with a time constant ofone second. The slit width was set to 1 nm. Fluorescenceemission and excitation spectra were recorded on aSPEX Fluorolog spectro¯uorometer (SPEX IndustriesInc.) at room temperature in 1 cm � 1 cm cuvettes.Sample concentrations were adjusted to an A of 0.0125to 0.025 at the absorption maximum. Spectra were theaverage of four scans recorded at a speed of 120 nm/min. The excitation slit width was adjusted to 1.7 nm,the emission slit width to 0.85 nm. Excitation ¯uor-escence spectra were recorded at 695 nm with an exci-

tation range from 600 to 685 nm and emission¯uorescence spectra were recorded between 620 and720 nm with an excitation wavelength of 610 nm.

Gel electrophoresis, electroblotting and amino acidmicrosequence analysis

SDS-PAGE was performed using a BioRad Mini-Pro-tean II dual slab cell system with the buffer systememployed by Laemmli (1970) as reported elsewhere(Ducret et al., 1996). For electroblotting, about ®ve to tentimes the amount of protein used for analytical SDS-PAGE was loaded onto SDS gels optimised to give thebest separation. The unstained gels were then directlyblotted onto PVDF membranes as described by Franket al. (1992). The protein band(s) of interest (stained withamido black) were ®nally excised from the membraneand used directly for protein sequencing. AutomatedEdman degradation was performed on a Knauer model810 amino acid sequencer (Dr Ing. H. Knauer, GmbH)using standard programs. The PTH amino acid deriva-tives were detected at 269 nm on an isocratic on-lineHPLC-system as described by Frank (1989).

Scanning transmission electron microscopy (STEM)

A Vacuum Generators HB-5 scanning transmissionelectron microscope was employed to obtain highresolution dark-®eld images from negatively stainedpreparations of both digested and untreated AP corereconstitutes, and for mass analysis of unstained speci-mens of the same samples. Gold coated copper grids,prepared with thin carbon ®lms by the standard pro-cedure for STEM microscopy (Fukami & Adachi, 1965),were used throughout. The AP core complexes, sus-pended to a concentration of 10 to 40 mg/ml in 900 mMKH2PO4 buffer (pH 7.0), 2 mM K2EDTA and 1 mMNaN3 with 0.2 M sucrose as stabilising agent, wereadsorbed to the freshly glow discharged carbon ®lms fortwo minutes. The grids were subsequently washed ontwo drops of the 900 mM KH2PO4 buffer (pH 7.0) toremove sucrose before protein stabilisation with 0.3%(v/v) glutaraldehyde for seven minutes. This was fol-lowed by a seven minute wash on 500 mM ammoniumacetate and a ®ve minute wash on 50 mM ammoniumacetate. The specimens were then either negativelystained with 0.75% (w/v) uranyl formate and air driedor washed on ®ve droplets of quartz bi-distilled water,to remove buffer salts and aldehyde, and freeze-dried atÿ80�C overnight in the microscope. The digital imagesrecorded from the negatively stained specimens werecontrast inverted using the SEMPER VI program pack-age (Synoptics Ltd., Cambridge, England). Single particleaveraging was carried out with the same program asdescribed by Frank et al. (1987). The STEM mass analyseswere performed following the procedures describedby Engel & Reichelt (1988) and MuÈ ller et al. (1992). Thelow dose, dark-®eld micrographs required for massmeasurement were recorded at 80 kV, a nominal magni-®cation of 200,000� and doses ranging between 300 and400 e/nm2 from the unstained samples. These digitalimages were evaluated using the specialised programpackage, IMPSYS, described by MuÈ ller et al. (1992). Inaddition, for the digested AP core reconstitute, someframes were repeatedly irradiated and recorded. Theirevaluation yielded the mass-loss relationship for bothAP core specimens and allowed accurate correction of


the data for this effect. Thus, the results presented areabsolute values.

Acknowledgements

We thank Mrs M Zoller and Mrs H. Frefel for excellentphotographic work. The project was supported by theSwiss National Foundation grant number 3142435.94and the Maurice E. MuÈ ller Foundation of Switzerland.

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Edited by R. Huber

(Received 22 September 1997; received in revised form 28 January 1998; accepted 29 January 1998)

Documents

Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120