Molecular composition of cyanobacterial phycobilisomes* · Proc. Natl. Acad.Sci. USA74(1977) 1637 n 30 c 20 L a (10 w a a. 20 a. 0 0 U 101 0 '5 4 E. C e e a 3cn x rL 2gj 'E)1n cn-J

Proc. Natl. Acad. Sci. USAVol. 74, No. 4, pp. 1635-1639, April 1977Cell Biology

Molecular composition of cyanobacterial phycobilisomes*(phycobiliproteins/polyacrylamide gel electrophoresis/cyanobacteria/chromatic adaptation)

N. TANDEAU DE MARSAC AND G. COHEN-BAZIREDepartement de Biochimie et Gknktique Microbienne, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France

Communicated by H. A. Barker, February 3, 1977

ABSTRACT Phycobilisomes isolated from eight differentspecies of cyanobacteria contain, in addition to the light-har-vesting phycobiliproteins, a small number of colorless poly-peptides with molecular weights higher than those of thechromopolypeptide subunits of the phycobiliproteins. In thephycobilisomes of the species examined, from four to nine col-orless polypeptides were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Those of highest molecularweight (70,000-120,000) also occurred in the washed membranefraction of the cell and may therefore be derived from the thy-lakoids, to which the phycobilisomes are attached in vivo.Colorless polypeptides of lesser molecular weight (30,000-70,000) appeared to be specific constituents of the phycobili-some. In strains of cyanobacteria that adapt chromatically, theirsynthesis, like that of the major phycobiliproteins, is regulatedby light quality.

In cyanobacteria, a major part of the light-harvesting pigmentsystem is located in a special organelle, the phycobilisome (1).Regular rows of phycobilisomes, each some 40 nm in diameter,are attached to the external surface of the thylakoid whichcontains the other elements of the photosynthetic apparatus.Three phycobiliproteins-allophycocyanin B (Xmax 671 nm),allophycocyanin (Xmax 650 nm), and phycocyanin (Xmax 620nm)-are always present in the phycobilisome (2). They areaccompanied in many cyanobacteria by other phycobiliproteinswith absorption maxima at shorter wavelengths: phycoerythrins(Amax 500-580 nm) or the recently discovered (3) pigmentphycoerythrocyanin (,max 568 nm). Quantum energy absorbedby any phycobilisomal pigment is channeled by radiationlesstransfer to the photochemical reaction centers of the thylakoid(4). Within the phycobilisome, radiationless energy transfertakes place through the sequence: phycoerythrin (or phycoer-ythrocyanin) -- phycocyanin - allophycocyanin o allo-phycocyanin B (ref. 5; Ley, Bryant, Glazer, and Butler, personalcommunication).

Phycobilisomes can be extracted from the cell in a seeminglyintact state with a phosphate buffer of high ionic strength andsubsequently separated from other cell components by differ-ential centrifugation (6). Dilution of the solvent causes rapiddisaggregation of the phycobilisomes, accompanied by theuncoupling of radiationless energy transfer between the con-stituent phycobiliproteins (7).

It has been reported that the protein content of phycobili-somes extracted from the red alga Porphyridium can be en-tirely accounted for by phycobiliproteins (8); and only tracesof other proteins have been detected in phycobilisomes ex-tracted from a cyanobacterium, Nostoc sp. (6). We report herethat the protein composition of cyanobacterial phy~obilisomesis in fact considerably more complex. About 15% of the totalprotein in phycobilisomes is accounted for by a small number

Abbreviation: NaDodSO4, sodium dodecyl sulfate.* A preliminary account of this work was presented at the Second In-ternational Symposium on Photosynthetic Prokaryotes, Dundee,Scotland, August 1976. This work will be part of the Doctoral dis-sertation of N. Tandeau de Marsac.

1635

of colorless polypeptides, all of higher molecular weight thanthe chromopolypeptide subunits of the phycobiliproteins.

MATERIALS AND METHODS

Biological Material. Phycobilisomes were isolated fromeight species of cyanobacteria maintained in the culture col-lection of our laboratory (Table 1). Cultures were grown pho-toautotrophically at room temperature (20-25°) in mediumBG-li (10) and harvested while still growing actively. Mostcultures were grown in white light (Osram white Universalfluorescent lamps). Some were grown in chromatic light pro-duced by the interposition of a green or red plastic filter (9)between the fluorescent light source and the culture vessel.

Extraction and Isolation of Phycobilisomes. Phycobili-somes were prepared by a procedure similar to that developedby Gray and Gantt (6). Organisms harvested by centrifugationwere resuspended in 0.5 M ammonium phosphate buffer (pH7.0) at a concentration of approximately 0.1 g (wet weight)/ml.The suspension was then broken in a French pressure cell under1300 atm. The extract was collected and incubated for 30 minat room temperature in the presence of 1% (vol/vol) TritonX-100. In a few experiments, the Triton X-100 treatment wasomitted. All subsequent operations were conducted at 4°C. Theextract was clarified by centrifugation at 30,000 X g for 30 min.Aliquots (1.5 ml) of the clarified supernatant were then layeredonto discontinuous sucrose gradients, prepared with 2, 5, 5, 4,and 3 ml, respectively, of 2.0, 1.0, 0.75, 0.5, and 0.25 M sucrosedissolved in 0.75 M Na,K phosphate buffer (pH 7.0). Aftercentrifugation at 65,000 X g for 15-16 hr, the phycobilisomefraction was eluted and freed of sucrose by passage through acolumn of Sephadex G-25 previously equilibrated with the samebuffer. Many phycobilisome preparations were subsequentlyconcentrated by precipitation with ammonium sulfate (30%saturation) and then chromatographed on a Bio-Gel A-15 col-umn equilibrated with the same buffer.

Analysis of Polypeptide Composition. Proteins were ana-lyzed on sodium dodecyl sulfate (NaDodSO4)/polyacrylamideslab gels with the discontinuous buffer system described byLaemmli (11) and the apparatus described by Studier (12). Gelswere prepared by diluting a stock solution containing 30%(wt/vol) acrylamide and 0.1% (wt/vol) N,N'-bismethylene-acrylamide. The resolving gel, 20% (wt/vol) acrylamide con-taining 0.375 M Tris-HCI (pH 8.8) and 0.1% (wt/vol) NaDod-SO4, was polymerized with 0.05% (vol/vol) N,N,N',N'-tetra-methylethylenediamine and 0.05% (wt/vol) ammonium per-sulfate. The stacking gel, 6% (wt/vol) acrylamide, contained0.125 M Tris-HCl (pH 6.8) and 0.1% NaDodSO4 and was po-lymerized with 0.06% (vol/vol) N,N,N',N'-tetramethylethy-lenediamine and 0.6% (wt/vol) ammonium persulfate.

Prior to electrophoresis, samples were dialyzed against 5 mMNa,K phosphate buffer (pH 7.0) or against distilled water andconcentrated, if necessary, with Aquacide III (Calbiochem) inorder to obtain a solution containing 0.5-1 mg of protein per ml.

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1636 Cell Biology: de Marsac and Cohen-Bazire

Table 1. Strain designations and some properties of cyanobacteria used as sources of phycobilisomes

Phycobiliproteins synthesizedt Photoregulation ofATCC* phycobiliprotein

no. PE PEC PC AP synthesis (9)

Unicellular cyanobacteriaSynechococcus 6312 27167 - - + +Synechocystis 6714 27178 - - + +Synechocystis 6808 27189 + - + + +Gloeobacter 7421 29082 + - + +Chamaesiphon 6605 37169 + - + + +

Filamentous cyanobacteriaLPPt group 7409 + - + + +Anabaena 6411 27898 - + + +Fremyella 7601 + - + + +

* American Type Culture Collection.t PE, phycoerythrin; PEC, phycoerythrocyanin; PC, phycocyanin; AP, allophycocyanin.I Lyngbya-Plectonema-Phormidium.

Samples were diluted with an equal volume of 0.065 M Tris-HCI (pH 6.8) containing 2% (wt/vol) NaDodSO4, 5% (vol/vol)2-mercaptoethanol, and 10% (vol/vol) glycerol and immersedin boiling water for 3 min. Samples (5-50 ,l) containing notmore than 15 ,gg of protein were electrophoresed for 15 hr ata constant voltage of 30 V in 0.025 M Tris buffer (pH 8.3)containing 0.192 M glycine and 0.1% (wt/vol) NaDodSO4. Thegels were fixed in 50% trichloroacetic acid for 2 hr and stainedfor 1 hr at room temperature with 0.1% (wt/vol) Coomassiebrilliant blue R250 dissolved in 50% (wt/vol) trichloroaceticacid. The destaining solution contained 5% (vol/vol) absolutemethanol and 7.5% (vol/vol) glacial acetic acid. Stained gelswere examined with an automatic gel scanner (Vernon) fittedwith a filter transmitting in the range 630-650 nm. The relativecontribution of each polypeptide peak was estimated by mea-surement of the total peak area of the gel scan.

Protein Determinations. Total protein was determined bythe method of Lowry et al. (13) with bovine serum albumin asstandard. The contents of phycoerythrin, phycocyanin, andallophycocyanin in isolated phycobilisomes were determinedby measuring the absorbancy at 565, 620, and 652 nm, re-

spectively, of samples diluted in 0.01 M Na phosphate buffer(pH 7.0) containing 0.15 M NaCl (10).

RESULTSFig. 1 shows NaDodSO4/acrylamide gel electropherograms ofphycobilisomes isolated from seven species of cyanobacteriagrown in white light. The polypeptides of group IV (molecularweight, 16,000-22,000) which were only partly resolved in theseelectropherograms, consisted of the a and ,B subunits of thephycobiliproteins, identified by their intrinsic color beforestaining. Polypeptides of higher molecular weight (groups I-III)were apparent only after Coomassie blue staining. The variousphycobilisome preparations examined contain four to ninemajor colorless polypeptides that accounted for 14-18% of thetotal stainable material on the gels. If the treatment with TritonX-100 was omitted from the preparative procedure, the yieldof phycobilisomes was greatly decreased; however, their mo-lecular composition was identical with that of phycobilisomesextracted in the presence of the detergent. Are the colorlesspolypeptides structural components of the phycobilisome,

Mr

120,000

70,000

a ..

6714 6312 6411 7421 6605 6808 7409FIG. 1. NaDodSO4/polyacrylamide gel electropherograms of isolated phycobilisomes prepared from seven different cyanobacteria grown

in white light. Molecular weights (Mr) were determined from a standard calibration curve using as markers: f3-galactosidase (130,000), bovineserum albumin (68,000), catalase (57,000), ovalbumin (43,000), chymotrypsinogen A (25,700), f-lactoglobulin (17,400), lysozyme (14,300). Theelectropherograms shown are taken from four separate gel electrophoreses, in which the extents of migration of the polypeptides differed.

30,000 --- --- _ -III

25,000

IV, I15,000 ----------

Proc. Nati. Acad. Sci. USA 74 (1977)

'-Npmgmk.4*NMNNM

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Proc. Natl. Acad. Sci. USA 74 (1977) 1637

30n

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0

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4 E.C

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2 gj1n'E)cn

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5 10 ISFRACTION NUMBER

FIG. 2. Polypeptide compositions of fractions eluted from adiscontinuous sucrose gradient overlaid with a cell-free extract ofstrain 7409 grown under white light. Each fraction was analyzed byNaDodSO4/polyacrylamide gel electrophoresis, the quantity of in-dividual polypeptides or groups of polypeptides being estimated fromstained gel scans. The amounts of total chromopolypeptides (0-0)and total colorless polypeptides (O--- -0) are expressed in arbitraryunits per fraction (1 ml) eluted from the sucrose gradient.

thylakoidal proteins detached together with the phycobilisomes,or merely contaminating soluble proteins? The results of a seriesof experiments undertaken in an attempt to answer this questionare summarized below.The polypeptide composition of phycobilisomes isolated by

elution from a sucrose gradient was unchanged both afterprecipitation with ammonium sulfate at 30% saturation andafter passage through a Bio-Gel A-15 molecular sieve. Suchexperiments were performed with phycobilisomes isolated fromfive different species of cyanobacteria.The isolated phycobilisomes of strain 7409 contained six

colorless polypeptides (Fig. 1). The relative amounts of thesecolorless polypeptides and of total chromopolypeptides weredetermined from gel scans on successive fractions eluted froma sucrose gradient after centrifugation of an extract of strain7409 (Fig. 2). The distributions through the gradient of theindividual colorless polypeptides are shown in Fig. 3. In theregion of the gradient that contains the phycobilisomes (frac-tions 7-11: 0.55-0.85 M sucrose) there was excellent correlationbetween the distributions of chromopolypeptides and colorlessphycobilisomal polypeptides.

Light quality affects differentially the rates of phycoerythrinand phycocyanin synthesis in strains 7409 and 7601 (9, 14).Phycobilisomes prepared from these strains after growth inwhite, red, and green light differed markedly in their phyco-biliprotein composition. "Green-light" phycobilisomes had ahigh phycoerythrin:phycocyanin ratio, "white-light" phyco-bilisomes had a somewhat lower phycoerythrin:phycocyaninratio, and "red-light" phycobilisomes were virtually devoid ofphycoerythrin. These light-induced modifications of the majorphycobilisomal light-harvesting proteins were accompaniedby marked changes in the relative concentrations of the colorlessgroup II polypeptides (Fig. 4).The results of a semiquantitative analysis of the composition

.5 IQ 15FRACTION NUMBER

FIG. 3. Distributions through a sucrose gradient of the individualcolorless polypeptides associated with the phycobilisomes of strain7409. Data from the experiment described in Fig. 2.

of phycobilisomes prepared from strain 7409 after growth inwhite, green, and red light are presented in Fig. 5 and Table2. In all three preparations, the group II polypeptides collec-tively accounted for the same fraction (approximately 10%) oftotal phycobilisomal protein. In red-light phycobilisomes, whichcontained no phycoerythrin, polypeptide 5 was barely de-tectable, polypeptides 3 and 4 accounting for over 95% of thegroup II components. In green-light phycobilisomes, of whichphycoerythrin was the major phycobiliprotein (51% of thetotal), polypeptide 5 accounted for 80% of the group II com-ponents. White-light phycobilisomes had an intermediatecomposition with respect both to phycobiliproteins and to groupII polypeptides.The physical integrity of the phycobiisome is maintained

only in buffers of high ionic strength. Consequently, if cells arebroken in buffers of low ionic strength, the constituent proteinsof the phycobilisome pass into solution and become mixed withother soluble cytoplasmic proteins in the resulting extract. Thebulk soluble proteins of such an extract can be subsequentlyseparated by differential centrifugation from the membranefraction, which contains the thylakoid-associated proteins. Fortwo cyanobacteria, we compared the polypeptide compositionof isolated phycobilisomes with the polypeptide compositionsof low-ionic-strength extracts and of the soluble and membranefractions prepared from them (Fig. 6). In both cyanobacteria,the group II polypeptides, as well as the bulk of the chromo-polypeptides, occurred in the soluble fraction of the low-ionic-strength extract. The group III polypeptide, present inthe phycobilisomes of only one of these strains (7601), was

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1638 Cell Biology: de Marsac and Cohen-Bazire

Mr

120,000

70,000 -

11

30,000 -I-25,000

IV I

____p15,000 --

R G7 4 0 9

11

III

IV

R G7 6 0 1

FIG. 4. NaDodSO4/polyacrylamide gel electropherograms ofisolated phycobilisomes prepared from two chromatically adaptingcyanobacteria, strains 7409 and 7601, after growth in red (R) and green(G) light. Note the analogous light-induced changes in the relativeamounts of the group II polypeptides. Mr, molecular weight.

likewise associated with the soluble fraction. On the other hand,the group I polypeptides of both strains were largely, andperhaps exclusively, located in the membrane fraction of thelow-ionic-strength extract.

DISCUSSIONIn view of earlier reports (6, 8) that nearly all the protein contentof isolated phycobilisomes is accounted for by phycobiliproteins,our observation that colorless polypeptides account for about15% of the total protein of the cyanobacterial phycobilisomewas wholly unexpected. In the seven cyanobacteria studied, thismaterial consisted of a small number of polypeptides: four tonine components are resolvable by NaDodSO4/polyacrylamidegel electrophoresis. It is improbable that these polypeptides arederived from contaminating soluble cytoplasmic proteins inthe preparations examined. The strongest evidence for theirspecific association with the phycobilisome was obtained by an

Il II m tv4-1~ ~ ~ ~ IIV120 70 30 25 15

Molecular weights (x 10 3) of marker proteins

FIG. 5. Scans of Coomassie blue-stained NaDodSO4/polyacryl-amide gel electropherograms of phycobilisomes prepared from cellsof strain 7409 after growth under white light, green light, and red light.Peaks 1-6 represent the colorless polypeptides of groups I (1 and 2),II (3 to 5), and III (6). The broad band containing two peaks (7 and8) of unequal height, extending over the molecular weight range16,000-22,000, reflects the overlapping absorbances of the chromo-polypeptides derived from the phycobiliproteins. The predominanceof phycoerythrin in "green-light" phycobilisomes is reflected by anincrease in absorbance at the higher end of the molecular weightrange, since the mean subunit molecular weight of it is greater thanthat of phycocyanin and allophycocyanin.

analysis of the polypeptide compositions of successive fractionseluted from a sucrose gradient after centrifugation of an extractof strain 7409. This analysis revealed a good correlation betweenthe distribution through the gradient of the chromopolypeptidesderived from the phycobiliproteins and of each of the six col-orless polypeptides associated with isolated phycobilisome.There is no obvious explanation for the difference between ourfindings concerning the molecular composition of the phyco-bilisome and those of earlier workers (6, 8). Nevertheless, thediscrepancy should be easily resolved. As our data show, col-orless proteins represent approximately 15% of total.phyco-

Table 2. Chemical composition of phycobilisomes isolated from strain 7409after growth with three light sources of different spectral character

% of total phycobilisomal protein*

All % of total group II % of totalcolorless Polypeptide groups: polypeptides* phycobiliproteinstpoly-

Grown under peptides I II III 3 4 5 AP PC PE

White light 17 3.5 9.5 4.0 42 37 21 21 69 10Green light 14 2.0 10.0 2.0 7 13 80 20 29 51Red light 18 4.0 10.0 4.0 53 43 4 20 80 0

* Estimated from scans of gel electropherograms (see Materials and Methods).t Estimated spectrophotometrically (see Materials and Methods). AP, allophycocyanin; PC, phycocyanin; PE, phycoerythrin.

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-.

W- -4

11

III

IV

A B C D A B63 1 2 7601

FIG. 6. NaDodSO4/polyacrylamide gel electropherograms ofcrude cell-free extracts (A), washed membrane fractions (B), solubleprotein fractions (C), and isolated phycobilisomes (D) prepared fromstrain 6312 grown under white light and from strain 7601 grown underred light. See text for explanations.

bilisomal protein. These components should be readily de-tectable by the analytical procedures described here in allpreparations of phycobilisomes, irrespective of their biologicalsource and mode of isolation.

In view of the function of the phycobilisome as a light-har-vesting organelle, two possible roles for its colorless proteinconstituents can be envisaged: attachment of the organelle toa specific site on the thylakoid membrane and positioning ofthe constituent light-harvesting pigments within the phycobi-lisome. Because the group I polypeptides appear also to bepresent in the washed membrane fraction prepared from cellsafter extraction with a buffer of low ionic strength, they mayserve to attach the phycobilisome to the thylakoid. The groupII polypeptides, on the other hand, are probably involved in theassembly and positioning of the phycobiliproteins. Like thephycobiliproteins, they are located exclusively in the solublefraction of extracts prepared at low ionic strength. Furthermore,in chromatically adapting strains, changes in the relative

amounts of the two major phycobiliproteins are correlated withchanges in the relative amounts of individual group II poly-peptides. Photoregulation thus governs the synthesis both ofchromoproteins and of certain colorless protein components ofthe phycobilisome.We thank Dr. R. Haselkorn for helpful discussions and Dr. R. Y.

Stanier for advice and encouragement. The skillful assistance of MissA. M. Castets is gratefully acknowledged. This work was supportedby grants from the "Centre National de la Recherche Scientifique"ERA no. 398 and by the "Delegation Generale a la Recherche Scien-tifique et Technique" (Contract 747.0573).

1. Gray, B. H., Lipschultz, C. A. & Gantt, E. (1973) "Phycobilisomesfrom a blue-green alga Nostoc sp.," J. Bacteriol. 116, 471-478.

2. Glazer, A. N. & Bryant, D. A. (1975) "Allophycocyanin B (Amax671, 618 nm): A new cyanobacterial phycobiliprotein," Arch.Microbiol. 104, 15-22.

3. Bryant, D. A., Glazer, A. N. & Eiserling, F. A. (1976) "Charac-terization and structural properties of the major biliproteins ofAnabaena sp.," Arch Microbiol. 110,61-75.

4. Haxo, F. T. (1960) "The wavelength dependence of photosyn-thesis and the role of accessory pigments," in Comparative Bio-chemistry of Photoreactive Pigments, ed. Allen, M. B. (Aca-demic Press, New York), pp. 339360.

5. Gantt, E. & Lipschultz, C. A. (1973) "Energy transfer in phyco-bilisomes from phycoerythrin to allophycocyanin," Biochim.Biophys. Acta 292,858-861.

6. Gray, B. H. & Gantt, E. (1975) "Spectral properties of phyco-bilisomes and phycobiliproteins from the blue-green alga Nostocsp.," Photochem. Photobiol. 21, 121-128.

7. Gantt, E., Lipschultz, C. A. & Zilinkas, B. (1976) "Further evi-dence for a phycobilisome model from selective dissociation,fluorescence emission, immunoprecipitation and electron mi-croscopy," Biochim. Biophys. Acta 430,375-388.

8. Gantt, E. & Lipschultz, C. A. (1974) "Phycobilisomes of Por-phyridium cruentum: pigment analysis," Biochemistry 13,2960-2966.

9. Tandeau de Marsac, N. (1977) "The occurrence and nature ofchromatic adaptation in cyanobacteria," J. Bacteriol., inpress.

10. Stanier, R. Y., Kunisawa, R., Mandel, M. & Cohen-Bazire, G.(1971) "Purification and properties of unicellular blue-greenalgae (order Chroococales)," Bacteriol. Rev. 35, 171-205.

11. Laemmli, U. K. (1970) "Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4," Nature 227,680-685.

12. Studier, F. W. (1973) "Analysis of bacteriophage T7 early RNAsand proteins on slab gels," J. Mol. Biol. 79, 237-248.

13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.(1951) "Protein measurement with the Folin phenol reagent,"J. Biol. Chem. 193, 265-275.

14. Bennett, A. & Bogorad, L. (1973) "Complementary chromaticadaptation in a filamentous blue-green alga," J. Cell Biol. 58,419-435.

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