Proc. Natl. Acad. Sci. USA Vol. 74, No. 4, pp. 1635-1639, April 1977 Cell Biology Molecular composition of cyanobacterial phycobilisomes* (phycobiliproteins/polyacrylamide gel electrophoresis/cyanobacteria/chromatic adaptation) N. TANDEAU DE MARSAC AND G. COHEN-BAZIRE Departement 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 different of colorless polypeptides, all of higher molecular weight than species of cyanobacteria contain, in addition to the light-har- the chromopolypeptide subunits of the phycobiliproteins. vesting phycobiliproteins, a small number of colorless poly- peptides with molecular weights higher than those of the chromopolypeptide subunits of the phycobiliproteins. In the MATERIALS AND METHODS phycobilisomes of the species examined, from four to nine col- orless polypeptides were resolved by sodium dodecyl sulfate/ Biological Material. Phycobilisomes were isolated from polyacrylamide gel electrophoresis. Those of highest molecular eight species of cyanobacteria maintained in the culture col- weight (70,000-120,000) also occurred in the washed membrane lection of our laboratory (Table 1). Cultures were grown pho- fraction of the cell and may therefore be derived from the thy- toautotrophically at room temperature (20-25°) in medium lakoids, to which the phycobilisomes are attached in vivo. BG-li (10) and harvested while still Colorless polypeptides of lesser molecular weight (30,000- growing actively. Most 70,000) appeared to be specific constituents of the phycobili- cultures were grown in white light (Osram white Universal some. In strains of cyanobacteria that adapt chromatically, their fluorescent lamps). Some were grown in chromatic light pro- synthesis, like that of the major phycobiliproteins, is regulated duced by the interposition of a green or red plastic filter (9) by light quality. between the fluorescent light source and the culture vessel. Extraction and Isolation of Phycobilisomes. Phycobili- In cyanobacteria, a major part of the light-harvesting pigment somes were prepared by a procedure similar to that developed system is located in a special organelle, the phycobilisome (1). by Gray and Gantt (6). Organisms harvested by centrifugation Regular rows of phycobilisomes, each some 40 nm in diameter, were resuspended in 0.5 M ammonium phosphate buffer (pH are attached to the external surface of the thylakoid which 7.0) at a concentration of approximately 0.1 g (wet weight)/ml. contains the other elements of the photosynthetic apparatus. The suspension was then broken in a French pressure cell under Three phycobiliproteins-allophycocyanin B (Xmax 671 nm), 1300 atm. The extract was collected and incubated for 30 min allophycocyanin (Xmax 650 nm), and phycocyanin (Xmax 620 at room temperature in the presence of 1% (vol/vol) Triton nm)-are always present in the phycobilisome (2). They are X-100. In a few experiments, the Triton X-100 treatment was accompanied in many cyanobacteria by other phycobiliproteins omitted. All subsequent operations were conducted at 4°C. The with absorption maxima at shorter wavelengths: phycoerythrins extract was clarified by centrifugation at 30,000 X g for 30 min. (Amax 500-580 nm) or the recently discovered (3) pigment Aliquots (1.5 ml) of the clarified supernatant were then layered phycoerythrocyanin (,max 568 nm). Quantum energy absorbed onto discontinuous sucrose gradients, prepared with 2, 5, 5, 4, by any phycobilisomal pigment is channeled by radiationless and 3 ml, respectively, of 2.0, 1.0, 0.75, 0.5, and 0.25 M sucrose transfer to the photochemical reaction centers of the thylakoid dissolved in 0.75 M Na,K phosphate buffer (pH 7.0). After (4). Within the phycobilisome, radiationless energy transfer centrifugation at 65,000 X g for 15-16 hr, the phycobilisome takes place through the sequence: phycoerythrin (or phycoer- fraction was eluted and freed of sucrose by passage through a ythrocyanin) -- phycocyanin - allophycocyanin o allo- column of Sephadex G-25 previously equilibrated with the same phycocyanin B (ref. 5; Ley, Bryant, Glazer, and Butler, personal buffer. Many phycobilisome preparations were subsequently communication). concentrated by precipitation with ammonium sulfate (30% Phycobilisomes can be extracted from the cell in a seemingly saturation) and then chromatographed on a Bio-Gel A-15 col- intact state with a phosphate buffer of high ionic strength and umn equilibrated with the same buffer. subsequently separated from other cell components by differ- Analysis of Polypeptide Composition. Proteins were ana- ential centrifugation (6). Dilution of the solvent causes rapid lyzed on sodium dodecyl sulfate (NaDodSO4)/polyacrylamide disaggregation of the phycobilisomes, accompanied by the slab gels with the discontinuous buffer system described by uncoupling of radiationless energy transfer between the con- Laemmli (11) and the apparatus described by Studier (12). Gels stituent phycobiliproteins (7). were prepared by diluting a stock solution containing 30% It has been reported that the protein content of phycobili- (wt/vol) acrylamide and 0.1% (wt/vol) N,N'-bismethylene- somes extracted from the red alga Porphyridium can be en- acrylamide. The resolving gel, 20% (wt/vol) acrylamide con- tirely accounted for by phycobiliproteins (8); and only traces taining 0.375 M Tris-HCI (pH 8.8) and 0.1% (wt/vol) NaDod- of other proteins have been detected in phycobilisomes ex- SO4, was polymerized with 0.05% (vol/vol) N,N,N',N'-tetra- tracted from a cyanobacterium, Nostoc sp. (6). We report here methylethylenediamine and 0.05% (wt/vol) ammonium per- that the protein composition of cyanobacterial phy~obilisomes sulfate. The stacking gel, 6% (wt/vol) acrylamide, contained is in fact considerably more complex. About 15% of the total 0.125 M Tris-HCl (pH 6.8) and 0.1% NaDodSO4 and was po- protein in phycobilisomes is accounted for by a small number lymerized with 0.06% (vol/vol) N,N,N',N'-tetramethylethy- Abbreviation: NaDodSO4, sodium dodecyl sulfate. lenediamine and 0.6% (wt/vol) ammonium persulfate. * A preliminary account of this work was presented at the Second In- Prior to electrophoresis, samples were dialyzed against 5 mM ternational Symposium on Photosynthetic Prokaryotes, Dundee, Na,K phosphate buffer (pH 7.0) or against distilled water and Scotland, August 1976. This work will be part of the Doctoral dis- concentrated, if necessary, with Aquacide III (Calbiochem) in sertation of N. Tandeau de Marsac. order to obtain a solution containing 0.5-1 mg of protein per ml. 1635 Downloaded by guest on October 7, 2021 1636 Cell Biology: de Marsac and Cohen-Bazire Proc. Nati. Acad. Sci. USA 74 (1977)

Table 1. Strain designations and some properties of cyanobacteria used as sources of phycobilisomes Phycobiliproteins synthesizedt Photoregulation of ATCC* phycobiliprotein no. PE PEC PC AP synthesis (9) Unicellular cyanobacteria Synechococcus 6312 27167 - - + + Synechocystis 6714 27178 - - + + Synechocystis 6808 27189 + - + + + Gloeobacter 7421 29082 + - + + Chamaesiphon 6605 37169 + - + + + Filamentous cyanobacteria LPPt 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- spectively, of samples diluted in 0.01 M Na phosphate buffer HCI (pH 6.8) containing 2% (wt/vol) NaDodSO4, 5% (vol/vol) (pH 7.0) containing 0.15 M NaCl (10). 2-mercaptoethanol, and 10% (vol/vol) glycerol and immersed in boiling water for 3 min. Samples (5-50 ,l) containing not more than 15 ,gg of protein were electrophoresed for 15 hr at RESULTS a constant voltage of 30 V in 0.025 M Tris buffer (pH 8.3) Fig. 1 shows NaDodSO4/acrylamide gel electropherograms of containing 0.192 M glycine and 0.1% (wt/vol) NaDodSO4. The phycobilisomes isolated from seven species of cyanobacteria gels were fixed in 50% trichloroacetic acid for 2 hr and stained grown in white light. The polypeptides of group IV (molecular for 1 hr at room temperature with 0.1% (wt/vol) Coomassie weight, 16,000-22,000) which were only partly resolved in these brilliant blue R250 dissolved in 50% (wt/vol) trichloroacetic electropherograms, consisted of the a and ,B subunits of the acid. The destaining solution contained 5% (vol/vol) absolute phycobiliproteins, identified by their intrinsic color before methanol and 7.5% (vol/vol) glacial acetic acid. Stained gels staining. Polypeptides of higher molecular weight (groups I-III) were examined with an automatic gel scanner (Vernon) fitted were apparent only after Coomassie blue staining. The various with a filter transmitting in the range 630-650 nm. The relative phycobilisome preparations examined contain four to nine contribution of each polypeptide peak was estimated by mea- major colorless polypeptides that accounted for 14-18% of the surement of the total peak area of the gel scan. total stainable material on the gels. If the treatment with Triton Protein Determinations. Total protein was determined by X-100 was omitted from the preparative procedure, the yield the method of Lowry et al. (13) with bovine serum albumin as of phycobilisomes was greatly decreased; however, their mo- standard. The contents of phycoerythrin, phycocyanin, and lecular composition was identical with that of phycobilisomes allophycocyanin in isolated phycobilisomes were determined extracted in the presence of the detergent. Are the colorless by measuring the absorbancy at 565, 620, and 652 nm, re- polypeptides structural components of the phycobilisome,

Mr 120,000

70,000

'-Npmgmk. - 4*NMNNM 11

30,000 ------_ - III 25,000

demoa .. IV, I

15,000 ------

6714 6312 6411 7421 6605 6808 7409 FIG. 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), bovine serum albumin (68,000), catalase (57,000), ovalbumin (43,000), chymotrypsinogen A (25,700), f-lactoglobulin (17,400), lysozyme (14,300). The electropherograms shown are taken from four separate gel electrophoreses, in which the extents of migration of the polypeptides differed. Downloaded by guest on October 7, 2021 Cell Biology: de Marsac and Cohen-Bazire Proc. Natl. Acad. Sci. USA 74 (1977) 1637

'5

4 E.C

e a n 30 3 cn aLc 20 x (10 w rL a a. 20 2 gj a. 'E)1n 0 cn 0 -J U 101 I O0 u

0 5 10 IS FRACTION NUMBER FIG. 2. Polypeptide compositions of fractions eluted from a discontinuous sucrose gradient overlaid with a cell-free extract of strain 7409 grown under white light. Each fraction was analyzed by NaDodSO4/polyacrylamide gel electrophoresis, the quantity of in- dividual polypeptides or groups of polypeptides being estimated from stained gel scans. The amounts of total chromopolypeptides (0-0) and total colorless polypeptides (O--- -0) are expressed in arbitrary units per fraction (1 ml) eluted from the sucrose gradient. 5 IQ 15 . thylakoidal proteins detached together with the phycobilisomes, FRACTION NUMBER or merely contaminating soluble proteins? The results of a series FIG. 3. Distributions through a sucrose gradient of the individual of experiments undertaken in an attempt to answer this question colorless polypeptides associated with the phycobilisomes of strain are summarized below. 7409. Data from the experiment described in Fig. 2. The polypeptide composition of phycobilisomes isolated by elution from a sucrose gradient was unchanged both after of phycobilisomes prepared from strain 7409 after growth in precipitation with ammonium sulfate at 30% saturation and white, green, and red light are presented in Fig. 5 and Table after passage through a Bio-Gel A-15 molecular sieve. Such 2. In all three preparations, the group II polypeptides collec- experiments were performed with phycobilisomes isolated from tively accounted for the same fraction (approximately 10%) of five different species of cyanobacteria. total phycobilisomal protein. In red-light phycobilisomes, which The isolated phycobilisomes of strain 7409 contained six contained no phycoerythrin, polypeptide 5 was barely de- colorless polypeptides (Fig. 1). The relative amounts of these tectable, polypeptides 3 and 4 accounting for over 95% of the colorless polypeptides and of total chromopolypeptides were group II components. In green-light phycobilisomes, of which determined from gel scans on successive fractions eluted from phycoerythrin was the major phycobiliprotein (51% of the a sucrose gradient after centrifugation of an extract of strain total), polypeptide 5 accounted for 80% of the group II com- 7409 (Fig. 2). The distributions through the gradient of the ponents. White-light phycobilisomes had an intermediate individual colorless polypeptides are shown in Fig. 3. In the composition with respect both to phycobiliproteins and to group region of the gradient that contains the phycobilisomes (frac- II polypeptides. tions 7-11: 0.55-0.85 M sucrose) there was excellent correlation The physical integrity of the phycobiisome is maintained between the distributions of chromopolypeptides and colorless only in buffers of high ionic strength. Consequently, if cells are phycobilisomal polypeptides. broken in buffers of low ionic strength, the constituent proteins Light quality affects differentially the rates of phycoerythrin of the phycobilisome pass into solution and become mixed with and phycocyanin synthesis in strains 7409 and 7601 (9, 14). other soluble cytoplasmic proteins in the resulting extract. The Phycobilisomes prepared from these strains after growth in bulk soluble proteins of such an extract can be subsequently white, red, and green light differed markedly in their phyco- separated by differential centrifugation from the membrane biliprotein composition. "Green-light" phycobilisomes had a fraction, which contains the thylakoid-associated proteins. For high phycoerythrin:phycocyanin ratio, "white-light" phyco- two cyanobacteria, we compared the polypeptide composition bilisomes had a somewhat lower phycoerythrin:phycocyanin of isolated phycobilisomes with the polypeptide compositions ratio, and "red-light" phycobilisomes were virtually devoid of of low-ionic-strength extracts and of the soluble and membrane phycoerythrin. These light-induced modifications of the major fractions prepared from them (Fig. 6). In both cyanobacteria, phycobilisomal light-harvesting proteins were accompanied the group II polypeptides, as well as the bulk of the chromo- by marked changes in the relative concentrations of the colorless polypeptides, occurred in the soluble fraction of the low- group II polypeptides (Fig. 4). ionic-strength extract. The group III polypeptide, present in The results of a semiquantitative analysis of the composition the phycobilisomes of only one of these strains (7601), was Downloaded by guest on October 7, 2021 1638 Cell Biology: de Marsac and Cohen-Bazire Proc. Natl. Acad. Sci. USA 74 (1977)

Mr 120,000 70,000 -

11 11 30,000 -I- 25,000

IV I III

____p 15,000 -- IV

II R G R G Il4-1~ ~ ~ ~ mIIVtv 120 70 30 25 15 7 4 0 9 7 6 0 1 Molecular weights (x 10 3) of marker proteins FIG. 4. NaDodSO4/polyacrylamide gel electropherograms of FIG. 5. Scans of Coomassie blue-stained NaDodSO4/polyacryl- isolated phycobilisomes prepared from two chromatically adapting amide gel electropherograms of phycobilisomes prepared from cells cyanobacteria, strains 7409 and 7601, after growth in red (R) and green ofstrain 7409 after growth under white light, green light, and red light. (G) light. Note the analogous light-induced changes in the relative Peaks 1-6 represent the colorless polypeptides of groups I (1 and 2), amounts of the group II polypeptides. Mr, molecular weight. II (3 to 5), and III (6). The broad band containing two peaks (7 and 8) of unequal height, extending over the molecular weight range with the soluble fraction. On the other 16,000-22,000, reflects the overlapping absorbances of the chromo- likewise associated hand, polypeptides derived from the phycobiliproteins. The predominance the group I of both strains were and polypeptides largely, of phycoerythrin in "green-light" phycobilisomes is reflected by an perhaps exclusively, located in the membrane fraction of the increase in absorbance at the higher end of the molecular weight low-ionic-strength extract. range, since the mean subunit molecular weight of it is greater than that of phycocyanin and allophycocyanin. DISCUSSION In view of earlier reports (6, 8) that nearly all the protein content analysis of the polypeptide compositions of successive fractions of isolated phycobilisomes is accounted for by phycobiliproteins, eluted from a sucrose gradient after centrifugation of an extract our observation that colorless polypeptides account for about of strain 7409. This analysis revealed a good correlation between 15% of the total protein of the cyanobacterial phycobilisome the distribution through the gradient of the chromopolypeptides was wholly unexpected. In the seven cyanobacteria studied, this derived from the phycobiliproteins and of each of the six col- material consisted of a small number of polypeptides: four to orless polypeptides associated with isolated phycobilisome. nine components are resolvable by NaDodSO4/polyacrylamide There is no obvious explanation for the difference between our gel electrophoresis. It is improbable that these polypeptides are findings concerning the molecular composition of the phyco- derived from contaminating soluble cytoplasmic proteins in bilisome and those of earlier workers (6, 8). Nevertheless, the the preparations examined. The strongest evidence for their discrepancy should be easily resolved. As our data show, col- specific association with the phycobilisome was obtained by an orless proteins represent approximately 15% of total.phyco-

Table 2. Chemical composition of phycobilisomes isolated from strain 7409 after growth with three light sources of different spectral character % of total phycobilisomal protein* All % of total group II % of total colorless Polypeptide groups: polypeptides* phycobiliproteinst poly- 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 10 Green light 14 2.0 10.0 2.0 7 13 80 20 29 51 Red 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. Downloaded by guest on October 7, 2021 Cell Biology: de Marsac and Cohen-Bazire Proc. Nati. Acad. Sci. USA 74 (1977) 1639 amounts of the two major phycobiliproteins are correlated with changes in the relative amounts of individual group II poly- peptides. Photoregulation thus governs the synthesis both of

-. chromoproteins and of certain colorless protein components of the phycobilisome. W- -4 We thank Dr. R. Haselkorn for helpful discussions and Dr. R. Y. 11 Stanier for advice and encouragement. The skillful assistance of Miss A. M. Castets is gratefully acknowledged. This work was supported by 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). III 1. Gray, B. H., Lipschultz, C. A. & Gantt, E. (1973)"Phycobilisomes from a blue-green alga Nostoc sp.," J. Bacteriol. 116, 471- 478. 2. Glazer, A. N. & Bryant, D. A. (1975) "Allophycocyanin B (Amax 671, 618 nm): A new cyanobacterial phycobiliprotein," Arch. IV Microbiol. 104, 15-22. 3. Bryant, D. A., Glazer, A. N. & Eiserling, F. A. (1976) "Charac- terization and structural properties of the major biliproteins of Anabaena 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- A B C D A B bilisomes from phycoerythrin to allophycocyanin," Biochim. 63 1 2 Biophys. Acta 292,858-861. 7601 6. Gray, B. H. & Gantt, E. (1975) "Spectral properties of phyco- FIG. 6. NaDodSO4/polyacrylamide gel electropherograms of bilisomes and phycobiliproteins from the blue-green alga Nostoc crude cell-free extracts (A), washed membrane fractions (B), soluble sp.," Photochem. Photobiol. 21, 121-128. protein fractions (C), and isolated phycobilisomes (D) prepared from 7. Gantt, E., Lipschultz, C. A. & Zilinkas, B. (1976) "Further evi- strain 6312 grown under white light and from strain 7601 grown under dence for a phycobilisome model from selective dissociation, red light. See text for explanations. fluorescence emission, immunoprecipitation and electron mi- croscopy," Biochim. Biophys. Acta 430,375-388. bilisomal protein. These components should be readily de- 8. Gantt, E. & Lipschultz, C. A. (1974) "Phycobilisomes of Por- tectable the analytical procedures described here in all phyridium cruentum: pigment analysis," Biochemistry 13, by 2960-2966. preparations of phycobilisomes, irrespective of their biological 9. Tandeau de Marsac, N. (1977) "The occurrence and nature of source and mode of isolation. chromatic adaptation in cyanobacteria," J. Bacteriol., in In view of the function of the phycobilisome as a light-har- press. vesting organelle, two possible roles for its colorless protein 10. Stanier, R. Y., Kunisawa, R., Mandel, M. & Cohen-Bazire, G. constituents can be envisaged: attachment of the organelle to (1971) "Purification and properties of unicellular blue-green a specific site on the thylakoid membrane and positioning of algae (order Chroococales)," Bacteriol. Rev. 35, 171-205. the constituent light-harvesting pigments within the phycobi- 11. Laemmli, U. K. (1970) "Cleavage of structural proteins during lisome. Because the group I polypeptides appear also to be the assembly of the head of bacteriophage T4," Nature 227, present in the washed membrane fraction prepared from cells 680-685. 12. F. W. of T7 RNAs after extraction with a buffer of low ionic strength, they may Studier, (1973) "Analysis bacteriophage early and proteins on slab gels," J. Mol. Biol. 79, 237-248. serve to attach the phycobilisome to the thylakoid. The group 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. II polypeptides, on the other hand, are probably involved in the (1951) "Protein measurement with the Folin phenol reagent," assembly and positioning of the phycobiliproteins. Like the J. Biol. Chem. 193, 265-275. phycobiliproteins, they are located exclusively in the soluble 14. Bennett, A. & Bogorad, L. (1973) "Complementary chromatic fraction of extracts prepared at low ionic strength. Furthermore, adaptation in a filamentous blue-green alga," J. Cell Biol. 58, in chromatically adapting strains, changes in the relative 419-435. Downloaded by guest on October 7, 2021