[32] Phycobilisomes

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[32] P h y c o b i l i s o m e s

By ALEXANDER N. GLAZER Introduction Phycobiliproteins are quantitatively the major proteins in cyanobacterial cells. They may account for up to 24% of the dry weight of the cells and well over half of the total soluble protein. 1-3 Their polypeptide chains carry covalently attached tetrapyrrole prosthetic groups (bilins) which endow the native proteins with intense colors and brilliant fluorescence. The properties of the individual phycobiliproteins are the subject of another chapter in this volume. 4 In intact cyanobacterial cells (and red algal chloroplasts) the phycobiliproteins are assembled into particles named phycobilisomes 5 which are attached in regular arrays to the external surface of the thylakoid membranes. Phycobilisome morphology varies with the organism of origin. In all cases, however, they consist of rods made of stacked disks that radiate from a central core. These particles have molecular weights of 7 × 10 6 to 15 × 10 6, contain between 300 and 800 bilin chromophores, and absorb light over much of the visible spectrum. Energy absorbed by any of these chromophores is efficiently transferred to terminal energy acceptors in the particle via energetically favorable radiationless pathways. It is believed that the excitation energy from the terminal acceptors is funneled into the reaction centers of photosystem 11.6 The preparation and characterization of phycobilisome-photosystem II complexes is described elsewhere in this volume. 7 Numerous recent reviews should be consulted for detailed information on phycobilisome morphology, 8,9 polypeptide composition and assembly, 1°-15 and energy-transfer dynamics. 13,16-19 I j. Myers and W. A. Kratz, J. Gen. Physiol. 39, 11 (1955). 2 A. Bennett and L. Bogorad, J. Cell Biol. 58, 419 (1973). 3 N. Tandeau de Marsac, Doctoral thesis. Universit~ Pierre et Marie Curie, Paris, 1978. 4 A. N. Glazer, this volume [31]. 5 E. Gantt and S. F. Conti, Brookhaoen Syrup. Biol. 19, 393 (1966). 6 A. Manodori and A. Melis, FEBS Lett. 181, 79 (1985). 7 E. Gantt, J. D. Clement-Metral, and B. M. Chereskin, this volume [30]. 8 E. Gantt, Int. Reo. Cytol. 66, 45 (1980). 9 G. Cohen-Bazire and D. A. Bryant, in "The Biology of Cyanobacteria" (N. G. Carr and B. A. Whitton, eds.), p. 143. Univ. of California Press, Berkeley, 1982. i0 A. N. Glazer, Annu. Rev. Microbiol. 36, 173 (1982).

METHODS IN ENZYMOLOGY,VOL. 167

Copyright © 1988 by Academic Press, Inc.

All fightsof reproductionin any formreserved.

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Preparation of Phycobilisomes All the procedures currently in use for the preparation of phycobilisomes are variants on the procedure originally developed by Gantt and Lipschultz 2° for the isolation of these particles from the unicellular red alga Porphyridium cruentum. The original and the majority of the derivative procedures retain certain major features in common: a cell breakage buffer containing sodium and/or potassium phosphate buffer at a concentration ranging from 0.6 to 1.0 M, a pH of 7-8, and a temperature of 1823 ° . Several representative procedures are described below.

Phycobilisomes from Various Cyanobacteria and Red Algae zl The entire procedure is carried out at room temperature (20-23 °) in 0.75 M potassium phosphate (pH 6.8-7.0). One to two grams (wet weight) of cells is suspended in 10 ml of buffer and then broken by passage through a French pressure cell at 10,000 psi. Triton X-100 is immediately added to the broken cells to a final concentration of 2% (or more) and the mixture incubated for 20 min with stirring. Centrifugation for 30 min at 25,000 g is employed to remove cell debris. The supernatant is removed with a syringe from underneath a floating chlorophyll-Triton layer. Aliquots of the supernatant are then layered (2-4 ml per tube) on a step gradient containing the following sucrose molarities in the 0.75 M phosphate buffer: 2.0 M (6 ml), 1.0 M (4 ml), 0.5 M (4 ml), and 0.25 M (8 ml). The gradients are centrifuged for 3 hr in an angle-head rotor (Beckman 42.1) at 42,000 rpm (136,000 g). Phycobilisomes are recovered from the 1.0 M sucrose layer by suction through a fiat-tipped syringe needle. The 1~ W. Wehrmeyer, in "Photosynthetic Prokaryotes: Cell Differentiation and Function" (G. C. Papageorgiou and L. Packer, eds.), p. 1. Elsevier, New York, 1983. ~2 A. N. Glazer, D. J. Lundell, G. Yamanaka, and R. C. Williams, Ann. Microbiol. (Inst. Pasteur) 13411, 159 (1983). ~3 A. N. Glazer, Annu. Rev. Biophys. Biophys. Chem. 14, 47 (1985). 14 B. A. Zilinskas and L. S. Greenwald, Photosynth. Res. 10, 7 (1986). ~5 A. N. Glazer, in " T h e Cyanobacteria" (P. Fay and C. Van Baalen, eds.), p. 69. Elsevier, Amsterdam, 1987. 16 A. N. Glazer, S. W. Yeh, S. P. Webb, and J. H. Clark, Science, 227, 419 (1985). 17 A. N. Glazer, C. Chan, R. C. Williams, S. W. Yeh, and J. H. Clark, Science 230, 1051 (1985). 18 A. R. Holzwarth, Photochem. Photobiol. 43, 707 (1986). ~9 I. Yamazaki, M. Mimuro, T. Murao, T. Yamazaki, K. Yoshihara, and Y. Fujita, Photochem. Photobiol. 39, 233 (1984). 20 E. Gantt and C. A. Lipschultz, J. Cell Biol. 54, 313 (1972). Zl E. Gantt, C. A. Lipschultz, J. Grabowski, and B. K. Zimmerman, Plant Physiol. 63, 615 (1979).

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phycobilisomes are either stored in the suspending medium or are diluted 3- to 4-fold with 0.75 M phosphate buffer and concentrated by centrifugation for 2.5 hr at 254,000 g. For many species, this procedure led to the recovery of 85-90% of the total phycobiliprotein in the 1.0 M sucrose layer. Cell breakage of some cyanobacteria, e.g., Synechococcus PCC 6301 (Anacystis nidulans), Synechococcus PCC 7002 (AgmeneUum quadruplicatum), was increased by pretreatment with lysozyme.

Preparation of Synechococcus PCC 6301 Phycobilisomes zz The variant procedure described here led to a greater preservation of the integrity of Synechococcus PCC 6301 phycobilisomes than was permitted by the first procedure. 2°,21 All buffers contain 1 m M 2-mercaptoethanol and 1 m M sodium azide. All operations are carried out at room temperature unless otherwise specified. Cells are harvested by centrifugation, washed twice with 0.65 M NaH2PO4/K2HPO4 (NaKPO4) buffer at pH 8.0, and resuspended in the same buffer at a concentration of 0.12 g wet weight/ml. The cells are broken by passage through a French pressure cell at 20,000 psi, then incubated for 30 min in the presence of 1% (v/v) Triton X-100. Cell debris is subsequently removed by centrifugation at 31,000 g for 30 rain at 18°. Centrifugation leads to the formation of a green membrane-detergent layer at the top of an intensely blue layer. The blue supernatant is removed carefully, leaving behind the membranedetergent layer and a trace amount of the blue layer on top of the pellet. Aliquots (1.1 ml) of the blue supernatant are layered on sucrose step gradients consisting of 1.0, 3.0, 3.0, 2.3, and 2.2 ml of 2.0, 1.0, 0.75, 0.5, and 0.25 M solutions of sucrose, respectively, all in 0.75 M NaKPO4, pH • 8.0, then centrifuged in a Spinco SW41 rotor at 24,000 rpm (98,000 g) for 13 hr at 18°. The phycobilisomes are recovered from the 0.75 M sucrose layer as a deep blue band, free of detectable chlorophyll, and are stable for at least 2 weeks when stored at 4° as a concentrated solution obtained directly from sucrose gradients. When it was desired to carry out small-scale phycobilisome preparations, the following pretreatment of cells was found to be convenient. 23 Cells are harvested by centrifugation, washed once with 30 mM sodium phosphate, pH 6.8, and resuspended at 0.1 g wet weight/ml in a spheroplasting medium containing 350 m M mannitol, 30 m M sodium phosphate, pH 6.8, 10 m M disodium EDTA, and 5 mg/ml egg white lysozyme. After 22 G. Yamanaka, A. N. Glazer, and R. C. Williams, J. Biol. Chem. 253, 8303 (1978). 23 G. Yamanaka and A. N. Glazer, Arch. Microbiol. 130, 23 (1981).

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3.5 hr of incubation at 37 °, spheroplasts are washed twice in 500 mM mannitol, 30 m M sodium phosphate, pH 6.8, and resuspended in a lysis buffer containing 0.65 M NaKPO4, pH 8.0, and 1 m M 2-mercaptoethanol. Samples are sonicated briefly to ensure good breakage, then solubilized by the addition of Triton X-100 to a final concentration of 1% (v/v). The remainder of the procedure is exactly as described above.

Preparation of Phycobilisomes of Pseudoanabaena

s p p . 24

Cells are collected by centrifugation and washed once with 0.75 M potassium phosphate buffer at pH 7.0 containing a mixture of three protease inhibitors, phenylmethylsulfonyl fluoride (5 × 10-4 M ) , p-chloromercuribenzoate (5 x 10-5 M), and EDTA (I mM). They are then resuspended in the same buffer at 1 g wet weight of cells/10 ml. The cells are broken by passage through the French pressure cell at 20,000 psi and Triton X-100 added to 2% (v/v). After 10 min, the suspension is centrifuged at 30,000 g for 10 min at 20°. One milliliter of the supernatant is layered on each discontinuous sucrose density gradient (0.5 M, 2 ml; 0.75 M, 3 ml; 1 M, 2 ml; 1.5 M, 2 ml), in 0.75 M potassium phosphate buffer at pH 7. Centrifugation is performed in a fixed-angle Beckman 50Ti rotor at 200,000 g (45,000 rpm) for 3 hr at 20°. The phycobilisomes recovered from the 0.75 M sucrose layer are dialyzed at ambient temperature against 0.75 M potassium phosphate buffer, pH 7, containing 5 × 10-4 M phenylmethylsulfonyl fluoride and 1 m M EDTA. For long-term storage, the phycobilisomes are precipitated by the addition of solid (NH4)2SO4 (14 g/ 100 ml). The precipitates are pelleted by centrifugation in Eppendorf tubes and the pellets stored at - 2 0 ° .

Preparation of Fremyella diplosiphon (Calothrix PCC 7601) Phycobilisomes z5,26 This procedure was developed for the rapid isolation of F. diplosiphon phycobilisomes, and its general applicability is not known. All steps are performed at room temperature. Cells are collected by continuous centrifugation and suspended at 1 g wet weight/15 ml in an extraction medium containing 0.75 M potassium phosphate buffer, pH 6.8, 1% (w/v) Triton X-100. After stirring for 1 hr the extract is centrifuged for 20 min at 27,000 24 G. Guglielmi and G. Cohen-Bazire, Protistologica 20, 393 (1984). 25 M. Rigbi, J. Rosinski, H. W. Siegelman, and J. C. Sutherland, Proc. Natl. Acad. Sci. U.S.A. 77, 1961 (1980). 26 j. Rosinski, J. F. Hainfeld, M. Rigbi, and H. W. Siegelman, Ann. Bot. 47, 1 (1981).

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g, and the sedimented material is discarded. Polyethylene glycol 6000 is added to the supernatant solution to 15% (w/v), the mixture is stirred for 1 hr and then centrifuged for 20 min at 27,000 g, and the supernatant is discarded. The sedimented material is suspended in 0.75 M potassium phosphate buffer, pH 6.8, 1% Triton X-100, 15% (w/v) polyethylene glycol 6000, the mixture centrifuged for 20 rain at 27,000 g, and the supernatant discarded. The purple sediment is suspended in 0.75 M potassium phosphate buffer, pH 6.8, and centrifuged for 15 min at 27,000 g; the supernatant is saved. The purple sediment is again extracted and centrifuged, and the supernatants are combined to give a stock solution of phycobilisomes, which is stored in the dark at room temperature. For further purification, z6 the phycobilisomes are suspended in 0.7 M potassium phosphate buffer, pH 6.8, applied to a Sepharose CL-4B column (2.5 x 40 cm) preequilibrated with the pH 6.8 phosphate buffer, and eluted with the same buffer. The peak fractions are used for further studies.

Rapid Procedure for Isolation of Phycobilisomes from Certain Organ&msz7 This procedure can be performed in 3-4 hr and does not require ultracentrifugation. It has been employed to isolate phycobilisomes on a large scale from Synechococcus PCC 6301, as well as two eukaryotic organisms, Porphyridium aerugineum and Cyanidium caldarium. The procedure was not applicable to the isolation of Porphyridium cruentum phycobilisomes; its range of applicability to other organisms is not known. Cells suspended in 1 M NaKPO4 buffer, pH 7.5, are lysed by passage through a French pressure cell (15,000 psi). Triton X-100 (1%, v/v) is added to the broken cell suspension, and the mixture is incubated at room temperature for 30 min. It is then centrifuged in a Sorvall RC5B centrifuge (SS34 rotor) at 20,000 rpm for 30 min. Both the membranous material and the phycobilisomes pellet during this centrifugation. The supernatant is discarded, and the pellet is resuspended in 0.6 M NaKPO4 buffer, pH 7.5. For homogeneous resuspension the pellet is dispersed with a ground-glass homogenizer. The suspension is incubated for 30 min after addition of Triton X-100 to 1% (v/v) and then centrifuged at 20,000 rpm for 30 min. During this centrifugation the membranes are pelleted while a large proportion of intact phycobilisomes remain in solution. The pellet is discarded and the supernatant diluted 10-fold with 1.0 M NaKPO4, pH 7.5, and spun at 20,000 rpm for 1 hr. Intact phycobilisomes pellet under these conditions. 27 A. Grossman and J. Brand, Carnegie Inst. Wash. Yearbook 82, 116 (1983).

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Fro. 1. Electron micrograph of glutaraldehyde-cross-linked phycobilisomes from wildtype Synechocystis PCC 6701 phycobilisomes. The three cylinders that make up the triangulax core, seen in face view, are 110 A in diameter. The stacked disks that make up the six rods are each 60 ,A thick. Uranyl formate negative stain. Magnification: ×290,000.

Characterization of Phycobilisomes

The degree of preservation of phycobilisome structure and function can be assessed by three complementary types of analyses. Transmission electron microscopy of glutaraldehyde-cross-linked negatively stained preparations (Fig. 1) provides a measure of the preservation of the phycobilisome structure at the level of gross morphology and reveals the degree of dissociation and/or aggregation of the particles. 28 Fluorescence emission spectroscopy provides a rapid and sensitive measure of the functional integrity of a phycobilisome preparation. At room temperature, the majority of intact phycobilisomes have a fluorescence emission maximum between 670 and 680 nm independent of excitation wavelength21 (Fig. 2). 28 A. N. Glazer, R. C. Williams, G. Yamanaka, and H. K. Schachman, Proc. Natl. Acad. Sci. U.S.A~ 76, 6162 (1979).

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FIG. 2. Absorption and fluorescence emission spectra of intact and dissociated Synechocystis PCC 6701 phycobilisomes. The solid lines in A and B show the absorption and emission spectra, respectively, of phycobilisomes in 0.75 M sodium potassium phosphate buffer, pH 8.0. The dashed line in B shows the fluorescence emission spectrum of phycobilisomes dialyzed to equilibrium against 10 mM NaKPO4, pH 8.0. The excitation wavelength ~vas 530 nm.

Significant emission with maxima at shorter wavelengths is diagnostic of partial dissociation. The extent of chlorophyll a emission on appropriate excitation can be used to monitor possible membrane contamination of the phycobilisome preparation since'pure phycobilisomes do not contain chlorophyll. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate provides a means of showing that adventitious polypeptides are absent from the phycobilisome preparation. A high-molecularweight polypeptide (75,000-120,000, depending on organismal source) is present in all phycobilisomes thus far examined. This polypeptide is believed to function in the attachment of the phycobilisome to the thylakoid membrane. 29-31Assessment of possible proteolytic degradation is another concern. It has been observed by many investigators that this polypeptide may be subject to partial proteolysis during the phycobilisome purification procedure. The occurrence and extent of such degradation is evident from inspection of the polypeptide pattern obtained from gel electrophoresis. 29 B. A. Zilinskas, Plant Physiol. 70, 1060 (1982). 30 E. Gantt, C. A. Lipschultz, and T. Redlinger, in "Molecular Biology of the Photosynthetic Apparatus" (K. E. Steinback, S. Bonitz, C. J. Arntzen, and L. Bogorad, eds.), p. 223. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1985. 3, E. Gantt, M. Mimuro, and C. A. Lipschultz, in "Hungarian-USA Binational Symposium on Photosynthesis" (M. Gibbs, ed.), p. 1. Salve Regina College, Newport, Rhode Island, 1986.

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PHYCOBILISOMES 9S 91

31 1 POLYPEPTIDE ASSOCIATED WITH THE CORE )ATION PRODUCT DERIVED rilE 99kDo POLYPEPTIDE

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FIG. 3. Separation of the polypeptides of Synechocystis PCC 6701 phycobilisomes by SDS-polyacrylamide gel electrophoresis. For abbreviations, see Fig. 4.

Figures 1-3 show the application of each of these means of characterization to the phycobilisomes of Synechocystis PCC 6701.32

Polypeptide Composition and Topology in Phycobilisomes Phycobilisomes have a complex polypeptide composition 33 (e.g., Fig. 3). Polypeptides which carry bilin chromophores are present as well as polypeptides, called linker polypeptides, 34 whose major function is in phycobilisome assembly rather than in light-energy absorption and transfer. Phycobilisomes from different organisms have distinctive polypeptide compositions. The diagrammatic representation of the Synechocystis 32 R. C. Williams, J. C. Gingrich, and A. N. Glazer, J. Cell Biol. 85, 558 (1980), 33 N. Tandeau de M~rsac and G. Cohen-Bazire, Proc. Natl. Acad. Sci. U.S.A. 74, 1635 (1977). D. J. Lundell, R. C. Williams, and A. N. Glazer, J. Biol. Chem. 256, 3580 (1981).

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FIG; 4. Schematic representation of the Synechocystis 6701 phycobilisome. A rod is made up of four hexameric phycobiliprotein complexes, each of which is attached through its specific linker polypeptide to the component adjacent to it in the phycobilisome. The manner in which the core cylinders are held together is not known. The abbreviations AP, PC, and PE are used for allophycocyanin, phycocyanin, and phycoerythrin, and aAP and /9~ , etc., for the a and/3 subunits of these proteins. Linker polypeptides are abbreviated L, with a superscript denoting the apparent size (× 10-3 D) and a subscript that specifies the location of the polypeptide: R, rod substructure; RC, rod-core junction; C, core; CM, coremembrane junction. For other details, see Ref. 13. The number of bilins present in each domain of the structure is indicated in the upper diagram. Abbreviations: PEB, phycoerythrobilin; PCB, phycocyanobilin. The fluorescence emission maximum, hrmax,is given for each of the eight subcomplexes isolated from this phycobilisome.

PCC 6701 phycobilisome shown in Fig. 4 indicates the location of the various polypeptide chains within the particle. 35 Acknowledgments Work in the author's laboratory was supported by National Science Foundation Grant DMB 8518066. 35 j. C. Gingrich, D. J. Lundell, and A. N. Glazer, J. Cell. Biochem. 22, 1 (1983).