A small multigene family encodes the rod-core linker polypeptides of Anabaena sp. PCC7120 phycobilisomes.

The cpc operon of Anabaena sp. PCC7120 is shown to encode ten genes: 5'-cpcB-cpcA-cpcC-cpcD-cpcE-cpcF- cpcG1-cpcG2-cpcG3-cpcG4-3'. The 3' portion of this operon includes four tandemly repeated genes encoding phycocyanin (PC)-associated, rod-core linker polypeptides of the phycobilisomes (PBS). The products of these four genes are most similar at their N termini, and overall are 50-61% identical and 68-76% similar to one another. The four CpcG proteins of Anabaena sp. PCC7120 are 41-47% identical and 62-65% similar to the single CpcG rod-core linker protein in Synechococcus sp. PCC7002. The N-terminal domains of the polypeptides are also more distantly related to the conserved domains of other types of rod-linker polypeptides associated with PC, phycoerythrin, and allophycocyanin (AP). Three of these rod-core linker proteins (CpcG1, CpcG2, and CpcG4) were demonstrated to occur in isolated PBS by N-terminal amino acid sequence analyses. These results indicate that previously proposed models for the PBS of Anabaena sp. are incorrect. It is suggested that the PBS of Anabaena sp. have eight peripheral rods, each of which interacts with the AP of the core via a specific rod-core linker (CpcG) polypeptide.     

Structure of the genes encoding the rod-core linker polypeptides of Mastigocladus laminosus phycobilisomes and functional aspects of the phycobiliprotein/linker-polypeptide interactions.

The 3' portion of the cpc operon in Mastigocladus laminosus encloses the genes 5'-cpcF-cpcG1-cpcG2-cpcG3 3'. The three cpcG genes encode different phycocyanin-associated rod-core linker polypeptides of the phycobilisomes with predicted 279, 247 and 254 amino acids in length. The gene products CpcG show a high similarity at their N-terminal domains (190 amino acids) and an overall identity of 47-53% to one another. Each of the three CpcG polypeptides is highly related to one of the four CpcG gene products of Anabaena sp. PCC 7120 (66-81% identity). It is suggested that these pairs of rod-core linker polypeptides mediate the same specific type of phycocyanin----allophycocyanin interaction in the similar phycobilisomes of M. laminosus and Anabaena sp. PCC 7120. The similarity of the CpcG1, CpcG2 and CpcG3 polypeptides to the single CpcG rod-core linker polypeptide of Synechococcus sp. PCC 7002 (36-41% identity) is lower. The rod-core linker polypeptides are more distantly related to the rod linker polypeptides associated with phycocyanin or phycoerythrin. However, six conserved domains were identified within the N-terminal 190 amino acids of these linker proteins, which bear similar amino acid sequences, including highly conserved basic amino acids. A similar amino acid sequence but with conserved acidic amino acids can be found in the beta subunits of phycocyanin, phycoerythrin and phycoerythrocyanin, which is protruding into the central cavity of the phycobiliprotein hexamers. It is suggested that these domains are sites of phycobiliprotein-hexamer/rod and rod-core linker interactions.     

Cyanobacterial phycobilisomes

Cyanobacterial phycobilisomes harvest light and cause energy migration usually toward photosystem II reaction centers. Energy transfer from phycobilisomes directly to photosystem I may occur under certain light conditions. The phycobilisomes are highly organized complexes of various biliproteins and linker polypeptides. Phycobilisomes are composed of rods and a core. The biliproteins have their bilins (chromophores) arranged to produce rapid and directional energy migration through the phycobilisomes and to chlorophyll a in the thylakoid membrane. The modulation of the energy levels of the four chemically different bilins by a variety of influences produces more efficient light harvesting and energy migration. Acclimation of cyanobacterial phycobilisomes to growth light by complementary chromatic adaptation is a complex process that changes the ratio of phycocyanin to phycoerythrin in rods of certain phycobilisomes to improve light harvesting in changing habitats. The linkers govern the assembly of the biliproteins into phycobilisomes, and, even if colorless, in certain cases they have been shown to improve the energy migration process. The Lcm polypeptide has several functions, including the linker function of determining the organization of the phycobilisome cores. Details of how linkers perform their tasks are still topics of interest. The transfer of excitation energy from bilin to bilin is considered, particularly for monomers and trimers of C-phycocyanin, phycoerythrocyanin, and allophycocyanin. Phycobilisomes are one of the ways cyanobacteria thrive in varying and sometimes extreme habitats. Various biliprotein properties perhaps not related to photosynthesis are considered: the photoreversibility of phycoviolobilin, biophysical studies, and biliproteins in evolution. Copyright 1998 Academic Press.     

CHARACTERIZATION OF PHYCOCYANIN-DEFICIENT PHYCOBILISOMES FROM A PIGMENT MUTANT OF PORPHYRIDIUM SP. (RHODOPHYTA)

The structure and function of phycobilisomes in the rhodophyte Porphyridium sp. were investigated by comparing the properties of the wild type with a pigment mutant called C12. When grown under low light, cells of C12 were bright orange, while wild-type cells were deep red. The results obtained from a characterization of purified phycobilisomes of the mutant C12 led us to propose the existence in Porphyridium sp. phycobilisomes of two types of rods, some containing only phycoerythrin and others containing phycoerythrin bound to phycocyanin, which is in turn linked to the core by the linker L_RC. By studying the partitioning of phycobiliproteins between phycobilisomes and pools of free phycobiliproteins, we found that phycocyanin in the C12 mutant was only present in the pool of free proteins and that its specific linker, L_RC, was totally absent. Phycoerythrin was present in the free pool and in the purified phycobilisomes as well. One of the three specific phycoerythrin linkers γ was missing. In light of the fact that in the C12 mutant, the linker L_RC is absent and that there is no phycocyanin bound to the phycobilisomes, we propose that the rods in the mutant contain only phycoerythrin. These phycobilisomes are nevertheless functional and exhibit an efficient excitation transfer from phycoerythrin directly to allophycocyanin. Electron microscopy showed the purified phycobilisomes of C12 to be less dense than those of the wild type. This change was attributed to the disappearance of the rods containing the combination phycocyanin/phycoerythrin. Light still regulates phycobiliprotein synthesis in the mutant, as shown by the change in the color of the culture, which turned green-yellow when cells were shifted from low light to high light growth conditions. Light also regulates the structure of the phycobilisomes, which have fewer rods under high light growth conditions.     

Significant energy transfer from CpcG2-phycobilisomes to photosystem I in the cyanobacterium Synechococcus sp. PCC 7002 in the absence of ApcD-dependent state transitions

Hemidiscoidal phycobilisomes (PBS), the major light harvesting complexes of photosynthesis in most cyanobacteria, are composed of rods and cores, which are linked by the linker CpcG1 (L(RC)). Another type of PBS, CpcG2-PBS exits and their function in energy transfer has not been fully understood. We measured growth rates, absorption cross-sections and quantum efficiency of photosystem I in mutant strains of Synechococcus PCC sp. 7002 lacking the linker CpcG2. Our results showed that energy transfer from CpcG2-PBS to PSI in the absence of state transitions could be significant under PBS-absorbing light and energy transfer from two types of PBS is independent to each other. Evidence also suggested that CpcG2 anchors the CpcG2-PBS to thylakoid membranes.     

Diversity and evolution of phycobilisomes in marine <Emphasis Type="Italic">Synechococcus</Emphasis>spp.: a comparative genomics study

Background  

Marine Synechococcus owe their specific vivid color (ranging from blue-green to orange) to their large extrinsic antenna complexes called phycobilisomes, comprising a central allophycocyanin core and rods of variable phycobiliprotein composition. Three major pigment types can be defined depending on the major phycobiliprotein found in the rods (phycocyanin, phycoerythrin I or phycoerythrin II). Among strains containing both phycoerythrins I and II, four subtypes can be distinguished based on the ratio of the two chromophores bound to these phycobiliproteins. Genomes of eleven marine Synechococcus strains recently became available with one to four strains per pigment type or subtype, allowing an unprecedented comparative genomics study of genes involved in phycobilisome metabolism.     

New linker proteins in phycobilisomes isolated from the cyanobacterium Gloeobacter violaceus PCC 7421

Two new linker proteins were identified by peptide mass fingerprinting in phycobilisomes isolated from the cyanobacterium Gloeobacter violaceus PCC 7421. The proteins were products of glr1262 and glr2806. Three tandem phycocyanin linker motifs similar to CpcC were present in each. The glr1262 product most probably functions as a rod linker connecting phycoerythrin and phycocyanin, while the glr2806 product may function as a rod-core linker. We have designated these two proteins CpeG and CpcJ, respectively. The morphology of phycobilisomes in G. violaceus has been reported to be a bundle-like shape with six rods, consistent with the proposed functions of these linkers.     

Growth and Chromatic Adaptation of Nostoc sp. Strain MAC and the Pigment Mutant R-MAC

A spontaneous, stable, pigmentation mutant of Nostoc sp. strain MAC was isolated. Under various growth conditions, this mutant, R-MAC, had similar phycoerythrin contents (relative to allophycocyanin) but significantly lower phycocyanin contents (relative to allophycocyanin) than the parent strain. In saturating white light, the mutant grew more slowly than the parent strain. In nonsaturating red light, MAC grew with a shorter generation time than the mutant; however, R-MAC grew more quickly in nonsaturating green light.

When the parental and mutant strains were grown in green light, the phycoerythrin contents, relative to allophycocyanin, were significantly higher than the phycoerythrin contents of cells grown in red light. For both strains, the relative phycocyanin contents were only slightly higher for cells grown in red light than for cells grown in green light. These changes characterize both MAC and R-MAC as belonging to chromatic adaptation group II: phycoerythrin synthesis alone photocontrolled.

A comparative analysis of the phycobilisomes, isolated from cultures of MAC and R-MAC grown in both red and green light, was performed by polyacrylamide gel electrophoresis in the presence of 8.0 molar urea or sodium dodecyl sulfate. Consistent with the assignment of MAC and R-MAC to chromatic adaptation group II, no evidence for the synthesis of red light-inducible phycocyanin subunits was found in either strain. Phycobilisomes isolated from MAC and R-MAC contained linker polypeptides with relative molecular masses of 95, 34.5, 34, 32, and 29 kilodaltons. When grown in red light, phycobilisomes of the mutant R-MAC appeared to contain a slightly higher amount of the 32-kilodalton linker polypeptide than did the phycobilisomes isolated from the parental strain under the same conditions. The 34.5-kilodalton linker polypeptide was totally absent from phycobilisomes isolated from cells of either MAC or R-MAC grown in green light.

     

Role of the Colorless Polypeptides in Phycobilisome Assembly in Nostoc sp

We have identified the function of the `extra' polypeptides involved in phycobilisome assembly in Nostoc sp. These phycobilisomes, as those of other cyanobacteria, are composed of an allophycocyanin core, phycoerythrin- and phycocyanin-containing rods, and five additional polypeptides of 95, 34.5, 34, 32, and 29 kilodaltons. The 95 kilodalton polypeptide anchors the phycobilisome to the thylakoid membrane (Rusckowski, Zilinskas 1982 Plant Physiol 70: 1055-1059); the 29 kilodalton polypeptide attaches the phycoerythrin- and phycocyanin-containing rods to the allophycocyanin core (Glick, Zilinskas 1982 Plant Physiol 69: 991-997). Two populations of rods can exist simultaneously or separately in phycobilisomes, depending upon illumination conditions. In white light, only one type of rod with phycoerythrin and phycocyanin in a 2:1 molar ratio is synthesized. Associated with this rod are the 29, 32, and 34 kilodalton colorless polypeptides; the 32 kilodalton polypeptide links the two phycoerythrin hexamers, and the 34 kilodalton polypeptide attaches a phycoerythrin hexamer to a phycocyanin hexamer. The second rod, containing predominantly phycocyanin, and the 34.5 and 29 kilodalton polypeptides, is synthesized by redlight-adapted cells; the 34.5 kilodalton polypeptide links two phycocyanin hexamers. These assignments are based on isolation of rods, dissociation of these rods into their component biliproteins, and analysis of colorless polypeptide composition, followed by investigation of complexes formed or not formed upon their recombination.     

Factors influencing the phycobilisome rod composition of the cyanobacterium Synechococcus sp. PCC 7942: Effects of reduced phycocyanin content, lack of rod-linkers, and over-expression of the rod-terminating linker

Four novel mutants with altered phycobilisomes were constructed in the cyanobacterium Synechococcus 7942 to study factors influencing the rod length and composition. These mutants show (1) reduced phycocyanin content, (2) reduced phycocyanin content combined with loss of the 33 kDa linker, (3) loss of the 30 kDa rod-linker and (4) overexpression of the 9 kDa rod terminating linker. For these mutants we determined the 33 to 27 kDa and 30 to 27 kDa linker ratios in the isolated phycobilisomes and compared these ratios with those in the wild type. The 30 kDa linker can be incorporated into the rods in absence of the 33 kDa linker. The incorporation of the 30 kDa linker is lower in absence of the 33 kDa linker. When the 30 kDa linker is missing, an increase in the level of the 33 kDa linker is seen, indicating that there could be an excess of the 33 kDa linker in the cells. Our results also show that a reduction in the phycocyanin content causes a decrease in the rod length simultaneously with a reduction of the 30/27 linker ratio, without altering the 33/27 ratio. Reduced phycocyanin content and absence of the 33 kDa linker cause a dramatic reduction in the incorporation of the 30 kDa linker into the rods in the mutant B2SMIKM. Over-expression of the 9 kDa linker results in a decreased incorporation of both the 33 and 30 kDa linkers into the rods, the effect being more pronounced for the 30 kDa linker. This result indicates that the level of the 9 kDa linker relative to those of the 33 and the 30 kDa linkers may be an important determinant of the phycobilisome rod length.     

Molecular architecture of a light-harvesting antenna. Comparison of wild type and mutant Synechococcus 6301 phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain C-phycocyanin and allophycocyanin in a molar ratio of approximately 3.8:1, a minor biliprotein, allophycocyanin B, and nonpigmented polypeptides of 75, 33, 30, and 27 kilodaltons. A nitrosoguanidine-induced mutant AN112 produces altered phycobilisomes with the molar ratio of C-phycocyanin to allophycocyanin reduced to approximately 1.4:1 and without any of the 33- and 30-kilodalton polypeptides. The mutant and wild type phycobilisomes contain the same molar amount of the 75- and 27-kilodalton polypeptides relative to allophycocyanin. As seen by electron microscopy, the allophycocyanin-containing core of the mutant and of the wild type phycobilisomes appears the same. In some views of the core, each of the two core units in the mutant particles can be seen to consist of four discs approximately 3 nm thick. In wild type phycobilisomes five or six rods, made up of two to six stacked discs (11.5 X 6 nm) are attached to the core. In the mutant, no such rods are seen; rather, single disc-shaped elements, ranging from two to six in number, are found attached. Spectroscopic measurements show that the assembly form of phycocyanin in the mutant phycobilisomes differs from that in the wild type particles but reveal no difference in the organization of the core elements. These results indicate that the portions of the rod substructures of wild type phycobilisomes, beyond the disc proximal to the core, are made up of phycocyanin and the 33- and 30-kilodalton polypeptides. Emission from phycocyanin is a significant component in the fluorescence from isolated Synechococcus 6301 phycobilisomes and indicates an upper limit of 94% for the efficiency of energy transfer from phycocyanin to allophycocyanin and allophycocyanin B in these particles.     

PHOTOREGULATION OF PHYCOBILISOME STRUCTURE DURING COMPLEMENTARY CHROMATIC ADAPTATION IN THE MARINE CYANOPHYTE PHORMIDIUM SP. C861

Changes in the molecular structure of phycobilisomes during complementary chromatic adaptation were studied in the marine cyanophyte Phormidium sp. C86. This strain forms phycoerythrin (PE)-less phycobilisomes under red light but synthesizes PE-rich phycobilisomes under green light. Analysis of phycobiliprotein composition and electron microscopic examination of phycobilisomes in ultra-thin sections of cells and of isolated phycobilisomes were performed for cells acclimated to red and green light, respectively. The structure of phycobilisomes formed under red light conditions was typically hemidiscoidal. Phycobilisomes in cells acclimated to green light were twice as large in size as those in cells acclimated to red light. This increase in phycobilisome size was a result of the increase in the molar ratio of antenna pigment (PE and phycocyanin) to allophycocyanin, from 3.5 to 11.3. Pigment composition and fine structure of phycobilisomes formed under green light were similar to those of “nonhemidiscoidal” phycobilisomes reported in Phormidium persicinum. These results suggest that changes occur not only in the molecular species of peripheral rods but also in the structure of rods and probably of cores in relation to their connection with rods during chromatic adaptation of Phormidium sp. C86.     

High-resolution crystal structures of trimeric and rod phycocyanin

The phycobilisome light-harvesting antenna in cyanobacteria and red algae is assembled from two substructures: a central core composed of allophycocyanin surrounded by rods that always contain phycocyanin (PC). Unpigmented proteins called linkers are also found within the rods and core. We present here two new structures of PC from the thermophilic cyanobacterium Thermosynechococcus vulcanus. We have determined the structure of trimeric PC to 1.35 Å, the highest resolution reported to date for this protein. We also present a structure of PC isolated in its intact and functional rod form at 1.5 Å. Analysis of rod crystals showed that in addition to the α and β PC subunit, there were three linker proteins: the capping rod linker (L_R^8.7), the rod linker (L_R), and only one of three rod-core linkers (L_RC, CpcG4) with a stoichiometry of 12:12:1:1:1. This ratio indicates that the crystals contained rods composed of two hexamers. The crystallographic parameters of the rod crystals are nearly identical with that of the trimeric form, indicating that the linkers do not affect crystal packing and are completely embedded within the rod cavities. Absorption and fluorescence emission spectra were red-shifted, as expected for assembled rods, and this could be shown for the rod in solution as well as in crystal using confocal fluorescence microscopy. The crystal packing imparts superimposition of the three rod linkers, canceling out their electron density. However, analysis of B-factors and the conformations of residues facing the rod channel indicate the presence of linkers. Based on the experimental evidence presented here and a homology-based model of the L_R protein, we suggest that the linkers do not in fact link between rod hexamers but stabilize the hexameric assembly and modify rod energy absorption and transfer capabilities.     

Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6301.

A procedure is described for the preparation of stable phycobilisomes from the unicellular cyanobacterium Synechococcus sp. 6301 (also known as Anacystis nidulans). Excitation of the phycocyanin in these particles at 580 nm leads to maximum fluorescence emission, from allophycocyanin and allophycocyanin B, at 673 nm. Electron microscopy shows that the phycobilisomes are clusters of rods. The rods are made up of stacks of discs which exhibit the dimensions of short stacks made up primarily of phycocyanin (Eiserling, F. A., and Glazer, A. N. (1974) J. Ultrastruct. Res. 47, 16-25). Loss of the clusters, by dissociation into rods under suitable conditions, is associated with loss of energy transfer as shown by a shift in fluorescence emission maximum to 652 nm. Synechococcus sp. 6301 phycobilisomes were shown to contain five nonpigmented polypeptides in addition to the colored subunits (which carry the covalently bound tetrapyrrole prosthetic groups) of the phycobiliproteins. Evidence is presented to demonstrate that these colorless polypeptides are genuine components of the phycobilisome. The nonpigmented polypeptides represent approximately 12% of the protein of the phycobilisomes; phycocyanin, approximately 75%, and allophycocyanin, approximately 12%. Spectroscopic studies that phycocyanin is in the hexamer form, (alpha beta)6, in intact phycobilisomes, and that the circular dichroism and absorbance of this aggregate are little affected by incorporation into the phycobilisome structure.     

Phycobilisome structure in the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC 7120.

Phycobilisomes of the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC7120 differ from typical tricylindrical, hemidiscoidal phycobilisomes in three respects. Firstly, size comparisons of the core-membrane linker phycobiliproteins (LCM) in different cyanobacteria by SDS/PAGE reveal an apparent molecular mass of 120 kDa for the LCM of M. laminosus and Anabaena sp. PCC7120. This observation suggests that the polypeptides of these species have four linker-repeat domains. Secondly, phycobilisomes of M. laminosus are shown to contain at least three, but most probably four, different rod-core linker polypeptides (LRC). These LRC, which attach the peripheral rods to the core and thereby make phycocyanin/allophycocyanin contacts, have been identified and characterized by N-terminal amino acid sequence analysis. Additionally, electron microscopy of phycobilisomes isolated from M. laminosus and Anabaena sp. PCC7120 reveals similar structures which differ from those of Calothrix sp. PCC7601 with their typical six, peripheral rods. Based upon protein-analytical results and a reinterpretation of the data of [Isono, T. & Katoh, T. (1987) Arch. Biochem. Biophys. 256, 317-324], we discuss structural implications of recent findings on the established hemidiscoidal model for the phycobilisomes of M. laminosus and Anabaena sp. PCC7120. Up to eight peripheral rods are suggested to radiate from a modified core substructure which contains two additional peripheral allophycocyanin hexamer equivalents that serve as the core-proximal discs for two peripheral rods.     

Phosphorylation of lymphocyte myosin catalyzed in vitro and in intact cells

Synechocystis 6701 phycobilisomes contain phycoerythrin, phycocyanin, and allophycocyanin in a molar ratio of approximately 2:2:1, and other polypeptides of 99-, 46-, 33.5-, 31.5-, 30.5-, and 27-kdaltons. Wild- type phycobilisomes consist of a core of three cylindrical elements in an equilateral array surrounded by a fanlike array of six rods each made up of 3-4 stacked disks. Twelve nitrosoguanidine-induced mutants were isolated which produced phycobilisomes containing between 0 and 53% of the wild-type level of phycoerythrin and grossly altered levels of the 30.5- and 31.5-kdalton polypeptides. Assembly defects in these mutant particles were shown to be limited to the phycoerythrin portions of the rod substructures of the phycobilisome. Quantitative analysis of phycobilisomes from wild-type and mutant cells, grown either in white light or chromatically adapted to red light, indicated a molar ratio of the 30.5- and 31.5-kdalton polypeptides to phycoerythrin of 1:6, i.e., one 30.5- or one 31.5-kdaltons polypeptide per (alpha beta)6 phycoerythrin hexamer. Presence of the phycoerythrin-31.5-kdalton complex in phycobilisomes did not require the presence of the 30.5- kdalton polypeptide. The converse situation was not observed. These and earlier studies (R. C. Williams, J. C. Gingrich, and A. N. Glazer. 1980. J. Cell Biol. 85:558-566) show that the average rod in wild type Synechocystis 6701 phycobilisomes consists of four stacked disk-shaped complexes: phycocyanin (alpha beta)6-27 kdalton, phycocyanin (alpha beta)6-33.5 kdalton, phycoerythrin (alpha beta)6-31.5 kdalton, and phycoerythrin-30.5 kdalton, listed in order starting with the disk proximal to the core.     

Evidence for a Transient Association of New Proteins with the Spirulina maxima Phycobilisome in Relation to Light Intensity

Environmental parameters are known to affect phycobilisomes. Variations of their structure and relative composition in phycobiliproteins have been observed. We studied the effect of irradiance variations on the phycobilisome structure in the cyanobacterium Spirulina maxima and discovered the appearance of new polypeptides associated with the phycobilisomes under an increased light intensity. In high light, the six rods of phycocyanin associated with the central core of allophycocyanin contained only one to two phycocyanin hexamers instead of the two to three they contained in low light. The concomitant disappearance of a 33-kD linker polypeptide was observed. Moreover, in high light three polypeptides of 29, 30, and 47 kD, clearly unrelated to linkers, were found to be associated with the phycobilisome fraction: protein labeling showed that a specific association of these polypeptides was induced by high light. One polypeptide, at least, would play the role of a chaperone protein. Not only the synthesis of these proteins, which appeared slightly increased in high light, but also their association with phycobilisome structure are light intensity dependent.     

Allophycocyanin I and the 95 Kilodalton Polypeptide : The Bridge between Phycobilisomes and Membranes

Allophycocyanin was isolated from dissociated phycobilisomes from Nostoc sp. and was separated into allophycocyanin I, II, III, and B as described elsewhere. If the separation of the proteins following phycobilisome isolation is done in the presence of the protease inhibitor, phenylmethylsulfonylfluoride, associated with allophycocyanin I are two colored polypeptides of 95 kilodalton (kD) and 80 kD, belonging to the class of Group I polypeptides as defined by Tandeau de Marsac and Cohen-Bazire (Proc Natl Acad Sci USA 1977 74: 1635-1639). Allophycocyanin I has a fluorescence maximum of 680 nanometers as do intact phycobilisomes and has thus been suggested to be the final emitter of excitation energy in phycobilisomes. Thylakoid membranes washed in low ionic strength buffer containing phenylmethylsulfonylfluoride lose all biliproteins, but retain the 95 kD and 80 kD polypeptides. As suggested by Tandeau de Marsac and Cohen-Bazire, these are likely to be the polypeptides involved in binding the phycobilisome to the membrane. As these polypeptides are isolated with allophycocyanin I, structural evidence is provided for placing allophycocyanin I as the bridge between the phycobilisome and the membrane. These Group I polypeptides and the 29 kD polypeptide (involved in rod attachment to the APC core) are particularly susceptible to proteolytic breakdown. It is thought that in vivo the active protease may be selectively attacking these polypeptides to detach the phycobilisome from the membrane and release the phycoerythrin and phycocyanin containing rods from the allophycocyanin core for greater susceptibility of the biliproteins to protease attack.     

Distinct roles of CpcG1 and CpcG2 in phycobilisome assembly in the cyanobacterium Synechocystis sp. PCC 6803

Structural role of the second copy of the rod–core linker CpcG, which was found by genome analysis, was studied in Synechocystis sp. PCC 6803 by gene disruption and fractionation of phycobilisome (sub)complexes. Disruption of cpcG2 (sll1471) resulted in a marked decrease in phycocyanin content both in the background of wild-type and cpcG1 (slr2051)-disruptant. The unique phycocyanin rod–CpcG2 complex without the major allophycocyanin components was isolated from the cpcG1-disruptant. By fluorescence analysis, it was proposed that CpcG2 protein connects the rods with a minor allophycocyanin component, to support energy transfer to Photosystem I.     

Structure, composition, and assembly of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 and of phycobilisomes in the derivative strain BO 9201.

The phycobiliproteins of the unicellular cyanobacterium Synechocystis sp. strain BO 8402 and its derivative strain BO 9201 are compared. The biliproteins of strain BO 8402 are organized in paracrystalline inclusion bodies showing an intense autofluorescence in vivo. These protein-pigment aggregates have been isolated. The highly purified complexes contain phycocyanin with traces of phycoerythrin, corresponding linker polypeptides LR35PC and LR33PE (the latter in a small amount), and a unique colored polypeptide with an M(r) of 55,000, designated L55. Allophycocyanin and the core linker polypeptides are absent. The substructure of the aggregates has been studied by electron microscopy. Repetitive subcomplexes of hexameric stacks of biliproteins form extraordinary long rods associated side by side in a highly condensed arrangement. Evidence that the linker polypeptides LR35PC and LR33PE stabilize the biliprotein hexamers is presented, while the location and function of the colored linker L55 remain uncertain. The derivative strain BO 9201 contains established hemidiscoidal phycobilisomes comprising phycoerythrin, phycocyanin, and allophycocyanin as well as the corresponding linker polypeptides. The core-membrane linker protein (LCM), and two polypeptides with M(r)s of 40,000 and 45,000 which are present in small amounts, exhibit strong cross-reactivity in Western blot (immunoblot) analysis using an antibody directed against the colored LCM of a Nostoc sp. In contrast, strain BO 8402 exhibits no polypeptide with a significant immunological cross-reactivity in Western blot analysis. Physiological and genetic implications of the unusual pigment compositions of both strains are discussed.