Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: an overview

Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.     

Structural organisation of phycobilisomes from Synechocystis sp. strain PCC6803 and their interaction with the membrane

In cyanobacteria, the harvesting of light energy for photosynthesis is mainly carried out by the phycobilisome — a giant, multi-subunit pigment-protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides such that light absorption and energy transfer to photosystem II are optimised. In this work we have studied, using single particle electron microscopy, the phycobilisome structure in mutants lacking either two or all three of the phycocyanin hexamers. The images presented give much greater detail than those previously published, and in the best two-dimensional projection maps a resolution of 13 Å was achieved. As well as giving a better overall picture of the assembly of phycobilisomes, these results reveal new details of the association of allophycocyanin trimers within the core. Insights are gained into the attachment of this core to the membrane surface, essential for efficient energy transfer to photosystem II. Comparison of projection maps of phycobilisomes with and without reconstituted ferredoxin:NADP oxidoreductase suggests a location for this enzyme within the complex at the rod-core interface.     

Changes in polypeptide composition of Synechocystis sp. strain 6308 phycobilisomes induced by nitrogen starvation.

Phycobilisomes isolated from actively growing Synechocystis sp. strain 6308 (ATCC 27150) consist of 12 polypeptides ranging in molecular mass from 11.5 to 95 kilodaltons. The phycobilisome anchor and linker polypeptides are glycosylated. Nitrogen starvation causes the progressive loss of phycocyanin and allophycocyanin subunits with molecular masses between 16 and 20 kilodaltons and of two linker polypeptides with molecular masses of 27 and 33 kilodaltons. Nitrogen starvation also leads to enrichment of four additional polypeptides with molecular masses of 46, 53, 57, and 61 kilodaltons and a transient enrichment of 35- and 41-kilodalton polypeptides in isolated phycobilisomes. The 57-kilodalton additional polypeptide was identified by immunoblotting as the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proteins with the same molecular weights as the additional polypeptides were also coisolated with the 12 phycobilisome polypeptides in the supernatant of nitrogen-replete Synechocystis thylakoid membranes extracted in high-ionic-strength buffer and washed with deionized water. These observations suggest that the additional polypeptides in phycobilisomes from nitrogen-starved cells may be soluble or loosely bound membrane proteins which associate with phycobilisomes. The composition and degree of association of phycobilisomes with soluble and adjacent membrane polypeptides appear to be highly dynamic and specifically regulated by nitrogen availability. Possible mechanisms for variation in the strength of association between phycobilisomes and other polypeptides are suggested.     

Phycobilisome structure and function

Phycobilisomes are aggregates of light-harvesting proteins attached to the stroma side of the thylakoid membranes of the cyanobacteria (blue-green algae) and red algae. The water-soluble phycobiliproteins, of which there are three major groups, tetrapyrrole chromophores covalently bound to apoprotein. Several additional protiens are found within the phycobilisome and serve to link the phycobiliproteins to each other in an ordered fashion and also to attach the phycobilisome to the thylakoid membrane. Excitation energy absorbed by phycoerythrin is transferred through phycocyanin to allophycocyanin with an efficiency approximating 100%. This pathway of excitation energy transfer, directly confirmed by time-resolved spectroscopic measurements, has been incorporated into models describing the ultrastructure of the phycobilisome. The model for the most typical type of phycobilisome describes an allophycocyanin-containing core composed of three cylinders arranged so that their longitudinal axes are parallel and their ends form a triangle. Attached to this core are six rod structures which contain phycocyanin proximal to the core and phycoerythrin distal to the core. The axes of these rods are perpendicular to the longitudinal axis of the core. This arrangement ensures a very efficient transfer of energy. The association of phycoerythrin and phycocyanin within the rods and the attachment of the rods to the core and the core to the thylakoid require the presence of several linker polypeptides. It is recently possible to assemble functionally and structurally intact phycobilisomes in vitro from separated components as well as to reassociate phycobilisomes with stripped thylakoids. Understanding of the biosynthesis and in vivo assembly of phycobilisomes will be greatly aided by the current advances in molecular genetics, as exemplified by recent identification of several genes encoding phycobilisome components.Combined ultrastructural, biochemical and biophysical approaches to the study of cyanobacterial and red algal cells and isolated phycobilisome-thylakoid fractions are leading to a clearer understanding of the phycobilisome-thylakoid structural interactions, energy transfer to the reaction centers and regulation of excitation energy distribution. However, compared to our current knowledge concerning the structural and functional organization of the isolated phycobilisome, this research area is relatively unexplored.     

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.     

Evolutionary Analysis of Phycobiliproteins: Implications for Their Structural and Functional Relationships

Phycobiliproteins, together with linker polypeptides and various chromophores, are basic building blocks of phycobilisomes, a supramolecular complex with a light-harvesting function in cyanobacteria and red algae. Previous studies suggest that the different types of phycobiliproteins and the linker polypeptides originated from the same ancestor. Here we retrieve the phycobilisome-related genes from the well-annotated and even unfinished cyanobacteria genomes and find that many sites with elevated d _N /d _S ratios in different phycobiliprotein lineages are located in the chromophore-binding domain and the helical hairpin domains (X and Y). Covariation analyses also reveal that these sites are significantly correlated, showing strong evidence of the functional-structural importance of interactions among these residues. The potential selective pressure driving the diversification of phycobiliproteins may be related to the phycobiliprotein-chromophore microenvironment formation and the subunits interaction. Sites and genes identified here would provide targets for further research on the structural-functional role of these residues and energy transfer through the chromophores. [Reviewing Editor: Dr. Rasmus Nielsen]     

Role of the PB-loop in ApcE and phycobilisome core function in cyanobacterium Synechocystis sp. PCC 6803

The phycobilisome (PBS) is a giant highly-structured pigment-protein antenna of cyanobacteria and red algae. PBS is composed of the phycobiliproteins and several linker polypeptides. The large core-membrane linker protein (L_CM or ApcE) influences many features and functions of PBS and consists of several domains including the chromophorylated PB-domain. Being homologous to the phycobiliprotein α-subunits this domain includes a so-called PB-loop insertion whose functions are still unknown. We have created the photoautotrophic mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with lacking PB-loop. Using various spectral techniques we have demonstrated that this mutation does not destroy the PBS integrity and the internal PBS excitation energy transfer pathways. At the same time, the deletion of the PB-loop leads to the decrease of connectivity between the PBS and thylakoid membrane and to the compensatory increase of the relative photosystem II content. Mutation provokes the violation of the thylakoid membranes arrangement, the inability to perform state transitions, and diminishing of the OCP-dependent non-photochemical PBS quenching. In essence, even such a minute mutation of the PBS polypeptide component, like the PB-loop deletion, becomes important for the concerted function of the photosynthetic apparatus.     

Spectroscopic properties of the C-phycocyanin-allophycocyanin conjugate and the isolated phycobilisomes from Spirulina platensis

C-phycocyanin (CPC) and allophycocyanin (APC) were purified from Spirulina platensis, then the CPC was attached covalently to the APC by reacting their ∈-amino groups. The excitation energy could be transferred from the CPC to the APC in the CPC-APC conjugate. Intact phycobilisomes (PBS), consisting of CPC, APC, colourless linker polypeptides, and APC B or Lcm, were isolated from S. platensis. Spectroscopic properties of the isolated PBSs kept at 20 °C for various times showed that the connection between the APC and the APC B or Lcm was looser than that between the CPC and the APC in the isolated PBSs. The CPC-APC conjugate was more stable than the isolated PBSs, and the linker polypeptides had a minor influence on the excitation energy transfer characteristic between different phycobiliproteins in the PBS.     

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.     

Spectroscopic properties of the C-phycocyanin-allophycocyanin conjugate and the isolated phycobilisomes from Spirulina platensis

C-phycocyanin (CPC) and allophycocyanin (APC) were purified from Spirulina platensis, then the CPC was attached covalently to the APC by reacting their ∈-amino groups. The excitation energy could be transferred from the CPC to the APC in the CPC-APC conjugate. Intact phycobilisomes (PBS), consisting of CPC, APC, colourless linker polypeptides, and APC B or Lcm, were isolated from S. platensis. Spectroscopic properties of the isolated PBSs kept at 20 °C for various times showed that the connection between the APC and the APC B or Lcm was looser than that between the CPC and the APC in the isolated PBSs. The CPC-APC conjugate was more stable than the isolated PBSs, and the linker polypeptides had a minor influence on the excitation energy transfer characteristic between different phycobiliproteins in the PBS. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

The supramolecular architecture, function, and regulation of thylakoid membranes in red algae: an overview

Red algae are a group of eukaryotic photosynthetic organisms. Phycobilisomes (PBSs), which are composed of various types of phycobiliproteins and linker polypeptides, are the main light-harvesting antennae in red algae, as in cyanobacteria. Two morphological types of PBSs, hemispherical- and hemidiscoidal-shaped, are found in different red algae species. PBSs harvest solar energy and efficiently transfer it to photosystem II (PS II) and finally to photosystem I (PS I). The PS I of red algae uses light-harvesting complex of PS I (LHC I) as a light-harvesting antennae, which is phylogenetically related to the LHC I found in higher plants. PBSs, PS II, and PS I are all distributed throughout the entire thylakoid membrane, a pattern that is different from the one found in higher plants. Photosynthesis processes, especially those of the light reactions, are carried out by the supramolecular complexes located in/on the thylakoid membranes. Here, the supramolecular architecture, function and regulation of thylakoid membranes in red algal are reviewed.     

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.     

Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

In Synechocystis sp. PCC 6803, the loop domain (aa 1–70) of the phycobilisome core-membrane linker, L_CM, was found to interact with the glycosyl transferase homolog, Sll1466. Growth of a Sll1466 knock-out mutant was slightly faster in low light, but strongly inhibited in high light; the phenotype is discussed in relation to the regulation of light energy transfer to photosystem II. At the molecular level, the mutant shows the following changes compared to the wild type: (1) a smaller size and higher mobility of phycobilisomes on the thylakoid membrane, and (2) a changed lipid composition of the thylakoid membrane, especially decreased amounts of digalactosyl diacylglycerol. These results indicate a profound regulatory role for Sll1466 in regulating photosynthetic energy transfer.     

Structure of the Thylakoids and Envelope Membranes of the Cyanelles of Cyanophora paradoxa

The cyanelles of Cyanophora paradoxa Korsch. are photosynthetically active obligate endosymbionts in which phycobiliproteins serve as the major accessory pigments. Freeze-fracture electron micrographs of thylakoids in isolated cyanelles reveal long parallel rows of particles covering most of the E-face, while a more random particle arrangement is evident in some areas. The center-to-center spacing of particles within these rows is about 10 nanometers. Their mean diameter was measured at 9.4 nanometers. The particles on the P-face have a mean diameter of 7.2 nanometers. Thylakoids that retained nearly the full complement of phycobiliproteins (determined spectrophotometrically and by gel electrophoresis) were isolated from the cyanelles. In thin sections of these preparations, rows of disc-shaped phycobilisomes are evident on the surface of the thylakoids. The spacing of the rows of phycobilisomes corresponds to that of the rows of E-face particles (approximately 45 nanometers, center to center). The periodicity of the disc-shaped phycobilisomes within a row is 10 nanometers suggesting a one-to-one association between phycobilisomes and E-face particles.

In addition, visualization of the protoplasmic surface (PS) of isolated thylakoids by freeze-etch electron microscopy shows that rows of disc-shaped phycobilisomes are aligned directly above rows of particles exhibiting two subunits, presumably the P-surface projections of the 10-nanometer intramembrane particles. These observations, together with earlier studies indicating that the 10-nanometer E-face particles probably represent photosystem II (PSII) complexes, suggest that phycobilisomes are positioned on the thylakoid surface in direct contact with PSII centers within the thylakoid membrane.

The inner envelope membrane of the cyanelles, observed in freeze-fracture replicas, resembles cyanobacterial plasma membranes and is dissimilar to the chloroplast envelope membranes of red or green algae. The envelope of isolated cyanelles exhibits two additional layers: (a) a 5- to 7-nanometer-thick layer that lies adjacent to the inner membrane and which seems to correspond to the peptidoglycan layer of cyanobacteria; and (b) a layer external to the purported peptidoglycan layer that exhibits fracture faces similar to those of the lipopolysaccharide layer of gram negative bacteria. Our findings indicate that the supramolecular architecture of cyanelles differs only slightly from free-living cyanobacteria to which they are presumably related.

     

Chromatic adaptation and the events involved in phycobilisome biosynthesis

Abstract. The major light-harvesting complex in cyanobacteria and red algae is the phycobilisome, a macromolecular complex that is attached to the surface of the photosynthetic membranes. The phycobilisome is composed of a number of different chromophoric polypeptides called phycobiliproteins and nonchromophoric polypeptides called linker proteins. Several environmental parameters modulate the synthesis, assembly and degradation of phycobilisome components. In many cyanobacteria, the composition of the phycobilisome can change to accommodate the prevalent wavelengths of light in the environment. This phenomenon is called complementary chromatic adaptation. Organisms that exhibit complementary chromatic adaptation must perceive the wavelengths of light in the environment and transduce the light signals into a sequence of biochemical events that result in altering the activities of genes encoding specific phycobiliprotein and linker polypeptides. Other environmental parameters such as light intensity and nutrient status can also have marked effects on both the number and composition of the phycobilisomes. The major concern of this article is the molecular events involved in chromatic adaptation. Most of the information concerning this process has been gained from studies involving the filamentous cyanobacterium Fremyella diplosiphon . However, also briefly considered are some of the complexities involved in phycobilisome biosynthesis and degradation; they include post-translational modification of phycobilisome polypeptides, the coordinate expression of chromophore and apobiliprotein, the specific degradation of phycobilisomes when cyanobacteria are deprived of macronutrients such as nitrogen, sulphur and phosphorus, and the assembly of the individual phycobilisome components into substructures of the light harvesting complex.     

The membrane-associated CpcG2-phycobilisome in Synechocystis: a new photosystem I antenna

The phycobilisome (PBS) is a supramolecular antenna complex required for photosynthesis in cyanobacteria and bilin-containing red algae. While the basic architecture of PBS is widely conserved, the phycobiliproteins, core structure and linker polypeptides, show significant diversity across different species. By contrast, we recently reported that the unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two types of PBSs that differ in their interconnecting "rod-core linker" proteins (CpcG1 and CpcG2). CpcG1-PBS was found to be equivalent to conventional PBS, whereas CpcG2-PBS retains phycocyanin rods but is devoid of the central core. This study describes the functional analysis of CpcG1-PBS and CpcG2-PBS. Specific energy transfer from PBS to photosystems that was estimated for cells and thylakoid membranes based on low-temperature fluorescence showed that CpcG2-PBS transfers light energy preferentially to photosystem I (PSI) compared to CpcG1-PBS, although they are able to transfer to both photosystems. The preferential energy transfer was also supported by the increased photosystem stoichiometry (PSI/PSII) in the cpcG2 disruptant. The cpcG2 disruptant consistently showed retarded growth under weak PSII light, in which excitation of PSI is limited. Isolation of thylakoid membranes with high salt showed that CpcG2-PBS is tightly associated with the membrane, while CpcG1-PBS is partly released. CpcG2 is characterized by its C-terminal hydrophobic segment, which may anchor CpcG2-PBS to the thylakoid membrane or PSI complex. Further sequence analysis revealed that CpcG2-like proteins containing a C-terminal hydrophobic segment are widely distributed in many cyanobacteria.     

Structure-function relationships and energy transfer in phycobiliprotein antennae

The present understanding of how interactions between chromophore and protein as well as between chromophore and chromophore in different aggregation states influence the spectral and excited state kinetic properties of phycobiliprotein antenna pigments is discussed. Properties of isolated phycobiliproteins from both cyanobacteria and red algae as well as from cryptophytes and of intact phycobilisomes are covered. The experimental results are discussed in terms of general principles for chromophore coupling and energy transfer. Some controversial topics in this field are outlined.     

Localization and quantitation of chloroplast enzymes and light-harvesting components using immunocytochemical methods

Seven chloroplast proteins were localized in Porphyridium cruentum (ATCC 50161) by immunolabeling with colloidal gold on electron microscope sections of log phase cells grown under red, green, and white light. Ribulose bisphosphate carboxylase labeling occurred almost exclusively in the pyrenoid. The major apoproteins of photosystem I (56-64 kD) occurred mostly over the stromal thylakoid region and also appeared over the thylakoids passing through the pyrenoid. Labeling for photosystem II core components (D2 and a 45 kD Chl-binding protein), for phycobilisomes (allophycocyanin, and a 91 kD L_cm linker) and for ATP synthase (β subunit) were predominantly present in the thylakoid region but not in the pyrenoid region of the chloroplast. Red light cells had increased labeling per thylakoid length for polypeptides of photosystem II and of phycobilisomes, while photosystem I density decreased, compared to white light cells. Conversely, green light cells had a decreased density of photosystem II and phycobilisome polypeptides, while photosystem I density changed little compared with white light cells. A comparison of the immunogold labeling results with data from spectroscopic methods and from rocket immunoelectrophoresis indicates that it can provide a quantitative measure of the relative amounts of protein components as well as their localization in specific organellar compartments.