SummaryBiological self-assembly is remarkable in its fidelity and in the efficient production of intricate molecular machines and functional materials from a heterogeneous mixture of macromolecules. The phycobilisome, a light-harvesting structure of cyanobacteria, presents the opportunity to study an in vivo assembly process in detail. The phycobilisome molecular architecture is defined, and crystal structures are available for all major proteins, as are a large sequence database (including a genome sequence) and effective genetic systems exist for some cyanobacteria. Recent studies on subunit interaction, covalent modification, and protein stability suggest a model for the earliest events in the phycobilisome assembly pathway. Partitioning of phycobilisome proteins between degradation and assembly is proposed to be controlled by the interaction equilibria between phycobilisome assembly partners, processing enzymes and chaperones. The model provides plausible explanations for existing observations and makes predictions that are amenable to direct experimental investigation. Function and structureIt is a common feature of photosynthetic systems that reaction centres are associated with antenna proteins that absorb a wider range of light energies and transmit it to the primary chlorophyll. Organisms that are capable of oxygenic photosynthesis have evolved two distinct architectures for their antennae. Plants and green algae use membrane proteins, as exemplified in the association of PSII reaction centres with a bed of chlorophyll a/b lightharvesting complexes. The chlorophyll a/b proteins expand the spectrum of light energy available for photosynthesis by coupling chlorophyll b chromophores (maximal absorbance near 665 nm) with chlorophyll a (maximal absorbance near 680 nm) (Barber, 1987).The cyanobacteria and chloroplasts of red algae have evolved membrane-peripheral antenna complexes that predominantly serve PSII reaction centres. These are large structures that can consist of 350-600 polypeptides, the majority bearing covalently attached bilin chromophores with the spectral properties required for light-harvesting. Bilins are linear tetrapyrroles that, as the molecules of primary function in energy transfer, have engendered terms for the proteins that bear them (the phycobiliproteins or biliproteins) and the large complexes in which they are found (the phycobilisomes). Thin sections of cyanobacteria show the presence of phycobilisome particles on the photosynthetic membrane surface ( Figs 1A and B). Like chlorophyll b in plants, biliproteins expand the range of light energy that can service photosynthetic electron transfer but do so to a greater extent because of the spectral features of the three major biliprotein classes: phycoerythrin (PE, max ¼ 565 nm), phycocyanin (PC, max ¼ 617 nm) and allophycocyanin (AP, max ¼ 650 nm). In function, the phycobilisome absorbs light energy in the 500-650 nm range, transduces it to Ϸ680 nm and transfers it to chlorophyll for photosynthesis at efficiencies that approach 100%...
Strain 4R is a phycocyanin-minus mutant of the unicellular cyanobacterium Synechocystis sp. strain 6803. Although it lacks the light-harvesting protein phycocyanin, 4R has normal levels of phycocyanin (cpc) transcripts. Sequence analysis of the cpcB gene encoding the phycocyanin  subunit shows an insertion mutation in 4R that causes early termination of translation. Other work has shown that the phycocyanin ␣ subunit and the linker proteins encoded on the cpc transcripts are all functional in 4R, yet the defective phycocyanin  subunit results in the complete absence of the ␣ subunit and the linkers. Phycocyanin-minus mutants were constructed in a wild-type background by interruption of cpcB and cpcA with an antibiotic resistance gene and were compared with the 4R strain. Immunoblot analysis of the mutants demonstrated that interruption of one subunit was accompanied by a complete absence of the unassembled partner subunit. Phycocyanin assembly begins with the formation of the ␣ heterodimer (the monomer) and continues through higher-order trimeric and hexameric aggregates that associate with linker proteins to form the phycobilisome rods. The results in this paper indicate that monomer formation is a critical stage in the biliprotein assembly pathway and that unassembled subunits are subject to stringent controls that prevent their appearance in vivo.Light harvesting in cyanobacteria is mediated by the phycobilisomes, which are complex protein structures located on the surface of the photosynthetic membrane (20). The major structural components of the phycobilisome are the biliproteins, which contain covalently attached bilin chromophores that constitute a resonance energy transfer pathway. Light energy in the 500-to 650-nm range can be absorbed by different classes of biliproteins and is rapidly and efficiently transferred through the phycobilisome to chlorophyll complexes in the photosynthetic membrane. The three major classes of biliproteins are distinguished by their spectral properties. The allophycocyanins (AP [ max ϭ 650 to 665 nm]) are located in the core of the phycobilisome, which is in direct contact with chlorophyll complexes in the membrane. Phycocyanin (PC [ max ϭ 617 nm]) is found in the rod substructures that are attached to the phycobilisome cores. A third major biliprotein, phycoerythrin (PE [ max ϭ 565 nm]), is synthesized in some cyanobacteria and is attached to PC at the periphery of the rod substructures. Each biliprotein has the same subunit organization that is based on a heterodimer (called a monomer by convention) composed of ␣ and  subunits (11,16,(37)(38)(39). Monomers are assembled into disc-like trimers with a central channel, which then stack to form a hexamer in the PC and PE biliproteins. Hexamers are associated with single copies of linker proteins that direct their assembly into the rod substructures. The organization of biliproteins within the phycobilisome structure establishes an energy transfer pathway, PE to PC to AP to chlorophyll, that operates at close to 100% effici...
Phycobilisomes, comprised of both chromophoric (phycobiliproteins) and non-chromophoric (linker polypeptides) proteins, are light-harvesting complexes present in the prokaryotic cyanobacteria and the eukaryotic red algae. Many cyanobacteria exhibit complementary chromatic adaptation, a process which enables these organisms to optimize absorption of prevalent wavelengths of light by altering the composition of the phycobilisome. To examine the mechanisms involved in adjusting the levels of phycobilisome components during complementary chromatic adaptation, we have isolated and sequenced genes encoding phycobiliprotein and linker polypeptides in the cyanobacterium Fremyella diplosiphon, analyzed their transcriptional characteristics (transcript sizes and abundance when F. diplosiphon is grown in different light qualities) and mapped transcript initiation and termination sites. Our results demonstrate that genes encoding phycobilisome components are often cotranscribed as polycistronic messenger RNAs. Light quality regulates the composition of the phycobilisome by causing changes in the abundance of transcripts encoding specific components, suggesting that regulation is at the level of transcription (although not eliminating the possibility of changes in mRNA stability). The work presented here sets the foundation for analyzing the evolution of the different phycobilisome components and exploring signal transduction from photoperception to activation of specific genes using in vivo and in vitro genetic technology.
Light harvesting in cyanobacteria is performed by the biliproteins, which are organized into membraneassociated complexes called phycobilisomes. Most phycobilisomes have a core substructure that is composed of the allophycocyanin biliproteins and is energetically linked to chlorophyll in the photosynthetic membrane. Rod substructures are attached to the phycobilisome cores and contain phycocyanin and sometimes phycoerythrin. The different biliproteins have discrete absorbance and fluorescence maxima that overlap in an energy transfer pathway that terminates with chlorophyll. A phycocyanin-minus mutant in the cyanobacterium Synechocystis sp. strain 6803 (strain 4R) has been shown to have a nonsense mutation in the cpcB gene encoding the phycocyanin  subunit. We have expressed a foreign phycocyanin operon from Synechocystis sp. strain 6701 in the 4R strain and complemented the phycocyanin-minus phenotype. Complementation occurs because the foreign phycocyanin ␣ and  subunits assemble with endogenous phycobilisome components. The phycocyanin ␣ subunit that is normally absent in the 4R strain can be rescued by heterologous assembly as well. Expression of the Synechocystis sp. strain 6701 cpcBA operon in the wild-type Synechocystis sp. strain 6803 was also examined and showed that the foreign phycocyanin can compete with the endogenous protein for assembly into phycobilisomes.Phycobilisomes from the unicellular cyanobacterium Synechocystis sp. strain 6803 consist of the AP and PC biliproteins. We have recently characterized a PC-minus mutant, Synechocystis sp. strain 6803 (strain 4R) (15). This strain synthesizes phycobilisome cores without rods because of a single-base insertion in the cpcB gene, producing a truncated PC  subunit that leads to a complete absence of both PC subunits. The PC-minus phenotype of 4R suggested that this transformable cyanobacterium (20) would be a suitable genetic host for the introduction of heterologous biliprotein genes in studies focused on early events of biliprotein biosynthesis. We are particularly interested in analyzing structural differences between PC and PE subunits that direct recognition-dependent processes such as subunit assembly and chromophore attachment. These questions can be addressed by site-directed mutagenesis and protein domain exchange in a heterologous transformation system that employs the PC-minus strain of Synechocystis sp. strain 6803 as a host for expression of the cpc and cpe genes from Synechocystis sp. strain 6701 (1, 2, 10, 12). This report is an analysis of Synechocystis sp. strain 6803 transformants that express the cpcBA operon from Synechocystis sp. strain 6701. The results validate the heterologous transformation system as a research tool and demonstrate the rescue of the endogenous PC ␣ subunit in 4R by heterologous assembly. MATERIALS AND METHODSBacterial strains and culture media. Synechocystis sp. strain 6803 (wild-type [WT] and 4R strains) were grown as previously described (15). Liquid and solid media were supplemented with glucose (20 mM)...
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