Accumulation of monomer and dimer photosystem (PS) II reaction center core complexes has been analyzed by two-dimensional Blue-native/SDS-PAGE in Synechocystis PCC 6803 wild type and in mutant strains lacking genes psbA, psbB, psbC, psbDIC/DII, or the psbEFLJ operon. In vivo pulse-chase radiolabeling experiments revealed that mutant cells assembled PSII precomplexes only. In ⌬psbC and ⌬psbB, assembly of reaction center cores lacking CP43 and reaction center complexes was detected, respectively. In ⌬psbA, protein subunits CP43, CP47, D2, and cytochrome b 559 were synthesized, but proteins did not assemble. Similarly, in ⌬psbD/C lacking D2, and CP43, the de novo synthesized proteins D1, CP47, and cytochrome b 559 did not form any mutual complexes, indicating that assembly of the reaction center complex is a prerequisite for assembly with core subunits CP47 and CP43. Finally, although CP43 and CP47 accumulated in ⌬psbEFLJ, D2 was neither expressed nor accumulated. We, furthermore, show that the amount of D2 is high in the strain lacking D1, whereas the amount of D1 is low in the strain lacking D2. We conclude that expression of the psbEFLJ operon is a prerequisite for D2 accumulation that is the key regulatory step for D1 accumulation and consecutive assembly of the PSII reaction center complex. The photosystem II (PSII)1 reaction center core (RCC) complex of higher plants, algae, and cyanobacteria can be subdivided into a heterodimer containing D1 and D2, the antenna proteins CP47 and CP43, and a large number of low molecular weight integral membrane proteins including the ␣ and ␤ subunits of cytochrome b 559 (␣ and ␤ cytochrome b 559 ) (1-3). The heterodimer and antenna proteins are essential for binding the prosthetic groups needed for energy and electron transfer (4) as well as for binding the multitude of plastid-encoded small subunits, e.g. Psb-H, -J, -K, -L, and Psb-T, which affect the function of PSII (5-8). Furthermore, plastid-encoded subunit psbZ has been shown to be required for attachment of CP26 during assembly of PSII-LHC supercomplexes, whereas the nucleusencoded subunit psbW was demonstrated to be required for RCC dimer formation (9 -11). The role of plastid-encoded subunits Psb-I, -M, and -N and the nucleus-encoded small subunits Psb-R, and X remains unclear. A striking feature of PSII is the fast turnover of the D1 protein that is believed to be required for PSII repair and restoration of its photochemical activity after photoinactivation (12, 13). Maintaining PSII function may require selective replacement of this central PSII subunit including an efficient apparatus to recognize inactive complexes, and remove damaged and insert a new D1 copy (5, 14, 15). Zhang et al. (16) suggested that D1 replacement in higher plants may occur cotranslationally in a PSII subcomplex consisting of at least D2 and CP47, hence eliminating the need for complete disassembly and de novo assembly from PSII subunits.Cyanobacteria are an excellent model organism to study PSII assembly. The strain used most frequently i...

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b 559 , with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and ␤-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q Y region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/ Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.Photosystem II (PSII) 3 is the light-driven water:plastoquinone oxidoreductase of oxygenic photosynthesis, responsible for producing most of the oxygen in the atmosphere (1). It is located in the thylakoid membrane of chloroplasts and cyanobacteria and is a multisubunit lipoprotein complex composed of both intrinsic and extrinsic proteins. Crystal structures of dimeric PSII protein complexes isolated from the thermophilic cyanobacteria Thermosynechococcus elongatus (2-5) and Thermosynechococcus vulcanus (6, 7) have revealed the organization of the 20 subunits within each monomeric complex and the positions of the various cofactors. These include 35 chlorophyll (Chl) a molecules, two pheophytin a molecules, 12 carotenoids, two heme molecules, one non-heme iron, two calcium ions, two chloride ions, three plastoquinones, 25 lipids, and the Mn 4 Ca cluster, which catalyzes water oxidation (4).We are interested in understanding how PSII is assembled from its individual components. Current models suggest a stepwise assembly in both cyanobacteria and chloroplasts involving distinct intermediates (8 -10). However, as with other membrane protein complexes, detailed analysis of PSII assembly complexes is hindered by their low abundance in the membrane, and until now, early assembly intermediates of PSII have not been isolated and biochemically characterized.Here, we describe the isolation of the CP47 and ...

Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680+, was similar to that of PSII complexes lacking the Mn4CaO5 cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that serves as a model to analyze prokaryotic cell differentiation, evolutionary development of plastids, and the regulation of nitrogen fixation. The cell wall is the cellular structure in contact with the surrounding medium. To understand the dynamics of the cell wall proteome during cell differentiation, the cell wall from Anabaena heterocysts was enriched and analyzed. In line with the recently proposed continuity of the outer membrane along the Anabaena filament, most of the proteins identified in the heterocyst cell-wall fraction are also present in the cell wall of vegetative cells, even though the lipid content of both membranes is different.

The proteome of a membrane compartment has been investigated by de novo sequence analysis after tryptic in gel digestion. Protein complexes and corresponding protein subunits were separated by a 2-D Blue Native (BN)/SDS-PAGE system. The transmembrane proteins of thylakoid membranes from a higher plant (Hordeum vulgare L.) were identified by the primary sequence of hydrophilic intermembrane peptide domains using nano ESI-MS/MS-analysis. Peptide analysis revealed that lysine residues of membrane proteins are primarily situated in the intermembrane domains. We concluded that esterification of lysine residues with fluorescent dyes may open the opportunity to label membrane proteins still localized in native protein complexes within the membrane phase. We demonstrate that covalent labelling of membrane proteins with the fluorescent dye Cy3 allows high sensitive visualization of protein complexes after 2-D BN/SDS-PAGE. We show that pre-electrophoretic labelling of protein subunits supplements detection of proteins by post-electrophoretic staining with silver and CBB and assists in completing the identification of the membrane proteome.