Abstract. We studied the assembly of photosystem II (PSII) in several mutants from Chlamydomonas reinhardtii which were unable to synthesize either one PSII core subunit (P6 [43 kD], D1, or D2) or one oxygen-evolving enhancer (OEE1 or OEE2) subunit. Synthesis of the PSII subunits was analyzed on electrophoretograms of cells pulse labeled with [14C]acetate. Their accumulation in thylakoid membranes was studied on immunoblots, their chlorophyll-binding ability on nondenaturating gels, their assembly by detergent fractionation, their stability by pulse-chase experiments and determination of in vitro protease sensitivity, and their localization by immunocytochemistry.In Chlamydomonas, the PSII core subunits P5 (47 kD), D1, and D2 are synthesized in a concerted manner while P6 synthesis is independent. P5 and P6 accumulate independently of each other in the stacked membranes. They bind chlorophyll soon after, or concomitantly with, their synthesis and independently of the presence of the other PSII subunits. Resistance to degradation increases step by step: beginning with assembly of P5, D1, and D2, then with binding of P6, and, finally, with binding of the OEE subunits on two independent high affinity sites (one for OEE1 and another for OEE2 to which OEE3 binds). In the absence of PSII cores, the OEE subunits accumulate independently in the thylakoid lumen and bind loosely to the membranes; OEE1 was found on stacked membranes, but OEE2 was found on either stacked or unstacked membranes depending on whether or not P6 was synthesized.p HOTOSYSTEM IX (PSII) ~ is a major protein complex of the photosynthetic apparatus in oxygen-evolving species. Light-harvesting chlorophyll-protein complexes (LHCs) transfer excitons to PSII cores where primary photochemistry occurs. PSII complexes (PSII cores with oxygen-evolving enhancer [OEE] subunits) are able to carry out the oxidation of water.The PSII core comprises five main intrinsic chloroplastencoded subunits P5, P6, D1, D2, and cytochrome b559 (59). Their molecular masses vary slightly from one species to another. Two subunits of 4%50 and 43-47 kD, called P5 and P6 in Chlamydomonas reinhardtii or by their molecular mass in higher plants (respectively encoded by psbB and psbC genes), bind most of the PSII core chlorophylls (58) and form the core antenna (9, 44). The chlorophyll-P5 and chlorophyll-P6 complexes-called, respectively, CPIII and CPIV in C. reinhardtii or CP47 and CP43 in higher plantscan be separated by electrophoresis at 4°C (13, 21). D1 and D2 of 32-35 kD (encoded, respectively, by psbA and psbD genes [18,48,64]) cooperate in the binding of the primary 1. Abbreviations used in this paper: LHC, light-harvesting complex; OEE, oxygen-evolving enhancer; PSII, photosystem II; WT, wild type. reactants (44) and show sequence homologies with the subunits L and M of the reaction center from purple bacteria (39, 53). Three extrinsic polypeptides encoded by nuclear genes (12, 60) are involved in oxygen evolution; OEE1 (29-33 kD) stabilizes the association of manganese ions ...

The molecular weight of the cytochrome b 6 f complex purified from Chlamydomonas reinhardtii thylakoid membranes has been determined by combining velocity sedimentation measurements, molecular sieving analyses, and determination of its lipid and detergent content. The complex in its enzymatically active form is a dimer. Upon incubation in detergent solution, it converts irreversibly into an inactive, monomeric form that has lost the Rieske iron-sulfur protein, the b 6 f-associated chlorophyll, and, under certain conditions, the small 32-residue subunit PetL. The results are consistent with the view that the dimer is the predominant form of the b 6 f in situ while the monomer observed in detergent solution is a breakdown product. Indirect observations suggest that subunit PetL plays a role in stabilizing the dimeric state. Delipidation is shown to be a critical factor in detergent-induced monomerization.

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Although the changes in organization of the lightharvesting antenna upon state transitions are well documented, possible changes in the organization of the photosynthetic electron transfer chain have not been directly investigated. Cytochrome b6/f (cyt b6/f), a major protein complex of this electron-transfer chain, has, however, been implicated in state transitions through its role in LHCIIkinase activation. State transitions are abolished in cyt b6/f mutants of green algae and higher plants due to the absence of LHCII reversible phosphorylation (4-8). Gal et al. (9) recently reported that the LHCII-kinase was, indeed, associated with cyt b6/f complexes.Whereas the PSII and PSI centers are well separated between the stacked and unstacked regions of the thylakoid membranes, cyt b6/f complexes are found in significant amounts in both membrane domains (10-13). The identity of the long-distance carrier between PSIl in the grana regions and PSI in the SL regions has been a matter of debate (14). It has been recently argued that the rapid diffusion ofplastoquinones, which transfer electrons between PSII and cyt b6/f complexes, is limited to small domains containing less than eight PSII centers (15,16). Therefore linear electron flow should be sustained by plastocyanin diffusing in the luminal space from its binding site on cyt b6df complexes in the stacked regions to PSI in the unstacked regions. The fraction of cyt b6/f complexes located in the unstacked regions next to PSI would then serve cyclic electron flow around PSI.There is a growing body of evidence that the ATP requirement of the photosynthetic cell controls state transitions (17)(18)(19) 8262The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.