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.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b 6 f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b 6 f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b 6 f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LH-CII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.State transition has been originally described as a process whereby organisms performing oxygenic photosynthesis respond to changes in the spectral quality of light by changing the relative sizes of their photosystem I (PSI) 1 and photosystem II (PSII) antennae (for review, see Ref. 1). This leads to a redistribution of excitation energy between these two photosystems, which optimizes the photosynthetic yield. The reorganization of the antennae involves the displacement of the mobile fraction of a peripheral antenna complex, the light-harvesting antenna of photosystem II (LHCII), within the thylakoid membrane. During transition from state I to state II induced by PSII light (650 nm), the mobile fraction of LHCII is displaced from the PSII-enriched grana region to the PSI-enriched stromal lamellae. In the presence of PSI light (700 nm), the reverse migration of LHCII from PSI to PSII occurs. The redistribution of excitation energy between the photosystems results in changes in room temperature fluorescence with high and low fluorescence levels in state I and II, respectively, and in drastic alterations of the fluorescence emission spectrum at low temperature. As a consequence of this redistribution of energy between the two photosystems, state transition regulates the...

In a previous study [BultC, L. & Wollman, F.-A. (1992) Eur: J. Biochem. 204, 327-3361, we identified a novel gamete-specific polypeptide of Chlamydomonas reinhardtii, Ma. This 66-kDa polypeptide reacts with antibodies to cytochrome f and accumulates in gametes only in conditions that promote destabilisation of the cytochrome bJf complex. Here, we show that Ma is not a modification product of cytochrome J but is part of protein M, a high-molecular-mass L-amino-acid oxidase located in the periplasm. It catalyzes oxidation of all L-amino acids tested, except cysteine. Using phenylalanine as a substrate, saturation of the enzymatic rate is reached at 2 pM. These characteristics suggest that protein M may operate in vivo as an efficient scavanger of ammonium from extracellular amino acids. The enzyme contains non-covalently bound FAD. It exists in two forms with essentially similar enzymatic properties, of 1.2-1.3 MDa and 0.9-1 . O MDa, respectively. The lighter form is an oligomer of Ma, while the heavier form contains, in addition to Ma, a second polypeptide of 135 kDa, Mp, in a molar ratio of 3 -4 MaMp. Both polypeptides are glycosylated.In a previous study Wollman, 1990, 1992), we have shown that cytochrome bdf is specifically removed from the thylakoid membrane during gametogenesis of Chlamydomonas reinhardtii, resulting in an inactivation of photosynthesis in aging gametes. In parallel with the removal of the subunits of the cytochrome bJf complex, we observed a continuous accumulation of a gamete-specific protein, which we called Ma, showing immunological cross-reactivity with cytochromef. This 66-kDa polypeptide, becomes one of the major polypeptides in gametes deprived of nitrogen for more than 50 h. The production of Ma and the disappearance of cytochrome bJf occurred only in conditions that promote starch accumulation during gametogenesis, i.e. they were prevented when acetate was omitted from the medium, or when mitochondrial respiration was impaired by mutations or inhibitors. Developmental control was also operating, since neither phenomenon was observed in nitrogen-starved cultures of mutant C4, which has a block in gametic differentiation (Bultt and Bennoun, 1990).In the present paper, we have further characterized the biochemical properties and the function of polypeptide Ma. In particular we have shown that the immunological crossreaction between cytochrome f and Ma is due to a short shared protein motif and we demonstrate that Ma is the major subunit of protein M, a gamete-specific L-amino-acid oxidase located in the periplasm. MATERIALS AND METHODS Strains and culture conditionsVegetative cells of the '137c' wild type and CW15 (Davies and Plaskitt, 1971) strains of C. reinhardtii were grown on Tris/acetate/phosphate medium (Harris, 1989) at 28 OC, under 500-1x continuous illumination. Gametogenesis was triggered by diluting 1 vol. of a vegetative culture in stationary phase (2 X 10' cells/ml) into 7-10 vol. of the same medium from which all sources of nitrogen were omitted (NO medium). Flasks ...

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A 15.2-kDa polypeptide, encoded by the nuclear gene PETO, was identified as a novel cytochrome b 6 f subunit in Chlamydomonas reinhardtii. The PETO gene product is a bona fide subunit, subunit V, of the cytochrome b 6 f complex, because (i) it copurifies with the other cytochrome b 6 f subunits in the early stages of the purification procedure, (ii) it is deficient in cytochrome b 6 f mutants accumulating little of the complex, and (iii) it colocalizes with cytochrome f, which migrates between stacked and unstacked membrane regions upon state transition. Sequence analysis and biochemical characterization of subunit V shows that it has a one transmembrane ␣-helix topology with two large hydrophilic domains extending on the stromal and lumenal side of the thylakoid membranes, with a lumenal location of the N terminus. Subunit V is reversibly phosphorylated upon state transition, a unique feature that, together with its topological organization, points to the possible role of subunit V in signal transduction during redoxcontrolled short term and long term adaptation of the photosynthetic apparatus in eukaryotes.