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.

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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 ...

We studied the process of photosynthetic inactivation during gametogenesis of the unicellular green alga Chlamydomonas reinhardtii. We show that it is caused by the selective destabilization of a single transmembrane protein complex, the cytochrome b6/f complex, which is initially accumulated in the thylakoid membranes of vegetative cells. This protein destabilization is controlled by the intracellular energy sources available in the gametes, i. e. the coupled electron flow in the mitochondria and the amount of starch accumulated in the chloroplast. It nevertheless requires the expression of gamete-specific proteins. The loss of cytochrome b6/f complexes during gametogenesis is prevented by the addition of cycloheximide, but is chloramphenicol insensitive. Therefore, it is likely to involve some translation product of nuclear origin, specifically expressed during gametogenesis. Among the new polypeptides specifically found in the gametes, we detected a soluble polypeptide Ma (approximate molecular mass of 63 kDa), which shared common epitopes with cytochromef. Its synthesis displays an antibiotic sensitivity typical of a nuclear-encoded polypeptide and is controlled by the same intracellular signals which control the destabilization of the cytochrome b6/fcomplexes in the thylakoid membranes.

A renewal of ribosomes has been previously reported to occur during gametogenesis in C. reinhardtii. In order to further characterize these new ribosomes, we performed pulse-labelling experiments on whole cells of C. reinhardtii, during gametogenesis and in the presence of various aminoglycosides known to alter translational accuracy: Hygromycin and Paromomycin are assumed to increase the rate of translational errors at the level of 80S and 70S ribosomes whereas Kasugamycin is assumed to induce the opposite effect. Three lines of evidence support an increased inaccuracy in protein translation during gametogenesis: (1) gamete cells displayed a higher sensitivity than vegetative cells to Hygromycin and Paromomycin; 4 micrograms/ml Hygromycin cancelled cytoplasmic protein synthesis in gametes but not in vegetative cells; Paromomycin induced the synthesis of new polypeptides of high molecular weight and of nuclear origin in gametes but not in vegetative cells. In addition, chloroplast protein synthesis was more sensitive to Hygromycin and Paromomycin in gametes than in vegetative cells. (2) Kasugamycin-sensitive alterations of thylakoid membranes were detected during gametogenesis. (3) 35S-misincorporation in the OEE3 polypeptide, of nuclear origin and normally devoid of sulphur containing amino acids, was more than three times higher in gametes than in vegetative cells. This increase was prevented by Kasugamycin, suggesting that 80S translation in gametes was more inaccurate than in vegetative cells. The possible significance of these changes occurring during gametogenic differentiation is discussed in light of the importance of a modulation of translational accuracy at particular stages of the life cycle in other lower eukaryotes.

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