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 ...

FtsH is the major thylakoid membrane protease found in organisms performing oxygenic photosynthesis. Here, we show that FtsH from Chlamydomonas reinhardtii forms heterooligomers comprising two subunits, FtsH1 and FtsH2. We characterized this protease using FtsH mutants that we identified through a genetic suppressor approach that restored phototrophic growth of mutants originally defective for cytochrome b 6 f accumulation. We thus extended the spectrum of FtsH substrates in the thylakoid membranes beyond photosystem II, showing the susceptibility of cytochrome b 6 f complexes (and proteins involved in the c i heme binding pathway to cytochrome b 6 ) to FtsH. We then show how FtsH is involved in the response of C. reinhardtii to macronutrient stress. Upon phosphorus starvation, photosynthesis inactivation results from an FtsH-sensitive photoinhibition process. In contrast, we identified an FtsH-dependent loss of photosystem II and cytochrome b 6 f complexes in darkness upon sulfur deprivation. The D1 fragmentation pattern observed in the latter condition was similar to that observed in photoinhibitory conditions, which points to a similar degradation pathway in these two widely different environmental conditions. Our experiments thus provide extensive evidence that FtsH plays a major role in the quality control of thylakoid membrane proteins and in the response of C. reinhardtii to light and macronutrient stress.

Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b  6  f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b  6  f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Clp chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b  6  f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b  6  f degradation pathway is activated.