The rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. Notably the bovine enzyme comprises up to 46 subunits, while 27 subunits could be considered as widely conserved among eukaryotic complex I. By combining proteomic and genomic approaches, we characterized the complex I composition from the unicellular green alga Chlamydomonas reinhardtii. After purification by blue-native polyacrylamide gel electrophoresis (BN-PAGE), constitutive subunits were analyzed by SDS-PAGE coupled to tandem mass spectrometry (MS) that allowed the identification of 30 proteins. We compared the known complex I components from higher plants, mammals, nematodes and fungi with this MS data set and the translated sequences from the algal genome project. This revealed that the Chlamydomonas complex I is likely composed of 42 proteins, for a total molecular mass of about 970 kDa. In addition to the 27 typical components, we have identified four new complex I subunit families (bovine ESSS, PFFD, B16.6, B12 homologues), extending the number of widely conserved eukaryote complex I components to 31. In parallel, our analysis showed that a variable number of subunits appears to be specific to each eukaryotic kingdom (animals, fungi or plants). Protein sequence divergence in these kingdom-specific sets is significant and currently we cannot exclude the possibility that homology between them exists, but has not yet been detected.

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Photosynthetic activities were analyzed in Chlamydomonas reinhardtii mitochondrial mutants affected in different complexes (I, III, IV, I ϩ III, and I ϩ IV) of the respiratory chain. Oxygen evolution curves showed a positive relationship between the apparent yield of photosynthetic linear electron transport and the number of active proton-pumping sites in mitochondria. Although no significant alterations of the quantitative relationships between major photosynthetic complexes were found in the mutants, 77 K fluorescence spectra showed a preferential excitation of photosystem I (PSI) compared with wild type, which was indicative of a shift toward state 2. This effect was correlated with high levels of phosphorylation of lightharvesting complex II polypeptides, indicating the preferential association of light-harvesting complex II with PSI. The transition to state 1 occurred in untreated wild-type cells exposed to PSI light or in 3-(3,4-dichlorophenyl)-1,1-dimethylureatreated cells exposed to white light. In mutants of the cytochrome pathway and in double mutants, this transition was only observed in white light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. This suggests higher rates of nonphotochemical plastoquinone reduction through the chlororespiratory pathway, which was confirmed by measurements of the complementary area above the fluorescence induction curve in dark-adapted cells. Photo-acoustic measurements of energy storage by PSI showed a stimulation of PSI-driven cyclic electron flow in the most affected mutants. The present results demonstrate that in C. reinhardtii mutants, permanent defects in the mitochondrial electron transport chain stabilize state 2, which favors cyclic over linear electron transport in the chloroplast.Metabolic processes of photosynthetic organisms depend on the regeneration of ATP through photosynthesis and respiration. Although these two processes are now well understood at the molecular and physiological levels, less is known about their mutual regulation. In eukaryotic cells, complex interactions between photosynthesis and respiration occur because both processes are linked by common key metabolites such as ADP/ATP, NAD(P)H, triose-P, and hexose-P (for review, see Hoefnagel et al., 1998).When the dependence of respiration on photosynthesis seems to rely essentially on the availability of substrates, the influence of respiration on photosynthesis is suggested to involve complex organizational changes in the PSs, known as state transitions. The transition from states 1 to 2 corresponds to the reversible transfer of a mobile pool of PSII lightharvesting complexes II (LHCII) from PSII to PSI along the thylakoid membrane (state 2 transition) and is triggered by persistent reduction of the plastoquinone (PQ) pool. This reduction causes the activation of an LHCII-kinase interacting with the quinine oxidizing site of cytochrome (Cyt) b6/f (for review, see Allen, 1992;Wollman, 2001). Due to its high affinity for the PSI-h subunit, phospho-LHCII then is bound preferential...

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In Chlamydomonas reinhardtii, the expression of the nit1 gene encoding nitrate reductase is dependent on the nature of the nitrogen source and on other environmental factors. We have fused the nit1 promoter region to the arylsulphatase (ars) reporter gene lacking its own promoter and introduced this chimeric construction (nit1/ars) into a wall-less strain of C. reinhardtii. A new and sensitive method, based on the use of alpha-naphthylsulphate as a substrate and a diazonium salt as a chromogenic post-coupling agent, was developed to detect the activity of arylsulphatase (an enzyme which is almost completely secreted in the culture medium) both in vitro and in agar plates. The transformants carrying nit1/ars did not express arylsulphatase when grown in ammonium-sufficient medium but readily accumulated the enzyme in ammonium-free medium either supplemented, or not supplemented, with nitrate or nitrite. The nit1/ars construct, however, was not expressed in the nit2 mutant lacking a specific transcription regulator controlling the expression of nit1. These results, together with the observation that the transcription of nit1/ars is initiated at the same sites as the nit1 endogenous gene, confirms the hypothesis that the regulation of nit1 expression takes place mainly at the transcriptional level. The expression of the ars gene from the nit1 promoter was high enough to allow direct measurements of arylsulphatase activities in pools of transformants without prior isolation of nit1/ars clones. This original procedure has permitted the analysis of the effects of nested deletions in the nit1 promoter region on the expression of the reporter gene. The results indicate that the -282 to -198 sequence is required for transcription to occur and that the -751 to -282 region contains several elements mediating nit1 expression.