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Regulatory roles of the cytochrome b6f complex in redox sensing and state transitions in Chlamydomonas reinhardtii

Abstract : Sunlight is a powerful but fluctuating energy source used by photosynthetic organisms to power the assimilation of atmospheric CO2 into reduced carbon compounds. To optimize photosynthetic efficiency in ever-changing light conditions, organisms of the green lineage regulate the distribution of energy input between the two photosystems through state transitions. This process is governed by the redox state of the membrane pool of quinones, liposoluble electron carriers. Through a yet unknown mechanism, the cytochrome (cyt) b6f complex detects these redox changes and transmits a signal to the state transition kinase Stt7 for its activation. In order to decipher the role of the cyt b6f, we devised a novel experimental strategy in Chlamydomonas reinhardtii combining the random mutagenesis of a chloroplast gene (petD coding for cyt b6f subunit IV) by error-prone PCR, chloroplast transformation and selection of clones on phototrophic growth. Analysis of variant sequences allowing proper assembly and function of cyt b6f revealed some interesting features on the mutational robustness of this subunit in the context of its large transmembrane protein complex. Transformants were then screened by chlorophyll fluorescence emission imaging on their ability to perform state transitions. Several state transition mutants were isolated, and sequencing revealed that mutations concentrated in the stromal fg loop of subunit IV. Site-directed mutagenesis of key fg loop residues showed that substitutions of Asn122, Tyr124 and Arg125 produced mutants that are blocked in State I independently of the redox state of the PQ pool and with no adverse effects on cyt b6f assembly and electron transfer activity. Protein-protein interaction studies provided evidence that the kinase domain of Stt7 interacts with the subunit IV fg loop and that its autophosphorylation activity depends on Arg125. We propose a model for the interaction between these two proteins as well as for the cyt b6f-mediated mechanism of Stt7 activation.
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Louis Dumas. Regulatory roles of the cytochrome b6f complex in redox sensing and state transitions in Chlamydomonas reinhardtii. Biochemistry, Molecular Biology. Aix Marseille University; CEA Cadarache, 2018. English. ⟨tel-01763249⟩

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