Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38–40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end. The middle part of the Tbc2 protein displays partial amino acid sequence identity with Crp1, a protein from Zea mays that is implicated in the processing and translation of the chloroplast petA and petD RNAs. The Tbc2 protein is enriched in chloroplast stromal subfractions and is associated with a 400-kD protein complex that appears to play a role in the translation of specifically the psbC mRNA

Contribution a l'etude genetique de l'appareil photosynthetique chez l'algue verte Chlamydomonas reinhardtii

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Evidence for nuclear control of the expression of the atpA and atpB chloroplast genes in Chlamydomonas

We analyzed three nuclear mutants of Chlamydomonas reinhardtii altered in the expression of the chloroplast genes atpA or atpB coding for the a or p subunit of the chloroplast ATP synthase. These mutants revealed the existence of three nuclear products controlling the expression of the two chloroplast genes: the first one acts on the translation of the atpA transcript, and the two others act specifically on the stability of either the atpB or the atpA mRNAs. The nuclear mutation responsible for the decreased stability of the atpB mRNA prevented translation of the corresponding polypeptide. In contrast, the mutation responsible for the decreased stability of the atpA mRNA had limited effect on the translation of the a subunit, thereby allowing its accumulation and assembly in an active ATP synthase. Although acting originally on the expression of only one of the two main coupling factor 1 subunits, the three mutations caused a change in the translation rate of the other subunit, as viewed in 5-min pulse labeling experiments. This is indicative of a concerted expression of the a and p subunits at an early post-translational step, or during translation, that may be critical for the assembly of the chloroplast ATP synthase

Régulation de la traduction chloroplastique chez Chlasmydomonas reinhardtii (cytochrome f et autres protéines CES...)

La biogenèse de l'appareil photo synthétique nécessite l'action concertée des génomes nucléaires et chloroplastiques pour produire en quantité stœchiométrique les sous-unités des complexes de la membrane photo synthétique, d'origine génétique double. Ce travail effectué sur l'organisme modèle Chlamydomonas reinhardtii présente un mécanisme original traduisant cette concertation: le processus CES (Contrôle par Epistasie de Synthèse), reposant sur une régulation de la traduction. En étudiant le gène chloroplastique petA codant le cytochrome f, une protéine majeure du complexe cytochrome b6f, nous montrons que sa traduction dépend de l'assemblage de la protéine au sein du complexe. En absence de ses partenaires d'assemblage, la synthèse du cytochrome f- mais pas sa stabilité est réduite. Nous présentons une caractérisation du mécanisme impliquant: I) une régulation de l'initiation de la traduction; II) un domaine régulateur porté par la protéine CES ; III) un effecteur ternaire, plutôt qu'une interaction directe entre le motif régulateur et la région 5' non-traduite du gène CES. Cet effecteur pourrait être un activateur traductionnel, qui piégé par la sous-unité non-assemblée, ne serait plus disponible pour initier la traduction. Nous avons caractérisé un activateur traductionnel, requis spécifiquement pour l'initiation de la traduction du gène petA, le gène TCA1, qui pourrait être l'effecteur du processus CES. Enfin, nous avons démontré l'existence d'autres protéines CES au sein des complexes de la membrane photosynthétique de C. reinhardtii. Le contrôle de la traduction par l'assemblage des sous-unités PsaA et PsaC du photosystème I, et des sous-unités D1 et P5 du photo système II repose sur le même mécanisme: une autorégulation négative de l'initiation de la traduction par la sous-unité CES non-assemblée. Ces observations posent la question d'une éventuelle généralisation du processus CES et de son mécanisme aux plantes supérieures, et à un autre organite, la mitochondrie.The biogenesis of the photosynthetic protein complexes requites the co-ordinate interaction of the nuclear and chloroplast genomes in order to produce in stoichiometric amounts their subunits. I contributed to the characterization of an unique mechanism of regulation: the CES process (Control by Epistasy of Synthesis) in the unicellular green alga Chlamydomonas reinhardtii. Using the chloroplast petA gene, encoding cytochrome f, a major subunit of the cytochrome b6f complex, we showed that petA translation depends on its assembly state: in the absence of its assembly partners, cytochrome f synthesis is inhibited at the level of translation initiation. Moreover, an autoregulation is exerted by a regulatory motif of the unassembled cytochrome f CES subunit. A ternary effector may "shuttle" the CES control between the regulatory motif of the protein and the 5' untranslated region of the CES (petA) transcript. I characterized the TCA1 nuclear encoded translational activator, specifically required for the initiation of the translation of the petA RNA. TCA1 may be the effector of the CES process: in our model, competitive binding of TCA1 to cytochrome f's regulatory motif would decrease petA translation efficiency. Finally, I have shown that this assembly-dependent regulation of translation also occurs for chloroplast-encoded subunits of other photosynthetic complexes of C. reinhardtii such as PsaA and PsaC subunits of Photosystem I, as well as D1 and P5 subunits of Photosystem II. In all cases, the CES mechanism is conserved. These observations raise interesting questions as to the possible generalization of the CES process to vascular plants, and to the mitochondrion.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Studies on the cytochrome b 6 / f complex. I. Characterization of the complex subunits in Chlamydomonas reinhardtiiChlamydomonas\ reinhardtii

International audienceWe have analyzed the heme-associated peroxidase activity in thylakoid membranes from the. green algae$Chlamydomonas\ reinhardtii$ after electrophoresis in the presence of sodium dodecyl sulfate. Besidescytochrome $f$ and cytochrome $b_6$, we observed peroxidase activity in two other bands, of 34 and 11 kDa, ofunknown origin. Characterization of the $b_6/f$ complex subunits was undertaken by means of a comparisonof the polypeptide deficiencies in several $b_6/f$ mutants with the polypeptide content of preparationsenriched in $b_6/f$ complexes. We conclude that the $b_6/f$ complex consists of five subunits. Usingsite-specific translation inhibitors, we show that cytochrome $f$, cytochrome $b_6$ and subunit IV are ofchloroplast origin, whereas the Rieske protein and probably subunit V are translated on cytoplasmicribosomes. A model of assembly of the complex is proposed: a cytochrome moiety, comprising the subunitsof chloroplast origin, is assembled in the thylakoid membranes prior to the insertion and assembly of thesubunits encoded in the nuclear genome