Ubiquity of the CES process using two model organisms, the yeast Saccharomyces cerevisiae and the green alga Chlamydomonas reinhardtii. – chloro/mito CES

Energy transduction in mitochondria or chloroplasts is performed by oligomeric proteins that comprise subunits of dual genetic origin, some of them being encoded by the organelle genome, whereas the others are nucleus-encoded. How the expression of the two genetic compartments is coordinated to produce the various subunits of a protein complex in the stoichiometry required for their functional assembly is a major issue in cell biology. During the last decade, partner 1 discovered a major regulatory process for chloroplast biogenesis using the green alga Chlamydomonas reinhardtii as a model system: an assembly-dependent regulation of translation of chloroplast genes that was called CES process for Control by Epistasy of Synthesis. In the absence of certain assembly partners, the rate of synthesis of some chloroplast-encoded subunits from the same protein complex, thereafter named CES subunits, is drastically reduced. CES is a key player in the biogenesis of the photosynthetic apparatus of C. reinhardtii since all major photosynthetic proteins contains CES subunits. Initially identified in this alga, CES is being recognised as a general feature of organelle gene expression since it has been more recently identified as a major process for the biogenesis of RuBisCO in the chloroplasts of higher plants and of Cytochrome OXidase in the mitochondria of yeast. Chloroplasts and mitochondria share a common protein, the ubiquitous proton-driven ATP synthase, comprising a membrane-anchored proton channel, (C)F0, and a catalytic sector responsible for ATP synthesis, (C)F1. Its biogenesis, a major interest of partner 2, should obey very specific constraints since unequal stoichiometries between subunits are required in both (C)F1 and (C)F0. For example, 10-14 copies of the organelle-encoded proteolipid subunit (Apt9p and AtpH in mitochondria and chloroplasts, respectively) assemble with only 1 copy of the other (C)F0 subunits. Biogenesis of (C)F0 should involve CES regulation both in yeast and in Chlamydomonas: in the former, the synthesis of subunits Atp6p and Atp8p is strongly reduced in strain defective for Atp9p expression. Most interestingly, the very same regulatory loop operates in the chloroplast of Chlamydomonas, where subunit AtpI shows decreased expression in strains lacking subunit AtpH, the chloroplast counterpart of Atp9p. Our project takes advantage of the unique opportunity provided by this similar regulatory loop, setting the rate of production of Atp6/I to the presence of subunit Atp9/H, to track the similarities and differences in the mechanism of the CES controls involved in (C)F0 biogenesis in these two distant organisms. We will perform a similar set of experiments, based on chimeric genes and mutated alleles of the genes coding the organelle-encoded ATP synthase subunits introduced in the appropriate organelle. At each step we will confront our results obtained in the two organisms. This dialog will help understand how similar features of this CES reflect the specific constraints on ATP synthase biogenesis, how CES evolved, its physiological significance and the mechanisms responsible for the specific 10-14:1 stoichiometry between AtpH/9 and the other CF0 subunits (Tasks 1 and 2). Our project also aims at understanding the molecular basis of the specific interaction that develops between unassembled CES subunits and the 5'UTR of their mRNA. We will test by biochemical approaches the role of nuclear-encoded factors controlling the post-transcriptional steps of CES subunits and also identify CES ternary effectors though appropriate genetic screen (Tasks 3 and 4). Carried out on C. reinhardtii and S. cerevisiae, because of their unique potential for genetic studies, our project, owing to the generality of the CES process in organelle biogenesis, will be of general significance for organelle biology as a whole but also in broad fields of translational regulation and assembly of oligomeric ptoteins. The two partners have an internationally recognised experience in photosynthesis/respiration and chloroplast/mitochondrion molecular genetics. Previous contributions of partner 1 were central in the discovery and characterization of the CES process, while partner 2 is one of the leaders in the field of ATP synthase biogenesis. Both laboratories group genetic, genomic, biochemical and spectroscopic competences. We have already established the basis for a successful characterisation of the CES process involved in ATP synthase biogenesis in the two distant organisms and for the identification of the epistatic effectors. We ask for ANR funding because this comparison between two organisms can obviously not been carried out by a single laboratory. This new and original project will require a tight collaboration that should reinforce the international leadership of the two partners in the field of protein assembly in organelles and should lead to experimental and conceptual cross-fertilisation.