A novel c-type cytochrome maturation system is required for oxygenic photosynthesis

Denis Saint-Marcoux1,2, Richard Kuras1,2, Lina Lezhneva1,2, Alizée Malnoë1,2, Jacqueline Girard-Bascou1,2, Genevieve Ephritikhine3,4, Jean Alric1,2, Fabrice Rappaport1,2, Francis-André Wollman1,2 and Catherine de Vitry1,2

1 CNRS, UMR 7141, Laboratoire de Physiologie Membranaire et Moléculaire du Chloroplaste, Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France 2 UPMC Univ Paris 06 3CNRS, UPR 2355, Institut des Sciences du Végétal, B‰t 22, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France 4Univ Paris-Diderot.

Our studies of Chlamydomonas mutants revealed that covalent binding of c type hemes localized on the electronegative side of the thylakoid membranes (case of heme ci' covalent binding to cytochrome b6) requires a specific maturation pathway named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)) implicating at least four chloroplast membranes proteins (Kuras et al. PNAS 2007). They define a new metabolic pathway (system IV), fully distinct from those previously implicated in cytochrome biogenesis in the past two decades. They are encoded by the genomes of all organisms performing oxygenic photosynthesis, whatever their phylogenetic distances. We confirmed the similar role of the CCB proteins in Arabidopsis and Chlamydomonas and thereby establish the generality of the CCB pathway in chloroplasts (Lezhneva et al. submitted). Further biochemical characterization of CCB factors was performed in Chlamydomonas. CCB1-4 factors are integral membrane proteins not extracted by chaotropic treatments. Interactions between CCB proteins were shown by their concerted accumulation in ccb mutants, their migration pattern after blue native electrophoresis and yeast 2-hybrid split ubiquitin experiments. Blue native experiments also indicate that b6f complexes are assembled in mutants devoid of covalent heme ci'. Nuclear ccb mutants preventing heme ci' binding and chloroplast mutants lacking the cysteine residue involved in covalent binding of heme ci' accumulate very little b6f complex and are non phototrophic. In our search for extragenic suppressors of ccb mutants, we obtained a particularly interesting revertant that is phototrophic, accumulates wild-type levels of b6f complex that may be devoid of covalent heme ci' and is very photosensitive only in presence of oxygen. This revertant should offer a unique possibility to test the role of heme ci'. Other ccb mutants are analysed to establish whether the four CCB factors represent the entire CCB pathway or to identify additional CCB partners.

e-mail address of presenting author: catherine.devitry@ibpc.fr