title: Maturation of pre-apocytochrome f in vivo. A site-directed mutagenesis study in Chlamydomonas reinhardtii.
abstract: The biosynthesis of cytochrome f is a multistep process which requires processing of the precursor protein and covalent ligation of a c-heme upon membrane insertion of the protein. The crystal structure of a soluble form of cytochrome f has revealed that one axial ligand of the c-heme is provided by the alpha-amino group of Tyr1 generated upon cleavage of the signal sequence from the precursor protein (Martinez S. E., Huang D., Szczepaniak A., Cramer W.A., and Smith J. L. (1994) Structure 2, 95-105). We therefore investigated, by site-directed mutagenesis, the possible interplay between protein processing and heme attachment to cytochrome f in Chlamydomonas reinhardtii. These modifications were performed by chloroplast transformation using a petA gene encoding the full-length precursor protein and also a truncated version lacking the C-terminal membrane anchor. We first substituted the two cysteinyl residues responsible for covalent ligation of the c-heme, by a valine and a leucine, and showed that heme binding is not a prerequisite for cytochrome f processing. In another series of experiments, we replaced the consensus cleavage site for the thylakoid processing peptidase, AQA, by an LQL sequence. The resulting transformants were nonphototrophic and displayed delayed processing of the precursor form of cytochrome f, but nonetheless both the precursor and processed forms showed heme binding and assembled in cytochrome b6f complexes. Thus, pre-apocytochrome f adopts a suitable conformation for the cysteinyl residues to be substrates of the heme lyase and pre-holocytochrome f folds in an assembly-competent conformation. In the last series of experiments, we compared the rates of synthesis and degradation of the various forms of cytochrome f in the four types of transformants under study: (i) the C terminus membrane anchor apparently down-regulates the rate of synthesis of cytochrome f and (ii) degradation of misfolded forms of cytochrome f occurs by a proteolytic system intimately associated with the thylakoid membranes.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D003545:Cysteine; D003580:Cytochromes / Q000096:biosynthesis* / Q000737:chemistry*; D045348:Cytochromes f; D017343:Genes, Plant; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D003580:Cytochromes; D011498:Protein Precursors; D011994:Recombinant Proteins; D006418:Heme; D045348:Cytochromes f; D003545:Cysteine