The overall objective of the research in this proposal is to investigate the biochemical steps that occur during the post-translational maturation of protein-cofactor complexes. The specific goal includes the cloning and characterization of at least one gene that is required for attachment of heme during the assembly of cyt f and cyt c6 in chloroplasts, and the determination of the function of the gene product. Cytochromes f and c6 are members of a highly conserved family of c-type cytochromes that are ubiquitous in all energy transducing membranes. The experiments will exploit Chlamydomonas, since it is well-suited for approaches involving both genetics and molecular biology. The work involves the generation and identification of tagged nuclear mutants that are defective in cyt c6 and cyt f accumulation, the demonstration that these are indeed heme attachment mutants and that the locus of interest is tagged, followed by the cloning of the corresponding gene on the basis of the tag. """"""""Tagged"""""""" mutants will be generated by random integration of the argininosuccinate lyase (ASL) gene into the nuclear genome by transformation of a recipient arg-minus strain. Mutants will be identified among the arg-plus transformants on a fluorescence video imaging system based on their loss of photosynthetic capacity and by biochemical assays for failure to accumulate holocyt c6 and holocyt f. Complementation of previously- characterized nuclear heme attachment mutants using an indexed genomic cosmid library or chloroplast mutants using cloned chloroplast DNA is proposed as a parallel strategy. The project has revelance to human health because the same principles apply to respirator cytochromes, and thus will enhance our understanding and hence treatment of mitochondrial myopathies.
