DESCRIPTION: The broad, long term research objective of this program is the understanding of the process of assembly of the chloroplast energy transducing membrane, with particular emphasis on how the cofactors and polypeptides are assembled to yield a specific functional arrangement in vivo. In this application, the emphasis is on the c-type cytochromes, soluble c6 and membrane-anchored f of the b6/f complex, and cyt b6, an integral membrane subunit of the b6/f complex. The b6/f complex is chosen for its relative compositional simplicity and well-defined cofactor binding sites. This complex is not essential for growth in the experimental organism Chlamydomonas reinhardtii which allows the research plan to exploit classical molecular genetics as well as biochemical approaches for the study of its assembly. A prime objective during the present project period is to undertake functional studies of two newly identified components of a putative thylakoid membrane-associated cytochrome c assembly complex, CcsA and Ccsl. The topology and orientation of these novel proteins will be analyzed by phoA-fusion analysis in E. coli combined with epitope exposure and protease susceptibility assays in situ with purified thylakoid membranes. The proteins, or a complex containing the two proteins, will be purified from C. reinhardtii strains that express biotin- or his6-tagged versions of the two proteins, in order to identify other components of the complex and to develop assays for the biochemical activities of the Ccs proteins. A second aim is to clone genes corresponding to the previously defined CCS2, 3 and 4 loci, which are required for the assembly of all chloroplast c-type cytochromes, and whose products are proposed to interact with Ccsl and CcsA. The genes will be identified from an indexed cosmid library on the basis of their ability to rescue non-photosynthetic ccs strains, or by virtue of their physical association wit ble sequences in ccs strains carrying ble-tagged alleles. In the case of cyt b6, the description of a unique assembly phenotype led to the definition of multiple CCB loci whose products are required specifically for heme insertion into one of the two heme binding sites (bH site). A third aim is to clone thes loci by applying the same methods described for the CCS loci. The increased knowledge of heme protein assembly in this model system is directly applicable to the understanding of the assembly of other cofactor-containing electron transfer complexes, especially the structurally more elaborate respiratory bc1 complex or phagocyte NADPH oxidase whose functions are affected in mitochondrial myopathies and chronic granulomatous disease, respectively.