MATURATION OF CHLOROPLAST C-TYPE CYTOCHROMES

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In eukaryotic cells, the biosynthesis of the electron transfer complexes of energy transducing membranes requires not only the regulated and coordinate expression of nuclear and organellar genes for the various macromolecular components, but also the correct targeting and processing of the pre-proteins, the association of cofactors with the apoproteins and the stoichiometric assembly of multisubunit structures. These post- translational events are the subject of our long term research program on thylakoid membrane biogenesis. This application focusses on the processing steps - especially heme attachment - involved in the maturation of photosynthetic c-type cytochromes (soluble cyt c6 and membrane-anchored cyt f). The maturation of nuclear-encoded, lumen- targeted cyt c6 includes specific proteolytic processing steps following translocation across the envelope- and thylakoid membranes, and heme attachment following the second translocation. Identifiable intermediates that define the maturation sequence include pre-apocyt c6, intermediate apocyt c6 and apocyt c6. Our previous work suggests that the conversion of apocyt c6 to holocyt c6 is specified by multiple nuclear- and chloroplast-encoded genes whose products function also in the pathway for maturation of organelle-encoded pre-apocyt f. The recognition that there are activities required for the maturation of both proteins simplifies the identification of mutants affected specifically at the post-translational events in c-type cytochrome biosynthesis. The further dissection of the pathway of cyt c6 and cyt f maturation is proposed here, with the goal of identifying the several genes and functions that are necessary at each step of the respective pathways. Parallel biochemical and molecular genetic approaches will be brought to bear on the problem, since the experimental organism, Chlamydomonas reinhardtii, is ideally suited for either strategy. specifically, "tagged" mutants defective in both cyt f and cyt c6 accumulation will be generated to enable the isolation of nuclear genes required for the maturation of chloroplast c-type cytochromes, and chloroplast gene disruption techniques will be exploited to identify the plastid loci. The biochemical functions and sequential action of the gene products at each stage in the pathway will be ascertained by in vivo and in vitro phenotypic characterization of each new tagged or site-directed mutant. The isolation of a cell-free system for heme attachment will permit in vitro analysis, while established techniques will be employed for the in vivo study. The proposed work has as its long term goal, the understanding of the general principles underlying the assembly of the cofactors in the photosynthetic electron transfer membrane. the project has relevance to human health because the same principles apply to respiratory cytochromes, and the anticipated results are expected to enhance our understanding and hence treatment of mitochondrial myopathies, many of which result from deficiencies in respiratory chain components. A unique aspect of this work is the expected identification of biochemical components and genes involves in intracellular heme transport. This is an unexplored area, but one that is ripe for genetic dissection in this organism.