The research proposed in this application is a comprehensive investigation of the chloroplast energy transducing H+-ATP synthase complex (CF-o-CF1 complex). This study is subdivided into three independent but interrelated general areas. The goals are: 1. (Part I) To physically locate the ATP synthase active site on CF1 and to sequence those sections of the CF1 polypeptides that make up the active site. Using base and ribose ring modified adenine nucleotide photoaffinity analogs, the vascular plant CF1 will be covalently modified. The subunits that bind the analogs will be determined. Modified subunits will be fragmented, and the covalently modified peptides will be isolated and sequenced. 2. (Part II) To study subunit-subunit interactions of CF1. Coupling factors with different properties will be isolated from two species of the alga Dunaliella. After partial dissociation and heterologous reconstitution, the properties of the coupling factors will be determined. Their subunit structures will be compared both chemically and immunologically. Their activities will be modulated by sulfhydryl-directed reagents, and the binding sites for the reagents will be determined. 3. (Part III) To study the biogenesis of the Chlamydomonas reinhardi CF-o-CF1 complex. Non-denatured subunits of the C. reinhardi CF1 complex will be isolated and reconstituted to regenerate an active complex. The CF-o-CF1 complex will be purified and its subunit composition and stoichiometry determined. Coupling factors from mutant strains of C. reinhardi, defective in phosphorylation, will be studied. Their defects will be established by examining the isolated proteins, the genes coding for the polypeptides, and their mRNAs. Finally, the mechanism for the directed insertion of the CF-o hydrophobic subunit III into the chloroplast thylakoid membrane will be examined in situ in a reconstituted system.
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