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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National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Wisconsin Madison
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United States
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Smart, E J; Selman, B R (1993) Complementation of a Chlamydomonas reinhardtii mutant defective in the nuclear gene encoding the chloroplast coupling factor 1 (CF1) gamma-subunit (atpC). J Bioenerg Biomembr 25:275-84
Selman-Reimer, S; Duhe, R J; Stockman, B J et al. (1991) L-1-N-methyl-4-mercaptohistidine disulfide, a potential endogenous regulator in the redox control of chloroplast coupling factor 1 in Dunaliella. J Biol Chem 266:182-8
Smart, E J; Selman, B R (1991) Isolation and characterization of a Chlamydomonas reinhardtii mutant lacking the gamma-subunit of chloroplast coupling factor 1 (CF1). Mol Cell Biol 11:5053-8
Duhe, R J; Selman, B R (1990) The dithiothreitol-stimulated dissociation of the chloroplast coupling factor 1 epsilon-subunit is reversible. Biochim Biophys Acta 1017:70-8
Theg, S M; Bauerle, C; Olsen, L J et al. (1989) Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J Biol Chem 264:6730-6
Olsen, L J; Theg, S M; Selman, B R et al. (1989) ATP is required for the binding of precursor proteins to chloroplasts. J Biol Chem 264:6724-9
Yu, L M; Selman, B R (1988) cDNA sequence and predicted primary structure of the gamma subunit from the ATP synthase from Chlamydomonas reinhardtii. J Biol Chem 263:19342-5
Yu, L M; Merchant, S; Theg, S M et al. (1988) Isolation of a cDNA clone for the gamma subunit of the chloroplast ATP synthase of Chlamydomonas reinhardtii: import and cleavage of the precursor protein. Proc Natl Acad Sci U S A 85:1369-73
Selman-Reimer, S; Selman, B R (1988) The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ. FEBS Lett 230:17-20
Selman-Reimer, S; Selman, B R (1988) The partial purification of a factor from Dunaliella salina that causes the rapid in situ inactivation of light-activated chloroplast coupling factor 1 (CF1). FEBS Lett 230:21-4

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