Abstract

In this proposal, we plan to investigate the energy transducing ATPase (CF0-CF1) complex associated with the thylakoid membranes of higher plants and the green algae Chlamydomonas reinhardi. Part I: Mechanistic problems related to the higher plant CF0-CF1 complex. Section A: Photoaffinity labels (2-azido adenine nucleotides) will be used to locate and identify the nucleotide regulatory binding site on membrane-bound CF1. Section B: (i) Distereomers of nucleoside phosphothioates will be used to determine the absolute stereochemistry of the ATPase reaction catalyzed by soluble CF1. (ii) Isotope trapping experiments will be performed to determine the kinetic mechanism of ATP synthesis and the binding constant for the first substrate to the ATP synthase active site. Section C: We will investigate the reversibility of the membrane-bound CF1 ATPase. Part A: We will investigate the mechanism of the reversible solvent-induced activation of the soluble ATPase using both physical and chemical methods. Part B: We will isolate and purify the CF0-CF1 complex from C. reinhardi. Part C: We plan to investigate the biochemical basis for the inability of isolated thylakoid membranes from C. reinhardi strain 2137 to catalyze photophosphorylation when cultured in the dark. Part D: We will measure the turnover of membrane-bound CF1 and the pool sizes of its subunits. SECTION D: We plan to isolate mutants of C. reinhardi defective in CF1 and characterize the resultant polypeptides.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031384-03
Application #
3279371
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-08-01
Project End
1986-07-31
Budget Start
1985-08-01
Budget End
1986-07-31
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
University of Wisconsin Madison
Department
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715

  Publications

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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