The long term objective of this project is to understand the physical mechanism for energy coupling by ion-translocating ATPases. For the immediate proposal, the proton-ATPase of yeast plasma membranes will be probed biochemically and genetically to determine which protein domains are involved with coupling proton transport to ATP hydrolysis. The yeast plasma membrane-ATPase closely resembles in structure and function the Na+, K+-ATPase and Ca2+-ATPase of higher eukaryotes and shares some sequence homology with these enzymes. A working hypothesis will be examined in which a local conformational change acts as the linkage between ATP hydrolysis and proton transport. This project will examine 1) the topography of the enzyme in the bilayer by correlating hydropathy profiles determined from the primary amino acid sequence with data from mapping studies using hydrophilic and hydrophobic probes; 2) localized conformational states of enzyme stopped at different stages of turnover by evaluating peptide profiles following limited proteolysis and protein cross-linking; 3) expression of the plasma membrane-ATPase gene in yeast and E. coli; 4) uncoupled ATPase mutants produced by localized random mutagenesis and 5) uncoupled enzymes produced by protein cross-linking and limited proteolysis. Biochemical methods will include protein labelling with photoactivatable probes, gel filtration, preparative gel electrophoresis, CNBr-cleavage, HPLC separation, amino acid analysis and micro-sequencing. Genetic methods will include large-scale- and mini-plasmid preparations, DNA fragment isolation, agarose gel electrophoresis, plasmid subcloning, yeast and bacterial transformation and DNA sequencing.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Physical Biochemistry Study Section (PB)
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Public Health Research Institute
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Valiakhmetov, Airat; Perlin, David S (2003) Molecular architecture of the phosphorylation region of the yeast plasma membrane H+-ATPase. J Biol Chem 278:6330-6
Soteropoulos, P; Valiakhmetov, A; Kashiwazaki, R et al. (2001) Helical stalk segments S4 and S5 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae are optimized to impact catalytic site environment. J Biol Chem 276:16265-70
Hasper, A; Soteropoulos, P; Perlin, D S (1999) Modification of the N-terminal polyserine cluster alters stability of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. Biochim Biophys Acta 1420:214-22
Soteropoulos, P; Perlin, D S (1998) Genetic probing of the stalk segments associated with M2 and M3 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae. J Biol Chem 273:26426-31
Soteropoulos, P; Wang, G; Perlin, D S (1998) Molecular genetic probing of energy coupling by the yeast plasma membrane proton pump. Acta Physiol Scand Suppl 643:115-22
Seto-Young, D; Bandell, M; Hall, M et al. (1998) Differential exposure of surface epitopes in the beta-strand region of LOOP1 of the yeast H+-ATPase during catalysis. J Biol Chem 273:18282-7
Wang, G; Perlin, D S (1997) Probing energy coupling in the yeast plasma membrane H+-ATPase with acetyl phosphate. Arch Biochem Biophys 344:309-15
Seto-Young, D; Hall, M J; Na, S et al. (1996) Genetic probing of the first and second transmembrane helices of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. J Biol Chem 271:581-7
Wang, G; Tamas, M J; Hall, M J et al. (1996) Probing conserved regions of the cytoplasmic LOOP1 segment linking transmembrane segments 2 and 3 of the Saccharomyces cerevisiae plasma membrane H+-ATPase. J Biol Chem 271:25438-45
Bandell, M; Hall, M J; Wang, G et al. (1996) Probing the cytoplasmic LOOP1 domain of the yeast plasma membrane H(+)-ATPase by targeted factor Xa proteolysis. Biochim Biophys Acta 1280:81-90

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