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.
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