The long-term objectives of this proposal are to understand the structure, mechanism and regulation of the vacuolar family of (H+)- ATPases (or V-ATPases). The V-ATPases are responsible for acidification of intracellular compartments in eukaryotic cells and serve an important function in a number of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, macro-molecular degradation and processing and the coupled transport of small molecules. Our previous studies have focused on a structural analysis of the V-ATPase of clathrin-coated vesicles and have provided insight into its intracellular distribution. We have also begun to address the function of subunits in the V-ATPase complex and the role of specific residues in catalysis and regulation. We have previously identified Cys254 of the 73 kDa A subunit as the residue responsible for the sensitivity of the V-ATPases to sulfhydryl reagents and have presented evidence for its involvement through disulfide bond formation in regulation of V-ATPase activity. To further define the structure of the nucleotide binding site located on the A subunit, the disulfide bonded partner of Cys254 will be identified and the sites of reaction with [32P]-2-azido-ATP will be determined. B subunit peptides labeled by [32P]-Bz-ATP will also be identified. The function of specific subunits in the peripheral V1 domain will be addressed by separation of the dissociated V1 polypeptides using protein chemical and immunochemical techniques and reassembly of the separated polypeptides in different combinations with the integral V0 domain using the protocol previously worked out in this laboratory. Reassembly of the integral V0 domain from its separated components will also be carried out in order to address the role of specific V0 subunits in proton conduction and the mechanism by which passive proton flux is regulated. Experiments to localize the site of bafilomycin binding will also be performed. Finally, the assembly pathway of the V-ATPase in MDBK cells will be determined by metabolic labeling of the V-ATPase with [35S] methionine and immunoprecipitation of the labeled pumps using both available monoclonal antibodies and polyclonal antisera prepared against synthetic peptides. These studies should provide significant new insights into this important class of (H+)-ATPases.
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