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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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Tufts University
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Collins, Michael P; Forgac, Michael (2018) Regulation of V-ATPase Assembly in Nutrient Sensing and Function of V-ATPases in Breast Cancer Metastasis. Front Physiol 9:902
McGuire, Christina M; Forgac, Michael (2018) Glucose starvation increases V-ATPase assembly and activity in mammalian cells through AMP kinase and phosphatidylinositide 3-kinase/Akt signaling. J Biol Chem 293:9113-9123
Cotter, Kristina; Liberman, Rachel; Sun-Wada, GeHong et al. (2016) The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer. Oncotarget 7:46142-46157
McGuire, Christina; Cotter, Kristina; Stransky, Laura et al. (2016) Regulation of V-ATPase assembly and function of V-ATPases in tumor cell invasiveness. Biochim Biophys Acta 1857:1213-1218
Stransky, Laura A; Forgac, Michael (2015) Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly. J Biol Chem 290:27360-9
Cotter, Kristina; Stransky, Laura; McGuire, Christina et al. (2015) Recent Insights into the Structure, Regulation, and Function of the V-ATPases. Trends Biochem Sci 40:611-622
Cotter, Kristina; Capecci, Joseph; Sennoune, Souad et al. (2015) Activity of plasma membrane V-ATPases is critical for the invasion of MDA-MB231 breast cancer cells. J Biol Chem 290:3680-92
Liberman, Rachel; Bond, Sarah; Shainheit, Mara G et al. (2014) Regulated assembly of vacuolar ATPase is increased during cluster disruption-induced maturation of dendritic cells through a phosphatidylinositol 3-kinase/mTOR-dependent pathway. J Biol Chem 289:1355-63
Liberman, Rachel; Cotter, Kristina; Baleja, James D et al. (2013) Structural analysis of the N-terminal domain of subunit a of the yeast vacuolar ATPase (V-ATPase) using accessibility of single cysteine substitutions to chemical modification. J Biol Chem 288:22798-808
Capecci, Joseph; Forgac, Michael (2013) The function of vacuolar ATPase (V-ATPase) a subunit isoforms in invasiveness of MCF10a and MCF10CA1a human breast cancer cells. J Biol Chem 288:32731-41

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