The application is for support to continue studies of the structure, mechanism, and regulation of proton-translocating ATPases in the V-type family. The V-type ATPases are responsible for acidification of intracellular compartments in eukaryotic cells and serve an important function in a variety of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, macromolecular processing, and degradation and coupled transport. V-ATPases in the plasma membrane of specialized cells also play a role in renal acidification, bone resorption, and tumor metastasis. Understanding how V-ATPases are regulated is important to understanding these processes. This laboratory has previously shown that the V-ATPase from clathrin- coated vesicles is organized into a peripheral V1 domain responsible for ATP hydrolysis and an integral Vo domain responsible for proton translocation. Chemical modification has been used to probe the structure of the nucleotide binding sites, this group has suggested that disulfide bond formation may play a role in regulation of V-ATPase activity in vivo. Reassembly studies have been used to test the function of individual subunits, including a protein shared between the V-ATPase and AP-2 adaptor complexes. More recently, they have begun mutagenesis studies of the yeast V-ATPase to identify residues important in V-ATPase activity.
Four specific aims will be pursued in the proposed studies. To determine the structure and function of the noncatalytic nucleotide binding sites on the B subunit, cysteine-scanning mutagenesis will be employed. The role of B subunit isoforms in activity and intracellular targeting of V-ATPases will also be tested. To further define the structure of the catalytic A subunit, residues participating in nucleotide binding will be identified by site-directed mutagenesis. The proximity and role of conserved A subunit cysteine residues in regulation of vacuolar acidification will also be further probed. Studies of the 100 kDa subunit will focus on identification of second-site suppressors of mutations demonstrated to affect function, on elucidation of the topography of the 100 kDa subunit, and on identification of mutations which confer concanamycin resistance. Finally, the arrangement and function of accessory subunits, including AP50 and the VMA6 gene product, will be investigated. These studies should provide further insight into the structure and regulation of this important family of H+-ATPase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034478-15
Application #
6018630
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1985-08-30
Project End
2001-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
15
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Tufts University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02111
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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