EXCEEDTHE SPACE PROVIDED. The long term objectives of this proposal are to determine the structure, mechanism and regulation of the vacuolar (H+)-ATPases (or V-ATPases). The V-ATPases are a family of ATP dependent proton pumps that function both in acidification of intracellular compartments and in proton transport across the plasma membrane of certain cells. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, viral and toxin entry, protein processing and degradation and coupled transport of small molecules. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis and tumor metastasis. The V-ATPases are composed of a peripheral V1 domain responsible for ATP hydrolysis and an integral VO domain responsible for proton transport. Although the V-ATPases operate by a rotary mechanism, the details of this mechanism remain uncertain. To further elucidate the arrangement of subunits within the V- ATPase complex and to gain insight into their function, a variety of approaches will be employed, including mutational analysis, cysteine-mediated crosslinking and electron microscopy. Purification of epitope tagged complexes expressed in yeast to determine the feasibility of crystallization trials will also be performed. Among the questions to be addressed are the mechanisms by which the ATPase activity of V1 and passive proton transport by VOare silenced upon dissociation of these domains (an important in vivo regulatory mechanism). Helical contacts between VOsubunits important for proton transport will be elucidated using disulfide-mediated crosslinking and residues critical for transport and inhibitor binding identified by mutagenesis. The role of the intracellular environment and of several critical domains of the V-ATPase in regulating in vivo dissociation will be explored, including the non-homologous region of subunit A and the N- terminal hydrophilic domain of subunit a. A novel screen will be employed to identify mutants blocked in this process. These studies will provide further insight into the structure, mechanism and regulation of the V- ATPases. Because of their role in such processes as cholesterol homeostasis, bone resorption, viral and toxin entry, renal acidification and tumor metastasis, the insights gained are directly relevant to such diverse human diseases as atherosclerosis, osteoporosis, influenza, anthrax, renal disease and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37GM034478-22
Application #
7027490
Study Section
Special Emphasis Panel (NSS)
Program Officer
Preusch, Peter C
Project Start
1985-08-30
Project End
2011-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
22
Fiscal Year
2006
Total Cost
$527,838
Indirect Cost
Name
Tufts University
Department
Physiology
Type
Schools of Medicine
DUNS #
039318308
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
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
Stransky, Laura A; Forgac, Michael (2015) Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly. J Biol Chem 290:27360-9
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
Toei, Masashi; Toei, Satoko; Forgac, Michael (2011) Definition of membrane topology and identification of residues important for transport in subunit a of the vacuolar ATPase. J Biol Chem 286:35176-86

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