The long term goals of this research are to determine the mechanism and regulation of the vacuolar (H+)- ATPases (V-ATPases). The V-ATPases are ATP-dependent proton pumps that function in both normal and disease processes, including membrane traffic, viral infection, urinary acidification, bone resorption and tumor invasion. V-ATPases are multisubunit complexes composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. V-ATPases are regulated in vivo by reversible dissociation of the V1 and V0 domains. The first objective of this proposal is to test the role of helical swiveling within the V0 domain in proton transport. We have obtained evidence for helical swiveling (rotation of a helix about its long axis) in both subunit a and the proteolipid subunits. We hypothesize that swiveling of helices containing transport critical residues functions in proton translocation through V0. The second objective of this proposal is to elucidate the mechanism by which glucose regulates V-ATPase assembly in yeast. We have developed a novel genetic screen for regulators of V-ATPase assembly and used this screen to identify the Ras/cAMP/PKA pathway as a key regulator. We hypothesize that there are novel regulators in addition to PKA that control V-ATPase assembly, and have recently identified protein phosphatase PP1 as one such regulator. We will test these hypotheses and achieve our objectives by pursuing the following Specific Aims.
Specific Aim 1 - To test the hypothesis that helical swiveling within the V0 domain functions in proton transport, we will determine the effect of intramolecular, disulfide-mediated cross-linking between adjacent helices within both subunit a and subunit c'on proton transport activity. We expect that if helical swiveling is required for proton transport, preventing helical swiveling by cross-linking adjacent helices within subunit a or subunit c'will inhibit activity.
Specific Aim 2 - To determine the mechanism by which glucose regulates V-ATPase assembly in yeast, we will identify and characterize additional novel regulators of V-ATPase assembly using our modified genetic screen. These regulators include both essential and non-essential genes whose mutation blocks V- ATPase dissociation or reverses PKA-mediated assembly. The proposed research is significant because it will greatly advance our understanding of the mechanism by which V-ATPases carry out proton transport and facilitate the identification of novel regulators of V-ATPase assembly. Because V-ATPases are highly conserved between yeast and mammalian cells, as is the use of regulated assembly to control V-ATPase activity, and because regulators such as glucose, PKA and aldolase control assembly in both yeast and higher eukaryotes, these studies will likely provide important insight into control of V-ATPase assembly in mammalian systems. These insights will in turn facilitate the development of therapies to modulate V-ATPase activity that could prove effective in the treatment of diseases, such as viral infection, osteoporosis and cancer, in which V- ATPases participate.

Public Health Relevance

The proposed research is relevant to human health because the V-ATPases, which are the focus of this proposal, are involved in many disease processes, including viral infection, osteoporosis and cancer. A better understanding of the mechanisms by which V-ATPases carry out proton transport and are regulated in vivo will facilitate the development of therapies for modulating V-ATPase activity that could prove effective in the treatment of diseases in which V-ATPases participate. The proposed research is also relevant to the mission of NIGMS by increasing our understanding of fundamental transport processes and their regulation as they relate to both normal and disease states.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM034478-29
Application #
8652319
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Anderson, Vernon
Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Tufts University
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02111
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
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
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
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
Kawasaki-Nishi, Shoko; Yamaguchi, Akihito; Forgac, Michael et al. (2007) Tissue specific expression of the splice variants of the mouse vacuolar proton-translocating ATPase a4 subunit. Biochem Biophys Res Commun 364:1032-6
Inoue, Takao; Forgac, Michael (2005) Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V1 domain and subunit a of the V0 domain. J Biol Chem 280:27896-903
Inoue, Takao; Wang, Yanru; Jefferies, Kevin et al. (2005) Structure and regulation of the V-ATPases. J Bioenerg Biomembr 37:393-8
Shen, Ruichao; Inoue, Takao; Forgac, Michael et al. (2005) Synthesis of photoactivatable acyclic analogues of the lobatamides. J Org Chem 70:3686-92
Wang, Yanru; Inoue, Takao; Forgac, Michael (2005) Subunit a of the yeast V-ATPase participates in binding of bafilomycin. J Biol Chem 280:40481-8
Shao, Elim; Forgac, Michael (2004) Involvement of the nonhomologous region of subunit A of the yeast V-ATPase in coupling and in vivo dissociation. J Biol Chem 279:48663-70

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