V-ATPases are ubiquitous and highly conserved proton pumps responsible for organelle acidification in virtually all eukaryotic cells and for proton export in a few cell types. Through their roles in organelle acidification, V-ATPases impact macromolecular degradation, protein sorting, pH homeostasis, and sequestration of ions and nutrients. Recent data has revealed that V-ATPases play a central role in multiple pathophysiological conditions. For example, they help defend against some types of neurodegeneration, but can promote tumor metastasis and osteoporosis. They are thus attractive drug targets, but their complexity also makes them difficult. V-ATPases are multisubunit enzymes comprised of a peripheral complex, the V1 sector, attached to an integral membrane complex, the Vo sector;interaction between these two sectors is a major target of enzyme regulation. We propose to exploit the unparalleled flexibility of the yeast V-ATPase model system to address several issues that are critically important but experimentally intractable in mammalian V-ATPases.
In Aim 1, we will test the hypothesis that the endosome/lysosome signaling lipid PI(3,5)P2 interacts directly with the Vo sector of the V-ATPase and regulates enzyme activity by stabilizing V1- Vo interactions. Depletion of this lipid iin mammals results in neurodegeneration;our experiments may indicate whether loss of organelle acidification is a potential cause.
In Aim 2, we will use compartment-specific ratiometric fluorescent probes to test the contributions of two different yeast subunit isoforms to pH regulation in vivo. Higher eukaryotic cells often have several V-ATPase subunit isoforms whose individual contributions to organelle pH control and regulation are unclear;results from the more experimentally tractable yeast system may serve as a paradigm for isoform-dependent pH control in other cells. Finally, we have found that both acute and chronic loss of V-ATPase activity triggers downregulation of the major plasma membrane proton exporter, suggesting an unexpected level of coordination between the major organelle and plasma membrane pH control mechanisms.
In Aim 3, we will test the hypothesis that loss of organelle acidification, possibly sensed at the endosome, induces ubiquitin-dependent internalization of proton export machinery at the plasma membrane as a compensatory mechanism. Mechanisms for balancing demands of organelle acidification, cytosolic pH control, and proton export are likely present in all cells but are largely unexplored. These experiments will begin to address how organelle acidification is sensed and preserved.

Public Health Relevance

V-ATPases present in all cells help control pH, degrade defective proteins, and respond to different types of environmental stress;because of their importance in these functions, they are potential drug targets for neurodegenerative diseases, cancer, and osteoporosis. The proposed experiments use a widely accepted model system to address how V-ATPases in different cellular locations respond to stress and how the activity of V-ATPases is coordinated with activity of other cellular proteins. Our experiments will provide insights into how V-ATPase activity might be increased or decreased to target specific tissues and disease processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050322-19
Application #
8415511
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Anderson, Vernon
Project Start
1994-03-01
Project End
2016-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
19
Fiscal Year
2013
Total Cost
$307,835
Indirect Cost
$114,835
Name
Upstate Medical University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Smardon, Anne M; Kane, Patricia M (2014) Loss of vacuolar H+-ATPase activity in organelles signals ubiquitination and endocytosis of the yeast plasma membrane proton pump Pma1p. J Biol Chem 289:32316-26
Li, Sheena Claire; Diakov, Theodore T; Xu, Tao et al. (2014) The signaling lipid PI(3,5)P? stabilizes V?-V(o) sector interactions and activates the V-ATPase. Mol Biol Cell 25:1251-62
Smardon, Anne M; Diab, Heba I; Tarsio, Maureen et al. (2014) The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast. Mol Biol Cell 25:356-67
Diakov, Theodore T; Tarsio, Maureen; Kane, Patricia M (2013) Measurement of vacuolar and cytosolic pH in vivo in yeast cell suspensions. J Vis Exp :
Diab, Heba I; Kane, Patricia M (2013) Loss of vacuolar H+-ATPase (V-ATPase) activity in yeast generates an iron deprivation signal that is moderated by induction of the peroxiredoxin TSA2. J Biol Chem 288:11366-77
Lin, Meng; Li, Sheena Claire; Kane, Patricia M et al. (2012) Regulation of vacuolar H+-ATPase activity by the Cdc42 effector Ste20 in Saccharomyces cerevisiae. Eukaryot Cell 11:442-51
Kane, Patricia M (2012) Targeting reversible disassembly as a mechanism of controlling V-ATPase activity. Curr Protein Pept Sci 13:117-23
Li, Sheena Claire; Diakov, Theodore T; Rizzo, Jason M et al. (2012) Vacuolar H+-ATPase works in parallel with the HOG pathway to adapt Saccharomyces cerevisiae cells to osmotic stress. Eukaryot Cell 11:282-91
Tarsio, Maureen; Zheng, Huimei; Smardon, Anne M et al. (2011) Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis. J Biol Chem 286:28089-96
Diakov, Theodore T; Kane, Patricia M (2010) Regulation of vacuolar proton-translocating ATPase activity and assembly by extracellular pH. J Biol Chem 285:23771-8

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