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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Anderson, Vernon
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Upstate Medical University
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Graham, Laurie A; Finnigan, Gregory C; Kane, Patricia M (2018) Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field. Traffic 19:385-390
Velivela, Swetha Devi; Kane, Patricia M (2018) Compensatory Internalization of Pma1 in V-ATPase Mutants in Saccharomyces cerevisiae Requires Calcium- and Glucose-Sensitive Phosphatases. Genetics 208:655-672
Banerjee, Subhrajit; Kane, Patricia M (2017) Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast. Mol Biol Cell 28:2518-2530
Kane, Patricia M (2016) Proton Transport and pH Control in Fungi. Adv Exp Med Biol 892:33-68
Smardon, Anne M; Nasab, Negin Dehdar; Tarsio, Maureen et al. (2015) Molecular Interactions and Cellular Itinerary of the Yeast RAVE (Regulator of the H+-ATPase of Vacuolar and Endosomal Membranes) Complex. J Biol Chem 290:27511-23
Deranieh, Rania M; Shi, Yihui; Tarsio, Maureen et al. (2015) Perturbation of the Vacuolar ATPase: A NOVEL CONSEQUENCE OF INOSITOL DEPLETION. J Biol Chem 290:27460-72
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
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
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
Diakov, Theodore T; Tarsio, Maureen; Kane, Patricia M (2013) Measurement of vacuolar and cytosolic pH in vivo in yeast cell suspensions. J Vis Exp :

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