V-ATPases are conserved proton pumps important to pH homeostasis. Located at the membrane of lysosomes, vacuoles and endosomes, V-ATPases sustain the acidic luminal pH needed for protein sorting and degradation;and for entry of viruses and bacterial toxins into host cells. Cells specialized for active proton secretion such as the 1-intercalated cells of the kidney nephron, express V-ATPases at the plasma membrane where they fine-tune the systemic acid-base balance. It is our goal to demonstrate the fundamental mechanisms that carefully regulate V-ATPase function and assembly in order to gain insight into how V- ATPases assist in controlling luminal, cytosolic, and extracellular pH. V-ATPases are dynamic structures that reversibly disassemble to control pH. In yeast and kidney cells, activity and V-ATPase assembly are coupled with glycolysis, but the mechanisms involved are unclear. Using yeast model systems, we have shown a novel link between V-ATPases and the glucose-fatty acid cycle, suggesting that glucose and lipid metabolism remodel pH homeostasis via effects on V-ATPases. It is our hypothesis that V-ATPase assembly is regulated as a means of maintaining cellular pH homeostasis when metabolism switches between glucose and fatty acids. We propose that a complex consisting of the V-ATPase pump and glycolytic enzymes functionally and structurally couples pH homeostasis and energy metabolism;and that metabolic control of this macromolecular structure regulates V-ATPase assembly.
Three specific aims will test this model:
Aim 1 will dissect the metabolic signals that link V-ATPase to the glycolytic pathway;
Aim 2 will elucidate the mechanisms by which regulation of glycolytic enzymes remodels V-ATPase assembly and activity;
and Aim 3 will establish how activation of the glucose-fatty acid cycle cross-talks to V-ATPases. In order to accomplish the aims proposed, this study will measure V-ATPase assembly and disassembly in vma mutants and metabolic mutants deficient in key steps of glycolysis, 2- oxidation of fatty acids, and the glucose-fatty acid cycle. Parallels will be established between intracellular levels of metabolic intermediates and dynamics of binding between V-ATPase and glycolytic enzymes enabling us to understand how V-ATPases assist cells in adjusting to metabolic changes. Because of the complexity involved in both comprehensive metabolic studies and regulation of V-ATPase pumps by reversible disassembly, S. cerevisiae is an outstanding system to address this mechanism at both the genetic and biochemical levels. By showing the contribution of V-ATPases, new insights into the mechanisms that tune fuel energy selection will emerge. Cancer cells use V-ATPases to regulate pH as a result of changes in metabolism;thus new knowledge on the mechanisms by which V-ATPases maintain pH homeostasis in cancer may be revealed. As pathophysiology of the glucose-fatty acid cycle results in metabolic disorders including diabetes and chronic kidney disease, our studies will also contribute towards their understanding.

Public Health Relevance

Diabetes is a major risk factor for the development and progression of chronic kidney disease (CKD). Diabetes and CKD are important public health problems;both are serious conditions associated with decreased quality of life and have disproportionate impact on certain racial and ethnic groups, especially African Americans, American Indians or Alaska Natives, and Hispanics. This study which focuses on a major problem seen in diabetes and CKD: interconnection between glucose and fats.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086495-04
Application #
8299555
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Chin, Jean
Project Start
2009-08-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$295,990
Indirect Cost
$99,970
Name
University of New Mexico
Department
Biochemistry
Type
Schools of Medicine
DUNS #
868853094
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Chan, Chun-Yuan; Parra, Karlett J (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J Biol Chem 289:19448-57
Rane, Hallie S; Bernardo, Stella M; Hayek, Summer R et al. (2014) The contribution of Candida albicans vacuolar ATPase subunit V?B, encoded by VMA2, to stress response, autophagy, and virulence is independent of environmental pH. Eukaryot Cell 13:1207-21
Parra, Karlett J; Chan, Chun-Yuan; Chen, Jun (2014) Saccharomyces cerevisiae vacuolar H+-ATPase regulation by disassembly and reassembly: one structure and multiple signals. Eukaryot Cell 13:706-14
Michel, Vera; Licon-Munoz, Yamhilette; Trujillo, Kristina et al. (2013) Inhibitors of vacuolar ATPase proton pumps inhibit human prostate cancer cell invasion and prostate-specific antigen expression and secretion. Int J Cancer 132:E1-10
Rane, Hallie S; Bernardo, Stella M; Raines, Summer M et al. (2013) Candida albicans VMA3 is necessary for V-ATPase assembly and function and contributes to secretion and filamentation. Eukaryot Cell 12:1369-82
Raines, Summer M; Rane, Hallie S; Bernardo, Stella M et al. (2013) Deletion of vacuolar proton-translocating ATPase V(o)a isoforms clarifies the role of vacuolar pH as a determinant of virulence-associated traits in Candida albicans. J Biol Chem 288:6190-201
Chan, Chun-Yuan; Prudom, Catherine; Raines, Summer M et al. (2012) Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p). J Biol Chem 287:10236-50