V-ATPases are ATP-driven proton pumps that are responsible for organelle acidification in all eukaryotic cells and are recruited to the plasma membrane for specialized functions in osteoclasts, kidney, and the male reproductive tract. V-ATPase activity is intimately linked to protein sorting in the endocytic and biosynthetic pathways, zymogen activation, and pH, calcium, and metal ion homeostasis. V-ATPase activity is also subverted to support metastasis of certain cancers and virus and toxin release from the endocytic pathway into the cytosol. V-ATPases are very highly conserved, and are comprised of a complex of peri- pheral membrane proteins containing the sites of ATP hydrolysis, V1, attached to a membrane complex containing the proton pore, Vo. V1 and Vo sectors must associate for proton pumping to occur, but they also reversibly dissociate under conditions of energy limitation. We propose to continue to use yeast as a model system to explore the structural basis of V-ATPase mechanism and regulation, along with the cellular context of V-ATPase activity, through the following three aims: 1) We will define the interactions of the V1 C and H subunits with the V1 and Vo sectors and probe how these interactions change during glucose deprivation. Work in the current grant period indicated that the V1 sector has two stator stalks, and we hypothesize that the C and H bridge these stalks to different regions of the membrane sector, and from this position, regulate reversible disassembly of V1 from Vo. 2) Reversible disassembly in response to glucose suggests that V- ATPase activity is aligned to the varying needs of the cell through poorly understood metabolic signals. We will explore the cellular basis of this alignment by testing the functional roles of several proteins recently found to interact with the Vo sector and exploiting the sensitivity of diploid cells to H subunit haploin- sufficiency as a means to identify gene products important for V1-Vo reassembly. 3) We will determine the extent to which the V-ATPase is both a general pH regulator in the cell and a pH sensor. Using recently developed methods for flexible and robust vacuolar and cytosolic pH measurement in yeast, we will deter- mine the extent to which V-ATPase activity affects overall pH homeostasis. These experiments may help explain the far-reaching physiological defects of yeast mutants lacking V-ATPase activity. We will also examine the activity of the V-ATPase itself in response to measured cytosolic and vacuolar pH changes.