PROJECT SUMMARY V-ATPases are versatile, highly conserved, multi-subunit proton pumps responsible for organelle acidification in virtually all eukaryotic cells. Complete loss of V-ATPase activity is lethal in all eukaryotes except fungi, but mutations in subunit isoforms are linked to distal renal tubule acidosis, infertility, deafness, and osteopetrosis. V-ATPase activity is subverted in cancer to support additional demands on cellular pH homeostasis and promote metastasis by creating an acidic extracellular environment. Endosomal acidification by V-ATPases also promotes entry of many viruses. There is a crucial need to understand the roles and regulation of V- ATPase subunit isoforms and enzyme subpopulations in order to target of V-ATPases in specific locations therapeutically. V-ATPases are regulated by reversible disassembly of the peripheral V1 subcomplex from the integral membrane Vo subcomplex, and RAVE/Rabconnectin-3 complexes play a critical role in this process. We will investigate the structure, mechanism, and subunit composition of the yeast RAVE and mammalian Rabconnectin-3 complexes, which appear to target specific V-ATPase subunit isoforms as part of their activity. Importantly, mutations in Rabconnectin-3 complexes have been associated with epilepsy and neurodegeneration but the underlying disease mechanism is unclear. We previously demonstrated in yeast that organelle-enriched phosphoinositide phospholipids bind differentially to the two a-subunit isoforms of the Vo subcomplex, providing organelle-specific inputs into V-ATPase localization and activity. Similar lipid interactions are observed with mammalian a-subunit isoforms in vitro, and we will extend these studies to characterizing the effects of the interactions in cultured mammalian cells. Finally, although V-ATPases must function in concert with other cellular mechanisms of pH homeostasis, the underlying mechanisms of this coordination are not understood. We will address this question in yeast, where we have discovered that acute or chronic loss of V-ATPase activity triggers endocytosis of a portion of the major H+ export pump, Pma1, from the plasma membrane. We will determine the mechanism of this vacuole to plasma membrane pH crosstalk. Reduced vacuole/lysosome acidification is an early step in aging in both yeast and mammalian cells. We will assess whether aging yeast cells display a loss in coordinated pH homeostasis or emerging defects in the V- ATPase itself and determine whether these processes can be manipulated.