Vacuolar H-+ translocating ATPases (V-ATPases) are highly conserved enzymes that play a central role in cell physiology stemming from their ability to acidify intracellular compartments, regulate cytosolic pH and calcium concentrations, and establish proton gradients that drive other transporters. These functions can be adapted to many different cellular contexts, and as a result, V-ATPase activity is linked to disease states as diverse as viral infection, metabolic acidosis due to impaired kidney function, and osteoporosis. Skp1 is a highly conserved protein that plays a central role in ubiquitin-dependent proteolysis as an essential member of the family of SCF E3 ubiquitin ligases in addition to a growing number of non-proteolytic roles. SCF complexes show specificity for a wide array of phosphorylated substrates and thus interact with many signal transductional pathways. Recently a new Skp1-containing complex, RAVE, was identified in yeast and shown to contain two other uncharacterized proteins. The RAVE complex does not appear to be an SCF ubiquitin ligase; instead, it appears to post-translationally regulate V-ATPase activity in response to extracellular conditions by modulating the extent of assembly of the V-ATPase complex. In the project proposed here, we will use the yeast Saccharomyces cerevisiae as a model system to examine the structural and functional basis for regulation of V-ATPase activity and assembly by the RAVE complex. We will examine how the three subunits of the RAVE complex interact with each other and with the peripheral V1 sector of the V-ATPase. We will determine whether protein components of RAVE are directly affected by changes in extracellular conditions, and how these changes might affect interaction with the V1 sector. We will develop a system for monitoring interactions between RAVE and the V-ATPase in vivo, using fluorescent derivatives of the two complexes. Finally, we will probe the molecular basis of RAVE action on the V-ATPase by developing an in vitro model for RAVE-dependent assembly of the peripheral and integral membrane sectors of the ATPase and using this model system to test the hypothesis that RAVE assists functional attachment of one of the V1 subunits.
