PROJECT SUMMARY V-ATPases are versatile, highly conserved, multi-subunit proton pumps responsible for organelle acidification in virtually all eukaryotic cells. Under the parent proposal, we are seeking to understand the regulation of V- ATPases, including the roles of subunit isoforms and enzyme subpopulations, in order to distinguish, and eventually target, V-ATPases in specific locations. 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. Visualizing the localization of V-ATPase subunits and regulators by wide-field and fluorescence microscopy is a critical component of this work. We have been able to visualize reversible release of V1 subunits from Vo in membranes as extracellular conditions vary. Recently, we have found that the V-ATPases become more disassembled as yeast cells age; this disassembly results in vacuole alkalinization but can be reversed by caloric restriction, an established mechanism for prolonging lifespan in multiple organisms. This result is exciting as mammalian cells show declines in lysosomal acidification in age that are poorly understood. We also observe glucose-dependent changes in association of the RAVE complex with the vacuolar membrane and have shown that loss of RAVE function shortens lifespan, consistent with its role in V-ATPase assembly. Much of our work continues to be in the yeast model system, and in addition to the inherent challenges of imaging in small cells, some of the proteins that signal and drive V-ATPase assembly changes are present at low levels in cells. We are requesting an administrative supplement for purchase of an improved camera, along with an updated workstation and software for its operation. These additions to an existing wide-field microscope will allow us to better detect and localize the proteins involved in V-ATPase regulation as cells respond to diverse conditions.