Summary The proton-pumping vacuolar ATPase (V-ATPase; V1Vo-ATPase) is an essential enzyme complex found in the endomembrane system of all eukaryotic organisms and in the plasma membrane of some animal cells. The V- ATPase functions in ATP hydrolysis-driven acidification of subcellular compartments or the extracellular space, a process vital for basic cellular processes including endocytosis, protein trafficking, bone remodeling, urine acidification, sperm maturation, and neurotransmitter release. Loss of V-ATPase in animal cells is embryonic lethal, while partial loss of function (or hyperactivity) has been linked to numerous human diseases such as renal tubular acidosis, osteoporosis, diabetes, male infertility, neurodegeneration, cancer, and AIDS. Fighting these diseases on a molecular level will require a detailed understanding of the structure, catalytic mechanism, and regulation of the eukaryotic V-ATPase complex. In cells, V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, a condition under which the complex dissociates into V1- ATPase and Vo proton channel sectors that are both functionally silenced. Despite its important role in V- ATPase function, the molecular mechanism of activity regulation by reversible disassembly is poorly understood, a gap in knowledge that is largely due to the lack of high-resolution structural information and a defined in vitro model system to study the process under controlled conditions. The immediate goal of this project is to obtain high-resolution structural and mechanistic information aimed at a better understanding of V- ATPase's catalytic mechanism and unique mode of regulation. We will address these aspects of V-ATPase structure and regulatory mechanisms with the following Specific Aims: (1) Atomic structures of lipid nanodisc reconstituted Vo and V1Vo and mechanism of proton pumping, and (2), Molecular determinants of V-ATPase regulation by reversible disassembly. We study the structure and regulation of the V-ATPase from the yeast Saccharomyces cerevisiae, a powerful model system for the mammalian enzyme due to the high level of conservation of the enzyme's structure and mechanism across species, the ease of genetic manipulation, and the ability to obtain highly purified enzyme with a defined subunit composition. The long-term objective of the project is to develop strategies aimed at either promoting or inhibiting the process of reversible disassembly of the human V-ATPase that will allow modulation of the activity of the enzyme in a tissue and subunit isoform dependent manner for therapeutic purposes. 1