Structure, Mechanism and Regulation of the V-ATPases

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EXCEEDTHE SPACE PROVIDED. The long term objectives of this proposal are to determine the structure, mechanism and regulation of the vacuolar (H+)-ATPases (or V-ATPases). The V-ATPases are a family of ATP dependent proton pumps that function both in acidification of intracellular compartments and in proton transport across the plasma membrane of certain cells. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, viral and toxin entry, protein processing and degradation and coupled transport of small molecules. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis and tumor metastasis. The V-ATPases are composed of a peripheral V1 domain responsible for ATP hydrolysis and an integral VO domain responsible for proton transport. Although the V-ATPases operate by a rotary mechanism, the details of this mechanism remain uncertain. To further elucidate the arrangement of subunits within the V- ATPase complex and to gain insight into their function, a variety of approaches will be employed, including mutational analysis, cysteine-mediated crosslinking and electron microscopy. Purification of epitope tagged complexes expressed in yeast to determine the feasibility of crystallization trials will also be performed. Among the questions to be addressed are the mechanisms by which the ATPase activity of V1 and passive proton transport by VOare silenced upon dissociation of these domains (an important in vivo regulatory mechanism). Helical contacts between VOsubunits important for proton transport will be elucidated using disulfide-mediated crosslinking and residues critical for transport and inhibitor binding identified by mutagenesis. The role of the intracellular environment and of several critical domains of the V-ATPase in regulating in vivo dissociation will be explored, including the non-homologous region of subunit A and the N- terminal hydrophilic domain of subunit a. A novel screen will be employed to identify mutants blocked in this process. These studies will provide further insight into the structure, mechanism and regulation of the V- ATPases. Because of their role in such processes as cholesterol homeostasis, bone resorption, viral and toxin entry, renal acidification and tumor metastasis, the insights gained are directly relevant to such diverse human diseases as atherosclerosis, osteoporosis, influenza, anthrax, renal disease and cancer.