Acidification of intracellular organelles, including clathrin-coated vesicles, lysosomes, chromaffin granules and other endomembrane compartments, has been shown to be mediated by a new class of proton translocating ATPases, termed vacuolar, or V-type, H+ATPases. In addition, these proton pumps are responsible for urinary acidification, as well as osteoclast-mediated bone reabsorption. The central aim of this proposal is to determine the structural basis of the differences in the regulation of these widely distributed pumps. In these studies, the proton pump of clathrin-coated vesicles will be used as paradigm for study of V-pumps present in organelles of constitutive pathways. Like all vacuolar type H+- ATPases, this enzyme is a large (greater than 850kDa) hetero-oligomer that is composed of between 10 and 15 different subunits. These components are organized into two general domains: an ATP-hydrolytic center (V1), that is peripheral to the membrane, and an intramembranous proton channel, (VO). Recently, the (V1), sector has been dissected biochemically and shown to have seven different subunits (termed A, B, C, D, E, F, and G). Additionally, a novel regulatory protein, with isoforms of 50- and 57-kDa, has been found to activate (V1), and functionally couple ATP-hydrolysis within V1 to proton movement through VO. Recombinant forms of these V1 components will be used to determine potential interactions with VO by directly assessing their effect on proton movement. It is expected that these studies will develop our understanding of a key site of regulation of V-type pumps, the V1/VO interface. In other aspects of this work, the isoform diversity of this regulatory protein and a 116-kDa polypeptide of VO will be studied using molecular and biochemical techniques to determine their potential roles in the function of pumps from organelles of regulatory pathways. In composite, these studies will provide a broad map of the structural basis for differences in V-pump function.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine B Study Section (GMB)
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Scherbenske, M James
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University of Texas Sw Medical Center Dallas
Internal Medicine/Medicine
Schools of Medicine
United States
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