Vacuolar H+-ATPase represents a ubiquitous class of ATP driven proton pumps found in the membrane of subcellular compartments of eukaryotic cells. The function of the vacuolar ATPase is to acidify the interior and at the same time energize the membranes of organelles such as clathrin coated vesicles, endosomes, lysosomes, chromaffin granules and Golgi derived vesicles. Very little is known about the catalytic mechanism of this multi subunit complex, mainly due to the lack of detailed structural information. This deficit exists despite the fact that a wide variety of human diseases as fundamental as cancer and osteoporosis are associated with an abnormal activity of the human vacuolar type ATPase. The applicant is studying the three-dimensional structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles by electron microscopy in conjunction with image reconstruction methods. His immediate goals are: 1) to obtain a three dimensional model of the vacuolar ATPase, and 2) to define the arrangement of the subunits within the complex by using monoclonal antibodies to tag the corresponding polypeptides. A longer-term objective is to image the complex at different stages in the catalytic cycle in order to resolve the large conformational changes that accompany catalytic turnover and energy coupling.
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