The objectives of this research are to determine the structure, ion transport properties and intracellular distribution of the ATP- dependent proton pump of clathrin-coated vesicles. This pump appears to play s crucial role in intracellular membrane traffic by providing the acidic environment required for ligand-receptor dissociation and receptor recycling following receptor-mediated endocytosis. In addition, the coated vesicle pump represents a new class of ATP-driven proton pumps which acidify a variety of intracellular compartments, including endosomes, lysosomes, Golgi and the vacuoles of plants and lower eukaryotes. Our work will focus on further structural characterization of the coated vesicle proton pump and on determination of its relationship to the proton pumps of other intracellular organelles. We have succeeded in isolation of the coated vesicle (H+)-ATPase and in reconstitution of this pump into artificial lipid vesicles. The purified enzyme contains nine polypeptides of molecular weight 17,000-100,000. On the basis of labeling studies we have suggested that the 73,000 dalton subunit is involved in ATP hydrolysis and the 17,000 dalton subunit forms part of a DCCD-inhibitable proton channel. Further studies are planned to characterize the reactive sites on these polypeptides. Subunit stoichiometry and proximity will be measured by amino acid analysis and covalent crosslinking, respectively. Subunits will be tested for glycosylation or peripheral membrane association, and the disposition of each subunit with respect to the membrane will be determined by labeling with membrane impermeant and hydrophobic reagents and by proteolysis. The ion transport properties of the intact complex and the isolated 17,080 dalton subunit will also be investigated. We have also succeeded in isolation of a series of monoclonal antibodies which recognize the native, detergent-solubilized (H+)- ATPase and immuneprecipitate this enzyme in an active form. These antibodies will be used to test the immunological relationship between the various intracellular proton pumps using both immunocytochemical techniques and by immuneprecipitation of crossreactive species from isolated organelles. These antibodies will also be used to probe for the presence of this pump in the plasma membrane.
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