Characterization of the V0 domain of the coated vesicle (H+)-ATPase.
The coated vesicle (H+)-ATPase is composed of two domains, a peripheral V1 domain containing the 73 (A subunit)-, 58 (B subunit)-, 40-, 34-, and 33-kDa subunits and an integral V0 domain containing the 100-, 38-, 19-, and 17 (c subunit)-kDa subunits (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973). In the present manuscript we characterize the V0 domain with respect to its structural and activity properties. Glycerol density gradient separation of solubilized coated vesicle membrane proteins reveals the presence of an excess of V0 domains which migrate with a molecular weight of 250,000 and contain the V0 polypeptides in the same stoichiometry as in the intact V1V0 complex. Like the c subunit in V1V0, the c subunit of the free V0 domain is labeled by [14C]N,N'-dicyclohexylcarbodiimide (DCCD) and is extracted by chloroform:methanol. In addition, a monoclonal antibody specific for the 100-kDa subunit of the intact (H+)-ATPase recognizes the 100-kDa subunit of V0. Tryptic cleavage of the V0 complex gives the same pattern of fragments for the 100- and 38-kDa subunits as in the intact complex, but with an increase in sensitivity, suggesting greater exposure of these subunits in free V0. Proton conduction was measured in reconstituted vesicles containing the V0 domain and in native vesicles stripped of V1. No DCCD-inhibitable proton conduction was observed in either preparation, suggesting that unlike the corresponding F0 domain of F1F0, the free V0 domain is not an open proton channel.
Zhang
J
J
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Myers
M
M
Forgac
M
M
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002725:Chloroform; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology* / Q000648:ultrastructure; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D000432:Methanol; D008970:Molecular Weight; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D046911:Macromolecular Substances; D004024:Dicyclohexylcarbodiimide; D002725:Chloroform; D006180:Proton-Translocating ATPases; D000432:Methanol