Proton conduction and bafilomycin binding by the V0 domain of the coated vesicle V-ATPase.
We have previously demonstrated that the V0 domain of the coated vesicle V-ATPase, a 250-kDa integral complex, does not form a functional proton channel (Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773-9778). In the present study we describe dissociation of the V0 complex and separation of the V0 subunits by gel filtration. Dicyclohexylcarbodiimide-inhibitable passive proton conductance of reconstituted vesicles containing reassembled V0 subunits was measured in response to a K+/valinomycin-generated membrane potential. We observed that reconstituted vesicles containing the 17/19-kDa subunits carried out passive proton transport, with the addition of the 38- and 100-kDa subunits increasing proton conductance. Reconstituted vesicles containing the 38- and/or 100-kDa subunits showed no proton transport. Partial separation of the 17- and 19-kDa subunits revealed that the 17-kDa subunit alone carried out proton transport, with increased conductance on the addition of the 19-kDa subunit. These results indicate that the V0 domain possesses the information necessary to form a dicyclohexylcarbodiimide-inhibitable passive proton channel. Bafilomycin binding by native and reassembled V0 complexes was also measured by their ability to protect V-ATPase activity against bafilomycin inhibition. The native V0 domain, the isolated 100-kDa subunit and the 100/38-kDa subunits were able to protect against inhibition by bafilomycin, suggesting that the binding site for bafilomycin resides on the 100-kDa subunit.
Zhang
J
J
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Feng
Y
Y
Forgac
M
M
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001665:Binding Sites; D001693:Biological Transport, Active; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D018942:Macrolides; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D018942:Macrolides; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases