PROTON CONDUCTION AND BAFILOMYCIN BINDING BY THE V-0 DOMAIN OF THE COATED VESICLE V-ATPASE

We have previously demonstrated that the V-0 domain of the coated vesi cle V-ATPase, a 250-kDa integral complex, does not form a functional p roton channel (Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Ch em. 267, 9773-9778). In the present study we describe dissociation of the V-0 complex and separation of the V-0 subunits by gel filtration. Dicyclohexylcarbodiimide-inhibitable passive proton conductance of rec onstituted Vesicles containing reassembled V-0 subunits was measured i n response to a K+/valinomycin-generated membrane potential. We observ ed that reconstituted vesicles containing the 17/19-kDa subunits carri ed out passive proton transport, with the addition of the 38- and 100- kDa subunits increasing proton conductance. Reconstituted vesicles con taining the 38- and/or 100-kDa subunits showed no proton transport. Pa rtial separation of the 17- and 19-kDa subunits revealed that the 17-k Da subunit alone carried out proton transport, with increased conducta nce on the addition of the 19-kDa subunit. These results indicate that the V-0 domain possesses the information necessary to form a dicycloh exylcarbodiimide-inhibitable passive proton channel. Bafilomycin bindi ng by native and reassembled V-0 complexes was also measured by their ability to protect V-ATPase activity against bafilomycin inhibition. T he native V-0 domain, the isolated 100-kDa subunit and the 100/38-kDa subunits were able to protect against inhibition by bafilomycin, sugge sting that the binding site for bafilomycin resides on the 100-kDa sub unit.