Abstract

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.

Highlights

  • We have previously demonstrated thatthe V, domain The coated vesicle V-ATPase is composed of two domains

  • Dicyclohexylcarbodiimide-inhibitable catalytic and noncatalytic nucleotide binding sites located on ucrtitpnreeonaangsnsipnttsttisosiaahvpnileenio.snW pier1ncrte7tr,goow/eot1raobia9etsns-ahiKekcnsrtDosg+vhena/eemvdpdsaaurbuldoclbitetntdhuoadoiannntmctirVicoote,eysnnccscodoiuannfuorbs-tcfrguthtiiearneetneundi3tcects8ooere-uwdanR.attvsaeneptescdidastmoiusmnc1setlsi0eeaevt0dssmiet-ucukpvbroteDrernesodadatitncaolseinienunsp-bo--tsd211ho5o07le00(muc,Ab3)ak-i8iDklan,i1Dnzac9adaco,tcoiBnao,mntnas(pdiiu1lnneb6fsxou,Csrne1,mu,vEi3bte)s,su.na,Tn(ra1iheft62sest)pe5.Vroe0Tfc,dhktimidesDvsoo1eaomll7ecyc-ciakao(uiDt3mnlia,oaprrn1ecl4emmosx,fuaa1tbwsih5nsu)eisn.t1VhTia0t,tsh0ihssse,eeu3rmisbe8ntsubr,tpnuel1oegic9ntdtrss,uaaialrbsaeVnlnea,dd vesicles containing the 38- andor 100-kDa subunits for the sensitivityof proton translocation to DCCD [17]

  • 17- and 19-kDa subunits revealed that the 17-kDa sub- vesicles contain a significant populationof V, domains notcomunit alone carried out proton transport, with increasedplexed with V, [16]

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Summary

Introduction

We have previously demonstrated thatthe V, domain The coated vesicle V-ATPase is composed of two domains.

Results
Conclusion

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