Proton conduction and bafilomycin binding by the V0 domain of the coated vesicle V-ATPase.
Proton conduction and bafilomycin binding by the V0 domain of the coated vesicle V-ATPase.
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发表时间：
1994-09
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通讯作者：
Jimin Zhang;Yu Feng;M. Forgac
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作者：
Jimin Zhang;Yu Feng;M. Forgac
我们以前已经证明，包被的囊泡V-ATPase的V0结构域是一个250 kDa的完整复合体，不形成一个功能质子通道(Zhang，J.，Myers，M.，和Forgac，M.(1992)J.Biol)。化学。267、9773-9778)。在本研究中，我们描述了V0复合体的解离和通过凝胶过滤分离V0亚基。测量了含有重组V0亚基的重组囊泡的二环己基碳二亚胺可抑制的被动质子电导，以响应K+/呋喃霉素产生的膜电位。我们观察到含有17/19 kDa亚基的重组囊泡进行被动质子转运，38 kDa和100 kDa亚基的加入增加了质子电导。含有38和/或100 kDa亚基的重组囊泡没有质子转运。17 kDa和19 kDa亚基的部分分离表明，17 kDa亚基单独进行质子转运，19 kDa亚基的加入增加了电导。这些结果表明，V0结构域具有形成二环己基碳二亚胺抑制的被动质子通道所需的信息。天然的和重组的V0复合体还通过保护V-ATPase活性对抗巴菲罗星抑制的能力来衡量巴菲罗星与巴菲霉素的结合。天然的V0结构域、分离的100 kDa亚基和100/38 kDa亚基均能抵抗巴菲霉素的抑制，提示巴非霉素的结合部位位于100 kDa亚基上。
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.