Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation
Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation
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DOI：
10.1074/jbc.m608253200
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发表时间：
2006-12-15
影响因子：
4.8
通讯作者：
Yokoyama, Ken
中科院分区：
文献类型：
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作者：
Imamura, Hiromi;Funamoto, Saeko;Yokoyama, Ken
液泡型H+-ATPase(V-ATPase或V型ATPase)是一种多亚单位复合体，由负责ATP水解的水溶性V1复合体和负责质子转运的包埋在膜中的V-O复合体组成。嗜热嗜热菌V-ATPase的V1亚基组成为A(3)B(3)df，其中A和B亚基形成六聚体环状结构。由D和F亚基组成的中心茎贯穿环。在这项研究中，我们通过从单体A和B亚基以及DF亚基在体外重组V1复合体来研究V1复合体的组装途径。将这些组分组装成V1复合体需要将ATP与A亚基结合，尽管ATP的水解不是必需的。在没有DF亚复合体的情况下，A和B单体以依赖于ATP结合的方式组装成A(1)B(1)和A(3)B(3)亚复合体，这表明A和B亚基之间依赖于ATP结合的相互作用是组装成V1复合体的关键步骤。利用荧光共振能量转移对A和B单体组装成A(1)B(1)杂二聚体的动力学分析表明，A亚基在与B亚基结合之前与ATP结合。荧光ADP类似物N-甲基邻氨基苯甲酰ADP(MANT-ADP)与单体A亚基的结合动力学也支持核苷酸与A亚基的快速结合。
Vacuolar-type H+-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V1 complex, responsible for ATP hydrolysis, and a membrane-embedded V-o complex, responsible for proton translocation. The V1 complex of Thermus thermophilus V-ATPase has the subunit composition of A(3)B(3)DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V1 complex by reconstituting the V1 complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V1 complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A(1)B(1) and A(3)B(3) subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V1 complex. Kinetic analysis of assembly of the A and B monomers into the A(1)B(1) heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP(mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.