From the Department of Microbiology, University of Osnabrück, Barbarastrasse 11, D-49069 Osnabrück, Germany.
From the Department of Microbiology, University of Osnabrück, Barbarastrasse 11, D-49069 Osnabrück, Germany.
J Biol Chem. 2013 Sep 6;288(36):25880-25894. doi: 10.1074/jbc.M113.484675. Epub 2013 Jul 17.
The ATP synthase (F(O)F1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c10γε and the stator ab2α3β3δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F(O)F1 complexes by affinity chromatography with the use of mutants synthesizing different sets of F(O)F1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F(O)F1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H(+)-translocating unit as intermediate product. Surprisingly, during the biogenesis of F(O)F1, F1 subunit δ is the key player in generating stable F(O). Subunit δ serves as clamp between ab2 and c10α3β3γε and guarantees that the open H(+) channel is concomitantly assembled within coupled F(O)F1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H(+)-translocating enzymes.
大肠杆菌的 ATP 合酶（F(O)F1）将质子穿过细胞质膜的转运与 ATP 的合成或水解偶联。这个纳米马达由转子 c10γε 和定子 ab2α3β3δ 组成。为了研究由膜整合和周围亲水亚基组成的这种多聚体酶复合物的组装，我们结合了使用突变体合成不同的 F(O)F1 亚基来进行分子间二硫键形成的最近邻分析或通过亲和层析纯化部分组装的 F(O)F1 复合物，以及使用突变体合成不同的 F(O)F1 亚基的方法。与延迟的体内组装系统一起，结果表明 F(O)F1 通过亚基复合物以模块化方式组装，从而防止形成功能性 H(+)转运单位作为中间产物。令人惊讶的是，在 F(O)F1 的生物发生过程中，F1 亚基 δ 是生成稳定 F(O)的关键因素。亚基 δ 作为 ab2 和 c10α3β3γε 之间的夹具，并保证在偶联的 F(O)F1 中同时组装开放的 H(+)通道，以维持对生存至关重要的低膜质子通透性，这是多聚体 H(+)转运酶组装的一般前提。
