Hardy Andrew W, Grabar Tammy Bohannon, Bhatt Deepa, Cain Brian D
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA.
J Bioenerg Biomembr. 2003 Oct;35(5):389-97. doi: 10.1023/a:1027363012727.
A homodimer of b subunits constitutes the peripheral stalk linking the F1 and F0 sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F1F0 ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F1F0 ATPase and no detectable ATP-driven proton pumping activity. Expression of the b(N2A,T6A,Q10A) subunit was also oxidative phosphorylation deficient, but the b(N2A,T6A,Q10A) protein was incorporated into an F1F0 complex. Single amino acid substitutions had minimal reductions in F1F0 ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F0, and that these interactions are strongest on the periplasmic side of the bilayer.
b亚基的同型二聚体构成了连接大肠杆菌ATP合酶F1和F0部分的外周柄。每个b亚基都有一个单膜结构域。通过系统诱变研究了对膜结构域的限制。替换膜细胞质侧近端的一段对F1F0 ATP合酶影响最小。然而，周质侧的多个取代导致酶复合物组装缺陷。这些突变体的氧化磷酸化不足以支持生长，生化研究显示几乎没有F1F0 ATP酶，也没有可检测到的ATP驱动的质子泵活性。b(N2A,T6A,Q10A)亚基的表达也存在氧化磷酸化缺陷，但b(N2A,T6A,Q10A)蛋白被整合到F1F0复合物中。单个氨基酸取代对F1F0 ATP合酶功能的降低最小。证据表明，b亚基膜结构域有几个相互作用位点有助于F0的组装，并且这些相互作用在双层膜的周质侧最强。
