DeLeon-Rangel Jessica, Zhang Di, Vik Steven B
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376, USA.
Arch Biochem Biophys. 2003 Oct 1;418(1):55-62. doi: 10.1016/s0003-9861(03)00391-6.
The importance of the second transmembrane span of subunit a of the ATP synthase from Escherichia coli has been established by two approaches. First, biochemical analysis of five cysteine-substitution mutants, four of which were previously constructed for labeling experiments, revealed that only D119C, found within the second transmembrane span, was deleterious to ATP synthase function. This mutant had a greatly reduced growth yield, indicating inefficient ATP synthesis, but it retained a significant level of ATP-driven proton translocation and sensitivity to N,N(')-dicyclohexyl-carbodiimide, indicating more robust function in the direction of ATP hydrolysis. Second, the entire second transmembrane span was probed by alanine-insertion mutagenesis at six different positions, from residues 98 to 122. Insertions at the central four positions from residues 107 to 117 resulted in the inability to grow on succinate minimal medium, although normal levels of membrane-bound ATPase activity and significant levels of subunit a were detected. Double mutants were constructed with a mutation that permits cross-linking to the b subunit. Cross-linked products in the mutant K74C/114iA were seen, indicating no major disruption of the a-b interface due to the insertion at 114. Analysis of the K74C/110iA double mutant indicated that K74C is a partial suppressor of 110iA. In summary, the results support a model in which the amino-terminal, cytoplasmic end of the second transmembrane span has close contact with subunit b, while the carboxy-terminal, periplasmic end is important for proton translocation.
通过两种方法确定了大肠杆菌ATP合酶a亚基第二个跨膜结构域的重要性。首先，对五个半胱氨酸取代突变体进行生化分析，其中四个先前是为标记实验构建的，结果表明只有位于第二个跨膜结构域内的D119C对ATP合酶功能有害。该突变体的生长产量大幅降低，表明ATP合成效率低下，但它保留了相当水平的ATP驱动的质子转运以及对N,N'-二环己基碳二亚胺的敏感性，表明在ATP水解方向上功能更强。其次，通过在六个不同位置（从第98位到第122位残基）进行丙氨酸插入诱变来探测整个第二个跨膜结构域。在第107位到第117位残基的中间四个位置插入导致无法在琥珀酸基本培养基上生长，尽管检测到正常水平的膜结合ATP酶活性和相当水平的a亚基。构建了一个允许与b亚基交联的突变体的双突变体。在突变体K74C/114iA中看到了交联产物，表明由于在114位的插入，a-b界面没有受到重大破坏。对K74C/110iA双突变体的分析表明，K74C是110iA的部分抑制子。总之，结果支持了一个模型，其中第二个跨膜结构域的氨基末端、细胞质末端与b亚基紧密接触，而羧基末端、周质末端对质子转运很重要。
