Mosher M E, White L K, Hermolin J, Fillingame R H
J Biol Chem. 1985 Apr 25;260(8):4807-14.
The uncE114 mutation from Escherichia coli strain KI1 (Nieuwenhuis, F. J. R. M., Kanner, B. I., Gutnick, D. L., Postma, P. W., and Van Dam, K. (1973) Biochim. Biophys. Acta 325, 62-71) was characterized after transfer to a new genetic background. A defective H+-ATPase complex is formed in strains carrying the mutation. Based upon the genetic complementation pattern of other unc mutants by a lambda uncE114 transducing phage, and complementation of uncE114 recipients by an uncE+ plasmid (pCP35), the mutation was concluded to lie in the uncE gene. The uncE gene codes for the omega subunit ("dicyclohexylcarbodiimide binding protein") of the H+-ATPase complex. The mutation was defined by sequencing the mutant gene. The G----C transversion found results in a substitution of Glu for Gln at position 42 of the omega subunit in the Fo sector of the H+-ATPase. The substitution did not significantly impair H+ translocation by Fo or affect inhibition of H+ translocation by dicyclohexylcarbodiimide. Wild-type F1 was bound by uncE114 Fo with near normal affinity, but the functional coupling between F1 and Fo was disrupted. The uncoupling was indicated by an H+-leaky membrane, even when saturating levels of wild-type F1 were bound. Disassociation of F1 from Fo under conditions of assay did partially contribute to the H+ leakiness, but the major contributor to the high H+ conductance was Fo with bound F1. The F1 bound to uncE114 membranes exhibited normal ATPase activity, but ATP hydrolysis was uncoupled from H+ translocation and was resistant to inhibition by dicyclohexylcarbodiimide. The F1 isolated from the uncE114 mutant was modified with partial loss of coupling function. However, this modification did not account for the uncoupled properties of the mutant Fo described above, since these properties were retained after reconstitution of mutant membrane (Fo) with wild-type F1.
将来自大肠杆菌KI1菌株（Nieuwenhuis, F. J. R. M., Kanner, B. I., Gutnick, D. L., Postma, P. W., and Van Dam, K. (1973) Biochim. Biophys. Acta 325, 62 - 71）的uncE114突变转移至新的遗传背景后对其进行了表征。携带该突变的菌株中会形成有缺陷的H⁺ - ATP酶复合体。基于λuncE114转导噬菌体对其他unc突变体的遗传互补模式，以及uncE⁺质粒（pCP35）对uncE114受体的互补作用，得出该突变位于uncE基因中的结论。uncE基因编码H⁺ - ATP酶复合体的ω亚基（“二环己基碳二亚胺结合蛋白”）。通过对突变基因进行测序确定了该突变。发现的G→C颠换导致H⁺ - ATP酶F₀区段的ω亚基第42位的谷氨酰胺被谷氨酸取代。该取代并未显著损害F₀的H⁺转运，也未影响二环己基碳二亚胺对H⁺转运的抑制作用。野生型F₁以接近正常的亲和力与uncE114 F₀结合，但F₁与F₀之间的功能偶联被破坏。即使结合了饱和水平的野生型F₁，H⁺泄漏的膜也表明存在解偶联现象。在测定条件下F₁与F₀的解离确实部分导致了H⁺泄漏，但高H⁺电导率的主要原因是结合了F₁的F₀。与uncE114膜结合的F₁表现出正常的ATP酶活性，但ATP水解与H⁺转运解偶联，并且对二环己基碳二亚胺的抑制作用具有抗性。从uncE114突变体中分离出的F₁发生了修饰，偶联功能部分丧失。然而，这种修饰并不能解释上述突变体F₀的解偶联特性，因为在用野生型F₁重构突变体膜（F₀）后，这些特性仍然保留。
