Eya S, Noumi T, Maeda M, Futai M
Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
J Biol Chem. 1988 Jul 25;263(21):10056-62.
Mutant alleles for the alpha subunit of H+-translocating ATPase (FoF1) were cloned from Escherichia coli strains isolated in this laboratory. Determination of their DNA sequence revealed four nonsense mutations (KF3 and KF9, Gln-20----end; KF24, Trp-111----end; KF2, Trp-231----end; KF70, Gln-252----end) and one missense mutation (KF45, Pro-143----Ser). The membranes of all the mutants except strain KF9 (KF3) had 50-70% of ATPase activities of the wild-type. Unlike the F1-ATPase of the wild-type, those of the mutants were insensitive to dicyclohexylcarbodiimide and were easier to solubilize from membranes. As membranes of strain KF24 had F1-ATPase activity, these results suggest that at least a part of the F1-binding sites could be formed without a region between residues 111 and the carboxyl terminus of the alpha subunit. However, normal interactions between Fo and F1 require regions between residues 252 and 271 (carboxyl terminus) and in the vicinity of Pro-143. Membranes of strain KF45 were capable of forming a low ATP-driven H+ gradient, whereas other membranes were not. The possibility that the region between residues 252 and 271 is involved in H+ translocation is discussed.
从本实验室分离得到的大肠杆菌菌株中克隆出了H⁺转运ATP酶（FoF1）α亚基的突变等位基因。对其DNA序列的测定揭示了四个无义突变（KF3和KF9，谷氨酰胺-20→终止；KF24，色氨酸-111→终止；KF2，色氨酸-231→终止；KF70，谷氨酰胺-252→终止）和一个错义突变（KF45，脯氨酸-143→丝氨酸）。除菌株KF9（KF3）外，所有突变体的膜具有野生型ATP酶活性的50 - 70%。与野生型的F1 - ATP酶不同，突变体的F1 - ATP酶对二环己基碳二亚胺不敏感，并且更容易从膜上溶解下来。由于菌株KF24的膜具有F1 - ATP酶活性，这些结果表明，至少部分F1结合位点可以在α亚基第111位残基与羧基末端之间的区域缺失的情况下形成。然而，Fo和F1之间的正常相互作用需要第252位至271位残基（羧基末端）之间以及脯氨酸-143附近的区域。菌株KF45的膜能够形成低ATP驱动的H⁺梯度，而其他膜则不能。文中讨论了第252位至271位残基之间的区域参与H⁺转运的可能性。
