Characterization of the Interface between γ and ε Subunits of Escherichia coli F1-ATPase

作者：Chunlin Tang, Roderick Capaldi · 发表于：Journal of Biological Chemistry · 年份：1996 · DOI：10.1074/jbc.271.6.3018 · 被引用次数：78 · 研究领域：ATP Synthase and ATPases Research、Mitochondrial Function and Pathology

The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the g...