CHARACTERIZATION OF THE INTERFACE BETWEEN GAMMA-SUBUNIT AND EPSILON-SUBUNIT OF ESCHERICHIA-COLI F1-ATPASE

The interaction faces of the gamma and epsilon subunits in the Escheri chia coli F-1-ATPase have been explored by a combination of cross-link ing and chemical modification experiments using several mutant epsilon subunits as follows: epsilon S10C, epsilon H38C, epsilon T43C, epsilo n S65C, epsilon S108C, and epsilon M138C, along with a mutant of the g amma subunit, gamma T106C. The replacement of Ser-10 by a Cys or Met-1 38 by a Cys reduced the inhibition of ECF(1) by the epsilon subunit, w hile the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimi de 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 epsilon T43C) and a Cys at position 106 of the gamma subunit (gamma T106C) also affected t he inhibition of ECF(1) by the epsilon subunit. The various epsilon su bunit mutants were reacted with TFPAM3, and the site(s) of cross-linki ng within the ECF(1) complex was determined. Previous studies have sho wn cross-linking from the Cys at positions 10 and 38 with the gamma su bunit 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. (199 2) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi , R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linkin g was found from a Cys at position 43 to the gamma subunit and from th e Cys at position 138 to a beta subunit. The site of cross-linking fro m Cys-10 of epsilon to the gamma subunit was localized by peptide mapp ing to a region of the gamma subunit between residues 222 and 242. Cro ss-linking from a Cys at position 38 and at position 43 was with the C -terminal part of the gamma subunit, between residues 202 and 286. ECF (1) treated with trypsin at pH 7.0 still binds purified epsilon subuni t, while enzyme treated with the protease at pH 8.0 does not. This ide ntifies sites around residue 70 and/or between 202 and 212 of the gamm a subunit as involved in epsilon subunit binding.