The transmembrane domain of subunit b of the Escherichia-coli F 1Fo ATP synthase is sufficient for H+- translocating activity together with subunits a and c

Abstract

Subunit b is indispensable for the formation of a functional H +-translocating Fo complex both in vivo and in vitro. Whereas the very C-terminus of subunit b interacts with F1 and plays a crucial role in enzyme assembly, the C-terminal region is also considered to be necessary for proper reconstitution of Fo into liposomes. Here, we show that a synthetic peptide, residues 1-34 of subunit b (b1-34) [Dmitriev, O., Jones, P.C., Jiang, W. & Fillingame, R.H. (1999) J. Biol. Chem. 274, 15598-15604], corresponding to the membrane domain of subunit b was sufficient in forming an active Fo complex when coreconstituted with purified ac subcomplex. H+ translocation was shown to be sensitive to the specific inhibitor N,N′-dicyclohexylcarbodiimide, and the resulting Fo complexes were deficient in binding of isolated F1. This demonstrates that only the membrane part of subunit b is sufficient, as well as necessary, for H+ translocation across the membrane, whereas the binding of F1 to Fo is mainly triggered by C-terminal residues beyond Glu34 in subunit b. Comparison of the data with former reconstitution experiments additionally indicated that parts of the hydrophilic portion of the subunit b dimer are not involved in the process of ion translocation itself, but might organize subunits a and c in Fo assembly. Furthermore, the data obtained functionally support the monomeric NMR structure of the synthetic b1-34.