Organization of the F0 sector of Escherichia coli proton-ATPase: the polar loop region of subunit c extends from the cytoplasmic face of the membrane

Abstract

The membrane-spanning F0 sector of the Escherichia coli H+-transporting ATP synthase (EC 3.6.1.34) contains multiple copies of subunit, c, a 79 amino acid residue protein that is thought to insert in the membrane like a hairpin with two membrane traversing .alpha.-helices. The center of the protein is much more polar than the putative transmembrane .alpha.-helices and had been postulated to play a crucial role in coupling H+ translocation through F0 to ATP synthesis in the membrane extrinsic, F1 sector of the complex. However, the direction of insertion of subunit c in the membrane has not been established. We show here that the "polar loop" lies on the F1 binding side of the membrane. A peptide corresponding to Lys34 .fwdarw. Ile46 of the polar loop was synthesized. Antisera were generated to the Lys34 .fwdarw. Ile46 cognate peptide, and the polyclonal antipeptide IgG was shown to bind to a crude F0 fraction by using enzyme-linked immunosorbent assays. The antipeptide serum did not bind tightly enough to F0 to disrupt function. However, a polyclonal antiserum made to purified, whole subunit c was shown to block the binding of F1 to the F0 exposed in F1-stripped membranes. Incubation of the antisubunit c serum with the peptide reduced the inhibitory effect of the antiserum on the binding of F1 to F0. The reversal of inhibition by the peptide was specific to the antisubunit c serum in that the peptide had no effect on inhibition of F1 binding to F0 by antiserum to subunit a of F0. We conclude that the antisubunit c serum blocks F1 binding to the cytoplasmic side of the inner membrane by recognizing epitope(s) in the Lys34 .fwdarw. Ile46 sequence.