Organization of the F₀ Sector of Escherichia coli H⁺-ATPase: The Polar Loop Region of Subunit c Extends from the Cytoplasmic Face of the Membrane

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 α-helices. The center of the protein is much more polar than the putative transmembrane α-helices and has 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 → Ile₄₆ of the polar loop was synthesized. Antisera were generated to the Lys₃₄ → 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 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 → Ile₄₆ sequence.