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The Cytoplasmic Loops of Subunit a of Escherichia coli ATP Synthase May Participate in the Proton Translocating Mechanism*S⃞

By Kyle J. Moore, Christine M. Angevine, Owen D. Vincent, Brian E. Schwem and Robert H. Fillingame

Abstract

Subunit a plays a key role in promoting H+ transport and the coupled rotary motion of the subunit c ring in F1F0-ATP synthase. H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F0 subunit c. H+ are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2–5 of subunit a based upon the chemical reactivity of Cys substituted into these helices. Here we substituted Cys into loops connecting TMHs 1 and 2 (loop 1–2) and TMHs 3 and 4 (loop 3–4). A large segment of loop 3–4 extending from loop residue 192 loop to residue 203 in TMH4 at the lipid bilayer surface proved to be very sensitive to inhibition by Ag+. Cys-161 and -165 at the other end of the loop bordering TMH3 were also sensitive to inhibition by Ag+. Further Cys substitutions in residues 86 and 93 in the middle of the 1–2 loop proved to be Ag+-sensitive. We next asked whether the regions of Ag+-sensitive residues clustered together near the surface of the membrane by combining Cys substitutions from two domains and testing for cross-linking. Cys-161 and -165 in loop 3–4 were found to cross-link with Cys-202, -203, or -205, which extend into TMH4 from the cytoplasm. Further Cys at residues 86 and 93 in loop 1–2 were found to cross-link with Cys-195 in loop 3–4. We conclude that the Ag+-sensitive regions of loops 1–2 and 3–4 may pack in a single domain that packs at the ends of TMHs 3 and 4. We suggest that the Ag+-sensitive domain may be involved in gating H+ release at the cytoplasmic side of the aqueous access channel extending through F0

Topics: Membrane Transport, Structure, Function, and Biogenesis
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2442323
Provided by: PubMed Central
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