Arrangement of the multicopy H+-translocating subunit c in the membrane sector of the Escherichia coli F1F0 ATP synthase.
The multicopy subunit c of the H+-transporting F1F0 ATP synthase of Escherichia coli is thought to fold across the membrane as a hairpin of two hydrophobic alpha-helices. The conserved Asp61, centered in the second transmembrane helix, is essential for H+ transport. In this study, we have made sequential Cys substitutions across both transmembrane helices and used disulfide cross-link formation to determine the oligomeric arrangement of the c subunits. Cross-link formation between single Cys substitutions in helix 1 provided initial limitations on how the subunits could be arranged. Double Cys substitutions at positions 14/16, 16/18, and 21/23 in helix 1 and 70/72 in helix 2 led to the formation of cross-linked multimers upon oxidation. Double Cys substitutions in helix 1 and helix 2, at residues 14/72, 21/65, and 20/66, respectively, also formed cross-linked multimers. These results indicate that at least 10 and probably 12 subunits c interact in a front-to-back fashion to form a ring-like arrangement in F0. Helix 1 packs at the interior and helix 2 at the periphery of the ring. The model indicates that the Asp61 carboxylate is centered between the helical faces of adjacent subunit c at the center of a four-helix bundle.
Jones
P C
PC
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Jiang
W
W
Fillingame
R H
RH
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D016376:Oligonucleotides, Antisense; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine