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 alpha-helices. The center of the protein is much more polar than the putative transmembrane alpha-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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Girvin
M E
ME
Department of Physiological Chemistry, University of Wisconsin, Madison 53706.
Hermolin
J
J
Pottorf
R
R
Fillingame
R H
RH
D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D006180:Proton-Translocating ATPases