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

Mark E. Girvin, Joe Hermolin, Richard Pottorf, Robert H. Fillingame

Research output: Contribution to journalArticle

34 Citations (Scopus)

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 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 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 → Ile46 sequence.

Original languageEnglish (US)
Pages (from-to)4340-4343
Number of pages4
JournalBiochemistry
Volume28
Issue number10
StatePublished - 1989
Externally publishedYes

Fingerprint

Cold Climate
Proton-Translocating ATPases
Escherichia coli
Cell Membrane
Membranes
Immune Sera
Peptides
Serum
Immunosorbents
Die casting inserts
Epitopes
Assays
Proteins
Immunoglobulin G
Adenosine Triphosphate
Enzyme-Linked Immunosorbent Assay
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Girvin, Mark E.; Hermolin, Joe; Pottorf, Richard; Fillingame, Robert H.

In: Biochemistry, Vol. 28, No. 10, 1989, p. 4340-4343.

Research output: Contribution to journalArticle

Girvin, Mark E. ; Hermolin, Joe ; Pottorf, Richard ; Fillingame, Robert H. / 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. In: Biochemistry. 1989 ; Vol. 28, No. 10. pp. 4340-4343.
@article{248e652d0d69489fb1eebf4be6a58cc2,
title = "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",
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 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 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 → Ile46 sequence.",
author = "Girvin, {Mark E.} and Joe Hermolin and Richard Pottorf and Fillingame, {Robert H.}",
year = "1989",
language = "English (US)",
volume = "28",
pages = "4340--4343",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "10",

}

TY - JOUR

T1 - Organization of the F0 sector of Escherichia coli H+-ATPase

T2 - The polar loop region of subunit c extends from the cytoplasmic face of the membrane

AU - Girvin, Mark E.

AU - Hermolin, Joe

AU - Pottorf, Richard

AU - Fillingame, Robert H.

PY - 1989

Y1 - 1989

N2 - 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 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 → Ile46 sequence.

AB - 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 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 → Ile46 sequence.

UR - http://www.scopus.com/inward/record.url?scp=0024473883&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024473883&partnerID=8YFLogxK

M3 - Article

C2 - 2475164

AN - SCOPUS:0024473883

VL - 28

SP - 4340

EP - 4343

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 10

ER -