Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm.
H(+)-transporting F1F0 ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F0 and F1. H(+) transport at the subunit a-c interface in transmembranous F0 drives rotation of a cylindrical c10 oligomer within the membrane, which is coupled to rotation of subunit γ within the α3β3 sector of F1 to mechanically drive ATP synthesis. F1F0 functions in a reversible manner, with ATP hydrolysis driving H(+) transport. ATP-driven H(+) transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag(+) and/or Cd(+2) with the substituted sulfhydryl groups. The H(+) transport pathway mapped via these Ag(+)(Cd(+2))-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H(+) release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd(+2) directly inhibits passive H(+) transport mediated by F0 reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H(+) transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H(+) transport likely interact in a single structural domain, which then functions in gating H(+) release to the cytoplasm.
Department of Biomolecular Chemistry, University of Wisconsin School Medicine and Public Health, Madison, WI 53706-1532.
P Ryan
PR
Steed
Department of Biomolecular Chemistry, University of Wisconsin School Medicine and Public Health, Madison, WI 53706-1532.
Kaitlin A
KA
Kraft
Department of Biomolecular Chemistry, University of Wisconsin School Medicine and Public Health, Madison, WI 53706-1532 rhfillin@wisc.edu.
Robert H
RH
Fillingame
D001692:Biological Transport; D002104:Cadmium / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D012834:Silver / Q000494:pharmacology
D011522:Protons; D002104:Cadmium; D012834:Silver; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases
F0 a–c subunits; F0 half channels; F1F0 ATP synthase; gating mechanism; proton transport