Subunits coupling H+ transport and ATP synthesis in the Escherichia coli ATP synthase. Cys-Cys cross-linking of F1 subunit epsilon to the polar loop of F0 subunit c.
Second site suppressor mutations at position 31 of F1 subunit epsilon recouple ATP-driven H+ translocation in the uncoupled Q42E mutant of subunit c of the Escherichia coli F1F0 ATP synthase (Zhang, Y., Oldenburg, M., and Fillingame, R. H. (1994) J. Biol. Chem. 269, 10221-10224). This finding suggests a functional interaction between subunit c and subunit epsilon during the coupling of H+ transport through F0 to ATP synthesis of F1. However, the physical proximity of the two subunits remained to be defined. In this study, Cys residues were introduced into residues in the polar loop region of subunit c surrounding Gln42 and at position 31 of subunit epsilon to see whether the subunits could be cross-linked. Disulfide bridge formation between subunit c and subunit epsilon was observed in membranes of three double mutants, i.e. cA40C/epsilon E31C, cQ42C/epsilon E31C, and cP43C/epsilon E31C, but not in wild type membranes or in membranes of the cA39C/epsilon E31C double mutant. These results indicate that the polar loop of subunit c and the region around residue 31 of subunit epsilon are physically close to each other in the F1F0 complex and support the hypothesis that these two subunits interact directly in the coupling of H+ transport to ATP synthesis. Disulfide cross-linking of the Q42C subunit c and E31C subunit epsilon leads to inhibition of ATPase coupled H+ transport, as might be expected in a model where the catalytic sites of the F1 ATPase alternate during H+ transport-coupled ATP hydrolysis/synthesis. However, a quantitative relationship between the extent of inhibition of transport and the extent of cross-linking could not be established by the methods used here, and the possibility remains that the epsilon-c cross-linked F1F0 complex retains residual H+ transporting activity.
Zhang
Y
Y
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706, USA.
Fillingame
R H
RH
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D002872:Chromosome Deletion; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017354:Point Mutation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine