Subunit a facilitates aqueous access to a membrane-embedded region of subunit c in Escherichia coli F1F0 ATP synthase.
Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occurs in the middle of the membrane at Asp-61 on transmembrane helix 2 of subunit c. Previously, the reactivity of cysteines substituted into F(0) subunit a revealed two regions of aqueous access, one extending from the periplasm to the middle of the membrane and a second extending from the middle of the membrane to the cytoplasm. To further characterize aqueous accessibility at the subunit a-c interface, we have substituted Cys for residues on the cytoplasmic side of transmembrane helix 2 of subunit c and probed the accessibility to these substituted positions using thiolate-reactive reagents. The Cys substitutions tested were uniformly inhibited by Ag(+) treatment, which suggested widespread aqueous access to this generally hydrophobic region. Sensitivity to N-ethylmaleimide (NEM) and methanethiosulfonate reagents was localized to a membrane-embedded pocket surrounding Asp-61. The cG58C substitution was profoundly inhibited by all the reagents tested, including membrane impermeant methanethiosulfonate reagents. Further studies of the highly reactive cG58C substitution revealed that NEM modification of a single c subunit in the oligomeric c-ring was sufficient to cause complete inhibition. In addition, NEM modification of subunit c was dependent upon the presence of subunit a. The results described here provide further evidence for an aqueous-accessible region at the interface of subunits a and c extending from the middle of the membrane to the cytoplasm.
Steed
P Ryan
PR
Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706, USA.
Fillingame
Robert H
RH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D019943:Amino Acid Substitution; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D005033:Ethylmaleimide / Q000378:metabolism; D008698:Mesylates / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism*; D017494:Proton Pumps / Q000378:metabolism; D012834:Silver / Q000378:metabolism; D013194:Staining and Labeling; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002250:Carbon Radioisotopes; D008698:Mesylates; D050505:Mutant Proteins; D021122:Protein Subunits; D017494:Proton Pumps; D013439:Sulfhydryl Reagents; D012834:Silver; C074502:methanethiosulfonate; D000255:Adenosine Triphosphate; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide