Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit.
The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.
D025181:ATP Synthetase Complexes; D003432:Cross-Linking Reagents / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000302:isolation & purification; D013388:Succinimides / Q000378:metabolism
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D013388:Succinimides; D010770:Phosphotransferases; D025181:ATP Synthetase Complexes; D006180:Proton-Translocating ATPases; C011240:dithiobis(succinimidylpropionate)