title: Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit.
abstract: 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.
mesh_terms: 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
chemical_list: 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)