Topology and function of "stalk" proteins in the bovine mitochondrial H+-ATPase.
Proton translocating ATPases comprise a hydrophilic sector F1, a membrane sector F0, and, in the case of bovine mitochondria, a connecting "stalk" which is believed to contain the oligomycin sensitivity-conferring protein (OSCP) and coupling factor 6 (F6). The present study was undertaken to verify the accessibility of F6 and OSCP to trypsin and to examine the functional consequences of such treatment. Our data show that F1 binds equally to trypsin-treated F0 and untreated F0, but the former complexes exhibit cold lability and only partial sensitivity to oligomycin. Furthermore, these complexes fail to exhibit ATP-driven proton translocation or ATP-32Pi exchange activity. Trypsinization of F0 does not, however, inhibit passive proton conductance through the membrane sector but actually enhances it. Immunological data indicate extensive degradation of OSCP under conditions where F6 proteolysis is insignificant. Intact H+-ATPase complexes are relatively resistant to both the structural and functional effects of trypsin. We conclude that OSCP is predominantly an extrinsic protein which is shielded by F1 in the native membrane. F6 may also be an extrinsic protein but is shielded from trypsinization by OSCP and/or other F0 polypeptides. The exposed, trypsin-sensitive segments of OSCP are not required for passive proton conductance through F0 but may be required for ATP-driven reactions. We propose that bovine mitochondrial OSCP is a functional analogue of subunit b in the Escherichia coli H+-ATPase.
Joshi
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Pringle
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D004579:Electron Transport; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D014357:Trypsin; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein