The uncoupling protein dimer can form a disulfide cross-link between the mobile C-terminal SH groups.
Isolated uncoupling protein (UCP) can be cross-linked, by various disulfide-forming reagents, to dimers. The best cross-linking is achieved with Cu2+-phenanthroline oxidation. Because cross-linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross-linking is prevented by SH reagent and reversed by SH-reducing reagents. In mitochondria, cross-linking of UCP with disulfide-forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide-bridge formation does not inhibit GTP-binding to UCP. Cross-linked UCP re-incorporated in proteoliposomes either before or after cross-linking fully retains the H1-transport function. Rapid cross-linking by membrane impermeant reagents indicates a surface localization of the C-terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide-bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross-linked UCP was cleaved by BrCN. The CB-7 C-terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys-292, removed cross-linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C-terminal peptide of 11 residues contains only cystein-304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S-S bridge formation. Obviously, the C-terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross-linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.
Klingenberg
M
M
Institut für Physikalische Biochemie, Universität München, Federal Republic of Germany.
Appel
M
M
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins; D006224:Cricetinae; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D003545:Cysteine / Q000378:metabolism; D004151:Dipeptides / Q000096:biosynthesis*; D004220:Disulfides / Q000096:biosynthesis*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007473:Ion Channels; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D024101:Mitochondrial Proteins; D010084:Oxidation-Reduction / Q000187:drug effects; D010618:Phenanthrolines / Q000494:pharmacology; D014357:Trypsin; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004151:Dipeptides; D004220:Disulfides; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010618:Phenanthrolines; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; C014741:bis(1,10-phenanthroline)copper(2+) ion; D006160:Guanosine Triphosphate; D014357:Trypsin; D019204:GTP-Binding Proteins; D003545:Cysteine; D003488:Cyanogen Bromide