The formation of a disulfide cross-link between the two subunits demonstrates the dimeric structure of the mitochondrial oxoglutarate carrier.
Isolated oxoglutarate carrier (OGC) can be cross-linked to dimers by disulfide-forming reagents such as Cu2+-phenanthroline and diamide. Acetone and other solvents increase the extent of Cu2+ -phenanthroline-induced cross-linking of OGC. Cross-linked OGC re-incorporated in proteoliposomes fully retains the oxoglutarate transport activity. The amount of cross-linked OGC calculated by densitometry of scanned gels depends on the method of staining, since cross-linked OGC exhibits a higher sensitivity to Coomassie brilliant blue as compared to silver nitrate. Under optimal conditions the formation of cross-linked OGC dimer (stained with Coomassie brilliant blue) amounts to 75% of the total protein. Approximately the same cross-linking efficiency was evaluated from Western blots. Cross-linking of OGC is prevented by SH reagents and reversed by SH-reducing reagents, which shows that it is mediated by disulfide bridge(s). The formation of S-S bridge(s) requires the native state of the protein, since it is suppressed by SDS and by heating. Furthermore, the extent of cross-linking is independent of OGC concentration indicating that disulfide bridge(s) must be formed between the two subunits of native dimers. The number and localization of disulfide bridge(s) in the cross-linked OGC were examined by peptide fragmentation and subsequent cleavage of disulfide bond(s) by beta-mercaptoethanol. Our experimental results show that cross-linking of OGC is accomplished by a single disulfide bond between the cysteines 184 of the two subunits and suggest that these residues in the putative transmembrane helix four are fairly close to the twofold axis of the native dimer structure.
Bisaccia
F
F
Department of Pharmaco-Biology, University of Bari, Italy.
Zara
V
V
Capobianco
L
L
Iacobazzi
V
V
Mazzeo
M
M
Palmieri
F
F
D000433:1-Propanol / Q000494:pharmacology; D000096:Acetone / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides; D004226:Dithioerythritol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000378:metabolism*; D010618:Phenanthrolines; D011510:Proteolipids / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D007656:Ketoglutaric Acids; D008081:Liposomes; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D010618:Phenanthrolines; D011510:Proteolipids; D013439:Sulfhydryl Reagents; C021723:oxoglutarate translocator; C020485:proteoliposomes; D003958:Diamide; D000096:Acetone; D004226:Dithioerythritol; D000433:1-Propanol; D003488:Cyanogen Bromide; C025205:1,10-phenanthroline