Translocation of loops regulates transport activity of mitochondrial ADP/ATP carrier deduced from formation of a specific intermolecular disulfide bridge catalyzed by copper-o-phenanthroline.
The cross-linking reagent copper-o-phenanthroline complex (Cu(OP)2) specifically caused a decrease in the amount of the 30-kDa ADP/ATP carrier in bovine submitochondrial particles associated predominantly with formation of a 60-kDa protein consisting of a cross-linked dimer of the carrier. However, Cu(OP)2 had no effect on mitochondria. The transport of ADP via the carrier through submitochondrial particle membranes was found to be inhibited in parallel with the progress of intermolecular cross-linking. Analysis of the cross-linked site showed that a disulfide bridge was formed only between two Cys56 residues in a pair of the first loops facing the matrix space. The transport inhibitor bongkrekic acid, which locks the m-state conformation of the carrier, had no effect on disulfide bridge formation catalyzed by Cu(OP)2, but carboxyatractyloside, which locks the c-state conformation by acting from the cytosolic side, completely inhibited the cross-linking. These results show that the ADP/ATP carrier functions as a dimer form, and a pair of the first loops protrudes into the matrix space in the m-state, but possibly intrudes into the membrane in the c-state. Thus, it is suggested that a pair of the first loops acts as a gate and that its opening and closing are regulated by their translocation.
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004220:Disulfides; D010618:Phenanthrolines; D000226:Mitochondrial ADP, ATP Translocases; C025205:1,10-phenanthroline