THE FORMATION OF A DISULFIDE CROSS-LINK BETWEEN THE 2 SUBUNITS DEMONSTRATES THE DIMERIC STRUCTURE OF THE MITOCHONDRIAL OXOGLUTARATE CARRIER

Isolated oxoglutarate carrier (OGC) can be cross-linked to dimers by d isulfide-forming reagents such as Cu2+-phenanthroline and diamide. Ace tone and other solvents increase the extent of Cu2+-phenanthroline-ind uced cross-linking of OGC. Cross-linked OGC re-incorporated in proteol iposomes fully retains the oxoglutarate transport activity. The amount of cross-linked OGC calculated by densitometry of scanned gels depend s 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 (sta ined with Coomassie brilliant blue) amounts to 75% of the total protei n. Approximately the same cross-linking efficiency was evaluated from Western blots. Cross-linking of OGC is prevented by SH reagents and re versed bq SH-reducing reagents, which shows that it is mediated by dis ulfide bridge(s). The formation of S-S bridge(s) requires the native s tate of the protein, since it is suppressed by SDS and by heating. Fur thermore, the extent of cross-linking is independent of OGC concentrat ion indicating that disulfide bridge(s) must be formed between the two subunits of native dimers. The number and localization of disulfide b ridge(s) in the cross-linked OGC were examined by peptide fragmentatio n and subsequent cleavage of disulfide bond(s) by beta-mercaptoethanol . Our experimental results show that cross-linking of OGC is accomplis hed by a single disulfide bond between the cysteines 184 of the two su bunits and suggest that these residues in the putative transmembrane h elix four are fairly close to the twofold axis of the native dimer str ucture.