THE RECONSTITUTED ADP ATP CARRIER ACTIVITY HAS AN ABSOLUTE REQUIREMENT FOR CARDIOLIPIN AS SHOWN IN CYSTEINE MUTANTS

Although the site-directed C73S mutation in the ADP/ATP carrier (AAC) AAC2 gene from Saccharomyces cerevisiae produced a glycerol-positive s train, indicating that the mutant AAC is active, on isolation and reco nstitution in egg yolk phosphatidylcholine, the C73S AAC had no transp ort activity, whereas the wild-type AAC was fully active. Only on addi tion of cardiolipin was an exchange activity with the C73S AAC obtaine d. The AACs isolated from the other cysteine mutants did not (C244S) o r only marginally (C271S) require cardiolipin for transport on reconst itution. [H-3]Carboxyatractylate binding as a measure of incorporated AAC molecules was unchanged on addition of cardiolipin in all mutants, indicating that cardiolipin does not increase the incorporation of th e AAC. It also shows that cardiolipin is required only for translocati on and not for binding. The activity of the C73S mutant AAC shows half -saturation with cardiolipin at 2% by weight or at 1.15 mol % in the p hosphatidylcholine vesicles. Other acidic phospholipids tested such as phosphatidylserine and phosphatidic acid did not activate. Among vari ous cardiolipin derivatives, the selectivity for cardiolipin is high. Only monolysocardiolipin still retains 12% activity. After removal of the bulk of phospholipid, the content of bound phospholipids was assay ed by P-31 NMR. By unmasking with SDS, in the wild-type AAC and in the C73S AAC, 6.4 mol and only 1.3 and 2.9 mol of bound cardiolipin/mol o f AAC dimer are found, respectively. Presumably, on isolation, cardiol ipin is lost from the more labile C73S mutant AAC. Although the absolu te requirement for cardiolipin is unique for the C73S AAC, it is concl uded that in this mutant, the unmasking of the cardiolipin requirement demonstrates a general cardiolipin requirement of the wild-type AAC a nd of AACs from other sources.