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 strain, indicating that the mutant AAC is active, on isolation and reconstitution in egg yolk phosphatidylcholine, the C73S AAC had no transport activity, whereas the wild-type AAC was fully active. Only on addition of cardiolipin was an exchange activity with the C73S AAC obtained. The AACs isolated from the other cysteine mutants did not (C244S) or only marginally (C271S) require cardiolipin for transport on reconstitution. [3H]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 the AAC. It also shows that cardiolipin is required only for translocation 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 phosphatidylcholine vesicles. Other acidic phospholipids tested such as phosphatidylserine and phosphatidic acid did not activate. Among various 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 assayed by 31P 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 of AAC dimer are found, respectively. Presumably, on isolation, cardiolipin is lost from the more labile C73S mutant AAC. Although the absolute requirement for cardiolipin is unique for the C73S AAC, it is concluded that in this mutant, the unmasking of the cardiolipin requirement demonstrates a general cardiolipin requirement of the wild-type AAC and of AACs from other sources.