Purification of Histidine-Tagged Mitochondrial ADP/ATP Carrier: Influence of the Conformational States of the C-Terminal Region

Abstract A functional recombinant mitochondrial ADP/ATP carrier from the yeast Saccharomyces cerevisiae that bears a six-histidine tag at the C-terminus, Anc2(His 6 )p, has been engineered to allow its purification by immobilized metal-ion affinity chromatography (IMAC). The tagged carrier was expressed at a level similar to that of unmodified Anc2p as determined by immunodetection and titration of the specific atractyloside binding sites. Anc2(His 6 )p, enriched by chromatography on hydroxyapatite of detergent extracts of mitochondria, was still contaminated by mitochondrial proteins and a large amount of ergosterol. It was highly purified after adsorption on Ni–NTA resin and elution by imidazole buffer, with a 90–95% overall yield. Anc2(His 6 )p interacted differently with immobilized ions depending on whether it was unliganded or bound to carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport, thus indicating that accessibility of the C-terminus is markedly influenced by the conformational state of the carrier. Fluorometric assays demonstrated that purified unliganded Anc2(His 6 )p was in a functional state since it underwent CATR- and BA-sensitive and ADP (or ATP)-induced conformational changes. Large-scale purification of Anc2(His 6 )p–CATR and Anc2(His 6 )p–BA complexes by IMAC will be of major interest for structural analysis of the ADP/ATP carrier.