Two distinct regions of the yeast mitochondrial ADP/ATP carrier are photolabeled by a new ADP analogue: 2-azido-3 '-O-naphthoyl-[beta-P-32]ADP. Identification of the binding segments by mass spectrometry

A novel photoactivatable radioactive ADP derivative, namely, 2-azido-3'-0-n aphthoyl-[beta-P-32]-ADP (2-azido-N-[P-32]ADP), was synthesized with the ai m at mapping the substrate binding site(s) of the yeast mitochondrial ADP/A TP carrier. It was used with mitochondria isolated from genetically modifie d strains of Saccharomyces cerevisiae, producing the native or the His-tagg ed Anc2p isoform of the carrier. In darkness, 2-azido-N-[P-32]ADP was rever sibly bound to the carrier in mitochondria, without being transported. Upon photoirradiation, only the ADP/ATP carrier was covalently radiolabeled amo ng all mitochondrial proteins. Specificity of labeling was demonstrated sin ce carboxyatractyloside (CATR), a potent inhibitor of ADP/ATP transport, to tally prevented the incorporation of the photoprobe. To localize the radioa ctive region(s), the purified photolabeled carrier was submitted to CNBr or hydroxylamine cleavage. The resulting fragments were characterized and ide ntified by SDS-PAGE, Western blotting, amino acid sequencing, and MALDI-MS and ESI-MS analyses. Two short photolabeled distinct segments, eight and ni ne residues long, were identified: S183-R191, located in the central part o f the ADP/ATP carrier; and I311-K318, belonging to its C-terminal end. Plau sible models of organization of the nucleotide binding site(s) of the carri er involving the two regions specifically labeled by 2-azido-N-[P-32]ADP ar e proposed.