A novel photoactivatable radioactive ADP derivative, na
mely, 2-azido-3'-
O-naphthoyl-[
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32P]ADP (2-azido-N-[
32P]ADP), was synthesized with the ai
m at
mapping the substrate binding site(s) of theyeast
mitochondrial ADP/ATP carrier. It was used with
mitochondria isolated fro
m genetically
modifiedstrains of
Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isofor
m of the carrier.In darkness, 2-azido-N-[
32P]ADP was reversibly bound to the carrier in
mitochondria, without beingtransported. Upon photoirradiation, only the ADP/ATP carrier was covalently radiolabeled a
mong all
mitochondrial proteins. Specificity of labeling was de
monstrated since carboxyatractyloside (CATR), apotent inhibitor of ADP/ATP transport, totally prevented the incorporation of the photoprobe. To localizethe radioactive region(s), the purified photolabeled carrier was sub
mitted to CNBr or hydroxyla
minecleavage. The resulting frag
ments were characterized and identified by SDS-PAGE, Western blotting,a
mino acid sequencing, and MALDI-MS and ESI-MS analyses. Two short photolabeled distinct seg
ments,eight and nine residues long, were identified: S183-R191, located in the central part of the ADP/ATPcarrier; and I311-K318, belonging to its C-ter
minal end. Plausible
models of organization of the nucleotidebinding site(s) of the carrier involving the two regions specifically labeled by 2-azido-N-[
32P]ADP areproposed.