Two distinct regions of the yeast mitochondrial ADP/ATP carrier are photolabeled by a new ADP analogue: 2-azido-3'-O-naphthoyl-[beta-32P]ADP. Identification of the binding segments by mass spectrometry
| Autor: | A.C. Dianoux, C. Fiore, Gérard Brandolin, Florence Noël, Kieffer S, Guy J.-M. Lauquin, Trézéguet, Michel Jaquinod |
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| Rok vydání: | 2000 |
| Předmět: |
Azides
Saccharomyces cerevisiae Photoaffinity Labels Mitochondrion Cleavage (embryo) Biochemistry Mass Spectrometry Adenosine Triphosphate Nucleotide Histidine Cyanogen Bromide Binding site chemistry.chemical_classification Binding Sites ATP transport biology Chemistry biology.organism_classification Peptide Fragments Recombinant Proteins Amino acid Mitochondria Adenosine Diphosphate Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization ATP–ADP translocase Carrier Proteins Mitochondrial ADP ATP Translocases Oligopeptides Protein Processing Post-Translational |
| Zdroj: | Biochemistry. 39(37) |
| ISSN: | 0006-2960 |
| Popis: | A novel photoactivatable radioactive ADP derivative, namely, 2-azido-3'-O-naphthoyl-[beta-(32)P]ADP (2-azido-N-[(32)P]ADP), was synthesized with the aim at mapping the substrate binding site(s) of the yeast mitochondrial ADP/ATP carrier. It was used with mitochondria isolated from genetically modified strains of Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isoform of the carrier. In darkness, 2-azido-N-[(32)P]ADP was reversibly bound to the carrier in mitochondria, without being transported. Upon photoirradiation, only the ADP/ATP carrier was covalently radiolabeled among all mitochondrial proteins. Specificity of labeling was demonstrated since carboxyatractyloside (CATR), a potent inhibitor of ADP/ATP transport, totally prevented the incorporation of the photoprobe. To localize the radioactive region(s), the purified photolabeled carrier was submitted to CNBr or hydroxylamine cleavage. The resulting fragments were characterized and identified by SDS-PAGE, Western blotting, amino acid sequencing, and MALDI-MS and ESI-MS analyses. Two short photolabeled distinct segments, eight and nine residues long, were identified: S183-R191, located in the central part of the ADP/ATP carrier; and I311-K318, belonging to its C-terminal end. Plausible models of organization of the nucleotide binding site(s) of the carrier involving the two regions specifically labeled by 2-azido-N-[(32)P]ADP are proposed. |
| Databáze: | OpenAIRE |
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