Two Distinct Regions of the Yeast Mitochondrial ADP/ATP Carrier Are Photolabeled by a New ADP Analogue: 2-Azido-3'-O-naphthoyl-[
详细信息 查看全文
详细信息 查看全文
- 作者：Anne-Christine Dianoux ; Florence Noë ; l ; Christelle Fiore ; Vé ; ronique Tré ; zé ; guet ; Sylvie Kieffer ; Michel Jaquinod ; Guy J.-M. Lauquin ; and Gé ; rard Brandolin
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：September 19, 2000
- 年：2000
- 卷：39
- 期：37
- 页码：11477 - 11487
- 全文大小：205K
- 年卷期：v.39,no.37(September 19, 2000)
- ISSN：1520-4995
文摘
A novel photoactivatable radioactive ADP derivative, namely, 2-azido-3'-O-naphthoyl-[
-32P]ADP (2-azido-N-[32P]ADP), was synthesized with the aim at mapping the substrate binding site(s) of theyeast mitochondrial ADP/ATP carrier. It was used with mitochondria isolated from genetically modifiedstrains of Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isoform 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 among allmitochondrial proteins. Specificity of labeling was demonstrated 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 submitted to CNBr or hydroxylaminecleavage. 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/ATPcarrier; and I311-K318, belonging to its C-terminal 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.
-32P]ADP (2-azido-N-[32P]ADP), was synthesized with the aim at mapping the substrate binding site(s) of theyeast mitochondrial ADP/ATP carrier. It was used with mitochondria isolated from genetically modifiedstrains of Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isoform 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 among allmitochondrial proteins. Specificity of labeling was demonstrated 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 submitted to CNBr or hydroxylaminecleavage. 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/ATPcarrier; and I311-K318, belonging to its C-terminal 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.