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

Biochemistry. 2000 Sep 19;39(37):11477-87. doi: 10.1021/bi000618l.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / analogs & derivatives*
  • Adenosine Diphosphate / chemical synthesis
  • Adenosine Diphosphate / chemistry
  • Adenosine Diphosphate / metabolism*
  • Adenosine Triphosphate / metabolism
  • Azides / chemistry
  • Azides / metabolism*
  • Binding Sites
  • Carrier Proteins / chemistry
  • Carrier Proteins / metabolism
  • Cyanogen Bromide
  • Histidine / genetics
  • Mass Spectrometry
  • Mitochondria / enzymology*
  • Mitochondrial ADP, ATP Translocases / chemistry
  • Mitochondrial ADP, ATP Translocases / metabolism*
  • Oligopeptides / chemistry
  • Oligopeptides / isolation & purification
  • Oligopeptides / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Peptide Fragments / metabolism*
  • Photoaffinity Labels / chemical synthesis
  • Photoaffinity Labels / chemistry
  • Photoaffinity Labels / metabolism*
  • Protein Processing, Post-Translational
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • 2-azido-3'-O-naphthoyl-ADP
  • Azides
  • Carrier Proteins
  • Oligopeptides
  • Peptide Fragments
  • Photoaffinity Labels
  • Recombinant Proteins
  • Histidine
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Mitochondrial ADP, ATP Translocases
  • Cyanogen Bromide