title: Yeast ADP/ATP carrier isoform 2: conformational dynamics and role of the RRRMMM signature sequence methionines.
abstract: The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure.
authors.affiliation: Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant (DSV), Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Biologie à Grande Echelle, Grenoble, F-38054, France; Université Joseph Fourier, Grenoble, F-38000, France; INSERM, U1038, Grenoble, F-38054, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Clémençon
authors.affiliation: Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant (DSV), Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Biologie à Grande Echelle, Grenoble, F-38054, France; Université Joseph Fourier, Grenoble, F-38000, France; INSERM, U1038, Grenoble, F-38054, France.
authors.forename: Martial
authors.initials: M
authors.lastname: Rey
authors.affiliation: Laboratoire de Physiologie Moléculaire et Cellulaire, UMR 5095 CNRS, Institut de Biochimie et Génétique Cellulaires, 1 rue Camille Saint-Saëns, F-33077 Bordeaux, France; Université Victor Segalen, Bordeaux, F-33076, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Trézéguet
authors.affiliation: Université Joseph Fourier, Grenoble, F-38000, France; CEA, DSV, Institut de Biologie Structurale, Grenoble, F-38054, France; Laboratoire de Spectrométrie de Masse des Protéines, UMR 5075 CNRS, Grenoble, F-38027, France. Electronic address: eric.forest@ibs.fr.
authors.forename: Eric
authors.initials: E
authors.lastname: Forest
authors.affiliation: Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant (DSV), Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Biologie à Grande Echelle, Grenoble, F-38054, France; Université Joseph Fourier, Grenoble, F-38000, France. Electronic address: ludovic.pelosi@cea.fr.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Pelosi
mesh_terms: D020816:Amino Acid Motifs; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D001865:Bongkrekic Acid / Q000494:pharmacology; D041961:Deuterium Exchange Measurement; D008715:Methionine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D035362:Transcription Factor TFIID / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; C112225:PET9 protein, S cerevisiae; D020033:Protein Isoforms; D029701:Saccharomyces cerevisiae Proteins; C090313:TAF14 protein, S cerevisiae; D035362:Transcription Factor TFIID; D001865:Bongkrekic Acid; D001278:Atractyloside; D000226:Mitochondrial ADP, ATP Translocases; D008715:Methionine; C003853:carboxyatractyloside