title: The human mitochondrial ADP/ATP carriers: kinetic properties and biogenesis of wild-type and mutant proteins in the yeast S. cerevisiae.
abstract: The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.
authors.affiliation: Laboratoire de Physiologie Moléculaire et Cellulaire, Institut de Biochimie et Génétique Cellulaires, 1, rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France.
authors.forename: Carine
authors.initials: C
authors.lastname: De Marcos Lousa
authors.forename: Véronique
authors.initials: V
authors.lastname: Trézéguet
authors.forename: Anne-Christine
authors.initials: AC
authors.lastname: Dianoux
authors.forename: Gérard
authors.initials: G
authors.lastname: Brandolin
authors.forename: Guy J-M
authors.initials: GJ
authors.lastname: Lauquin
mesh_terms: D033741:Adenine Nucleotide Translocator 1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D033742:Adenine Nucleotide Translocator 2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D033781:Adenine Nucleotide Translocator 3 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002244:Carbon / Q000378:metabolism; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D008239:Lysine / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D000226:Mitochondrial ADP, ATP Translocases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017246:Ophthalmoplegia, Chronic Progressive External / Q000201:enzymology / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D012331:RNA, Fungal / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
chemical_list: D033741:Adenine Nucleotide Translocator 1; D033742:Adenine Nucleotide Translocator 2; D033781:Adenine Nucleotide Translocator 3; D010446:Peptide Fragments; D012331:RNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D002244:Carbon; D000226:Mitochondrial ADP, ATP Translocases; D008239:Lysine; D000409:Alanine