title: Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: toward a molecular understanding of the transport mechanism.
abstract: BACKGROUND
  Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters.
  
  
  RESULTS
  In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate.
  
  
  CONCLUSIONS
  Taken together, these data provide a comprehensive molecular characterization of a chloroplast ATP/ADP transporter.
authors.affiliation: CEA, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Deniaud
authors.forename: Pankaj
authors.initials: P
authors.lastname: Panwar
authors.forename: Annie
authors.initials: A
authors.lastname: Frelet-Barrand
authors.forename: Florent
authors.initials: F
authors.lastname: Bernaudat
authors.forename: Céline
authors.initials: C
authors.lastname: Juillan-Binard
authors.forename: Christine
authors.initials: C
authors.lastname: Ebel
authors.forename: Norbert
authors.initials: N
authors.lastname: Rolland
authors.forename: Eva
authors.initials: E
authors.lastname: Pebay-Peyroula
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D060365:Chloroplast Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003902:Detergents; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: C106084:AATP1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060365:Chloroplast Proteins; D003902:Detergents; D010710:Phosphates; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases