Structure-function relationships of the C-terminal end of the Saccharomyces cerevisiae ADP/ATP carrier isoform 2.
The adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in the cellular energy metabolism. Two regions of Anc2p from Saccharomyces cerevisiae are specifically photolabeled using a photoactivable ADP derivative; they are the central matrix loop, m2, and the C-terminal end. To get more insights into the structure-function relationships of the C-terminal region during nucleotide transport, we have developed two independent approaches. In the first we have deleted the last eight amino acids of Anc2p (Anc2pDeltaCter) and demonstrated that the C-terminal end of Anc2p plays an essential role in yeast growth on a non-fermentable carbon source. This resulted from impaired nucleotide binding properties of the Anc2pDeltaCter variant in line with conversion of ADP binding sites from high to low affinity. In the second we probed the ligand-induced conformational changes of Anc2p C-terminal end (i) by assessing its accessibility to anti-C-terminal antibodies and (ii) by measuring intrinsic fluorescence changes of an Anc2p mutant containing only one tryptophan residue located at its C-terminal end (Anc2p3Y-u). We show that the C-terminal region is no further accessible to antibodies when Anc2p binds non-transportable analogues of ADP. Besides, Trp-316 fluorescence is highly increased upon ligand binding, suggesting large conformational changes. Taken together, our results highlight the involvement of the Anc2p C-terminal region in nucleotide recognition, binding, and transport.
Clémençon
Benjamin
B
Laboratoire de Biochimie et Biophysique des Systè Intégrés, Institut de Recherches en Technologies et Sciences du Vivant, UMR 5092 CNRS-Commissariat à l'Energie Atomique-Université Joseph Fourier, F-38054 Grenoble cedex 9, France.
Rey
Martial
M
Dianoux
Anne-Christine
AC
Trézéguet
Véronique
V
Lauquin
Guy J-M
GJ
Brandolin
Gérard
G
Pelosi
Ludovic
L
D000244:Adenosine Diphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D007700:Kinetics; D008024:Ligands; D008928:Mitochondria / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship
D008024:Ligands; C112225:PET9 protein, S cerevisiae; D020033:Protein Isoforms; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D000226:Mitochondrial ADP, ATP Translocases