Functional characterization and purification of a Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein.
A recombinant fusion protein combining the mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p), both from Saccharomyces cerevisiae, has been genetically elaborated with the aim of increasing the polar surface area of the carrier to facilitate its crystallization. The gene encoding the his-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2. The chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, which demonstrated its transport activity. The kinetic exchange properties of Anc2-Cyc1(His6)p and the wild type his-tagged carrier Anc2(His6)p were very similar. However, Anc2-Cyc1(His6)p restored cell growth less efficiently than Anc2(His6)p which correlates with the lower amount found in mitochondria. Purification of Anc2-Cyc1(His6)p in complex with carboxyatractyloside (CATR), a high affinity inhibitor of ADP/ATP transport, was achieved by combining ion-exchange chromatography and ion-metal affinity chromatography in the presence of LAPAO, an aminoxide detergent. As characterized by absorption in the visible range, heme was found to be present in isolated Anc2-Cyc1(His6)p, giving the protein a red color. Large-scale purification of Anc2-Cyc1(His6)p-CATR complex opens up novel possibilities for the use of crystallographic approaches to the yeast ADP/ATP carrier.
Dassa
Emmanuel Philippe
EP
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Département de Réponse et Dynamique Cellulaires, UMR 5092 CNRS-CEA-Université Joseph Fourier, CEA-Grenoble, 17 Avenue des Martyrs, 38054 Grenoble Cedex 9, France.
Dahout-Gonzalez
Cécile
C
Dianoux
Anne-Christine
AC
Brandolin
Gérard
G
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001278:Atractyloside / Q000031:analogs & derivatives*; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D003460:Crystallization; D045304:Cytochromes c / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D006418:Heme; D006639:Histidine; D007700:Kinetics; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
C014059:CYC1 protein, S cerevisiae; D015335:Molecular Probes; C112225:PET9 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D001278:Atractyloside; D006418:Heme; D006639:Histidine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D000226:Mitochondrial ADP, ATP Translocases; C003853:carboxyatractyloside