Functional characterization and purification of a Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein |
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Authors: | Dassa Emmanuel Philippe Dahout-Gonzalez Cécile Dianoux Anne-Christine Brandolin Gérard |
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Affiliation: | 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. |
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Abstract: | 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. |
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Keywords: | ADP/ATP carrier Mitochondria S. cerevisiae cyc 1 Carboxyatractyloside |
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