Purification of histidine-tagged mitochondrial ADP/ATP carrier: influence of the conformational states of the C-terminal region.
A functional recombinant mitochondrial ADP/ATP carrier from the yeast Saccharomyces cerevisiae that bears a six-histidine tag at the C-terminus, Anc2(His(6))p, has been engineered to allow its purification by immobilized metal-ion affinity chromatography (IMAC). The tagged carrier was expressed at a level similar to that of unmodified Anc2p as determined by immunodetection and titration of the specific atractyloside binding sites. Anc2(His(6))p, enriched by chromatography on hydroxyapatite of detergent extracts of mitochondria, was still contaminated by mitochondrial proteins and a large amount of ergosterol. It was highly purified after adsorption on Ni-NTA resin and elution by imidazole buffer, with a 90-95% overall yield. Anc2(His(6))p interacted differently with immobilized ions depending on whether it was unliganded or bound to carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport, thus indicating that accessibility of the C-terminus is markedly influenced by the conformational state of the carrier. Fluorometric assays demonstrated that purified unliganded Anc2(His(6))p was in a functional state since it underwent CATR- and BA-sensitive and ADP (or ATP)-induced conformational changes. Large-scale purification of Anc2(His(6))p-CATR and Anc2(His(6))p-BA complexes by IMAC will be of major interest for structural analysis of the ADP/ATP carrier.
Fiore
C
C
Laboratoire de Biophysique et Biochimie des Systèmes Intégrés, CNRS-UMR 314, CEA-Grenoble, 17 Rue des Martyrs, Grenoble, France.
Trézéguet
V
V
Roux
P
P
Le Saux
A
A
Noël
F
F
Schwimmer
C
C
Arlot
D
D
Dianoux
A C
AC
Lauquin
G J
GJ
Brandolin
G
G
D000900:Anti-Bacterial Agents / Q000737:chemistry; D001278:Atractyloside / Q000031:analogs & derivatives / Q000737:chemistry; D001865:Bongkrekic Acid / Q000737:chemistry; D002846:Chromatography, Affinity; D005453:Fluorescence; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006639:Histidine / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D005656:Fungal Proteins; D020033:Protein Isoforms; D001865:Bongkrekic Acid; D001278:Atractyloside; D006639:Histidine; D000226:Mitochondrial ADP, ATP Translocases; C003853:carboxyatractyloside