Characterization of the ATP-translocating properties of the predicted Arabidopsis thaliana mitochondrial adenine nucleotide translocator 2

In this study we demonstrate that the predicted Arabidopsis thaliana (L.) Heynh. mitochondrial adenine nucleotide translocator 2 (ANT2) mediates ATP translocation. The demonstration that recombinantly produced ANT2 mediates ATP uptake into proteoliposomes, confirms previous sequence-based identification of the protein as a member of the adenine nucleotide translocator family. Expressed in Saccharomyces cerevisiae with a C-terminal His6 tag, localization was confirmed in yeast membrane extracts. The recombinant protein was solubilized with Triton X-100, enriched by immobilized metal affinity chromatography, and reconstituted into liposomes. Functionality of the reconstituted protein was confirmed by demonstration of pyridoxal 5′-phosphate-sensitive [3H]ATP uptake. Transport assays showed first order kinetic uptake of ATP with an approximate Km value of 15 µmol·L–1. Competition assays indicated that the reconstituted protein had highest specificity for ATP. Overall, these results indicate that Arabidopsis ANT2 is an adenine nucleotide translocator.