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 nucleo- tide 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 ad- enine 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 immobi- lized metal affinity chromatography, and reconstituted into liposomes. Functionality of the reconstituted protein was con- firmed by demonstration of pyridoxal 5'-phosphate-sensitive ( 3 H)ATP uptake. Transport assays showed first order kinetic uptake of ATP with an approximate Km value of 15 mmolL -1 . Competition assays indicated that the reconstituted protein had highest specificity for ATP. Overall, these results indicate that Arabidopsis ANT2 is an adenine nucleotide transloca- tor.

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