NOTE / NOTE Characterization of the ATP-translocating properties of the predicted Arabidopsis thaliana mitochondrial adenine nucleotide translocator 2 Olesya A. Kharenko and Michele C. Loewen Abstract: 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 K m value of 15 mmolÁL –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. Key words: adenine nucleotide translocator 2, Arabidopsis thaliana, functional characterization, mitochondria, recombinant expression. Re ´sume ´: Dans cette e ´tude, les auteurs de ´montrent leur pre ´diction a ` l’effet que le transporteur du nucle ´otide ade ´nine (ANT2) mitochondrial intervient dans le transport de l’ATP. Que l’ANT2 produit par recombinaison intervienne dans l’ab- sorption de l’ATP dans des prote ´oliposomes confirme l’identification base ´e sur les se ´quences de la prote ´ine comme mem- bre de la famille des transporteurs du nucle ´otide ade ´nine. Les auteurs confirment sa pre ´sence dans des extraits de membranes de levure, lorsqu’on l’exprime chez le Saccharomyces cerevisiae, avec un marqueur C-terminal His6. On a so- lubilize ´ la prote ´ine recombinante avec du Triton X-100 et on l’a enrichie par chromatographie d’affinite ´ sur me ´tal immobi- lise ´, avant de la reconstituer dans des liposomes. Les auteurs ont ainsi confirme ´ la fonctionnalite ´ de la prote ´ine reconstitue ´e en de ´montrant l’absorption du pyridoxal 5’ [ 3 H]ATP sensible au phosphate. Des essais de transport montrent une absorption cine ´tique de premier ordre de l’ATP, avec une valeur Km approximative de 15 mmolÁL –1 . Les essais de compe ´tition indiquent que la prote ´ine reconstitue ´e posse `de une plus grande affinite ´ pour l’ATP. En ge ´ne ´ral, ces re ´sultats montrent que l’ANT2 de l’Arabidopsis constitue un transporteur du nucle ´otide ade ´nine. Mots-cle ´s : transporteur 2 du nucle ´otide ade ´nine, Arabidopsis thaliana, caracte ´risation fonctionnelle, mitochondrie, expres- sion de recombinaison. [Traduit par la Re ´daction] Introduction Adenine nucleotide translocators (ANTs) are a family of proteins that play a major role in energy metabolism, cata- lyzing ATP/ADP exchange across membranes (Kunji 2004). In plants, different ANTs are localized to the inner mem- branes of the endoplasmic reticulum, mitochondria, and chloroplasts (Millar and Heazlewood 2003; Leroch et al. 2008). Various physiological processes and metabolic path- ways are strictly dependent on the ATP pools available and decrease of ANT activity can lead to complete inhibition of cellular metabolism (Neuhaus et al. 1997; Klingenberg 2008). This makes ANT activity essential to cellular growth and development. Arabidopsis thaliana (L.) Heynh. is a known plant model system, the genome of which has been completely se- quenced (Picault et al. 2004). To date, one endoplasmic re- ticulum (ER-ANT1), two plastidic (At1g80300 and At1g15500), and at least three mitochondrial [At5g13490 Received 16 December 2009. Accepted 21 April 2010. Published on the NRC Research Press Web site at botany.nrc.ca on 2 July 2010. O.A. Kharenko and M.C. Loewen. 1 Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada. 1 Corresponding author (e-mail: Michele.Loewen@nrc-cnrc.gc.ca). 685 Botany 88: 685–690 (2010) doi:10.1139/B10-037 Published by NRC Research Press