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Despite the fundamental importance and high level of compartmentation of mitochondrial nucleotide metabolism in plants, our knowledge concerning the transport of nucleotides across intracellular membranes remains far from complete. Study of a previously uncharacterized Arabidopsis (Arabidopsis thaliana) gene (At4g01100) revealed it to be a novel adenine nucleotide transporter, designated ADNT1, belonging to the mitochondrial carrier family. The ADNT1 gene shows broad expression at the organ level. Green fluorescent protein-based cell biological analysis demonstrated targeting of ADNT1 to mitochondria. While analysis of the expression of beta-glucuronidase fusion proteins suggested that it was expressed across a broad range of tissue types, it was most highly expressed in root tips. Direct transport assays with recombinant and reconstituted ADNT1 were utilized to demonstrate that this protein displays a relatively narrow substrate specificity largely confined to adenylates and their closest analogs. ATP uptake was markedly inhibited by the presence of other adenylates and general inhibitors of mitochondrial transport but not by bongkrekate or carboxyatractyloside, inhibitors of the previously characterized ADP/ATP carrier. Furthermore, the kinetics are substantially different from those of this carrier, with ADNT1 preferring AMP to ADP. Finally, isolation and characterization of a T-DNA insertional knockout mutant of ADNT1, alongside complementation and antisense approaches, demonstrated that although deficiency of this transporter did not seem to greatly alter photosynthetic metabolism, it did result in reduced root growth and respiration. These findings are discussed in the context of a potential function for ADNT1 in the provision of the energy required to support growth in heterotrophic plant tissues.