Identification of the human mitochondrial ATP-Mg/Pi transporter

The functions of several members of the mitochondrial transporter family found in genome sequences are unknown. At the same time there are other transport activities observed in intact mitochondria that have yet to be associated with specific proteins. An example is the reversible counterexchange of ATP-Mg for Pi that accounts for the net uptake or efflux of ATP-Mg, as Pi recycles rapidly through the membrane via the phosphate carrier. By screening human ESTs with the sequence of the human ADP/ATP carrier (AAC1) we selected clones encoding proteins of unknown function and containing Ca 2 + -binding EF-hand motifs in their sequences. The corresponding cDNAs (accession numbers AJ619961, AJ619962 and AJ619963) encode three proteins (named APC1-3) with 66 –75% identical amino acids, and with Ca 2 + -binding motifs in their N-terminal domains and the characteristic features of the mitochondrial carrier family in their C-terminal domains. They were overexpressed in E. coli, purified and reconstituted into liposomes. The recombinant proteins APC1 and APC2 transported ATP-Mg, phosphate, ATP, ADP and, less effieciently, AMP in an electroneutral H + - compensated counterexchange. The APC-mediated transport was inhibited by mercurials, bathophenanthroline, tannic acid and bromocresol purple. Little inhibition was observed with carboxyatractyloside and bonkrekate (powerful inhibitors of the AAC1). The green fluorescence (GFP) protein fused to APC1-3 was found to be targeted to mitochondria. The transport properties of APC1 and APC2 and their targeting to mitochondria demonstrate that they are responsible for the ATP-Mg/Pi exchange described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one isoform is present in all the tissues investigated. By screening the human genome databases with the cDNAs of APC1, APC2 and APC3, the corresponding genes (SLC25A24, SLC25A23 and SLC25A26, respectively) were found. They were located on three chromosomes, 1p13.3, 19p13.3 and 9q34.13; contained10exons separated by nine introns; and all the splicing junctions occurred in the same nucleotide regions, indicating a triplication of a common ancestral gene. The main function of the APC isoforms is probably to catalyze the net uptake or efflux of adenine nucleotides into or from the mitochondria, thus explaining the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations.