The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF‐hand Ca²⁺‐binding motifs in their N‐terminal domains. Both proteins and their C‐terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H⁺. Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca²⁺ on the external side of the inner mitochondrial membrane, where the Ca²⁺‐binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca²⁺ through a mechanism independent of Ca²⁺ entry into mitochondria, and suggest a novel mechanism of Ca²⁺ regulation of the aspartate/malate shuttle.

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites, nucleotides, and cofactors through this membrane and thereby connect and/or regulate cytoplasm and matrix functions. ATP-Mg is transported in exchange for phosphate, but no protein has ever been associated with this activity. We have isolated three human cDNAs that encode proteins of 458, 468, and 489 amino acids with 66–75% similarity and with the characteristic features of the mitochondrial carrier family in their C-terminal domains and three EF-hand Ca²⁺-binding motifs in their N-terminal domains. These proteins have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties and their targeting to mitochondria demonstrate that they are isoforms of the ATP-Mg/P_i carrier described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one isoform was present in all of the tissues investigated. Because phosphate recycles via the phosphate carrier in mitochondria, the three isoforms of the ATP-Mg/P_i carrier are most likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and hence for the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations.

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.

The Ca²⁺-sensitive dehydrogenases of the mitochondrial matrix are, so far, the only known effectors to allow Ca²⁺ signals to couple the activation of plasma membrane receptors to the stimulation of aerobic metabolism. In this study, we demonstrate a novel mechanism, based on Ca²⁺-sensitive metabolite carriers of the inner membrane. We expressed in Chinese hamster ovary cells aralar1 and citrin, aspartate/glutamate exchangers that have Ca²⁺-binding sites in their sequence, and measured mitochondrial Ca²⁺ and ATP levels as well as cytosolic Ca²⁺ concentration with targeted recombinant probes. The increase in mitochondrial ATP levels caused by cell stimulation with Ca²⁺-mobilizing agonists was markedly larger in cells expressing aralar and citrin (but not truncated mutants lacking the Ca²⁺-binding site) than in control cells. Conversely, the cytosolic and the mitochondrial Ca²⁺ signals were the same in control cells and cells expressing the different aralar1 and citrin variants, thus ruling out an indirect effect through the Ca²⁺-sensitive dehydrogenases. Together, these data show that the decoding of Ca²⁺ signals in mitochondria depends on the coordinate activity of mitochondrial enzymes and carriers, which may thus represent useful pharmacological targets in this process of major pathophysiological interest.

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier protein family, most of which have not yet been functionally identified. Here the identification of the mitochondrial carrier for S ‐adenosylmethionine (SAM) Sam5p is described. The corresponding gene has been overexpressed in bacteria and the protein has been reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, (i) the Sam5p–GFP protein was found to be targeted to mitochondria; (ii) the cells lacking the gene for this carrier showed auxotrophy for biotin (which is synthesized in the mitochondria by the SAM‐requiring Bio2p) on fermentable carbon sources and a petite phenotype on non‐fermentable substrates; and (iii) both phenotypes of the knock‐out mutant were overcome by expressing the cytosolic SAM synthetase (Sam1p) inside the mitochondria.