The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and cofactors across the inner mitochondrial membrane. In Saccharomyces cerevisiae, NAD ؉ is synthesized outside the mitochondria and must be imported across the permeability barrier of the inner mitochondrial membrane. However, no protein responsible for this transport activity has ever been isolated or identified. In this report, the identification and functional characterization of the mitochondrial NAD ؉ carrier protein (Ndt1p) is described. Mitochondria contain in their matrix the universal hydrogen transfer coenzyme NAD ϩ , which serves to transfer hydrogen from substrates to the respiratory chain by oxidative phosphorylation. In addition to its well known role as a coenzyme in redox reactions, NAD ϩ exerts other important functions in mitochondria. In Saccharomyces cerevisiae, mitochondrial NADH has been shown to participate in Fe/S protein biogenesis (1) and to be the source for NADPH (2), which is required in mitochondria for oxidative stress protection and for specific biogenesis reactions. NAD ϩ has been shown also to serve critical regulatory functions in gene transcription, enzyme activity, and other important processes through ADP-ribosylation and deacetylation reactions (3-5). In addition, the increase in the NAD ϩ /NADH ratio in mitochondria seems to be important for the extension of the life-span of yeast by calorie restriction (6). The enzymes of NAD ϩ biosynthesis are generally believed to be localized outside the mitochondria (Refs. 7-9, but see Refs. 10 and 11), therefore, NAD ϩ must be imported into these organelles. For a long time nicotinamide adenine dinucleotides were known to be unable to cross the inner membranes of mitochondria (12). However, NAD ϩ has been shown to be taken up by intact plant mitochondria, the uptake being concentration-and temperature-dependent and specifically inhibited by an azido derivative of NAD ϩ (13, 14). Moreover, using human cultured cells harvested under quiescent conditions (6 -8 days after medium change), Rustin et al. (15) observed a depletion of mitochondrial NAD ϩ and an influx of NAD ϩ into the mitochondrial matrix of these cells after adding external NAD ϩ to digitonin-permeabilized cells.These studies contradicted the notion of mitochondrial inner membrane impermeability to pyridine coenzymes and led to the hypothesis that NAD ϩ is transported into mitochondria by a carrier-mediated system. However, the one or more proteins responsible for the observed transport activities have not been hitherto isolated or identified. In this study we provide evidence that the gene products of YIL006W and YEL006W, named Ndt1p and Ndt2p, respectively, are two isoforms of the mitochondrial NAD ϩ transporter in S. cerevisiae. These proteins are 373 and 335 amino acids long, respectively, possess the characteristics of the MCF, 2 and display a high degree (70%) of homology. Ndt1p was overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles,...

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.

Mitochondrial carriers are a family of proteins that transport metabolites, nucleotides, and cofactors across the inner mitochondrial membrane thereby connecting cytosolic and matrix functions. The essential cofactor coenzyme A (CoA) is synthesized outside the mitochondrial matrix and therefore must be transported into mitochondria where it is required for a number of fundamental processes. In this work we have functionally identified and characterized SLC25A42, a novel human member of the mitochondrial carrier family. The SLC25A42 gene (Haitina, T., Lindblom, J., Renström, T., and Fredriksson, R., 2006, Genomics 88, 779 -790) was overexpressed in Escherichia coli, purified, and reconstituted into phospholipid vesicles. Its transport properties, kinetic parameters, and targeting to mitochondria demonstrate that SLC25A42 protein is a mitochondrial transporter for CoA and adenosine 3,5-diphosphate. SLC25A42 catalyzed only a counter-exchange transport, exhibited a high transport affinity for CoA, dephospho-CoA, ADP, and adenosine 3,5-diphosphate, was saturable and inhibited by bongkrekic acid and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A42 is to import CoA into mitochondria in exchange for intramitochondrial (deoxy)adenine nucleotides and adenosine 3,5-diphosphate. This is the first time that a mitochondrial carrier for CoA and adenosine 3,5-diphosphate has been characterized biochemically.The mitochondrial carrier family, or the solute carrier family 25 (SLC25), 3 comprises a large group of proteins that transport a variety of substrates across the inner mitochondrial membrane and, in a few cases, across other membranes (1, 2). Common structural features of the mitochondrial carrier family members consist in a tripartite structure (three repeats of ϳ100 amino acids), the presence of two transmembrane ␣-helices separated by hydrophilic loops in each repeat, and the presence of a signature motif at the C terminus of the first helix in each repeat (Ref. 3 and references therein). The SLC25 family is by far the largest of the currently known 43 SLC families. The Saccharomyces cerevisiae genome contains 35 members, that of Arabidopsis thaliana 58, and the human genome at least 48 SLC25 members. Until now, nearly 30 members and isoforms of this family have been identified in humans. These include the uncoupling protein and the carriers for ADP/ATP, phosphate, 2-oxoglutarate/malate, citrate, carnitine/acylcarnitine, dicarboxylates, ornithine and other basic amino acids, oxodicarboxylates, deoxynucleotides and thiamine pyrophosphate, aspartate-glutamate, glutamate, S-adenosylmethionine, ATPMg/Pi, pyrimidine nucleotides, and adenine nucleotides in peroxisomes (see Ref. 1 for a review and Refs. 4 -8). The present investigation was undertaken to identify the function of SLC25A42, a novel member of the SLC25 family recently found in the human genome (9). SLC25A42 is 318 amino acids long and is highly expressed in virtually all tissues, in most at higher levels than ma...

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