CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca(2+)-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

Dihydrolipoamide dehydrogenase (DLD, E3) is a flavoprotein common to pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenases. We found two novel DLD mutations (p.I40Lfs*4; p.G461E) in a 19year-old patient with lactic acidosis and a complex amino- and organic aciduria consistent with DLD deficiency, manifesting progressive exertional fatigue. Muscle biopsy showed mitochondrial proliferation and lack of DLD cross-reacting material. Riboflavin supplementation determined the complete resolution of exercise intolerance with the partial restoration of the DLD protein and disappearance of mitochondrial proliferation in the muscle. Morphological and functional studies support the riboflavin chaperon-like role in stabilizing DLD protein with rescue of its expression in the muscle.

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.

The human SLC25A42 protein, ortholog of mitochondrial carrier Leu5p of S. cerevisiae, transports Coenzyme A and Adenosine 3’,5’-diphosphate G. Fiermonte, E. Paradies, S. Todisco, C.M.T. Marobbio, M.A Di Noia, and F. Palmieri Department of Pharmaco-Biology, Laboratory and Molecular Biology, University of Bari, Bari, Italy The essential cofactor Coenzyme A (CoA) is required in many intra-mitochondrial metabolic pathways. The CoA is synthesized outside the mitochondrial matrix, therefore must be transported into mitochondria. In S. cerevisiae, the mitochondrial carrier Leu5p is involved in the accumulation of CoA in the mitochondrial matrix. In fact, deletion of LEU5 (leu5) causes a reduction of mitochondrial coenzyme A (CoA) levels and growth defect on YP supplemented with glycerol or other non fermentative carbon sources. The closest relatives of Leu5p in human are SLC25A16 (37% identity) and SLC25A42 (31% identity). In this study we provide direct evidence that SLC25A42 is a novel transporter of CoA. SLC25A42 is localized in the mitochondrial inner membrane and is highly expressed in virtually all tissues. This protein was overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles, and shown to transport CoA, dephospho-CoA, Adenosine 3’,5’-diphosphate (PAP), and (deoxy)adenine nucleotides with high specificity and by a counter-exchange mechanism. The expression of SLC25A42 protein in LEU5 cells fully restores the phenotype of the LEU5 strain, indicating that the main function of both proteins is probably to catalyze the entry of CoA into mitochondria in exchange for adenine nucleotides and PAP.

The 2-oxoglutarate carrier (OGC) belongs to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. Initially, OGC was characterized by determining substrate specificity, kinetic parameters of transport, inhibitors and molecular probes that form covalent bonds with specific residues. It was shown that OGC specifically transports oxoglutarate and certain carboxylic acids. The substrate specificity combination of OGC is unique, although many of its substrates are also transported by other mitochondrial carriers. The abundant recombinant expression of bovine OGC in Escherichia coli and its ability to functionally reconstitute into proteoliposomes made it possible to deduce the individual contribution of each and every residue of OGC to the transport activity by a complete set of cys-scanning mutants. These studies give experimental support for a substrate binding site constituted by three major contact points on the even-numbered α-helices and identifies other residues as important for transport function through their crucial positions in the structure for conserved interactions and the conformational changes of the carrier during the transport cycle. The results of these investigations have led to utilize OGC as a model protein for understanding the transport mechanism of mitochondrial carrier

Mitochondrial carriers are a superfamily 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. Although several members of this family have been characterized, the functions of many others still remains unknown. For instance, the carrier responsible for coenzyme A (COA) transport into mitochondria has not yet been identified. CoA is an essential cytosolic-synthesized cofactor required in many intra-mitochondrial pathways, such as Krebs cycle, fatty acid β-oxidation and fatty acid synthesis (for the activation of mitochondrial ACP). In this work, we have identified SLC25A42 as the human gene responsible for the transport of CoA into mitochondria. The protein encoded by SLC25A42 is localized in the inner mitochondrial membrane and is ubiquitously expressed, aIthough at different levels. Its functional characterization has been carried out on bacterially expressed protein, upon its purification and reconstitution into phospholipid vesicles. In the reconstituted system the recombinant protein, exhibited high transport affinity for CoA, dephospho-CoA, ADP and adenosine 3',5'-diphosphate (PAP). The main physiological role of SLC25A42 is to import CoA into mitochondria in exchange for intra-mitochondrial adenine nucleotides and/or PAP. The export of PAP out of the mitochondria is crucial, since the catabolism of this cytotoxic molecule, produced in the organelles by the transfer of the 4'-phosphopantetheine prosthetic group of CoA to ACP, takes place in the cytosol. This is the first time that a mitochondrial carrier for CoA and PAP has been identified and characterized biochemically.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.