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. mitochondrial carrier | glucose and glutamine metabolism | Warburg effect | metabolic reprogramming | diabetes M itochondria couple respiratory oxidation of nutrients to ATP synthesis through an electrochemical proton gradient. Proton leak allows partial uncoupling of oxidative phosphorylation, producing heat. Through this mechanism, Uncoupling protein (UCP)1, a member of the mitochondrial carrier family (MCF), regulates adaptive thermogenesis in mammals. In 1997 a protein similar to UCP1 was cloned and named UCP2 (1) based on the assumption that the sequence homology implied a similar function. Whereas UCP1 has a clear-cut uncoupling activity relevant to nonshivering thermogenesis, this is not the case for UCP2. UCP2 has been involved in numerous physiopathological conditions including metabolic disorders, inflammation, ischemic shock, cancer, and aging. Furthermore, changes in UCP2 expression affect metabolic functions (2, 3). It has been suggested that these metabolic actions of UCP2 are due to a mild UCP1-like uncoupling activity (4, 5) that, combined with the generally low levels of UCP2 expression, would regulate the release of reactive oxygen species (ROS) (6) without significantly affecting energy conservation. Although fatty acid-dependent proton transport mediated by UCP2 was reported in reconstituted liposomes (7), a mounting body of evidence argues against UCP2 having an uncoupling activity in vivo (8, 9) and sugge...

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.

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 16 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates, and cofactors across the inner membrane. Here the identification and functional characterization of the mitochondrial GTP/GDP carrier (Ggc1p) is described. The ggc1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported GTP and GDP and, to a lesser extent, the corresponding deoxynucleotides and the structurally related ITP and IDP by a counter-exchange mechanism. Transport was saturable with an apparent K m of 1 M for GTP and 5 M for GDP. It was strongly inhibited by pyridoxal 5-phosphate, bathophenanthroline, tannic acid, and bromcresol purple but little affected by the inhibitors of the ADP/ATP carrier carboxyatractyloside and bongkrekate. Furthermore, in contrast to the ADP/ATP carrier, the Ggc1p-mediated GTP/GDP heteroexchange is H ؉ -compensated and thus electroneutral. Cells lacking the ggc1 gene had reduced levels of GTP and increased levels of GDP in their mitochondria. Furthermore, the knock-out of ggc1 results in lack of growth on nonfermentable carbon sources and complete loss of mitochondrial DNA. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria, where it is required for important processes such as nucleic acid and protein synthesis, in exchange for intramitochondrially generated GDP.In the mitochondrial matrix, GTP is required as an energy source for protein synthesis; as a substrate for the synthesis of tRNA, mRNA, rRNA, and RNA primers; and as a phosphate group donor for the activity of GTP-AMP phosphotransferase (1) and G proteins (2, 3). In several organisms, GTP is synthesized in the mitochondria by succinyl-CoA ligase, which catalyzes the conversion of succinyl-CoA to succinate with the generation of GTP, and by nucleoside diphosphate kinase, which catalyzes the transfer of the ␥ phosphate from ATP to a nucleoside diphosphate to yield nucleotide triphosphates. In Saccharomyces cerevisiae, however, succinyl-CoA ligase produces ATP instead of GTP (4), and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space and absent in the matrix (5). These observations imply that in S. cerevisiae GTP has to be imported into the mitochondria probably via a carrier system embedded in the inner mitochondrial membrane.Despite the importance of GTP in mitochondrial metabolism, the transport of guanine nucleotides has not been characterized in yeast mitochondria, nor has any mitochondrial protein responsible for this transport been identified. There are only two indirect observations that suggest that GTP is transported across the inner mitochondrial membrane of S. cerevisiae. First, mitochondri...

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria–localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death–inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.

SummaryThe malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae . On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. Saccharomyces cerevisiae has two principal pathways for replenishing the intermediates of the Krebs cycle that have been withdrawn for biosynthesis. They are the glyoxylate cycle and the carboxylation of pyruvate to oxaloacetate, catalyzed by two cytosolic isozymes of pyruvate carboxylase. (Mammalian pyruvate carboxylase is in the mitochondrial matrix.) These processes require traffic of substrates across the inner mitochondrial membrane. S. cerevisiae encodes 35 members of the mitochondrial carrier family (1-4), including the dicarboxylate and succinate-fumarate carriers (5, 6). The former catalyzes the import into mitochondria of succinate (or malate) in exchange for phosphate, producing a net uptake of succinate and supply of a substrate to the Krebs cycle (7). The latter exchanges external succinate for fumarate and is required for gluconeogenesis (6,8). Yeast gene disruption strains (⌬-DIC1 and ⌬-SFC1) cannot grow on either ethanol or acetate as sole carbon source, but they grow well on glycerol, pyruvate and other nonfermentable substrates (7, 9). Growth of the ⌬-DIC1 strain on ethanol or acetate is restored by both low concentrations of oxaloacetate and other compounds that start the tricarboxylate cycle (and the oxidation of the acetyl-CoA unconsumed by the glyoxylate cycle) by generating either intramitochondrial oxaloacetate or other Krebs cycle intermediates (7). Therefore, does oxaloacetate enter yeast mitochondria, and if so, how?The S. cerevisiae gene OAC1 1 (formerly known as PMT or YKL120w) encodes a member of the mitochondrial carrier family of hitherto unknown function that is 29% identical to the yeast DIC, its closest relative in yeast and 30% identical to the bovine 2-oxoglutarate/malate carrier. OAC and the dicarboxylate carrier are the only carriers that cluster on a phylogenetic tree with the bovine oxoglutarate/malate carrier (10, 11). Disruption of the OAC1 gene produced no phenotype on rich glycerol medium (12), and its transcript level was higher in synthetic than in rich medium (13).We have overexpressed the OAC in E. coli, reconstituted it into phospholipid vesicles, and shown that it transports oxaloacetate, sulfate, and thiosulfate both in vitro and in vivo. One of its main functions is probably to carry oxaloacetate produced by cytoplasmic pyruvate carboxylase into the mitochondrial matrix.

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