The mitochondrial carriers (MCs) shuttle a variety of metabolites across the inner mitochondrial membrane (i.m.m.). In man they are encoded by the SLC25 genes. Some MCs have isoforms encoded by different SLC25 genes, whereas the phosphate carrier has two variants arising from an alternative splicing of SLC25A3. Six MCs have been sequenced after purification, and many more have been identified from their transport and kinetic properties following heterologous over-expression and reconstitution into liposomes. All MCs of known function belong to the same protein family, since their polypeptide chains consist of three tandemly related sequences of about 100 amino acids, and the repeats of the different carriers are homologous. They probably function as homodimers, each monomer being folded in the membrane into six transmembrane segments. The functional information obtained in studies with mitochondria and/or the reconstituted system has helped to gain an insight into the physiological role of the MCs in cell metabolism, as have tissue distribution, the use of knock-out mice (and/or yeast) and over-expression in human cell lines (or yeast) of individual carriers and isoforms. At the same time, the cloning and functional identification of many SLC25 genes has made it possible (i) to identify the genes (and their defects) responsible for some diseases, e.g. Stanley syndrome and Amish microcephaly, and (ii) where the genes were already known, to characterize the function of the gene products and hence understand the molecular basis and the symptoms of the diseases, e.g. hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) syndrome and type II citrullinemia. It is likely that further extension and functional characterization of the SLC25 gene family will elucidate other diseases caused by MC deficiency.
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 Ca 2+ -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 unidenti®ed 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 Ca 2+ on the external side of the inner mitochondrial membrane, where the Ca 2+ -binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca 2+ through a mechanism independent of Ca 2+ entry into mitochondria, and suggest a novel mechanism of Ca 2+ regulation of the aspartate/malate shuttle.
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...
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