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.
Ca(2+) signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca(2+) entry in mitochondria via the Ca(2+) uniporter followed by Ca(2+) activation of three dehydrogenases in the mitochondrial matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca(2+) signals via a mechanism that does not require the entry of Ca(2+) in mitochondria, a mechanism depending on the activity of Ca(2+)-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC) and the ATP-Mg/P(i) carriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both types of CaMCs are activated by Ca(2+) in the intermembrane space and function together with the Ca(2+) uniporter in decoding the Ca(2+) signal into a mitochondrial response.
Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralardeficient (Aralar ؊/؊ ) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar ؊/؊ mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar ؊/؊ mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar ؊/؊ mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.
The deposition of L L-amyloid peptide (AL L), the hyperphosphorylation of tau protein and the death of neurons in certain brain regions are characteristic features of Alzheimer's disease. It has been proposed that the accumulation of aggregates of AL L is the trigger of neurodegeneration in this disease. In support of this view, several studies have demonstrated that the treatment of cultured neurons with AL L leads to the hyperphosphorylation of tau protein and neuronal cell death. Here we report that lithium prevents the enhanced phosphorylation of tau protein at the sites recognized by antibodies Tau-1 and PHF-1 which occurs when cultured rat cortical neurons are incubated with AL L. Interestingly, lithium also significantly protects cultured neurons from AL L-induced cell death. These results raise the possibility of using chronic lithium treatment for the therapy of Alzheimer's disease.z 1999 Federation of European Biochemical Societies.
Aralar1 and citrin were identified as calcium binding aspartate/glutamate carriers (AGC) in mitochondria. The presence of calcium binding motifs facing the extramitochondrial space allows the regulation of the transport activity of these carriers by cytosolic calcium and provides a new mechanism to transduce calcium signals in mitochondria without the requirement of calcium entry in the organelle. We now report the complete characterization of a second subfamily of human calcium binding mitochondrial carriers named SCaMC (short calcium-binding mitochondrial carriers). We have identified three SCaMC genes in the human genome. All code for highly conserved proteins (about 70-80% identity), of about 500 amino acids with a characteristic mitochondrial carrier domain at the C terminus, and an N-terminal extension harboring four EF-hand binding motifs with high similarity to calmodulin. All SCaMC proteins were found to be located exclusively in mitochondria, and their N-terminal extensions were dispensable for the correct mitochondrial targeting of the polypeptides. SCaMC-1 is the human orthologue of the rabbit Efinal protein, which was reported to be located in peroxisomes, and SCaMC-2 is the human orthologue of the rat MCSC protein, described as up-regulated by dexamethasone in AR42J cells. One of the SCaMC genes, SCaMC-2, has four variants generated by alternative splicing, resulting in proteins with a common C terminus but with variations in their N-terminal halves, including the loss of one to three EF-hand motifs. These results make SCaMC one of most complex subfamilies of mitochondrial carriers and suggest that the large number of isoforms and splice variants may confer different calcium sensitivity to the transport activity of these carriers.
The glutamate-glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate-glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo (13)C nuclear magnetic resonance labeling with (13)C(2)acetate or (1-(13)C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.
Disruption of the Krebs cycle is a hallmark of cancer. IDH1 and IDH2 mutations are found in many neoplasms, and germline alterations in SDH genes and FH predispose to pheochromocytoma/paraganglioma and other cancers. We describe a paraganglioma family carrying a germline mutation in MDH2, which encodes a Krebs cycle enzyme. Whole-exome sequencing was applied to tumor DNA obtained from a man age 55 years diagnosed with multiple malignant paragangliomas. Data were analyzed with the two-sided Student's t and Mann-Whitney U tests with Bonferroni correction for multiple comparisons. Between six- and 14-fold lower levels of MDH2 expression were observed in MDH2-mutated tumors compared with control patients. Knockdown (KD) of MDH2 in HeLa cells by shRNA triggered the accumulation of both malate (mean ± SD: wild-type [WT] = 1±0.18; KD = 2.24±0.17, P = .043) and fumarate (WT = 1±0.06; KD = 2.6±0.25, P = .033), which was reversed by transient introduction of WT MDH2 cDNA. Segregation of the mutation with disease and absence of MDH2 in mutated tumors revealed MDH2 as a novel pheochromocytoma/paraganglioma susceptibility gene.
We have identified a new calcium-dependent subfamily of mitochondrial carrier proteins with members inSaccharomyces cerevisiae, Caenorhabditis elegans, and various mammalian species. The members of this subfamily have a bipartite structure: a carboxyl-terminal half with the characteristic features of the mitochondrial solute carrier superfamily and an amino-terminal extension harboring various EF-hand domains. A member of this subfamily (that we have termed Aralar) was cloned from a human heart cDNA library. The corresponding cDNA comprises an open reading frame of 2037 base pairs encoding a polypeptide of 678 amino acids. The carboxyl-terminal half of Aralar (amino acids 321-678) has high similarity with the oxoglutarate, citrate, and adenine nucleotide carriers (28 -29% identity), whereas the amino-terminal half (amino acids 1-320) contains three canonical EF-hands. Aralar amino-terminal half was shown to bind calcium by 45 Ca 2؉ overlay and calcium-dependent mobility shift assays. The subcellular localization of the protein in COS cells transfected with Aralar was exclusively mitochondrial. Antibodies against Aralar amino-terminal fusion protein recognized a 70-kDa protein in brain mitochondrial fractions. Northern blot analysis showed that the protein was expressed in heart, brain, and skeletal muscle. The domain structure, mitochondrial localization, and presence in excitable tissues suggests a possible function of Aralar as calcium-dependent mitochondrial solute carrier.
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