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ABSTRACT: The mitochondrial transporter of aspartate-glutamate Aralar/AGC1 is a regulatory component of the malate-aspartate shuttle. Aralar-deficiency in mouse and human causes a shutdown of brain shuttle activity and global cerebral hypomyelination. A lack of neurofilament-labelled processes is detected in the cerebral cortex, but whether different types of neurons are differentially affected by Aralar-deficiency is still unknown. We have now found that Aralar-knockout (Aralar-KO) postnatal mice show hyperactivity, anxiety-like behaviour and hyperreactivity with a decrease of dopamine (DA) in terminal-rich regions. The striatum is the brain region most affected in terms of size, amino acid and monoamine content. We find a decline in vesicular monoamine transporter-2 (VMAT2) levels associated with increased DA metabolism through MAO activity (DOPAC/DA ratio) in Aralar-KO striatum. However, no decrease in DA or in the number of nigral tyrosine hydroxylase-positive cells was detected in Aralar-KO brainstem. Adult Aralar-hemizygous mice presented also increased DOPAC/DA ratio in striatum and enhanced sensitivity to amphetamine. Our results suggest that Aralar-deficiency causes a fall in GSH/GSSG ratio and VMAT2 in striatum that might be related to a failure to produce mitochondrial NADH and to an increase of ROS in the cytosol. The results indicate that the nigrostriatal dopaminergic system is a target of Aralar-deficiency. ©2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/jnc.12096.
Journal of Neurochemistry 12/2012; · 4.06 Impact Factor
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ABSTRACT: Mitochondrial carriers (MC) form a highly conserved family involved in solute transport across the inner mitochondrial membrane in eukaryotes. In mammals, ATP-Mg/Pi carriers, SCaMCs, form the most complex subgroup with four paralogs, SCaMC-1, -2, -3 and -3L, and several splicing variants. Here, we report the tissue distribution and subcellular localization of a mammalian-specific SCaMC paralog, 4930443G12Rik/SCaMC-1Like (SCaMC-1L), which displays unanticipated new features. SCaMC-1L proteins show higher amino acid substitution rates than its closest paralog SCaMC-1. In mouse, SCaMC-1L expression is restricted to male germ cells and regulated during spermatogenesis but unexpectedly its localization is not limited to mitochondrial structures. In mature spermatids SCaMC-1L is detected in the mitochondrial sheath but in previous differentiation stages appears associated to cytosolic granules which colocalize with specific markers of the chromatoid body (CB) in post-meiotic round spermatids and inter-mitochondrial cement (IMC) in spermatocytes. The origin of this atypical distribution was further investigated by transient expression in cell lines. Similarly to male germ cells, in addition to mitochondrial and cytosolic distribution, a fraction of SCaMC-1L-expressing COS-7 cells display cytosolic SCaMC-1L-aggregates which exhibit aggresomal-like features as the CB. Our results indicate that different regions of SCaMC-1L hinder its import into mitochondria and this apparently favours the formation of cytosolic aggregates in COS-7 cells. This mechanism could be also operational in male germ cells and explain the incorporation of SCaMC-1L into germinal granules.
PLoS ONE 01/2012; 7(7):e40470. · 4.09 Impact Factor
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ABSTRACT: The deficiency in the mitochondrial aspartate/glutamate transporter Aralar/AGC1 results in a loss of the malate-aspartate NADH shuttle in the brain neurons, hypomyelination, and additional defects in the brain metabolism. We studied the development of cortico/hippocampal local field potential (LFP) in Aralar/AGC1 knockout (KO) mice. Laminar profiles of LFP, evoked potentials, and unit activity were recorded under anesthesia in young (P15 to P22) Aralar-KO and control mice as well as control adults. While LFP power increased 3 to 7 times in both cortex and hippocampus of control animals during P15 to P22, the Aralar-KO specimens hardly progressed. The divergence was more pronounced in the CA3/hilus region. In parallel, spontaneous multiunit activity declined severely in KO mice. Postnatal growth of hippocampal-evoked potentials was delayed in KO mice, and indicated abnormal synaptic and spike electrogenesis and reduced output at P20 to P22. The lack of LFP development in KO mice was accompanied by the gradual appearance of epileptic activity in the CA3/hilus region that evolved to status epilepticus. Strikingly, CA3 bursts were poorly conducted to the CA1 field. We conclude that disturbed substrate supply to neuronal mitochondria impairs development of cortico-hippocampal LFPs. Aberrant neuronal electrogenesis and reduced neuron output may explain circuit dysfunction and phenotype deficiencies.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 09/2011; 32(2):306-17. · 5.46 Impact Factor
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ABSTRACT: The aralar/AGC1 knockout (KO) mouse shows a drastic decrease in brain aspartate and N-acetylaspartate levels and global hypomyelination, which are attributed to the lack of neuron-produced NAA used by oligodendrocytes as precursor of myelin lipid synthesis. In addition, these mice have a gradual drop in brain glutamine synthesis. We show here that hypomyelination is more pronounced in gray than in white matter regions. We find a lack of neurofilament-labelled processes in hypomyelinated fiber tracks from cerebral cortex but not from those of the cerebellar granule cell layer, which correspond to Purkinje neurons. Therefore, the impaired development or degeneration of neuronal processes in cerebral cortex is independent of hypomyelination. An increase in O4-labelled, immature oligodendrocytes is observed in gray and white matter regions of the aralar KO brain, suggesting a block in maturation compatible with the lack of NAA supplied by neurons. However, no defects in oligodendrocyte maturation were observed in in-vitro-cultured mixed astroglial cultures. We conclude that the primary defect of pyramidal neurons in cerebral cortex is possibly associated with a progressive failure in glutamatergic neurotransmission and may be among the main causes of the pathology of aralar/AGC1 deficiency.
Journal of Neuroscience Research 05/2011; 89(12):2008-17. · 2.74 Impact Factor
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ABSTRACT: In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.
Cellular and Molecular Life Sciences CMLS 01/2011; 68(7):1183-206. · 6.57 Impact Factor
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ABSTRACT: 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.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 01/2011; 31(1):90-101. · 5.46 Impact Factor
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ABSTRACT: The mitochondrial aspartate-glutamate carriers (AGC) aralar (SLC25A12) and citrin (SLC25A13) are components of the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents from NADH to the mitochondrial matrix. Aralar is the main AGC isoform present in the adult brain, and it is expressed mainly in neurons. To search for the other AGC isoform, citrin, in brain glial cells, we used a citrin knockout mouse in which the lacZ gene was inserted into the citrin locus as reporter gene. In agreement with the low citrin levels known to be present in the adult mouse brain, beta-galactosidase expression was very low. Surprisingly, unlike the case with astroglial cultures that express citrin, no beta-galactosidase was found in brain glial cells. It was confined to neuronal cells within discrete neuronal clusters. Double-immunolabelling experiments showed that beta-galactosidase colocalized not with glial cell markers but with the pan-neuronal marker NeuN. The deep cerebellar nuclei and a few midbrain nuclei (reticular tegmental pontine nuclei; magnocellular red nuclei) were the regions where beta-galactosidase expression was highest, and it was up-regulated in fasted mice, as was also the case for liver beta-galactosidase. The results support the notion that glial cells have much lower AGC levels and MAS activity than neurons.
Journal of Neuroscience Research 11/2009; 88(5):1009-16. · 2.74 Impact Factor
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ABSTRACT: Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic
Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate α-ketoglutarate
(αKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that αKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in αKG efflux through the oxoglutarate
carrier and in a drop in MAS activity. Inhibition of αKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in
lactate observed in the stimulated brain in vivo.
Journal of Biological Chemistry 03/2009; 284(11):7091-7099. · 4.77 Impact Factor
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ABSTRACT: Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.
Journal of Biological Chemistry 02/2009; 284(11):7091-9. · 4.77 Impact Factor
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ABSTRACT: The ADP/ATP and ATP-Mg/Pi carriers are widespread among eukaryotes and constitute two systems to transport adenine nucleotides in mitochondria. ADP/ATP carriers carry out an electrogenic exchange of ADP for ATP essential for oxidative phosphorylation, whereas ATP-Mg/Pi carriers perform an electroneutral exchange of ATP-Mg for phosphate and are able to modulate the net content of adenine nucleotides in mitochondria. The functional interplay between both carriers has been shown to modulate viability in Saccharomyces cerevisiae. The simultaneous absence of both carriers is lethal. In the light of the new evidence we suggest that, in addition to exchange of cytosolic ADP for mitochondrial ATP, the specific function of the ADP/ATP carriers required for respiration, both transporters have a second function, which is the import of cytosolic ATP in mitochondria. The participation of these carriers in the generation of mitochondrial membrane potential is discussed. Both are necessary for the function of the mitochondrial protein import and assembly systems, which are the only essential mitochondrial functions in S. cerevisiae.
Mitochondrion 02/2009; 9(2):79-85. · 3.62 Impact Factor
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ABSTRACT: Aralar, the mitochondrial aspartate-glutamate carrier present in beta-cells, is a component of the malate-aspartate NADH shuttle (MAS). MAS is activated by Ca2+ in mitochondria from tissues with aralar as the only AGC isoform with an S0.5 of approximately 300 nm. We have studied the role of aralar and its Ca2+-binding EF-hand motifs in glucose-induced mitochondrial NAD(P)H generation by two-photon microscopy imaging in INS-1 beta-cells lacking aralar or expressing aralar mutants blocked for Ca2+ binding. Aralar knock-down in INS-1 beta-cell lines resulted in undetectable levels of aralar protein and complete loss of MAS activity in isolated mitochondria and in a 25% decrease in glucose-stimulated insulin secretion. MAS activity in mitochondria from INS-1 cells was activated 2-3-fold by extramitochondrial Ca2+, whereas aralar mutants were Ca2+ insensitive. In Ca2+-free medium, glucose-induced increases in mitochondrial NAD(P)H were small (1.3-fold) and unchanged regardless of the lack of aralar. In the presence of 1.5 mm Ca2+, glucose induced robust increases in mitochondrial NAD(P)H (approximately 2-fold) in cell lines with wild-type or mutant aralar. There was a approximately 20% reduction in NAD(P)H response in cells lacking aralar, illustrating the importance of MAS in glucose action. When small Ca2+ signals that resulted in extremely small mitochondrial Ca2+ transients were induced in the presence of glucose, the rise in mitochondrial NAD(P)H was maintained in cells with wild-type aralar but was reduced by approximately 50% in cells lacking or expressing mutant aralar. These results indicate that 1) glucose-induced activation of MAS requires Ca2+ potentiation; 2) Ca2+ activation of MAS represents a larger fraction of glucose-induced mitochondrial NAD(P)H production under conditions where suboptimal Ca2+ signals are associated with a glucose challenge (50 versus 20%, respectively); 3) inactivation of EF-hand motifs in aralar prevents activation of MAS by small Ca2+ signals. The results suggest a possible role for aralar and MAS in priming the beta-cell by Ca2+-mobilizing neurotransmitter or hormones.
Journal of Biological Chemistry 12/2008; 284(1):515-24. · 4.77 Impact Factor
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ABSTRACT: The SCaMCs (small calcium-binding mitochondrial carriers) constitute a subfamily of mitochondrial carriers responsible for the ATP-Mg/P(i) exchange with at least three paralogues in vertebrates. SCaMC members are proteins with two functional domains, the C-terminal transporter domain and the N-terminal domain which harbours calcium-binding EF-hands and faces the intermembrane space. In the present study, we have characterized a shortened fourth paralogue, SCaMC-3L (SCaMC-3-like; also named slc25a41), which lacks the calcium-binding N-terminal extension. SCaMC-3L orthologues are found exclusively in mammals, showing approx. 60% identity to the C-terminal half of SCaMC-3, its closest paralogue. In mammalian genomes, SCaMC-3 and SCaMC-3L genes are adjacent on the same chromosome, forming a head-to-tail tandem array, and show identical exon-intron boundaries, indicating that SCaMC-3L could have arisen from an SCaMC-3 ancestor by a partial duplication event which occurred prior to mammalian radiation. Expression and functional data suggest that, following the duplication event, SCaMC-3L has acquired more restrictive functions. Unlike the broadly expressed longer SCaMCs, mouse SCaMC-3L shows a limited expression pattern; it is preferentially expressed in testis and, at lower levels, in brain. SCaMC-3L transport activity was studied in yeast deficient in Sal1p, the calcium-dependent mitochondrial ATP-Mg/P(i) carrier, co-expressing SCaMC-3L and mitochondrial-targeted luciferase, and it was found to perform ATP-Mg/P(i) exchange, in a similar manner to Sal1p or other ATP-Mg/P(i) carriers. However, metabolite transport through SCaMC-3L is calcium-independent, representing a novel mechanism involved in adenine nucleotide transport across the inner mitochondrial membrane, different to ADP/ATP translocases or long SCaMC paralogues.
Biochemical Journal 11/2008; 418(1):125-33. · 4.90 Impact Factor
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ABSTRACT: Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K(m) of 0.20 +/- 0.03 mM and 0.28 +/- 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca2+ dependent. Ca2+ increases the V(max) with a S(0.5) of 15 muM, and no changes in the K(m) for ATP-Mg. Glucose sensing in yeast generates Ca2+ transients involving Ca2+ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H+-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca2+-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.
Molecular Microbiology 06/2008; 69(3):570-85. · 5.01 Impact Factor
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ABSTRACT: Aralar, the Ca(2+)-dependent mitochondrial aspartate-glutamate carrier expressed in brain and skeletal muscle, is a member of the malate-aspartate NADH shuttle. Disrupting the gene for aralar, SLC25a12, in mice has enabled the discovery of two new roles of this carrier. On the one hand, it is required for synthesis of brain aspartate and N-acetylaspartate, a neuron-born metabolite that supplies acetate for myelin lipid synthesis; and on the other, it is essential for the transmission of small Ca(2+) signals to mitochondria via an increase in mitochondrial NADH.
Journal of Neuroscience Research 12/2007; 85(15):3359-66. · 2.74 Impact Factor
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ABSTRACT: Ca(2+) regulation of the Ca(2+) binding mitochondrial carriers for aspartate/glutamate (AGCs) is provided by their N-terminal extensions, which face the intermembrane space. The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle. We report that their N-terminal extensions contain up to four pairs of EF-hand motifs plus a single vestigial EF-hand, and have no known homolog. Aralar and citrin contain one fully canonical EF-hand pair and aralar two additional half-pairs, in which a single EF-hand is predicted to bind Ca(2+). Shuttle activity in brain or skeletal muscle mitochondria, which contain aralar as the major AGC, is activated by Ca(2+) with S(0.5) values of 280-350 nm; higher than those obtained in liver mitochondria (100-150 nm) that contain citrin as the major AGC. We have used aralar- and citrin-deficient mice to study the role of the two isoforms in heart, which expresses both AGCs. The S(0.5) for Ca(2+) activation of the shuttle in heart mitochondria is about 300 nm, and it remains essentially unchanged in citrin-deficient mice, although it undergoes a drastic reduction to about 100 nm in aralar-deficient mice. Therefore, aralar and citrin, when expressed as single isoforms in heart, confer differences in Ca(2+) activation of shuttle activity, probably associated with their structural differences. In addition, the results reveal that the two AGCs fully account for shuttle activity in mouse heart mitochondria and that no other glutamate transporter can replace the AGCs in this pathway.
Journal of Biological Chemistry 04/2007; 282(10):7098-106. · 4.77 Impact Factor
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ABSTRACT: 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.
Physiological Reviews 02/2007; 87(1):29-67. · 26.87 Impact Factor
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ABSTRACT: Aralar, the neuronal Ca(2+)-binding mitochondrial aspartate-glutamate carrier, has Ca(2+) binding domains facing the extramitochondrial space and functions in the malate-aspartate NADH shuttle (MAS). Here we showed that MAS activity in brain mitochondria is stimulated by extramitochondrial Ca(2+) with an S(0.5) of 324 nM. By employing primary neuronal cultures from control and aralar-deficient mice and NAD(P)H imaging with two-photon excitation microscopy, we showed that lactate utilization involves a substantial transfer of NAD(P)H to mitochondria in control but not aralar-deficient neurons, in agreement with the lack of MAS activity associated with aralar deficiency. The increase in mitochondrial NAD(P)H was greatly potentiated by large [Ca(2+)](i) signals both in control and aralar-deficient neurons, showing that these large signals activate the Ca(2+) uniporter and mitochondrial dehydrogenases but not MAS activity. On the other hand, small [Ca(2+)](i) signals potentiate the increase in mitochondrial NAD(P)H only in control but not in aralar-deficient neurons. We concluded that neuronal MAS activity is selectively activated by small Ca(2+) signals that fall below the activation range of the Ca(2+) uniporter and plays an essential role in mitochondrial Ca(2+) signaling.
Journal of Biological Chemistry 02/2006; 281(2):1039-47. · 4.77 Impact Factor
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ABSTRACT: Sal1p is a mitochondrial protein that belongs to the SCaMC (short calcium-binding mitochondrial carrier) subfamily of mitochondrial carriers. 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 have studied its transport activity, finding that it is a carboxyatractyloside-resistant ATP-Mg carrier. Mitochondria from a disruption mutant of SAL1 have a 50% reduction in the uptake of ATP. We have also found a clear stimulation of ATP-transport activity by calcium, with an S(0.5) of approx. 30 microM. Our results also suggest that Sal1p is a target of the glucose-induced calcium signal which is non-essential in wild-type cells, but becomes essential for transport of ATP into mitochondria in yeast lacking ADP/ATP translocases.
Biochemical Journal 01/2006; 392(Pt 3):537-44. · 4.90 Impact Factor
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Md Abdul Jalil,
Laila Begum,
Laura Contreras,
Beatriz Pardo,
Mikio Iijima,
Meng Xian Li,
Milagros Ramos,
Patricia Marmol,
Masahisa Horiuchi,
Kyoko Shimotsu,
Shiro Nakagawa,
Akiko Okubo,
Munefumi Sameshima,
Yasushi Isashiki,
Araceli Del Arco,
Keiko Kobayashi, Jorgina Satrústegui,
Takeyori Saheki
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ABSTRACT: 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 aralar-deficient (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.
Journal of Biological Chemistry 10/2005; 280(35):31333-9. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 07/2004; 279(23):24701-13. · 4.77 Impact Factor
