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Departamento de Biología Molecular Centro de Biología Molecular "Severo Ochoa" UAM-CSIC, Facultad de Ciencias, Universidad Autónoma, Madrid; and Área de Bioquímica, Centro Regional de Investigaciones Biomédicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, Toledo, Spain
ABSTRACT I. INTRODUCTION II. CALCIUM BINDING MITOCHONDRIAL CARRIER GENE AND PROTEINS III. ASPARTATE-GLUTAMATE CARRIERS A. Mammalian AGCs 1. Development and tissue distribution of aralar/AGC1 and citrin/AGC2 2. Import of AGC precursor proteins to mitochondria 3. Transport activity 4. Functional roles of AGCs in urea cycle and the MAS 5. Mutations and polymorphisms A) CITRIN, AGC2, AND SLC25a13. B) ARALAR, AGC1, AND SLC25A12. B. Nonmammalian AGCs 1. Transport activity 2. Role of yeast AGC in amino acid biosynthesis and in shuttling reducing equivalents to mitochondria C. Ca2+ Regulation of the MAS 1. Ca2+ activation of MAS in liver 2. Ca2+ regulation of MAS in heart 3. Ca2+ regulation of citrin and aralar A) ROLE OF ARALAR AND THE MALATE-ASPARTATE NADH SHUTTLE IN TRANSDUCTION OF SMALL CA2+ SIGNALS TO MITOCHONDRIA. B) EFFECTS OF LARGE CA2+ SIGNALS ON THE ARALAR-MAS PATHWAY. D. Other Mechanisms of Ca2+ Regulation of NAD(P)H Transfer to Mitochondria Independent of Ca2+ Entry 1. Mitochondrial glycerol-3-phosphate dehydrogenases 2. External NAD(P)H dehydrogenases in fungi and plants E. Interplay Between AGCs, GPS, and the Ca2+ Uniporter in Regulation of Mitochondrial Metabolism in Response to Cytosolic Ca2+ Signals IV. ATP-MG/Pi CARRIERS, SCAMC GENES, AND PROTEINS A. Mammalian SCaMCs 1. Extended diversity of mammalian SCaMCs 2. Targeting of ATP-Mg/Pi precursor proteins to mitochondria 3. Transport activity of mammalian ATP-Mg/Pi carriers A) NET INFLUX AND EFFLUX OF ADENINE NUCLEOTIDES IN MITOCHONDRIA. B) TRANSPORT ACTIVITY OF THE ATP-Mg/Pi CARRIER. C) Ca2+ REGULATION OF ATP-Mg/Pi TRANSPORT IN MAMMALIAN MITOCHONDRIA. B. Hormonal and Developmental Changes in AdN Content in Liver Mitochondria 1. Hormonal regulation of liver mitochondria AdN content 2. Role of liver ATP-Mg/Pi transport in early postnatal development 3. ATP-Mg/Pi transport in extrahepatic tissues A) ISCHEMIA. C. Nonmammalian ATP-Mg/Pi Carriers 1. Transport activity of yeast Sal1p 2. Ca2+ regulation of ATP-Mg/Pi transport in yeast mitochondria 3. Physiological role of yeast Sal1p V. EVOLUTION OF MECHANISMS FOR CALCIUM SIGNALING IN MITOCHONDRIA VI. CONCLUSIONS GRANTS ACKNOWLEDGMENTS REFERENCES
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ABSTRACT |
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I. INTRODUCTION |
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Mitochondrial carriers (MCs) are integral proteins of the mitochondrial inner membrane that function in the shuttling of metabolites, nucleotides, and cofactors between the cytosol and mitochondria (408), a subset of which are the Ca2+-binding MCs (CaMCs) (95). CaMCs fall into two groups: the aspartate/glutamate carriers (AGC) (92, 95, 195, 286) and the ATP-Mg/Pi carriers, also named SCaMC (for short CaMC) (94, 122). The two mammalian AGCs, aralar (AGC1) and citrin (AGC2), are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC2, is also a member of the urea cycle (217). The transport reaction catalyzed by the AGCs is the only irreversible step in the malate-aspartate shuttle (217, 218) and a main point of regulation. The ATP-Mg/Pi carriers exchange cytosolic ATP-Mg for mitochondrial Pi and are involved in loading newly formed mitochondria with adenine nucleotides and modulating mitochondrial adenine nucleotide content in response to hormones (10, 13). The activity of both types of CaMCs is stimulated by Ca2+ acting on the external face of the inner mitochondrial membrane (286, 163, 276).
To perform a role in transducing Ca2+ signals to mitochondria, CaMCs must be poised for activation under normal cell conditions. The existing data indicate that the reaction catalyzed by these carriers is energetically favored under resting conditions but limited by the low activity of the carrier. Ca2+ activation removes this limitation, allowing the transport reaction to occur. Available data indicate that such processes occur at the expense of relatively small Ca2+ signals, with limited capacity to activate Ca2+ entry in mitochondria, and may be responsible for the energizing role of low levels of hormones and neurotransmitters. These carriers exist in organisms such as Saccharomyces cerevisiae that lack a Ca2+ uniporter. In particular, S. cerevisiae Sal1p appears to be the most basic CaMC, in fact the only mechanism conveying Ca2+ signals to mitochondria in this organism.
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II. CALCIUM BINDING MITOCHONDRIAL CARRIER GENE AND PROTEINS |
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35 kDa, six transmembrane spanning segments, and a tripartite structure (408) that catalyze the transport of metabolites, nucleotides and derivatives, and cofactors in mitochondria. The CaMC are a subset of MCs that have long hydrophilic amino-terminal extensions harboring EF-hand Ca2+-binding motifs that face the intermembrane space (92, 95) (Fig. 1).
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35 members of the MC superfamily (117), and database analysis revealed that the genome of C. elegans also contained a number of similar sequences. A S. cerevisiae gene, YNL083w (364) (Table 1), and three sequences in C. elegans code for putative proteins containing sequence motifs corresponding to EF-hands (92), a property not described so far in members of the MC family. EF-hands (where EF stands for the E and F alpha helices of parvalbumin) are Ca2+-binding domains present in different Ca2+-binding proteins (269), and their presence in a putative MC protein opened up the possibility that the protein might be involved in the handling of Ca2+, or in Ca2+-regulated metabolite transport in mitochondria. The realization of the potential implications of this finding was the starting point for the cloning and characterization of all members of the CaMC subfamily. Soon after, it became clear that CaMCs are not involved in Ca2+ transport.
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The ATP-Mg/Pi carriers were first described as CaMCs with short amino-terminal extensions or SCaMCs and are represented by three main isoforms: SCaMC 1–3 (91, 94, 122) with a number of variants arising from alternative splicing. The identification of the transporter function of SCaMCs was carried out by Palmieri’s group (122) after reconstitution of the recombinant proteins into proteoliposomes. Throughout this manuscript the acronyms SCaMC 1–3 will be used to designate the particular isoforms of the ATP-Mg/Pi carrier, rather than the acronyms used previously (APC 1–3, from ATP-Mg/Pi carrier, Ref. 122), to avoid confusions with the well-known APC protein, adenomatous polyposis coli, a tumor suppressor multifunctional protein that is mutated in a majority of colon cancers (160).
The transporter function of the AGCs was reported long ago (27). These carriers are important for the urea cycle and malate-aspartate NADH shuttle and have been the subject of a number of studies including purification and functional reconstitution in proteoliposomes. However, Ca2+ regulation of the AGCs had escaped attention.
An ATP transport pathway different from the adenine nucleotide translocases was first described in fetal liver mitochondria where it was found to be important for the net uptake of adenine nucleotides immediately after birth (299, 300, 373). A few years later, a series of careful studies carried out by Aprille and co-workers (276) and Haynes and co-workers (163) resulted in the discovery that the ATP-Mg/Pi carrier was regulated by Ca2+ from the external side of the inner mitochondrial membrane (163, 276). In retrospect, this seminal finding set the stage for the identification of MCs endowed with Ca2+ regulation properties.
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III. ASPARTATE-GLUTAMATE CARRIERS |
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Aralar/AGC1 and citrin/AGC2 are coded for SLC25a12 and SLC25a13, in human chromosomes 2q31 (80, 339) and 7q21.3 (358), respectively. The two genes have the same genomic structure, with 18 exons (358), also conserved in mice (180, 359).
1. Development and tissue distribution of aralar/AGC1 and citrin/AGC2
Aralar, citrin, or both isoforms are present in early preimplantation embryos, as lactate supports development from the two-cell stage onwards, through the operation of the malate-aspartate NADH shuttle (214). Thus the AGCs are present very early in mouse development. Aralar and citrin have an overlapping distribution in embryogenesis (93, 358). Both isoforms are expressed strongly in branchial arches, dermomyotome, limb, and tail buds at early embryonic stages. The characteristic strong expression of citrin in liver is acquired postnatally, while that of aralar in heart and skeletal muscle begins during embryonic development (93). In the adult mouse, aralar is highly expressed in almost every tissue including brain, skeletal muscle, and heart (95) as well as in cells from the immune and hematopoietic tissues both at embryonic stages (embryonic liver) and in adults (Table 2). Many tissues express both isoforms, except hepatocytes from adult mouse liver where citrin is the only detectable AGC (93).
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2. Import of AGC precursor proteins to mitochondria
Like other MCs, AGC precursor proteins are synthesized in the cytosol and then imported to mitochondria. Targeting information in CaMCs is contained in their carboxy-terminal half, and all amino-truncated variants are targeted into mitochondrial membranes (91, 92, 94, 96, 286).
The mechanisms of import of MC precursor proteins and CaMCs have been discussed in detail in recent reviews (96, 196, 309). The insertion into the inner membrane of members of MC family is mediated by the TIM22 (translocase of the inner membrane) complex or carrier translocase (Fig. 2B). The TIM22 pathway contains components at the inner membrane as well as in the intermembrane space, referred to as the small Tim proteins, Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p (198). These small Tims act as chaperone-like molecules to guide hydrophobic precursors across the aqueous intermembrane space, whereas the inner membrane complex mediates their insertion into the inner membrane (88, 89, 412). The small Tim proteins assemble into 70-kDa complexes, Tim8p with Tim13p and Tim9p with Tim10p in a 3:3 ratio (Fig. 2B) (88, 89, 197). These 70-kDa complexes have different substrate specificities, with the classical MC precursor proteins binding predominantly to the Tim9p-Tim10p complex (89, 289). In contrast, the Tim8p-Tim13p complex binds to the human and yeast AGC precursor proteins through their long hydrophilic amino-terminal domains (318). Pathway selection seems to be determined by the presence of the hydrophilic amino-terminal extension of CaMCs; thus the import of full-length aralar decreases 80% in mitochondria that lack the Tim8p-Tim13p complex, and shortening of the aralar amino-terminal domain results in preferential binding of aralar to the Tim9p-Tim10p complex (318). Mutations in the locus for human Tim8p (DDP1/TIMM8a locus) cause Mohr-Tranebjaerg syndrome (MTS/DFN-1, deafness/dystonia syndrome) (198, 317), and a lymphoblast cell line derived from an MTS patient shows decreased mitochondrial NADH levels due to defects in the MAS where AGC is the rate-limiting component (318). The Tim8p-Tim13p complex probably also participates in SCaMCs import as suggested by the differences in import efficiency detected for overexpressed full-length and amino-truncated versions of SCaMC3 protein (91).
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The AGCs aralar and citrin catalyze the exchange of aspartate with glutamate plus a proton, as demonstrated in intact mitochondria (215, 216, 218) and in proteoliposomes reconstituted with the native purified protein (97, 98) or recombinant AGCs (286). Thus there is one positive charge that moves across the membrane together with the two amino acids, and the exchange is electrophoretic (114, 215–218, 352). This property of the AGC is rather uncommon among the MC family, only shared by the adenine nucleotide translocases and the uncoupling proteins. Because the aspartate-glutamate exchange reaction is strongly dependent on the membrane potential, the release of aspartate in exchange with glutamate is strongly favored (97, 98, 215, 216, 218, 286). Thus, in energized mitochondria, this carrier is essentially unidirectional. The affinity for glutamate is lower than that for aspartate (Km values of 0.2 and 0.05 mM, respectively), both for the native or recombinant AGCs (97, 99, 286), and these values do not change with the membrane potential (286). The effect of the membrane potential is a pronounced increase in transport activity that depends on an electrogenic modulation of the transport rate constant of the negatively charged carrier-aspartate complex, and not to a change in the substrate affinity of the carrier (286). The two AGCs also catalyze 1:1 electroneutral homoexchange reactions with aspartate, glutamate, and cysteine sulfinate and are strongly inhibited by pyridoxal phosphate (45, 101, 284, 286).
4. Functional roles of AGCs in urea cycle and the MAS
AGCs are required for the transfer of mitochondrial aspartate to the cytosol, an important step in urea synthesis (Fig. 3) and in gluconeogenesis from lactate and amino acids. Arginino-succinate synthetase (ASS), the cytosolic enzyme condensing citrulline with aspartate, uses preferentially but not exclusively, mitochondrial versus cytosolic aspartate (250, 319, 334, 413). For recent reviews on this subject, see References 333 and 334. The role of citrin in the urea cycle is discussed in section IIIA5.
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-ketoglutarate carrier (OGC) (Fig. 4). The presence of the AGCs in this cycle provides a unidirectional substrate flow along the shuttle that functions in the direction of reducing equivalent transfer from cytosol to mitochondria. MAS is important in reducing equivalent transfer in many tissues including liver, particularly human liver, where other NADH shuttle systems such as the glycerol-3-phosphate shuttle (GPS) have low activity, 1/20 of that in rodents (330).
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40% of the ATP production derived from glycolysis, but within days oxidative phosphorylation increases to provide 93% of the ATP as fatty acid utilization increases (see Ref. 369 for review). In neonatal hearts, lactate oxidation accounts for the majority of oxygen consumption (127). MAS activity of neonatal porcine heart mitochondria is about three times higher than that of adult heart (346, 347), in which fatty acid metabolism bypasses the NADH shuttles. Strikingly, the activity of the heart mitochondrial Ca2+ uniporter undergoes a similar decrease along postnatal life in the rat (32). In contrast to the developmental changes in MAS activity, mRNA and protein levels of AGCs in porcine and mouse heart are higher in adult than in neonates (35, 347), although a decrease in steady-state aralar mRNA levels was observed from postnatal day 9 onward (35). Studies of the levels of all other MAS members indicated that those of OGC decrease postnatally in pig and rabbit (141, 347) and probably limit shuttle function in adult heart (347). An upregulation of MAS could be of potential interest in heart failure and myocardium hypertrophy, when substrate utilization shifts away from fatty acids toward glucose and lactate (328, 369).
Glucose metabolism drives the stimulation of insulin secretion in pancreatic 
-cells through increased ATP levels that lead to closure of KATP channels (237, 238, 251). Lactate dehydrogenase activity is low in pancreatic -cells (177, 178, 354), and NADH shuttle systems are important for glucose-induced activation of mitochondrial metabolism and insulin secretion (112, 135, 377). Among NADH shuttle systems, MAS is particularly important, as insulin secretion is normal in mice with no functional mitochondrial FAD-GPDH, the mitochondrial member of the glycerol-phosphate shuttle, whereas it is blocked when MAS activity is inhibited in these transgenic mice (119). Aralar is the only AGC present in islets and -cells (93, 326), and its levels or activity appear to limit MAS function in these cells. Thus overexpression of aralar in INS-1E or pancreatic -cell leads to increases in glucose-induced reduction of mitochondrial NAD(P)H, mitochondrial membrane potential, and ATP levels along with an increase in glucose-induced insulin secretion (326). Glucose utilization was not changed by aralar overexpression, but lactate formation was decreased, suggesting that MAS activity regulates the fraction of pyruvate being either oxidized in mitochondria or reduced to lactate (326). On the other hand, aralar overexpression did not modify insulin secretion when pyruvate, rather than glucose, was used as fuel, showing that it contributes primarily upstream of mitochondria to efficiently couple glycolysis to mitochondrial metabolism (326). MAS is also important in neurons, especially during lactate utilization, and this is discussed in section IIIA5B.
Interestingly, MAS is also required in early embryos. Studies on the preimplantation mouse embryo carried out more than 30 years ago have shown that the zygote has an absolute requirement for pyruvate to complete the first cleavage division, whereas lactate and glucose are only capable to support development from the two- and eight-cell stages, respectively (56, 57). Mouse zygotes can use both lactate and pyruvate (213), but only pyruvate supports the first cleavage. In zygotes, but not in blastocysts, lactate oxidation and NAD/NADH ratios decreased with increasing lactate concentration, suggesting that lactate utilization by lactate dehydrogenase was limited due to an inability to provide NAD for the reaction (213). Although zygotes express all four MAS enzymes, the inability to transfer reducing equivalents to mitochondria was found to be due to a lack of MAS activity, aspartate being the rate-limiting factor probably due to the high Km of the cytosolic aspartate aminotransferase (214). This suggests that AGC levels or activity may limit MAS function at this particular developmental stage. Thus addition of exogenous aspartate enabled mouse zygotes to utilize lactate and develop normally (214).
5. Mutations and polymorphisms
Much of the actual roles of aralar and citrin in humans and rodents have been unveiled through the discovery of human diseases involving the two carriers and with the development of mouse models carrying disruptions in the two genes.
A) CITRIN, AGC2, AND SLC25a13. A deficiency in citrin, the AGC2 isoform specifically expressed in liver, kidney, and heart encoded by gene SLC25A13, causes type 2 citrullinemia (CTLN2), an adult-onset liver disease predominantly found in the Japanese population (195). Unlike classical citrullinemia in children, which is associated with a deficiency in ASS, in CTLN2 the ASS gene is normal and ASS deficiency is only found in liver, but not in other organs that express ASS. Moreover, the residual amount of enzyme present in the patients displays normal kinetic properties (333, 335). The disorder was first reported in a group of patients with high serum citrulline and ammonia levels and neurological symptoms that closely resemble those of hepatic encephalopathy. However, a different disease, neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), was found in a number of neonates with idiopathic neonatal hepatitis that carry the same mutations in citrin found in CTLN2 patients (280, 392). These neonates have multiple metabolic abnormalities including aminoacidemia, galactosemia, hypoproteinemia, hypoglycemia, and cholestasis. NICCD is usually not severe, and it can be managed by nutritional manipulation, but a few patients develop CTLN2 after 10 years or more (332).
In CTLN2 patients, mutations in citrin cause a complete lack of citrin protein (195). The generation of citrin-knockout mice has revealed many of the consequences of citrin deficiency (359). Liver mitochondria from citrin(–/–) mice had a drastic reduction of malate-aspartate NADH shuttle activity and aspartate efflux with no compensatory changes in the glycerophosphate NADH shuttle. Gluconeogenesis from lactate, but not from pyruvate, was severely impaired, in agreement with MAS requirement for gluconeogenesis from reduced substrates. Urea production from ammonia was severely reduced in liver from citrin(–/–) mice, and this was strongly alleviated in the presence of asparagine, indicating that aspartate was the major limiting factor. All of these metabolic characteristics are consistent with a primary failure in aspartate efflux from mitochondria and malate-aspartate NADH shuttle activity (359). However, despite the deficits in AGC-dependent pathways, citrin(–/–) mice failed to show any overt pathological consequences. In particular, these mice had normal serum ammonia and citrulline levels and responded to increases in protein load in their diet by increasing urea formation. This suggests that mitochondrial-derived aspartate is probably not essential for the ASS reaction in mice (265, 359).
The characteristic decline in hepatic ASS levels and activity of CTLN2 patients has been attributed to the defect in the transport of aspartate from mitochondria to the cytosol that could inhibit the ASS reaction and destabilize the liver enzyme (286). ASS protein has full activity in NICCD neonates (381) and declines gradually during the life of CTLN2 patients. Whether the shortage of aspartate by itself destabilizes ASS or other factors participate in this defect is still unknown, but this particular defect in hepatic ASS levels is also absent in the citrin(–/–) mouse (359). Species-specific differences in gene regulation and in alternative metabolic pathways have been proposed to explain the different phenotypes. In particular, the glycerol phosphate shuttle is 20-fold more active in rodent than in human liver (330), suggesting that this shuttle could compensate for the lack of MAS activity in mice. There is indirect evidence that, to compensate for the lack of MAS, CTLN2 patients rely heavily on an alternative NADH shuttle, the malate-citrate shuttle pathway, which is used for the transfer of acetyl CoA from mitochondria to the cytosol for fatty acid synthesis (332, 333). Fat accumulation that could be supported by increased malate-citrate shuttle activity has been observed in the livers of patients with citrin deficiencies (both NICCD and CTLN2) (175, 334, 381, 392). The defects in ureogenesis in the citrin(–/–) liver could be partially corrected by the administration of asparagine, which also normalized the high levels of citrulline and low levels of ornithine (265, 359). These findings suggest that the aspartate arising from asparagine hydrolysis could serve as a substrate for the ASS reaction. In addition, pyruvate administration corrected the defect in ureogenesis, by lowering the cytosolic NADH/NAD ratio (265).
B) ARALAR, AGC1, AND SLC25A12. Aralar is an ubiquitous AGC isoform highly expressed in brain and skeletal muscle. The liver parenchyma seems to be the only adult tissue devoid of aralar, although it is present in Kupffer cells, and also in the embryonic liver (93), a major hematopoietic organ.
The existence of aralar-knockout mice (180) has revealed new functions of this carrier, particularly a role in the supply of brain N-acetyl aspartate (NAA) for myelin lipid synthesis (Fig. 5). Aralar–/– mouse embryos are produced in normal numbers, as expected from the overlap of aralar and citrin expression in fetal tissues (35, 93). However, soon after birth these mice develop severe growth defects, neuromuscular deficiencies, and a reduced myelination in the central nervous system (CNS), but not in peripheral nerves. Brain and muscle mitochondria from aralar-knockout mice have a complete loss of MAS activity, a drastic decrease in respiration on glutamate plus malate, with no compensatory increase in respiration on pyruvate plus malate, and a pronounced decrease in aspartate efflux from mitochondria (180). Aralar deficiency results in a large drop of NAA and aspartate in the brain and primary neuronal cultures derived from Aralar–/– mice, together with a drop in myelin lipids and similar reductions in the levels of the major proteins in myelin, myelin basic protein (MBP), and myelin-associated oligodendrocyte basic protein (MOBP).
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80%) reduction in the brain acetate concentrations (235), showing that the lack of NAA results in a lack of acetyl groups for myelin lipid synthesis. The fall in brain aspartate levels and the lack of aspartate production in brain mitochondria from Aralar–/– mice indicate that the major route of aspartate production in the CNS is mitochondrial and that it depends on aralar for aspartate efflux to the cytosol. Indeed, the CNS pathology in aralar-null mice appears at about the time where the blood-brain barrier develops, preventing the entry of aspartate and other amino acids into most regions of the adult brain except for selected circumventricular regions (302). Therefore, from the first week of life onward, brain cells have to rely on their own endogenous production of aspartate, which takes place mostly in neurons, the brain cells with highest aralar expression (303). NAA is derived from neuronal aspartate, and its synthesis is catalyzed by aspartate-N-acetyltransferase in brain mitochondria (236, 334) and microsomes, which have four to five times higher activity than mitochondria (231). Thus the fall in NAA in brain and in cultured neurons from Aralar–/– mice is clearly associated with the defect in aspartate production.
The important brain functions underscored from the study of aralar-knockout mice may be relevant for two human diseases of the nervous system. A strong linkage and association of the gene for aralar, SLC25A12, with autism has been reported (304, 353; but see Ref. 46). Two polymorphisms that corresponded to single nucleotide polymorphisms (SNPs) located in flanking intronic sequences of exons 4 and 16 of SLC25A12 were associated with the disease, although the functional relevance of these SNPs is yet unknown (304, 353). The expression of a number of genes is regulated by cis-acting elements, and inherited variation in gene expression may contribute to disease (52). An altered regulation of gene expression without variations in its coding sequence could affect cellular functions when the levels of the gene product are limiting, as appears to be the case for aralar, as an overexpression of this protein in -cells increases MAS function (326).
Impaired aralar activity could also contribute to the pathology of Mohr-Tranebjaerg syndrome (MTS/DFN-1, deafness/dystonia syndrome) caused by mutations in the locus for human Tim8p (DDP1/TIMM8a locus) (198, 317; as discussed in sect. IIIA2). Together with Tim13, Tim8p forms a 70-kDa complex specifically utilized by AGCs (318), and possibly also the SCaMCs (89) on their way to the inner membrane, a complex with prominent expression in large neurons in the brain (318). The mutations in Tim8p in MTS/DFN-1 patients appear to result in impaired import of aralar, as a lymphoblast cell line derived from a MTS patient shows decreased mitochondrial NADH levels due to defects in MAS activity (318).
Thus genetic variations that change the expression of aralar, or an impaired import of aralar in mitochondria, could lead to changes in NADH shuttle function, respiration on glutamate and malate, aspartate synthesis by neuronal mitochondria, and NAA production, which could be related to these CNS diseases. A decrease in NAA in different brain regions of autistic patients has been reported (130, 281).
A glutamate-aspartate MC is present in all known eukaryotic genomes (96). In most higher eukaryotes AGCs are highly similar to the mammalian genes and have EF-hand Ca2+ binding motifs in equivalent positions. A prominent exception to this rule is S. cerevisiae, in which the long amino-terminal extension of the agc1p protein lacks Ca2+-binding motifs (69) (Table 1).
The carboxy-terminal domain of yeast agc1p, that encompasses the whole MC homologous region, has been expressed in bacteria and reconstituted in phospholipid vesicles. Its transport properties were found to be similar to those of the mammalian AGCs in terms of substrate specificity, affinity, inhibitor sensitivity, and voltage dependence but differed from mammalian AGCs in that yeast agcp is able to catalyze the uniport of aspartate, and to a greater extent of glutamate, in addition to the aspartate/glutamate exchange (69).
The carboxy-terminal domains of agc1p and mammalian AGCs are highly similar, suggesting that both arise from a common ancestor. This ancestor probably duplicated in mammals to give rise to the AGCs that catalyze a strict asp/glut exchange, and glutamate carriers (GCs) that catalyze the uniport of glutamate driven by the pH gradient (123), and evolved into agc1p in S. cerevisiae, that catalyzes both transport activities (69). The second of these activities, the uniport of glutamate, is thought to be required to provide mitochondrial glutamate for transamination reactions and as a carbon and nitrogen source for ornithine synthesis, as discussed below (69).
2. Role of yeast AGC in amino acid biosynthesis and in shuttling reducing equivalents to mitochondria
The activity of agc1p as a glutamate uniporter is probably responsible for changes in amino acid levels and labeling from [13C]acetate in yeast agc1p deletion mutants. These mutants had reduced valine, ornithine, and citrulline levels (69) that were explained as a consequence of reduced uptake of glutamate in mitochondria, with subsequent reduction in mitochondrial glutamate levels, since glutamate dehydrogenase is not mitochondrial but cytosolic in S. cerevisiae (169). Mitochondrial glutamate is required for transamination reactions within mitochondria (valine synthesis), and as carbon and nitrogen source for ornithine synthesis, all of which are reduced in agc1p mutants (69).
The existence of a malate-aspartate NADH shuttle in S. cerevisiae has been questioned for a long time, because yeast mitochondria harbor external NADH dehydrogenases that oxidize NADH on the external face of the inner mitochondrial membrane (233) and lack complex I in the respiratory chain. However, all the components of the shuttle exist in yeast, particularly the MCs for -ketoglutarate (-KG) and malate exchange, Odc1 and Odc2, oxodicarboxylate carriers that can transport -KG, -ketoadipate, and malate (285, 386, 396), and the aspartate-glutamate exchanger agc1p. Growth on oleic acid produces large amounts of NADH in peroxisomes that require shuttle systems for reducing equivalent transfer to mitochondria. MAS is the shuttle system involved in the transfer of reducing equivalents from the cytosol to the mitochondria in this particular condition, as neither the odc1
odc2 double mutants (386) or agc1p deletion mutants (69) grow on oleic acid. Because growth on ethanol, but not on acetate, is unimpaired (69), this defect is not due to defects in gluconeogenesis, but rather to defect in NADH shuttle function.
In this section we review evidence gathered many years ago suggesting that the MAS is regulated by Ca2+. The mechanism was long considered to rely on Ca2+ entry in mitochondria through the uniporter and subsequent activation of mitochondrial dehydrogenases, or on direct activation by calmodulin. After the molecular identification of the AGCs, it has been shown that the shuttle is regulated by Ca2+ from the external side of the inner mitochondrial membrane. However, the interactions between the Ca2+ uniporter-mediated Ca2+ signaling in mitochondria and the mechanism mediated by the AGCs are complex and may vary from tissue to tissue.
1. Ca2+ activation of MAS in liver
It has been known for a long time that -adrenergic agents stimulate gluconeogenesis in hepatocytes. The mechanism is independent of cAMP and related to changes in cytosolic Ca2+ concentration ([Ca2+]i) (205, 372, 379). Increased gluconeogenesis is observed with reduced substrates, such as glycerol, xylitol, and sorbitol, that require the transfer of reducing equivalents to mitochondria, but is minimal or absent with oxidized substrates such as pyruvate or dihydroxyacetone phosphate (193, 226). The proposed sites of regulation are at the MAS or the GPS (194, 225, 379).
According to some groups, stimulation of rat hepatocytes or perfused liver with -adrenergic agonists such as phenylephrine leads to sustained activations of MAS and GPS, or to a transient stimulation of MAS followed by a stable stimulation of GPS (194, 225, 372, 379). Increased MAS activity was typically inhibited by the transaminase inhibitor aminooxyacetate. The mechanisms responsible for increases in MAS activity were thought to involve either changes in intermediates affecting AGC activity or an increase in the mitochondrial membrane potential caused by a direct stimulation of respiration that would increase the driving force of the AGC and shuttle activity (225). In fact, -adrenergic agonists transiently increased the cytosolic/mitochondrial aspartate concentration gradient and strongly reduced glutamate and especially -ketoglutarate concentrations (226, 370, 372), a finding thought to reflect a rapid activation of -KG dehydrogenase (-KGDH) activity (379). Careful studies by LaNoue’s group (371) showed that the mitochondrial membrane potential does not change significantly in glucagon-stimulated hepatocytes, arguing in favor of changes in AGC activity at the expense of changes in intermediates. The fall in -KG concentrations was regarded as the mechanism responsible for -adrenergic agonist-dependent increase in liver AGC activity (370, 371). The effect of the decrease in -KG levels was explained by the finding that -KG was a strong competitive inhibitor of aspartate formation (competitive with respect to oxaloacetate) via glutamate transamination in isolated liver mitochondria (371).
Hepatocyte activation by Ca2+-mobilizing agents was thought to induce Ca2+ entry in mitochondria coupled to Ca2+ activation of -KGDH activity. Glucagon, which increases intracellular cAMP levels, synergistically stimulates net inflow of Ca2+ to hepatocytes through receptor-activated Ca2+ channels (4, 121, 167) and net uptake of Ca2+ by hepatocyte mitochondria (4, 9, 121, 167, 264). As Ca2+ increases the affinity for -KG of -KGDH (246, 273), Ca2+ entry in mitochondria would result in a reduction of -ketoglutarate levels, with subsequent activation of glutamate transamination and aspartate efflux from mitochondria, in spite of a concurrent decrease in oxaloacetate levels brought about by the increase in the NADH/NAD ratio (370). Studies of LaNoue’s group have shown that there is a preferential channeling of external glutamate to mitochondrial transaminase and to the active site of -KGDH, suggesting microcompartmentation or channeling between -KGDH and the mitochondrial transaminase (351, 361 but see Ref. 267), especially in liver. This could explain the high sensitivity of liver AGC activity to a decrease of mitochondrial -KG levels caused by Ca2+-induced activation of -KGDH. However, to the best of our knowledge, a demonstration of this mechanism with the use of Ca2+ uniporter blockers was never reported.
It should be noted that experiments carried out by Sugano’s group (155) in perfused rat liver indicated that MAS activation by -adrenergic agonists and vasopressin is completely suppressed by calmodulin antagonists (155), whereas agonist-evoked Ca2+ signaling was not modified. This was interpreted as a direct effect of calmodulin on MAS activity. While the molecular basis for this effect is still unknown, it would be reasonable to propose a cytosolic site for calmodulin-dependent effects of Ca2+, in contrast to the interpretations based on a mitochondrial matrix Ca2+ effect on -KGDH.
2. Ca2+ regulation of MAS in heart
Liver is not the only organ where Ca2+ activates MAS activity. Evidence for this was obtained in kidney (350) and heart (342). However, the activation by Ca2+ of MAS activity in heart has proven to be elusive. This is due to the fact that in the heart, Ca2+ has opposing actions on the two carriers of MAS, an activating role for the AGC, which is observed only under specific conditions, and an inhibitor role for the OGC. As a result, the overall activating role on the AGC or on MAS is not always apparent.
In studies with heart mitochondria, physiological increases in matrix Ca2+ resulted in a marked activation of -KGDH and in a decreased accumulation of -KG (409). However, Scaduto (342) found that under conditions of MAS operation (incubation with glutamate plus malate) the decrease in -KG levels led to an inhibition rather than an increase of aspartate formation. Stimulation by Ca2+ of aspartate efflux in rat heart mitochondria was only observed after imposing a rise in mitochondrial NADH/NAD ratio through addition of pyruvate (together with malate and glutamate), to decrease OAA levels and basal aspartate efflux rate [reduced to 30% of control levels (342)]. Because -KG is a competitive inhibitor of OAA transamination to aspartate, Ca2+-induced changes in -KG levels controlled aspartate formation only under conditions where OAA levels were very low, as those provided by pyruvate. In the absence of pyruvate, the influence of Ca2+ on AGC activity was attributed to a balance between an inhibitory effect, caused by the increase in the NADH/NAD ratio and consequent reduction in OAA levels, and a stimulatory effect caused by a decrease in -KG levels (342).
Regarding the OGC, in heart mitochondria there is a strict competition for -KG between -KGDH and this carrier (Fig. 4), by virtue of the similarities of their apparent Km values. An apparent Km of 1.5 mM for -KG on the matrix side of the OGC has been reported (360), while the Km for -KG in the case of -KGDH is 0.67 mM (219) so that both steps will be very sensitive to the concentration of -KG in the mitochondrial matrix. Consequently, activation or inhibition of OGC transport will lead to a reduction or stimulation in -KGDH activity, respectively (219). Conversely, in perfused rat heart and isolated heart mitochondria, increased -KGDH activity results in a decrease in -KG efflux from mitochondria (278).
The relevance of the effects of Ca2+ on competition for -KG has been underscored by a recent study in which heart metabolism was studied at both basal and high work loads (279). Lewandowski’s group (279) showed that high work loads, which lead to increases in mitochondrial Ca2+ (see sect. III, C3B and E), resulted in a drastic reduction in OGC and MAS activity (to 20% of basal levels). Consistently, the decreased transfer of reducing equivalents to mitochondria was accompanied by increased tissue lactate levels (279).
On the basis of the above findings, it is interesting to note that under physiological conditions of high cytosolic and mitochondrial Ca2+ signals, MAS activity is activated in the liver but not in the heart. There are several reasons that could account for this difference. The competition for -KG between -KGDH and the OGC has been detected in heart but not in the liver. There is only one OGC gene (283), with no known variants, so that the difference between heart and liver is unlikely due to kinetic differences among isoforms. Tissue specificity could arise from differences between OGC levels in liver and heart; however, the activity and expression levels of OGC in heart are severalfold higher than in liver (171).
Another reason is related to microcompartmentation and substrate channeling, which may differ in heart and liver. In liver but not in heart mitochondria, flux along -KGDH is higher when -KG arises from glutamate by transamination rather than being provided directly as a substrate (219, 361). As indicated above, interaction between mitochondrial transaminase and -KGDH with preferential -KG channeling between the two enzymes explains why Ca2+ activates MAS through stimulation of -KGDH in the liver. Experiments in intact mitochondria and in reconstituted systems suggest that -KGDH can be bound to mitochondrial aspartate aminotransferase and that mitochondrial malate dehydrogenase can have a higher affinity for this binary complex than for any of the two constituents alone (120). It has been argued that this ternary complex could facilitate direct transfer of oxaloacetate to the transaminase, activation of malate dehydrogenase due to a decrease in the Km of the enzyme within the complex, and direct transfer of -KG to -KGDH (120). The formation or stability of this complex may be less important in heart than in liver mitochondria, and this may explain the lack of a clear activation of the AGC upon Ca2+ stimulation of -KGDH in heart.
3. Ca2+ regulation of citrin and aralar
The mechanism described above for the activation of liver MAS by Ca2+, i.e., activation of -KGDH by Ca2+ uptake on the Ca2+ uniporter (CU--KGDH), has long been the accepted explanation of MAS activation by Ca2+ mobilizing agents (370, 371). The discovery of the AGCs and the existence of a mechanism to activate AGC by Ca2+ acting on the external face of mitochondria (95, 286) therefore came as a surprise.
It is self-evident that if this Ca2+ activation mechanism of the AGC is physiologically relevant it would need to operate under conditions where the mitochondrial Ca2+ uniporter is not active. This question has been addressed recently in liver mitochondria that contain a single AGC isoform, citrin, and in brain and skeletal muscle mitochondria, that also have a single AGC isoform, aralar. Ca2+ activation of reconstituted MAS activity has been studied in isolated rat and mouse mitochondria incubated with ruthenium red (RR), under conditions preventing Ca2+ uptake in mitochondria (287).
Liver ACG coupled to MAS activity was found to be modestly (1.5-fold) activated by extramitochondrial Ca2+, with an S0.5 (free Ca2+ concentration for half-maximal activation) of 120 nM, close to the resting [Ca2+]i (79). On the other hand, activation by Ca2+ of brain and skeletal muscle MAS activity was more pronounced (3-fold) with an S0.5 of 324 ± 114 nM for brain mitochondria (287), with no changes in the Km for glutamate (79). These S0.5 values are substantially lower than the Km of the Ca2+ uniporter of mitochondria [10–20 µM, (143, 145, 190)], suggesting that Ca2+ activation of citrin- and aralar-coupled MAS activity may contribute to NADH reduction in mitochondria at Ca2+ concentrations where the Ca2+ uniporter is still inactive.
Unlike the CU--KGDH mechanism, in which the control on AGC activity is secondary to an increase in matrix aspartate level, i.e., through mass action ratio, Ca2+ activation of the AGC from the external face of the inner membrane leads to an increase in AGC flux and MAS activity at constant substrate concentrations, as expected for a classical regulatory reaction.
A) ROLE OF ARALAR AND THE MALATE-ASPARTATE NADH SHUTTLE IN TRANSDUCTION OF SMALL CA2+ SIGNALS TO MITOCHONDRIA. The role of aralar-MAS pathway in transduction of small Ca2+ signals to mitochondria has been studied in neurons. Pardo et al. (287) have employed primary neuronal cultures from control and aralar-deficient mice and NAD(P)H imaging with two-photon excitation microscopy to study MAS activity. Lactate utilization involves NADH shuttle activity and is important in neurons, as part of the astrocyte-neuron lactate shuttle (292). Lactate is produced by astrocytes and taken up by neurons that use it as an oxidizable substrate, especially during periods of high activity (187, 293, 294). Addition of 20 mM lactate caused an increase in cytosolic and mitochondrial NAD(P)H and a substantial transfer of NAD(P)H to mitochondria in control, but not in aralar-deficient neurons (287), indicating that MAS is the main NADH shuttle in neurons. This was also confirmed by the marked increase of the lactate-to-pyruvate ratio observed in the culture medium of aralar-deficient neurons (Pardo and Satrústegui, unpublished observations).
To study the effect of small [Ca2+]i signals on MAS activity, neurons were incubated in Ca2+-free medium plus 100 µM EGTA and exposed to lactate together with 100 µM ATP or different Ca2+-mobilizing agonists (Fig. 6). ATP-induced [Ca2+]i transients were small, with departures of 
100 nM from resting values that lasted 1 min at most and were not accompanied by any detectable increase in mitochondrial Ca2+ concentration ([Ca2+]mit) measured with rhod 2, in control or aralar-deficient neurons. However, this small Ca2+ signal resulted in a remarkable potentiation of lactate-dependent increase in mitochondrial NAD(P)H fluorescence in control neurons, which was notably absent in aralar-deficient neurons. Other Ca2+-mobilizing agonists, such as carbachol (50 µM) or thapsigargin (1 µM), also potentiated in a significant way the response to lactate in control neurons (287).
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Synaptosomal mitochondria start taking up Ca2+ at a global [Ca2+]i of 350–400 nM (241). This indicates that Ca2+ signaling through the aralar-MAS pathway in neuronal mitochondria, which displays an S0.5 of 324 nM, would be significant only below these Ca2+ concentrations, which is exactly what Pardo et al. found. On the other hand, the fact that mitochondria respond to [Ca2+]i signals with a substantial increase in NAD(P)H through a Ca2+ uniporter-independent pathway was new and surprising, especially because the magnitude of the response via the aralar-MAS pathway is very large in neurons, not far from that evoked by the activity of the Ca2+ uniporter-mitochondrial dehydrogenases pathway (see sect. IIIC3B). The availability of aralar-knockout animals will make it possible to assess the contribution of this pathway to Ca2+ signaling in different tissues. These studies may provide novel insights in Ca2+ signaling in mitochondria.
The role of the citrin-MAS pathway in transduction of Ca2+ signals to liver mitochondria has not been investigated so far, but based on the very low S0.5 for Ca2+ of citrin (79), it may be relevant at small departures from the basal Ca2+ concentration, which are below the range of the Ca2+ uniporter. Larger Ca2+ signal increases would be relayed to the mitochondrial matrix, resulting in Ca2+ activation of -KGDH, decrease in -KG, and subsequent stimulation of aspartate efflux from mitochondria (370, 371) and MAS activity. Indirect evidence that this may be so is examined in section IIIE.
B) EFFECTS OF LARGE CA2+ SIGNALS ON THE ARALAR-MAS PATHWAY. Pardo et al. (287) studied the increase in mitochondrial NAD(P)H in neurons in response to large [Ca2+]i signals brought about by depolarization-dependent activation of voltage-operated Ca2+ channels (VOCC) in the presence of millimolar external Ca2+ concentrations. In addition to Ca2+-mediated MAS activation, which was abolished in aralar-deficient neurons, it was expected that VOCC-dependent Ca2+ entry would result in Ca2+ uptake by mitochondria and subsequent dehydrogenase activation (110) in both control and aralar-deficient neurons.
There was no difference between the increases in [Ca2+]i and [Ca2+]mit obtained after lactate plus KCl addition in control and aralar-deficient neurons, in agreement with the lack of effect of aralar overexpression on Ca2+ homeostatic mechanisms reported earlier (222). The increase in the mit/cyt NAD(P)H fluorescence ratio obtained in the presence of KCl was significantly larger than that obtained by lactate addition itself, but in this case, there were no differences between control and aralar-deficient neurons (287). As the increase in mitochondrial NAD(P)H obtained with high K+ was expected to result from the additive effects of Ca2+ activation on MAS activity and Ca2+ activation of mitochondrial dehydrogenases, the lack of difference between wild-type and aralar-deficient neurons indicated that MAS activity contributes very little to the increase in mitochondrial NAD(P)H under high-K+ depolarization, suggesting that MAS activity may be inhibited under conditions allowing mitochondrial Ca2+ uptake.
Studies of MAS activity in isolated mitochondria in the presence or absence of 200 nM RR showed that Ca2+ activation of MAS was diminished if no RR was present (Fig. 7). This concentration of RR inhibits mitochondrial Ca2+ uptake completely (Fig. 7). Thus, in neurons, Ca2+ activation of MAS is blunted when Ca2+ is allowed to enter the mitochondria (287). This is possibly due to the effect of mitochondrial Ca2+ on the affinity for -KG of -KGDH and the competition for -KG between -KGDH and the OGC (Fig. 4), as described earlier for the heart MAS activity (see sect. IIIC2). Another factor that may blunt MAS activation when all mitochondrial dehydrogenases are activated by Ca2+ is the thermodynamic constraint of proton gradient formation. If the proton electrochemical gradient obtained under those conditions is close to maximal and cannot be increased, this would limit the entry of reducing equivalents into mitochondria through MAS. This situation has been described in -cells incubated with different fuel secretagogues that bypass glucose metabolism, each of them setting a maximal hyperpolarization of the mitochondrial membrane that correlated with insulin secretion (7).
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