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.

Citrullinaemia (CTLN) is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase (ASS). Adult-onset type II citrullinaemia (CTLN2) is characterized by a liver-specific ASS deficiency with no abnormalities in hepatic ASS mRNA or the gene ASS (refs 1-17). CTLN2 patients (1/100,000 in Japan) suffer from a disturbance of consciousness and coma, and most die with cerebral edema within a few years of onset. CTLN2 differs from classical citrullinaemia (CTLN1, OMIM 215700) in that CTLN1 is neonatal or infantile in onset, with ASS enzyme defects (in all tissues) arising due to mutations in ASS on chromosome 9q34 (refs 18-21). We collected 118 CTLN2 families, and localized the CTLN2 locus to chromosome 7q21.3 by homozygosity mapping analysis of individuals from 18 consanguineous unions. Using positional cloning we identified a novel gene, SLC25A13, and found five different DNA sequence alterations that account for mutations in all consanguineous patients examined. SLC25A13 encodes a 3.4-kb transcript expressed most abundantly in liver. The protein encoded by SLC25A13, named citrin, is bipartite in structure, containing a mitochondrial carrier motif and four EF-hand domains, suggesting it is a calcium-dependent mitochondrial solute transporter with a role in urea cycle function.

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.

Aralar, the neuronal Ca2؉ -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 aralardeficient neurons, in agreement with the lack of MAS activity associated with aralar deficiency. The AGCs are one of the transporters responsible for the malateaspartate NADH shuttle (MAS). Because of the electrogenic nature of Asp/Glu exchange (20, 21), the AGC reaction is irreversible under physiological conditions and thus a potential site for regulation. The first aim of this work was to explore the potential of aralar as a brain AGC isoform to regulate MAS activity in brain at low Ca 2ϩ concentrations, below those required for the function of the mitochondrial Ca 2ϩ uniporter. MAS activity in brain mitochondria was found to have Ca 2ϩ -activation properties adequate for this purpose.The second aim of this work was to study the role of the aralar-MAS pathway in the supply of reducing equivalents to neuronal mitochondria. Aralar is expressed postnatally in rat and mouse brain, and it is located in neurons. Both MAS activity and aralar expression are acquired in parallel during neuronal maturation (22,23). Aralar is important for neuronal function as underscored by the finding that alterations in aralar gene and protein are associated with central nervous system diseases such as Mohr-Tranebjaerg syndrome, in which there is an impaired targeting of aralar to mitochondria (24), and autism (25). Aralar null mice also exhibit prominent motor coordination defects along with deficient myelination (26).We have employed primary neuronal cultures from control and aralar-deficient mice (26) and two-photon excitation microscopy imaging of NAD(P)H to monitor the transfer of reducing equivalents from cytosol to mitochondria. We show that MAS is the main pathway to transfer reducing equivalents to neuronal mitochondria. High [Ca 2ϩ ] i signals activate the Ca 2ϩ uniporter-mitochondrial dehydrogenases signaling pathway, whereas small [Ca 2ϩ ] i signals selectively activate MAS activity in neurons. We conclude that the aralar-MAS pathway plays an * This work was supported in part by Direcció n General de Investigació n del Ministerio de Ciencia y Tecnología Grant BMC2002-02072, Comunidad de Madrid Grant 08.5/ 0024/2003, Fondo de Investigaciones Sanitarias del Ministerio de Sanidad y Consumo 01/0395 (to J. S.), an institutional grant from the Fundació n Ramó n Areces to the Centro de Biología Molecular 'Severo Ochoa,' and by a Grant-in-aid for Scientific Research 16390100 from the Japan Society for the Promotion of Science (to K. K.). The costs of publicatio...

Deficiency of citrin, a liver-type mitochondrial aspartate-glutamate carrier (AGC), encoded by the SLC25A13 gene on chromosome 7q21.3, causes autosomal recessive disorders: adult-onset type II citrullinemia (CTLN2) and neonatal hepatitis associated with intrahepatic cholestasis (NICCD). So far, we have described 12 SLC25A13 mutations: 11 were from Japan and one from Israel. Three mutations found in Chinese and Vietnamese patients were the same as those in Japanese patients. In the present study, we identified a novel mutation IVS6+1G>C in a Japanese CTLN2 patient and widely screened 12 SLC25A13 mutations found in Japanese patients in control individuals from East Asia to confirm our preliminary results that the carrier frequency was high in Asian populations. Mutations 851-854del and 1638-1660dup were found in all Asian countries tested, and 851-854del associated with 290-haplotype in microsatellite marker D7S1812 was especially frequent. Other mutations frequently detected were IVS11+1G>A in Japanese and Korean, S225X in Japanese, and IVS6+5G>A in Chinese populations. We found a remarkable difference in carrier rates in China (including Taiwan) between north (1/ 940) and south (1/48) of the Yangtze River. We detected many carriers in Chinese (64/4169 = 1/65), Japanese (20/1372 = 1/69) and Korean (22/2455 = 1/112) populations, suggesting that over 80,000 East Asians are homozygotes with two mutated SLC25A13 alleles.