Citrin and aralar1 are Ca2+-stimulated aspartate/glutamate transporters in mitochondria

Abstract

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.

AGC1 Deficiency Causes Infantile Epilepsy, Abnormal Myelination, and Reduced N-Acetylaspartate

“…SLC25A12 encodes the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1). The specific SLC25A12 homozygous mutation identified is c.1058G>A, which results in an Arg353Gln substitution in the 678 amino acid AGC1 protein (Palmieri et al 2001). Moreover, SLC25A12 is located in one of the large regions of homozygosity shared by both affected children that we had demonstrated by genome-wide SNP microarray analysis (Supplemental Table 2).…”

“…Human wild-type and mutant AGC1 proteins were overexpressed as inclusion bodies in the cytosol of Escherichia coli C0214, solubilized, and purified as described (Palmieri et al 2001;Fiermonte et al 2009). The transport activities of the recombinant purified proteins were assayed as described (Palmieri et al 1995(Palmieri et al , 2001). The amount of both wild-type and mutant AGC1 incorporated into liposomes was about 20 % of the protein added to the reconstitution mixture.…”

Mitochondrial aspartate glutamate carrier (citrin) deficiency as the cause of adult-onset type II citrullinemia (CTLN2) and idiopathic neonatal hepatitis (NICCD)

“…In collaboration with two other groups of researchers, we have found that citrin and aralar are isoforms of aspartate glutamate carriers (AGCs) (Palmieri et al 2001). Recombinant citrin and aralar reconstituted into liposomes showed electrogenic exchange of aspartate for glutamate and H ϩ .…”

Brief Report: High Frequency of Biochemical Markers for Mitochondrial Dysfunction in Autism: No Association with the Mitochondrial Aspartate/Glutamate Carrier SLC25A12 Gene

“…This gene spreads over about 110 kb and is expressed primarily as 2.9 and 3.2 kb mRNA species, which encode a 678 amino-acid protein that is a calcium-dependent aspartate/glutamate mitochondrial carrier (AGC1). AGC1 plays an important role in the malate/aspartate shuttle, regulating the cytosolic redox state that controls conversion of lactate to pyruvate (Palmieri et al, 2001). We therefore examined whether, in autistic patients, this gene influences an intermediate biochemical phenotype characteristic of mitochondrial dysfunction, defined by plasma lactate levels and lactate/ pyruvate ratio distribution, and hence might play a role in the etiology of autism.…”