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S-adenosyl-L-methionine (SAM) is a coenzyme and the most commonly used methyl-group donor for the modification of metabolites, DNA, RNA and proteins. SAM biosynthesis and SAM regeneration from the methylation reaction product S-adenosyl-L-homocysteine (SAH) take place in the cytoplasm. Therefore, the intramitochondrial SAM-dependent methyltransferases require the import of SAM and export of SAH for recycling. Orthologous mitochondrial transporters belonging to the mitochondrial carrier family have been identified to catalyze this antiport transport step: Sam5p in yeast, SLC25A26 (SAMC) in humans, and SAMC1-2 in plants. In mitochondria SAM is used by a vast number of enzymes implicated in the following processes: the regulation of replication, transcription, translation, and enzymatic activities; the maturation and assembly of mitochondrial tRNAs, ribosomes and protein complexes; and the biosynthesis of cofactors, such as ubiquinone, lipoate, and molybdopterin. Mutations in SLC25A26 and mitochondrial SAM-dependent enzymes have been found to cause human diseases, which emphasizes the physiological importance of these proteins.

Itaconic acid (IA) is a naturally occurring dicarboxylic acid with applications in the manufacture of polymers. IA can be produced by fermentation using the fungi Aspergillus terreus or Ustilago maydis as biocatalysts. Indirect evidence has suggested that the mitochondrial carriers U. maydis Um_Mtt1 and A. terreus At_MttA export mitochondrially synthesized cis-aconitate to the cytosol for IA synthesis using malate as a countersubstrate. Here, by assaying the transport features of recombinant Um_Mtt1 and At_MttA in reconstituted liposomes, we find that both proteins efficiently transport cis-aconitate, but malate is well transported only by Um_Mtt1 and 2-oxoglutarate only by At_MttA. Bioinformatic analysis shows that Um_Mtt1 and At_MttA form a distinctive mitochondrial carrier subfamily. Our data show that although fulfilling the same physiological task, Um_Mtt1 and At_MttA have different biochemical features.

Neonatal epileptic encephalopathies with suppression bursts (SBs) are very severe and relatively rare diseases characterized by neonatal onset of seizures, interictal electroencephalogram (EEG) with SB pattern and very poor neurological outcome or death. Their etiology remains elusive but they are occasionally caused by metabolic diseases or malformations. Studying an Arab Muslim Israeli consanguineous family, with four affected children presenting a severe neonatal epileptic encephalopathy, we have previously identified a mutation in the SLC25A22 gene encoding a mitochondrial glutamate transporter. In this report, we describe a novel SLC25A22 mutation in an unrelated patient born from first cousin Algerian parents and presenting severe epileptic encephalopathy characterized by an EEG with SB, hypotonia, microcephaly and abnormal electroretinogram. We showed that this patient carried a homozygous p.G236W SLC25A22 mutation which alters a highly conserved amino acid and completely abolishes the glutamate carrier's activity in vitro. Comparison of the clinical features of patients from both families suggests that SLC25A22 mutations are responsible for a novel clinically recognizable epileptic encephalopathy with SB.

Summary The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspar- tate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identi- fied Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomy- ces cerevisiae . On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

Summary We describe the identification and functional characterization of two Arabidopsis mitochondrial basic amino acid carriers (BAC), AtmBAC1 and AtmBAC2, which are related to the yeast ornithine (Orn) carrier Ort1p, also known as Arg11p. The arg11 mutant requires arginine (Arg) supplementation because it fails to export sufficient ornithine from the mitochondrion to the cytosol where it is converted to arginine. AtmBAC1 and, to a lesser extent, AtmBAC2 partially replaced the function of Ort1p in yeast arg11. The more efficient putative carrier, AtmBAC1, was expressed in E. coli, purified, and reconstituted into phospholipid vesicles, where it transported the basic l-amino acids arginine, lysine, ornithine and histidine (in order of decreasing affinity). AtmBAC1 recognized l-histidine whereas both yeast Ort1p and the mammalian ortholog ORNT1p do not. Also different from ORNT1p, AtmBAC1 did not transport citrulline. AtmBAC1 appeared to be more stereospecific than the yeast and mammalian ornithine carriers, exhibiting greater preference for the l-forms of arginine, lysine and ornithine. By RT-PCR, both AtmBAC1 and AtmBAC2 transcripts were detected in stems, leaves, flowers, siliques, and seedlings. Expression of AtmBAC1 in seedlings is consistent with its involvement in Arg breakdown in early seedling development, i.e. delivery of Arg to mitochondrial arginase. The Km (0.19 mm) for Arg uptake by AtmBAC1 was close to the value we previously determined for the saturable component of Arg uptake into intact mitochondria from soybean seedling cotyledons.

Sengers syndrome is characterized by congenital cataracts, hypertrophic cardiomyopathy, mitochondrial myopathy, and lactic acidosis, but no abnormalities have been found with routine mitochondrial biochemical diagnostics (the determination of pyruvate oxidation rates and enzyme measurements). In immunoblot analysis, the protein content of the mitochondrial adenine nucleotide translocator 1 (ANT1) was found to be strongly reduced in the muscle tissues of two unrelated patients with Sengers syndrome. In addition, low residual adenine nucleotide translocator activity was detected upon the reconstitution of detergent-solubilized mitochondrial extracts from the patients' skeletal or heart muscle into liposomes. Sequence analysis and linkage analysis showed that ANT1 was not the primary genetic cause of Sengers syndrome. We propose that transcriptional, translational, or posttranslational events are responsible for the ANT1 deficiency associated with the syndrome.