Multiple mitochondrial DNA deletions are associated with clinically heterogeneous disorders transmitted as mendelian traits. Dominant missense mutations were found in the gene encoding the heart and skeletal muscle-specific isoform of the adenine nucleotide translocator (ANT1) in families with autosomal dominant progressive external opthalmoplegia and in a sporadic patient. We herein report on a sporadic patient who presented with hypertrophic cardiomyopathy, mild myopathy with exercise intolerance and lactic acidosis but no ophthalmoplegia. A muscle biopsy showed the presence of numerous ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. Molecular analysis revealed a C to A homozygous mutation at nucleotide 368 of the ANT1 gene. The mutation converted a highly conserved alanine into an aspartic acid at codon 123 and was absent in 500 control individuals. This is the first report of a recessive mutation in the ANT1 gene. The clinical and biochemical features are different from those found in dominant ANT1 mutations, resembling those described in ANT1 knockout mice. No ATP uptake was measured in proteoliposomes reconstituted with protein extracts from the patient's muscle. The equivalent mutation in AAC2, the yeast ortholog of human ANT1, resulted in a complete loss of transport activity and in the inability to rescue the severe Oxidative Phosphorylation phenotype displayed by WB-12, an AAC1/AAC2 defective strain. Interestingly, exposure to reactive oxygen species (ROS) scavengers dramatically increased the viability of the WB-12 transformant, suggesting that increased redox stress is involved in the pathogenesis of the disease and that anti-ROS therapy may be beneficial to patients.
To shed light on the metabolic role of two mitochondrial transporters for basic amino acids in Arabidopsis, we compared their functional properties in liposomes and expression during germination. Recombinant and purified BAC2, as previously reported for BAC1, transported various basic L-amino acids upon reconstitution in phospholipid vesicles. Both displayed highest affinity for arginine with similar Km and Vmax. However, BAC2 transported citrulline for which BAC1 had little or no affinity. Furthermore, BAC2 was less stereospecific than BAC1, transporting D-arginine and D-lysine at significant rates, and displayed a striking alkaline pH optimum (pH 8.0) whereas BAC1 activity was unaltered from pH 7.0 to 9.0. By semi-quantitative RT-PCR BAC1 transcript levels were found to be higher than those of BAC2 in germinated seeds. However, BAC2 expression transiently increased 2 days after germination. Disruption of the Arabidopsis arginase structural genes (ARGAH1 or ARGAH2) accentuated the increases of transcript levels of BAC1 at germination and of BAC2 2 days after germination and from 6 days on. Early expression of BAC1 and BAC2 is consistent with the delivery of arginine, released from seed reserves, to mitochondrial arginase and the export of ornithine. Increase of BAC2 transcript levels later in seedling development is consistent with roles in NO, polyamine or proline metabolism--processes involving arginine, citrulline and/or ornithine.
Despite the fundamental importance and high level of compartmentation of mitochondrial nucleotide metabolism in plants, our knowledge concerning the transport of nucleotides across intracellular membranes remains far from complete. Study of a previously uncharacterized Arabidopsis (Arabidopsis thaliana) gene (At4g01100) revealed it to be a novel adenine nucleotide transporter, designated ADNT1, belonging to the mitochondrial carrier family. The ADNT1 gene shows broad expression at the organ level. Green fluorescent protein-based cell biological analysis demonstrated targeting of ADNT1 to mitochondria. While analysis of the expression of b-glucuronidase fusion proteins suggested that it was expressed across a broad range of tissue types, it was most highly expressed in root tips. Direct transport assays with recombinant and reconstituted ADNT1 were utilized to demonstrate that this protein displays a relatively narrow substrate specificity largely confined to adenylates and their closest analogs. ATP uptake was markedly inhibited by the presence of other adenylates and general inhibitors of mitochondrial transport but not by bongkrekate or carboxyatractyloside, inhibitors of the previously characterized ADP/ATP carrier. Furthermore, the kinetics are substantially different from those of this carrier, with ADNT1 preferring AMP to ADP. Finally, isolation and characterization of a T-DNA insertional knockout mutant of ADNT1, alongside complementation and antisense approaches, demonstrated that although deficiency of this transporter did not seem to greatly alter photosynthetic metabolism, it did result in reduced root growth and respiration. These findings are discussed in the context of a potential function for ADNT1 in the provision of the energy required to support growth in heterotrophic plant tissues.
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