Summary: The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying different substrate specificities, patterns of gene expression and even non‐mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans‐membrane domain proteins. The number in other sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomyces cerevisiae and comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of specific symmetry‐related amino acid triplets. We assessed the predictive power of these triplets by comparing predictions with experimentally determined data for Arabidopsis MCs, and applied these predictions to the not yet functionally characterized mitochondrial carriers of the grass, Brachypodium distachyon, and the alga, Ostreococcus lucimarinus. We additionally studied evolutionary aspects of the plant MCF by comparing sequence data of the Arabidopsis MCF with those of Saccharomyces cerevisiae and Homo sapiens, then with those of Brachypodium distachyon and Ostreococcus lucimarinus, employing intra‐ and inter‐genome comparisons. Finally, we discussed the importance of the approaches of global gene expression analysis and in vivo characterizations in order to address the relevance of these vital carrier proteins.

The flux of a variety of metabolites, nucleotides and coenzymes across the inner membrane of mitochondria is catalysed by a nuclear-coded superfamily of secondary transport proteins called MCs (mitochondrial carriers). The importance of MCs is demonstrated by their wide distribution in all eukaryotes, their role in numerous metabolic pathways and cell functions, and the identification of several diseases caused by alterations of their genes. MCs can easily be recognized in databases thanks to their striking sequence features. Until now, 22 MC subfamilies, which are well conserved throughout evolution, have been functionally characterized, mainly by transport assays upon heterologous gene expression, purification and reconstitution into liposomes. Given the significant sequence conservation, it is thought that all MCs use the same basic transport mechanism, although they exhibit different modes of transport and driving forces and their substrates vary in nature and size. Based on substrate specificity, sequence conservation and carrier homology models, progress has recently been made in understanding the transport mechanism of MCs by new insights concerning the existence of a substrate-binding site in the carrier cavity, of cytosolic and matrix gates and conserved proline and glycine residues in each of the six transmembrane α-helices. These structural properties are believed to play an important role in the conformational changes required for substrate translocation.

The mitochondrial aspartate–glutamate carrier isoform 1 (AGC1), specific to neurons and muscle, supplies aspartate to the cytosol and, as a component of the malate–aspartate shuttle, enables mitochondrial oxidation of cytosolic NADH, thought to be important in providing energy for neurons in the central nervous system. We describe AGC1 deficiency, a novel syndrome characterized by arrested psychomotor development, hypotonia, and seizures in a child with a homozygous missense mutation in the solute carrier family 25, member 12, gene SLC25A12, which encodes the AGC1 protein. Functional analysis of the mutant AGC1 protein showed abolished activity. The child had global hypomyelination in the cerebral hemispheres, suggesting that impaired efflux of aspartate from neuronal mitochondria prevents normal myelin formation.

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.