Arabidopsis mitochondria have two basic amino acid transporters with partially overlapping specificities and differential expression in seedling development

Abstract

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.

Molecular Identification of an ArabidopsisS-Adenosylmethionine Transporter. Analysis of Organ Distribution, Bacterial Expression, Reconstitution into Liposomes, and Functional Characterization

“…This approach has been used for the identification of mitochondrial carriers from yeast (Palmieri et al, 2006a) and mammals (Palmieri, 2004), and has previously allowed the mitochondrial carriers for dicarboxylate/tricarboxylates and basic amino acids to be identified in Arabidopsis (Picault et al, 2002;Hoyos et al, 2003;Palmieri et al, 2006b). For the closely related sequence of SAMC2, which was also cloned and expressed in E. coli in this study, no biochemical data are available because we were incapable of renaturing and/or reconstituting it functionally.…”

Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site

“…2). A tryptophan is also found in other mitochondrial carriers in the equivalent position of Trp-224: the carnitine/acyl-carnitine transporters (42)(43)(44)(45)(46); the yeast Ymc1p and Ymc2p, which are implicated in oleic acid and glutamate metabolism (47); and the Arabidopsis thaliana basic amino acid carriers BAC1 and BAC2 (48,49) (see alignments in Refs. 6, 8, and 13).…”

Proline Metabolism and Its Implications for Plant-Environment Interaction

“…Basic Amino Acid Carrier1 (BAC1) and BAC2 have been shown to be mitochondrial transporters that move ornithine and arginine out of the mitochondria (Figure 2). Although BAC1 and BAC2 are similar, their pH optimum, substrate specificities and gene expression differ, with BAC2 being less specific for basic amino acids (Hoyos et al, 2003;Palmieri et al, 2006). Genevestigator data and our own experiments also show that BAC2 is induced 10-15 fold by ABA or Connections of proline synthesis to nitrogen assimilation by GS and GOGAT; the N-acetyl-glutamate pathway (chloroplast); the oxidative pentose phosphate pathway (chloroplast); GABA metabolism (cytoplasm and mitochondria); and, ornithine-arginine metabolism (mitochondria).…”