Identification of a mitochondrial transporter for basic amino acids in Arabidopsis thaliana by functional reconstitution into liposomes and complementation in yeast

Abstract

These authors contributed equally to this work. SummaryWe describe the identi®cation 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 suf®cient ornithine from the mitochondrion to the cytosol where it is converted to arginine. Atm-BAC1 and, to a lesser extent, AtmBAC2 partially replaced the function of Ort1p in yeast arg11. The more ef®cient putative carrier, AtmBAC1, was expressed in E. coli, puri®ed, and reconstituted into phospholipid vesicles, where it transported the basic L-amino acids arginine, lysine, ornithine and histidine (in order of decreasing af®nity). 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 stereospeci®c 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,¯owers, 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 K m (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.

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.…”

“…Control cultures with empty vector were processed in parallel. Inclusion bodies were purified on a Suc density gradient (Fiermonte et al, 1993), washed at 4°C with Tris-EDTA buffer (10 mM Tris/HCl, 1 mM EDTA, pH 7.0), then twice with a buffer containing Triton X-114 (3%, w/v), 1 mM EDTA, and 10 mM PIPES/NaOH, pH 7.0, and once again with Tris-EDTA buffer (Palmieri et al, 2001a;Picault et al, 2002;Hoyos et al, 2003). The SAMC1 and SAMC2 proteins were solubilized in 1.6% sarkosyl (w/v).…”

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

“…The identity of purified SLC25A33 and SLC25A36 was assessed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry of trypsin digests of the corresponding band excised from a Coomassie-stained gel (28,29). The amount of pure SLC25A33 and SLC25A36 was estimated by laser densitometry of stained samples using carbonic anhydrase as the protein standard.…”

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).…”