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

Abstract

Despite much study of the role of S-adenosylmethionine (SAM) in the methylation of DNA, RNA, and proteins, and as a cofactor for a wide range of biosynthetic processes, little is known concerning the intracellular transport of this essential metabolite. Screening of the Arabidopsis (Arabidopsis thaliana) genome yielded two potential homologs of yeast (Saccharomyces cerevisiae) and human SAM transporters, designated as SAMC1 and SAMC2, both of which belong to the mitochondrial carrier protein family. The SAMC1 gene is broadly expressed at the organ level, although only in specialized tissues of roots with high rates of cell division, and appears to be up-regulated in response to wounding stress, whereas the SAMC2 gene is very poorly expressed in all organs/tissues analyzed. Direct transport assays with the recombinant and reconstituted SAMC1 were utilized to demonstrate that this protein displays a very narrow substrate specificity confined to SAM and its closest analogs. Further experiments revealed that SAMC1 was able to function in uniport and exchange reactions and characterized the transporter as highly active, but sensitive to physiologically relevant concentrations of S-adenosylhomocysteine, S-adenosylcysteine, and adenosylornithine. Green fluorescent protein-based cell biological analysis demonstrated targeting of SAMC1 to mitochondria. Previous proteomic analyses identified this protein also in the chloroplast inner envelope. In keeping with these results, bioinformatics predicted dual localization for SAMC1. These findings suggest that the provision of cytosolically synthesized SAM to mitochondria and possibly also to plastids is mediated by SAMC1 according to the relative demands for this metabolite in the organelles.

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

“…The plastid AdoMet transporter, named SAMT1, is not a member of the FBT family but is part of the MCF (15,16). MCF proteins are also responsible for AdoMet transport in yeast (13) and human mitochondria (14).…”

“…Transport of AdoMet across the plasma membrane does not occur to a significant extent in mammalian cells, but transport of AdoMet is essential in several organelles. For example, the yeast (13) and human (14) mitochondrial AdoMet transporters and the Arabidopsis AdoMet plastid (and mitochondria) transporter (15,16) have been functionally characterized. They belong to the mitochondrial carrier protein family (MCF), a class of protein mediating the transport of various substrates (reviewed in Ref.…”

The Import of S-Adenosylmethionine into the Golgi Apparatus Is Required for the Methylation of Homogalacturonan

“…The genes encoding SAM transporters have now been cloned in yeast, human, and Arabidopsis (Marobbio et al, 2003;Agrimi et al, 2004;Bouvier et al, 2006;Palmieri et al, 2006), and belong to the mitochondrial carrier family. One of the interesting features of these carriers is that they are inhibited by SAH.…”

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

“…Reconstitution of SLC25A33 and SLC25A36 into LiposomesThe recombinant proteins in lauric acid were reconstituted into liposomes by cyclic removal of the detergent with a hydrophobic column of Amberlite beads (Fluka), as previously described (18,19) with some modifications. The composition of the initial mixture used for reconstitution was 50 l of purified protein (ϳ20 g of SLC25A33 or 30 g of SLC25A36), 60 l of 10% Triton X-114, 100 l of 10% phospholipids (L-␣-phosphatidylcholine from egg yolk; Sigma) in the form of sonicated liposomes, 10 mM substrate except where otherwise indicated, 0.6 mg (SLC25A33) or 1 mg (SLC25A36) of cardiolipin, 10 mM PIPES-NaOH, pH 7.0 (SLC25A33) or 5 mM MES-NaOH, 5 mM PIPES-NaOH, pH 6.25 (SLC25A36), and water to a final volume of 700 l. After vortexing, this mixture was recycled 13-fold through an Amberlite column (3.2 ϫ 0.5 cm) pre-equilibrated with a buffer containing 10 mM PIPES-NaOH at pH 7.0 (SLC25A33) or 5 mM MES-NaOH, 5 mM PIPES-NaOH at pH 6.25 (SLC25A36) and the substrate at the same concentration as in the starting mixture.…”