Molecular Identification of an ArabidopsisS-Adenosylmethionine Transporter. Analysis of Organ Distribution, Bacterial Expression, Reconstitution into Liposomes, and Functional Characterization (original) (raw)
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Biochemical Journal, 2004
The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.
Specificity and genetics of S adenosylmethionine transport in Saccharomyces cerevisiae
Journal of Bacteriology
The specificity of a transport system for S-adenosylmethionine was determined through the use of structurally related derivatives. Of the compounds tested, the analogues S-adenosylethionine and S-inosylmethionine and the naturally occurring compounds S-adenosyl-(5')-3-methylthiopropylamine and S-adenosylhomocysteine competitively inhibited uptake of the sulfonium compound. Ki values for these compounds indicate that the order of affinity for the
Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter
Embo Journal, 2001
The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier protein family, most of which have not yet been functionally identi-®ed. Here the identi®cation of the mitochondrial carrier for S-adenosylmethionine (SAM) Sam5p is described. The corresponding gene has been overexpressed in bacteria and the protein has been reconstituted into phospholipid vesicles and identi®ed by its transport properties. In con®rmation of its identity, (i) the Sam5p±GFP protein was found to be targeted to mitochondria; (ii) the cells lacking the gene for this carrier showed auxotrophy for biotin (which is synthesized in the mitochondria by the SAM-requiring Bio2p) on fermentable carbon sources and a petite phenotype on non-fermentable substrates; and (iii) both phenotypes of the knock-out mutant were overcome by expressing the cytosolic SAM synthetase (Sam1p) inside the mitochondria.
Adenine nucleotide transport in plants: much more than a mitochondrial issue
Trends in Plant Science, 2011
Adenine nucleotides play a vital role in plant metabolism and physiology, essentially representing the major energy currency of the cell. Heterotrophic cells regenerate most of the ATP in mitochondria, whereas autotrophic cells also possess chloroplasts, representing a second powerhouse for ATP regeneration. Even though the synthesis of these nucleotides is restricted to a few locations, their use is nearly ubiquitous across the cell and thereby highly efficient systems are required to transport these molecules into and out of different compartments. Here, we discuss the location, biochemical characterization and evolution of corresponding transport systems in plants. We include recent scientific findings concerning organellar transporters from plants and algae and also focus on the physiological importance of adenine nucleotide exchange in these cells.
2021
Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, to date, no Golgi SAM transporter has been identified in plants. In this work, we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knock-out of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesised polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, the NMR revealed the existence of pectin ‘egg-box’ structures in intact cell walls, and showed that their formation is enhanced by reduced methyl-esterification. These changes in wall architecture were linked to subst...
THE PLANT CELL ONLINE, 2000
In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake ( fcy2 ) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family ( AtPUP1 to AtPUP15 ) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant K i ؍ 20 to 35 M), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.
Bioscience, biotechnology, and biochemistry, 2015
S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.
THE PLANT CELL ONLINE, 1999
All flowering plants produce S -methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that L -SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( ف 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, L -SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given L -35 S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis ( S -adenosylMet:Met S -methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora , maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( ف 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5 -phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase. White, R.H. (1982). Analysis of dimethyl sulfonium compounds in algae. J. Mar. Res. 40, 529-536. Wong, A.D., Swiader, J.M., and Juvick, J.A. (1995). Nitrogen and sulfur fertilization influences aromatic flavor components in Shrunken2 sweet maize kernels.
The Biochemical journal, 2014
Homocysteine S-methyltransferases (HMTs) are widely distributed enzymes that convert homocysteine (Hcy) into methionine (Met) using either S-adenosylmethionine (AdoMet) or the plant secondary product S-methylmethionine (SMM) as methyl donor. AdoMet is chirally and covalently unstable, with racemization of natural (S,S)-AdoMet yielding biologically inactive (R,S)-AdoMet and depurination yielding S-ribosylmethionine (S-ribosylMet). The apparently futile AdoMet-dependent reaction of HMTs was assigned a role in repairing chiral damage to AdoMet in yeast: yeast HMTs strongly prefer (R,S)- to (S,S)-AdoMet and thereby limit (R,S)-AdoMet build-up [Vinci and Clarke (2010) J. Biol. Chem. 285, 20526-20531]. In the present study, we show that bacterial, plant, protistan and animal HMTs likewise prefer (R,S)- over (S,S)-AdoMet, that their ability to use SMM varies greatly and is associated with the likely prevalence of SMM in the environment of the organism and that most HMTs cannot use S-ribosy...