Folate metabolism in plants: an Arabidopsis homolog of the mammalian mitochondrial folate transporter mediates folate import into chloroplasts.
ABSTRACT The distribution of folates in plant cells suggests a complex traffic of the vitamin between the organelles and the cytosol. The Arabidopsis thaliana protein AtFOLT1 encoded by the At5g66380 gene is the closest homolog of the mitochondrial folate transporters (MFTs) characterized in mammalian cells. AtFOLT1 belongs to the mitochondrial carrier family, but GFP-tagging experiments and Western blot analyses indicated that it is targeted to the envelope of chloroplasts. By using the glycine auxotroph Chinese hamster ovary glyB cell line, which lacks a functional MFT and is deficient in folates transport into mitochondria, we showed by complementation that AtFOLT1 functions as a folate transporter in a hamster background. Indeed, stable transfectants bearing the AtFOLT1 cDNA have enhanced levels of folates in mitochondria and can support growth in glycine-free medium. Also, the expression of AtFOLT1 in Escherichia coli allows bacterial cells to uptake exogenous folate. Disruption of the AtFOLT1 gene in Arabidopsis does not lead to phenotypic alterations in folate-sufficient or folate-deficient plants. Also, the atfolt1 null mutant contains wild-type levels of folates in chloroplasts and preserves the enzymatic capacity to catalyze folate-dependent reactions in this subcellular compartment. These findings suggest strongly that, despite many common features shared by chloroplasts and mitochondria from mammals regarding folate metabolism, the folate import mechanisms in these organelles are not equivalent: folate uptake by mammalian mitochondria is mediated by a unique transporter, whereas there are alternative routes for folate import into chloroplasts.
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ABSTRACT: Abstract Mitochondrial carriers transport inorganic ions, nucleotides, amino acids, keto acids and cofactors across the mitochondrial inner membrane. Structurally they consist of three domains, each containing two transmembrane α-helices linked by a short α-helix and loop. The substrate binds to three major contact points in the central cavity. The class of substrate (e.g., adenine nucleotides) is determined by contact point II on transmembrane α-helix H4 and the type of substrate within the class (e.g., ADP, coenzyme A) by contact point I in H2, whereas contact point III on H6 is most usually a positively charged residue, irrespective of the type or class. Two salt bridge networks, consisting of conserved and symmetric residues, are located on the matrix and cytoplasmic side of the cavity. These residues are part of the gates that are involved in opening and closing of the carrier during the transport cycle, exposing the central substrate binding site to either side of the membrane in an alternating way. Here we revisit the plethora of mutagenesis data that have been collected over the last two decades to see if the residues in the proposed binding site and salt bridge networks are indeed important for function. The analysis shows that the major contact points of the substrate binding site are indeed crucial for function and in defining the specificity. The matrix salt bridge network is more critical for function than the cytoplasmic salt bridge network in agreement with its central position, but neither is likely to be involved in substrate recognition directly.Molecular Membrane Biology 03/2013; 30(2):149-159. · 3.13 Impact Factor
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ABSTRACT: The effect of methotrexate (MTX), a folate analogue and specific competitive inhibitor of dihydrofolate reductase (DHFR), is assessed (concentrations: 0.001, 0.01, 0.1, 1 and 10 μM) on germinating grass pea (Lathyrus sativus L.) seedlings in relation to radicle length, mitotic index, total RNA content and DHFR activity. Response of callus growth of the species is also studied following MTX treatments. Furthermore, the effect of MTX on seedlings treated with colchicine (0.5%, 8 h) and 5-formyl tetrahydrofolate (CF; 10 mM) are also analyzed. The objective of the present study is to evaluate the effectivity of the drug MTX on a plant species with the view to use plant system as a model for screening antifolate drugs. Results suggest that MTX possesses distinct role in inhibiting plant cell division, RNA synthesis and DHFR activity; although, at low concentration (0.001 μM) it shows stimulatory effect.Nucleus 07/2014;
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ABSTRACT: Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family (MCF) have, however, been identified in Saccharomyces cerevisiae (Leu5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters whereas non-flowering plants have only one. The homologs from maize (GRMZM2G161299, GRMZM2G420119) and Arabidopsis (At1g14560, At4g26180) all complemented the growth defect of the S. cerevisiae leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual import assays with purified pea mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently-transformed tobacco BY-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located MCF carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.Plant physiology 04/2013; · 6.56 Impact Factor