Folates are essential cofactors in one-carbon metabolism and are vital in homocysteine remethylation, methyl group metabolism, and purine and thymidylate synthesis. Folates are thus crucial to maintain health and to reduce chronic disease risk, yet folate intake is suboptimal in segments of the US population and throughout the rest of the world. As plants are major dietary folate sources, raising plant folate levels by engineering would directly benefit health. This requires knowledge of plant folate synthesis pathways and transport steps. Folates consist of p-aminobenzoate (pABA), pterin, and glutamate moieties, pABA is made in plastids, the pterin is formed in the cytosol, and the two are coupled, reduced and glutamylated in mitochondria. Folates i themselves are found throughout plant cells. These observations show that plants must have systems toI move folates and their precursors in and out of organelles. Nothing is known about the transporters involved. Genomics can now be applied to this problem because folate or pterin transporters have been cloned from the parasite Leishmania and from mammalian mitochondria, and plants have homologs of these carriers. Accordingly, specific Aim 1 is to characterize Arabidopsis folate/pterin transporter homologs by functional expression in Leishmania or bacteria, by transport assays, and by subcellular localization.
Aim 2 is to use a new HPLC-electrochemical detection method to quantify the types of folates in plastids, mitochondria and vacuoles, and thereby define the in-vivo substrates for organellar transporters. In addition, transport assays will be used to show which types of folates vacuoles import, especially whether they are polyglutamyl forms. pABA transport is not understood in any organism. In plants, pABA exists predominantly as its glucose ester (pABA-GIc), which is made in the cytosol and may be stored in vacuoles. It may thus be that mitochondria or vacuoles import pABA-GIc, and that this is carrier-mediated.
Aim 3 is therefore to establish whether mitochondria or vacuoles take up pABA-GIc, and to characterize the glucosyltransferase(s) that produce pABA-GIc. The folate/pterin carriers of Leishmania are almost certainly plant-derived (Leishmania arose via endosymbiosis with a plant-like ancestor) and are critical to the efficacy of antifolate chemotherapy against these parasites. Studies of the cognate plant carriers could thus help develop new chemotherapies for leishmaniasis. I
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