Transport of Folates and Their Precursors in Plants

叶酸是单碳代谢必不可少的辅助因子，在同型半胱氨酸再甲基化、甲基代谢、嘌呤和胸苷酸合成中至关重要。因此，叶酸对于维持健康和降低慢性疾病风险至关重要，但在美国和世界其他地区，叶酸的摄入量并不理想。由于植物是膳食中叶酸的主要来源，通过工程手段提高植物叶酸水平将直接有益于健康。这需要了解植物叶酸合成途径和运输步骤。叶酸由对氨基苯甲酸酯（pABA）、蝶呤（pterin）和谷氨酸（glutamate）组成，pABA在质体中生成，蝶呤（pterin）在细胞质中形成，两者在线粒体中偶联、还原和谷氨酸化。叶酸本身存在于植物细胞中。这些观察结果表明，植物必须具有将叶酸及其前体进出细胞器的系统。有关运送者的情况一无所知。基因组学现在可以应用于这个问题，因为叶酸或蝶呤转运体已经从寄生虫利什曼原虫和哺乳动物线粒体中克隆出来，植物有这些载体的同源物。因此，特定的目的1是通过利什曼原虫或细菌中的功能表达，通过运输测定和亚细胞定位来表征拟南芥叶酸/蝶呤转运蛋白同源物。目的2是使用一种新的hplc -电化学检测方法来定量质体、线粒体和液泡中叶酸的类型，从而确定细胞器转运体的体内底物。此外，运输试验将用于显示液泡进口的叶酸类型，特别是它们是否是聚谷氨酰基形式。pABA在任何生物体中的转运尚不清楚。在植物中，pABA主要以葡萄糖酯（pABA- gic）的形式存在，它在细胞质中产生，并可能储存在液泡中。这可能是线粒体或液泡输入pABA-GIc，这是载体介导的。因此，目的3是确定线粒体或液泡是否吸收pABA-GIc，并表征产生pABA-GIc的糖基转移酶。利什曼原虫的叶酸/蝶呤载体几乎肯定是植物来源的（利什曼原虫是通过与植物样祖先的内共生而产生的），并且对抗叶酸化疗对这些寄生虫的疗效至关重要。因此，对同源植物载体的研究可以帮助开发新的利什曼病化疗方法。

Description: 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