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首页> 外文期刊>Biochemical Pharmacology >Structure-function analysis of the highly conserved charged residues of the membrane protein FT1, the main folic acid transporter of the protozoan parasite Leishmania.
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Structure-function analysis of the highly conserved charged residues of the membrane protein FT1, the main folic acid transporter of the protozoan parasite Leishmania.

机译：膜蛋白FT1（原生动物寄生虫利什曼原虫的主要叶酸转运蛋白）的高度保守带电残基的结构功能分析。

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摘要

The main plasma membrane folate transporter FT1 of Leishmania belongs to the novel FBT family which is part of the major facilitator superfamily. We have investigated the role of the 10 most conserved charged amino acids of FBTs by site directed mutagenesis. The functions of the mutated proteins were tested for their capacity to transport FA, to sensitize methotrexate resistant cells to methotrexate, for protein production, and for protein localisation. Of the 10 conserved charged amino acids that were mutated to neutral amino acids, all had effects on FT1 transport activities. Only four of the 10 initial mutants (K116L, K133L, R497L, and D529V) retained between 15% and 50% of FT1 activity. The R497 residue was shown to be involved in substrate binding. When the charged conserved residues at position 124, 134, 179, 514, 537 and 565 were changed to neutral amino acids, this led to inactive proteins but the generation of new mutants D124E, R134K, D514E and D537E regained between 20% and 50% of wild-type FT1 activity suggesting that the charge is important for protein function. The mutated protein D179E had, under our standard experimental conditions, no activity, while E565D was completely inactive. The differential activity of the mutated proteins was due either to changes in the apparent K(m) or V(max). Mutagenesis experiments have revealed that charged amino acids were essential for FT1 stability or activity and led to a plausible model for the transport of folic acid through FT1.
机译：利什曼原虫的主要质膜叶酸转运蛋白FT1属于新的FBT家族，是主要促进子超家族的一部分。我们已经通过定点诱变研究了FBT的10个最保守带电氨基酸的作用。测试了突变蛋白的功能，以测定其转运FA的能力，使耐甲氨蝶呤的细胞对甲氨蝶呤敏感，用于蛋白质生产以及用于蛋白质定位的能力。在突变为中性氨基酸的10个保守带电氨基酸中，所有这些都对FT1转运活性有影响。 10个初始突变体（K116L，K133L，R497L和D529V）中只有四个保留了FT1活性的15％至50％。显示R497残基参与底物结合。当位置124、134、179、514、537和565的带电保守残基变为中性氨基酸时，这会导致蛋白失活，但新突变体D124E，R134K，D514E和D537E的生成又恢复了20％至50％野生型FT1活性的变化表明电荷对蛋白质功能很重要。在我们的标准实验条件下，突变的蛋白质D179E没有活性，而E565D完全没有活性。突变蛋白的差异活性是由于表观K（m）或V（max）的变化。诱变实验表明，带电荷的氨基酸对于FT1的稳定性或活性至关重要，并导致了通过FT1转运叶酸的合理模型。

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