Mechanism of Plasma Membrane Mediated Na+/H+ Exchange and Salt Tolerance in Plants, Animals and Yeast

土壤盐分是降低植物生长和生产力的主要因素。大多数农作物对盐胁迫很敏感。然而，盐生植物的耐盐性差异很大，盐生植物对盐分具有抵抗力。钠/氢逆向转运体(NHAs，或交换器)是一种膜蛋白，可以从植物和酵母的细胞内清除盐分，而在哺乳动物中，它们可以清除质子，以换取细胞外的Na。质膜植物NHA，SOS1的过表达提高了植物的耐盐性，使它们能够生活在更盐分、更干旱的土壤中。该研究计划将1短期了解质膜Na/H逆向转运蛋白在植物、动物和酵母NHAS中的分子转运机制，2长期设计和改进在植物中更活跃的SOS1类蛋白。实验将在SOS1蛋白和相当于S.pombe的Sod2(SpNHE1)蛋白和hNHE1上进行。我们有一株酵母NHA基因敲除的盐敏感菌株。野生型或突变型NHAS的回归恢复了耐盐性，并允许测试功能、表达、靶向和耐盐性。实验分为A、关于质膜耐盐蛋白、Na/H逆向转运蛋白及其调控的基础知识的实验和B、旨在提高蛋白质的表达和活性的实验。我们也有C；实验来了解NHA的结构和它们的运输机制。我们将1，使用氨基酸的定点突变，并确认它们是否在活性上是关键的，取代假定的孔衬和阳离子配位残基。我们已经开发了一种拟南芥SOS1蛋白的表达系统。在这里，我们将验证在阳离子转运的功能和调节中起关键作用的残基。我们将影响对SOS1的修改和修改的组合，以增强活性。这包括自身抑制域的突变、磷酸化氨基酸的突变(磷仿制)、调节蛋白结合区的突变和进化中显示的氨基酸的突变，以促进SOS1样蛋白的耐盐性。将在酵母和植物中检测对赋予耐盐性的能力的影响。3，我们将定义哺乳动物Na/H交换器的低温EM结构。在这里，我们已经生产了大量纯化和活性的全长蛋白，与我们的合作伙伴瑞士，我们将确定第一个脊椎动物Na/H反向转运蛋白的完整结构。我们将定义相关酵母耐盐蛋白Sod2(SpNHE1)中的关键残基，并将与SOS1相比较定义结构和拓扑。这包括分析被认为是阳离子配位的氨基酸。总体而言，该项目将使我们了解耐盐性和Na/H逆向转运蛋白的工作原理，如何提高它们的活性，并将使我们能够更好地设计耐盐性蛋白，以了解和提高植物的耐盐性。每个项目都将献给一名学生或博士后研究员。

Soil salinity is a major factor in reducing plant growth and productivity. Most crop plants are sensitive to salt stress. However, salt tolerance varies greatly and halophytes are resistant to salt. Sodium/Hydrogen Antiporters (NHAs, or exchangers) are membrane proteins that remove salt from inside the cells of plants and yeast, while in mammals they remove protons in exchange for extracellular Na. Overexpression of plasma membrane plant NHA, SOS1, improves salt tolerance in plants allowing them to live in saltier, more arid soils. This research program will 1, short term, understand molecular transport mechanisms of plasma membrane Na+/H+ antiport in plants, animal and yeast NHAs and 2, long term, design and improve SOS1-like proteins that are more active in plants. Experiments will be on SOS1 proteins and on S. pombe equivalent, sod2 (SpNHE1) protein and hNHE1. We have a yeast NHA knockout salt sensitive strain. Return of wild or mutant NHAs returns salt tolerance and allows testing of function, expression, targeting and salt tolerance. Experiments are divided into A; those on fundamental understanding of plasma membrane salt tolerance proteins, Na+/H+ antiporters and their regulation, and B; on experiments designed to improve expression and activity of the proteins. We also have C; experiments to understand the structure of NHA's and their mechanism of transport. We will 1, use site-specific mutagenesis of amino acids and confirm if they are critical in activity, replacing putative pore lining and cation coordinating residues. We have developed an expression system for the SOS1 Arabidopsis protein. Here we will verify residues critical in function and regulation of cation transport. We will 2, affect modifications and combinations of modifications to SOS1 to enhance activity. This includes mutations to the autoinhibitory domain, to phosphorylated amino acids (phosphomimetics), to regulatory protein binding regions and to amino acids shown evolutionarily, to promote saline resistance in SOS1-like proteins. Effects on the ability to confer salt tolerance will be examined in yeast and plants. 3, We will define the cryo-EM structure of a mammalian Na+/H+ exchanger. Here we have produced mg amounts of purified and active full-length protein and with our collaborator is Switzerland, we will determine the first complete structure of a vertebrate Na+/H+ antiport protein. We will 4, define residues critical in the related yeast salt tolerance protein sod2 (SpNHE1), and will define structure and topology in comparison with SOS1. This includes analysis of amino acids proposed to be in cation coordination. Overall, the project will allow us to understand how salt tolerance and Na+/H+ antiporter proteins work, how to improve their activity and will allow us to better design salt tolerance proteins to understand and improve salt tolerance in plants. Each of these projects would each be dedicated to a student or postdoctoral fellow.