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。大多数作物对盐胁迫很敏感。然而，盐生植物的耐盐性差异很大，对盐有抗性。钠/氢反转运蛋白（NHAs，或交换剂）是一种膜蛋白，可以从植物和酵母细胞内去除盐，而在哺乳动物中，它们可以去除质子以换取细胞外的Na。质膜植物NHA、SOS1的过度表达提高了植物的耐盐性，使它们能够在更盐、更干旱的土壤中生存。本项目短期目标是了解植物、动物和酵母NHAs中质膜Na+/H+反转运的分子转运机制，长期目标是设计和改进植物中活性更高的sos1样蛋白。实验将在SOS1蛋白和S. pombe等效蛋白、sod2 （SpNHE1）蛋白和hNHE1上进行。我们有一个酵母NHA敲除盐敏感菌株。野生或突变的NHAs恢复耐盐性，并允许测试功能，表达，靶向性和耐盐性。实验分为A类；对质膜耐盐蛋白、Na+/H+反转运蛋白及其调控的基本认识；旨在改善蛋白质表达和活性的实验。我们还有C；通过实验了解NHA的结构及其转运机制。我们将1，使用位点特异性突变氨基酸，并确认它们是否在活性中至关重要，取代假定的孔隙内衬和阳离子配位残基。我们开发了一个SOS1拟南芥蛋白的表达系统。在这里，我们将验证残基关键的功能和调控阳离子运输。我们将影响SOS1的修改和修改组合，以增强活动。这包括自抑制结构域、磷酸化氨基酸（拟磷物质）、调节蛋白结合区和进化上显示的氨基酸的突变，以促进sos1样蛋白的盐抗性。对授予耐盐能力的影响将在酵母和植物中进行研究。我们将定义哺乳动物Na+/H+交换剂的低温电镜结构。在这里，我们已经生产了mg量的纯化和活性全长蛋白，并与瑞士合作，我们将确定脊椎动物Na+/H+反港蛋白的第一个完整结构。我们将定义相关酵母耐盐蛋白sod2 （SpNHE1）的关键残基，并将定义与SOS1比较的结构和拓扑结构。这包括分析被认为是阳离子配位的氨基酸。总的来说，该项目将使我们了解耐盐性和Na+/H+反转运蛋白是如何工作的，如何提高它们的活性，并使我们能够更好地设计耐盐蛋白，以了解和提高植物的耐盐性。这些项目中的每一个都将致力于一个学生或博士后。

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.