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敏感。然而，耐盐性差异很大，盐生植物耐盐。钠/氢反向转运蛋白（NHA，或交换剂）是从植物和酵母细胞内移除盐的膜蛋白，而在哺乳动物中，它们移除质子以交换细胞外的Na。质膜植物NHA，SOS 1的过表达提高了植物的耐盐性，使它们能够生活在更咸，更干旱的土壤中。 该研究计划将1，短期内， 了解植物、动物和酵母NHAs中质膜Na+/H+反向转运的分子转运机制 2、长期设计和改良植物中活性更高的SOS 1样蛋白。 实验将在SOS 1蛋白和S。pombe等价物、sod 2（SpNHE 1）蛋白和hNHE 1。我们有一个酵母NHA敲除盐敏感菌株。野生型或突变型NHA的恢复恢复了耐盐性，并允许测试功能、表达、靶向和耐盐性。实验分为A;对质膜耐盐蛋白、Na+/H+反向转运蛋白及其调节的基本理解;和B;设计用于改善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，使用氨基酸的位点特异性诱变，并确认它们是否在活性中是关键的，替换推定的孔衬里和阳离子配位残基。我们已经开发了一个表达系统的SOS 1拟南芥蛋白。在这里，我们将验证在阳离子转运的功能和调节的关键残基。 我们将2、影响修饰和修饰的组合以增强SOS 1的活性。这包括对自身抑制结构域、磷酸化氨基酸（磷酸模拟物）、调节蛋白结合区和进化上显示的氨基酸的突变，以促进SOS 1样蛋白的耐盐性。将在酵母和植物中检查对赋予耐盐性的能力的影响。 3、确定哺乳动物Na ~+/H ~+交换器的低温电镜结构。在这里，我们已经产生了毫克量的纯化和活性全长蛋白质，我们的合作者是瑞士，我们将确定脊椎动物Na+/H+反向转运蛋白的第一个完整结构。 我们将4，定义相关的酵母耐盐蛋白sod 2（SpNHE 1）中的关键残基，并将定义与SOS 1相比的结构和拓扑结构。这包括分析提出的阳离子配位的氨基酸。 总的来说，该项目将使我们能够了解耐盐性和Na+/H+反向转运蛋白如何工作，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.