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

植物、动物和酵母质膜介导的Na/H交换和耐盐性机制

• 批准号: RGPIN-2020-03932
• 负责人: Fliegel, Larry
• 金额: $ 2.4万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716724
• 关键词: Mechanism; Plasma; Membrane; Mediated; Na+

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.

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敏感。然而，耐盐性差异很大，盐生植物对盐具有抵抗力。钠/氢逆向转运蛋白（NHAs，或交换器）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钠。质膜植物 NHA、SOS1 的过度表达可提高植物的耐盐性，使它们能够生活在含盐量更高、更干旱的土壤中。 该研究计划将 1、短期、 了解植物、动物和酵母 NHA 质膜 Na+/H+ 反向转运的分子转运机制 2、从长远来看，设计和改进在植物中更活跃的SOS1样蛋白。 实验将针对 SOS1 蛋白和粟酒裂殖酵母等效物、sod2 (SpNHE1) 蛋白和 hNHE1。我们有酵母 NHA 敲除盐敏感菌株。野生或突变 NHA 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；对质膜耐盐蛋白、Na+/H+逆向转运蛋白及其调节有基本了解的那些；B；旨在提高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，并确认它们是否对活性至关重要，取代假定的孔衬里和阳离子协调残基。我们开发了 SOS1 拟南芥蛋白的表达系统。在这里，我们将验证对阳离子运输的功能和调节至关重要的残留物。 2、影响SOS1的修饰和修饰组合以增强活性。这包括对自抑制结构域、磷酸化氨基酸（磷酸化）、调节蛋白结合区和进化显示的氨基酸的突变，以促进 SOS1 样蛋白的盐水耐受性。将在酵母和植物中检查对赋予耐盐性的能力的影响。 3, 我们将定义哺乳动物 Na+/H+ 交换器的冷冻电镜结构。在这里，我们已经生产了毫克量的纯化和活性全长蛋白质，并且与我们的合作者瑞士，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。 4、我们将定义相关酵母耐盐蛋白 sod2 (SpNHE1) 中关键的残基，并将定义与 SOS1 相比的结构和拓扑。这包括对提议进行阳离子配位的氨基酸的分析。 总体而言，该项目将使我们了解耐盐性和Na+/H+逆向转运蛋白如何发挥作用，如何提高其活性，并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。这些项目中的每一个都将专门针对一名学生或博士后研究员。