Collaborative Research: Arabidopsis NHX-like Vacuolar Antiporters: Structure and Function

从细菌、藻类、真菌和蠕虫到高等植物和人类，阳离子/质子反转运蛋白在整个生物王国中细胞的pH和Na离子稳态中起着重要作用。这些蛋白是存在于不同细胞的质膜和内膜中的完整膜蛋白。尽管对哺乳动物和细菌质膜结合钠/质子反转运蛋白氨基酸残基的功能分析正在兴起，但对参与阳离子/质子交换、pH感应、离子特异性等的特定氨基酸知之甚少。迄今为止，还没有液泡或溶酶体转运体（动物、酵母或植物）的拓扑结构、结构和作用模式被表征。本研究的主要目的是解决这一关键信息的缺乏。这项研究将采用多学科方法，结合生物化学、分子生物学、膜转运、酵母和植物的异种表达以及晶体学。具体目标是：(i)确定拟南芥液泡反转运蛋白AtNHX1和AtNHX5的拓扑结构；（ii） AtNHX1和AtNHX5的结构/功能分析，以深入了解蛋白质中负责离子结合、离子选择性、pH调节和运输活性的关键氨基酸和区域；（三）确定其三维结构。广泛影响：植物液泡阳离子/质子反转运蛋白已被证明在离子稳态和植物发育中发挥重要作用。AtNHX1和AtNHX5的异位过表达已被证明在拟南芥和其他植物中具有耐盐性。了解反转运蛋白的作用模式以及确定影响其活性的因素将增强我们生产耐盐植物的能力。这些植物将为世界上受盐度影响的大片地区的作物生产提供重要的工具。此外，该项目的性质为从高中学生和教师到博士后学员的各个层次的跨学科培训提供了极好的机会。

Cation/proton antiporters play a major role in pH and Na ion homeostasis of cells throughout the biological kingdom, from bacteria, algae, fungi and worms to higher plants and humans. These proteins are integral membrane proteins residing in the plasma membranes and endomembranes of different cells. Although the functional analysis of amino acid residues of mammals and bacteria plasma membrane-bound sodium/proton antiporters is emerging, little is known about the specific amino acids that are involved in cation/proton exchange, pH sensing, ion specificity, etc. There is no vacuolar or lysosomal transporter (animal, yeast or plant) whose topology, structure and mode(s) of action have been characterized to date. The main purpose of this research is to address this lack of critical information. The research will use a multidisciplinary approach that will combine biochemistry, molecular biology, membrane transport, heterologous expression in yeast and plants, and crystallography. The specific goals are: (i) Determine the topology of the Arabidopsis vacuolar antiporters AtNHX1 and AtNHX5; (ii) Structure/functional analysis of AtNHX1 and AtNHX5 in order to gain insight into key amino acids and regions of the protein responsible for ion binding, ion selectivity, pH regulation and transport activity; and (iii) Determine their three-dimensional structure. Broader impacts: Plant vacuolar cation/proton antiporters have been shown to play important roles in ion homeostasis and plant development. The ectopic overexpression of AtNHX1 and AtNHX5 has been shown to confer salt tolerance in Arabidopsis and other plants. An understanding of the antiporter mode(s) of action together with the identification of factors affecting its activity will augment our capability of producing plants with enhanced salt tolerance. These plants would provide an important tool for crop production in large areas of the world affected by salinity. In addition, the nature of this project provides excellent opportunity for interdisciplinary training at all levels from high school students and teachers to post-doctoral trainees.