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.
