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

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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.