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Functional and structural analysis of transmembrane segment IV of Na+/H+ exchanger of Schizosaccharomyces pombe

Sod2 is the plasma membrane Na+/H+ exchanger of fission yeast S. pombe. It plays an important role in providing salt tolerance to S. pombe by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acids residues of TMIV (126FQINFLGSLLIAGCITSTDPVLSALI152) in activity of this protein using alanine scanning mutagenesis, and examining LiCl or NaCl tolerance in Sod2‐deficient S. pombe. We identified two amino acids, T144 and V147, critical for the function of Sod2. The mutations T144A and V147A resulted in a defective sod2 protein that did not confer salt tolerance when reintroduced into to S. pombe. Sod2 protein with other mutations in TMIV, exported either sodium or lithium as efficiently as the wild type Sod2 or had intermediate effects on salt tolerance of S. pombe. When we mutated T144 to aspartic acid, lysine or serine, the mutant proteins were impaired in their ability to allow growth in both lithium and sodium containing media, comparable to the knock out sod2 yeast (sod2::ura4). Western blot analysis showed that mutants T144A and V147A were expressed at comparable levels as the wild type sod2. To determine the structure of TMIV, we expressed and purified TMIV as a fusion protein with maltose binding protein. The TMIV peptide was released by cleaving the fusion protein with tobacco etch virus and re‐purified by HPLC. The structure is being determined by NMR.