Functional Analysis of Conserved Transmembrane Charged Residues and a Yeast Specific Extracellular Loop of the Plasma Membrane Na(+)/H(+) Antiporter of Schizosaccharomyces pombe

The Na(+)/H(+) exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sodium in exchange for an extracellular proton. We examined the functional role of acidic amino acids of a yeast specific periplasmic extracellular loop 6 (EL6) and of Glu(74) and Arg(77) of transmembrane segment 3. Glu(74) and Arg(77) are conserved in yeast species while Glu(74) is conserved throughout various phyla. The mutation E74A caused a minor effect, while mutation R77A had a larger effect on the ability of SpNHE1 to confer salt tolerance. Mutation of both residues to Ala or Glu also eliminated the ability to confer salt tolerance. Arg(341) and Arg(342) were also necessary for SpNHE1 transport in S. pombe. Deletion of 3 out of 4 acidic residues (Asp(389), Glu(390), Glu(392), Glu(397)) of EL6 did not greatly affect SpNHE1 function while deletion of all did. Replacement of EL6 with a segment from the plant Na(+)/H(+) exchanger SOS1 also did not affect function. We suggest that EL6 forms part of a cation coordination sphere, attracting cations for transport but that the region is not highly specific for the location of acidic charges. Overall, we identified a number of polar amino acids important in SpNHE1 function.