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 ( Sp NHE1) 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 Sp NHE1 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 Sp NHE1 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 Sp NHE1 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 Sp NHE1 function.