Functional study of the Saccharomyces cerevisiae Nha1p C-terminus

Saccharomyces cerevisiae cells possess an alkali metal cation antiporter en coded by the NHA1 gene. Nha1p is unique in the family of yeast Na+/H+ antip orters on account of its broad substrate specificity (Na+, Li+, K+) and its long C-terminus (56% of the whole protein). In order to study the role of the C-terminus in Nha1p function, we constructed a series of 13 truncated N HA1 versions ranging from the complete one (2958 nucleotides, 985 amino aci ds) down to the shortest version (1416 nucleotides, 472 amino acids), with only 41 amino acid residues after the last putative transmembrane domain. T runcated NHA1 versions were expressed in an S. cerevisiae alkali metal cati on-sensitive strain (B31; ena1-4 Delta nha1 Delta). We found that the entir e Nha1p C-terminus domain is not necessary for either the proper localizati on of the antiporter in the plasma membrane or the transport of all four su bstrates (we identified rubidium as the fourth Nha1p substrate). Partial tr uncation of the C-terminus of about 70 terminal amino acids improves the to lerance of cells to Na+, Li+ and Rb+ compared with cells expressing the com plete Nha1p. The presence of the neighbouring part of the C-terminus (amino acids 883-928), rich in aspartate and glutamate residues, is necessary for the maintenance of maximum Nha1p activity towards sodium and lithium. In t he case of potassium, the participation of the long C-terminus in the regul ation of intracellular potassium content is demonstrated. We also present e vidence that the Nha1p C-terminus is involved in the cell response to sudde n changes in environmental osmolarity.