Role of the glutamic and aspartic residues in Na⁺-ATPase function in theZrENA1gene ofZygosaccharomyces rouxii

Abstract

The effect of replacement of negatively charged amino acid residues on the function of Na+ transport proteins of the salt-tolerant yeast Zygosaccharomyces rouxii was examined by heterologous expression of mutant proteins in a strain of Saccharomyces cerevisiae, RH16.6, that lacks native Na+-ATPase activity due to null mutations of ENA1, ENA2, ENA3, and ENA4. Mutants of Na+/H+ antiporter gene (ZrSOD2) and Na+-ATPase gene (ZrENA1) of Z. rouxii were generated by site-directed mutagenesis. The significance of two aspartic residues arranged in tandem (D265 and D266) was demonstrated in Z. rouxii Na+/H+ antiporter. Some Z. rouxii Na+-ATPase mutant genes, namely E778A, D852A, and E981A present in the transmembrane domains (TMDs) and D736A, D743A, D748A, D749A, D759A, and D760A present in the cytoplasmic space were constructed. A lower level of salt tolerance was bestowed by the mutant genes D852A and E981A present in TMDs and D748A and D749A present in cytoplasmic space, compared with the wild-type gene (ZrENA1).

Identification of Conserved Prolyl Residue Important for Transport Activity and the Substrate Specificity Range of Yeast Plasma Membrane Na+/H+ Antiporters

“…These residues are conserved in the antiporters of prokaryotes and of some lower eukaryotes, despite an overall low similarity in their primary sequences (28). The tandem of aspartyl residues identified in the Spsod2p study were shown to be vital for the function of the Z. rouxii Sod2 antiporter (Asp 265 -Asp 266 ) (31), and mutations of conserved aspartyl residues 266 and 267 to asparagine reduced the sodium transport activity also in C. albicans Cnh1p and S. cerevisiae Nha1p (17,26). Interestingly, in the case of ScNha1 (D266,267N) antiporter, the potassium efflux activity was not changed, and it was another aspartyl residue (Asp 241 ) whose mutation affected K ϩ extrusion (26).…”