Role of the glutamic and aspartic residues in Na+-ATPase function in the ZrENA1 gene of Zygosaccharomyces rouxii.
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).
Watanabe
Yasuo
Y
Laboratory of Biochemistry, Department of Applied Bioscience, Faculty of Agriculture, Ehime University, 3-5-8 Tarumi, Matsuyama-shi, 790-8566, Japan. watanabe@agr.ehime-u.ac.jp
Shimono
Yasutaka
Y
Tsuji
Hiromitsu
H
Tamai
Youichi
Y
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000502:physiology*; D001483:Base Sequence; D004352:Drug Resistance, Microbial; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005800:Genes, Fungal; D018698:Glutamic Acid / Q000737:chemistry / Q000502:physiology*; D017136:Ion Transport; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D012965:Sodium Chloride / Q000378:metabolism / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D020068:Zygosaccharomyces / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; C434279:SOD2 protein, Zygosaccharomyces rouxii; D017923:Sodium-Hydrogen Exchangers; D001224:Aspartic Acid; D018698:Glutamic Acid; D012965:Sodium Chloride