Functional analysis of amino acid residues essential for activity in the Na+/H+ exchanger of fission yeast.
We identified amino acid residues important for activity of sod2, the Na+/H+ antiporter of Schizosaccharomyces pombe. We mutated all eight His residues of sod2 into Arg. Only His367-->Arg affected function and resulted in complete inability of sod2 to allow growth of S. pombe in LiCl-containing medium. Mutant S. pombe (H367R) could not expel sodium in acidic (pH 4.0) medium and were defective in their ability to alkalinize external medium. When His367 was replaced by Asp, sodium export of S. pombe was suppressed at acidic pH while the sodium-dependent proton influx at pH 6.1 was increased compared to wild type. We also mutated three residues conserved in putative membrane regions of various eukaryotic and prokaryotic Na+/H+ exchangers. S. pombe containing Asp241-->Asn and Asp266, 267-->Asn mutations had greatly impaired growth in LiCl-containing medium. In addition, sodium-dependent proton influx at external pH 6. 1 was impaired. Sodium export from S. pombe cells at external pH 4.0 was also almost completely abolished by the D266,267N mutation; however, the D241N mutant protein retained almost normal Na+ export. The results demonstrate that His367, Asp241, and Asp266,267 are important in the function of the eukaryotic Na+/H+ exchanger sod2.
Dibrov
P
P
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Young
P G
PG
Fliegel
L
L
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects; D004252:DNA Mutational Analysis; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011522:Protons; D012568:Schizosaccharomyces / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics
D000596:Amino Acids; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D008094:Lithium; D012964:Sodium