Dibrov P, Young P G, Fliegel L
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1998 Jun 9;37(23):8282-8. doi: 10.1021/bi9801457.
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.
我们鉴定出了对粟酒裂殖酵母的Na⁺/H⁺逆向转运蛋白sod2活性至关重要的氨基酸残基。我们将sod2的所有八个组氨酸残基突变为精氨酸。只有His367→Arg影响功能，并导致sod2完全无法使粟酒裂殖酵母在含LiCl的培养基中生长。突变型粟酒裂殖酵母（H367R）在酸性（pH 4.0）培养基中无法排出钠，并且在碱化外部培养基的能力上存在缺陷。当His367被天冬氨酸取代时，粟酒裂殖酵母在酸性pH下的钠输出受到抑制，而与野生型相比，在pH 6.1时钠依赖性质子内流增加。我们还对各种真核和原核Na⁺/H⁺交换体的假定膜区域中保守的三个残基进行了突变。含有Asp241→Asn和Asp266、267→Asn突变的粟酒裂殖酵母在含LiCl的培养基中的生长受到极大损害。此外，在外部pH 6.1时钠依赖性质子内流受损。D266、267N突变几乎完全消除了粟酒裂殖酵母细胞在外部pH 4.0时的钠输出；然而，D241N突变蛋白保留了几乎正常的Na⁺输出。结果表明，His367、Asp241和Asp266、267对真核Na⁺/H⁺交换体sod2的功能很重要。
