CAJ | 학술논문

出芽酵母SNF1蛋白激酶参与葡萄糖阻遏和细胞胁迫应答.TOS3可通过磷酸化激活SNF1,参与SNF1调控的信号途径.本研究利用PCR方法扩增tos3基因的蛋白质编码序列,克隆到多拷贝表达载体pYES2/NTA上构建真核表达载体,转化酵母细胞并诱导TOS3过量表达.带HIS6标签的重组TOS3蛋白通过免疫印迹得以鉴定.进一步研究了过量表达TOS3对细胞热胁迫耐受性的影响,发现在热胁迫处理条件下,TOS3过量表达可恢复△snf1突变体细胞的生长缺陷,表明TOS3可能通过不依赖SNF1的其他信号途径参与细胞对热胁迫应答的调控.
출아효모SNF1단백격매삼여포도당조알화세포협박응답.TOS3가통과린산화격활SNF1,삼여SNF1조공적신호도경.본연구이용PCR방법확증tos3기인적단백질편마서렬,극륭도다고패표체재체pYES2/NTA상구건진핵표체재체,전화효모세포병유도TOS3과량표체.대HIS6표첨적중조TOS3단백통과면역인적득이감정.진일보연구료과량표체TOS3대세포열협박내수성적영향,발현재열협박처리조건하,TOS3과량표체가회복△snf1돌변체세포적생장결함,표명TOS3가능통과불의뢰SNF1적기타신호도경삼여세포대열협박응답적조공.
In Saccharomyces cerevisiae, SNF1 protein kinase is required for transcription of glucose-repressed genes and general stress responses. TOS3 activates SNF1 by phosphorylation of its activation-loop threonine. In this study, the ORF of tos3 was amplified by PCR, and cloned into multicopy expression vector pYES2/NTA to construct recombinant expression plasmid pYES2/NTA-tos3. The recombinant plasmid was transformed into yeast cells and TOS3 protein was overexpressed. The His6 tagged fusion protein was analyzed by immunoblot. The effect of TOS3 overexpression on cell growth under heat stress treatment was also studied. The results showed that the growth deficiency of △snf1 under heat stress treatment could be compensated by TOS3 overexpression. Our data indicate that TOS3 may regulate heat stress response through a pathway independent of SNF1.