酵母PMT1基因参与内质网应激的调控机制

Role of Saccharomyces cerevisiae PMT1 in endoplasmic reticulum stress response

【目的】内质网应激(Endoplasmic reticulum stress,ERS)可激活细胞保护性信号级联反应——未折叠蛋白质反应(Unfolded protein response,UPR)。研究表明,酵母细胞中的UPR信号通路由转录因子Hac1p和ERS感应因子Ire1p共同介导。前期研究发现:蛋白质-O-甘露糖转移酶1(Protein-O-mannosyltransferase 1,PMT1)基因缺失能延长酵母细胞的复制性寿命,其机制与上调UPR通路活性相关。本文进一步探讨PMT1基因缺失在酵母ERS反应中的作用。【方法】观察PMT1基因与IRE1或HAC1基因双缺失酵母菌株(pmt1?hac1?和pmt1?ire1?)在ERS反应条件下的克隆形成能力;通过比色法检测各菌株的细胞增殖活性;RT-PCR检测各菌株UPR通路下游部分靶基因的转录水平。【结果】与对照菌株比较,PMT1基因缺失菌株(pmt1?)在ERS反应条件下生长较慢,而HAC1和IRE1单基因缺失菌株(hac1?和ire1?)在ERS反应条件下无法存活;在hac1?或ire1?菌株的基础上进一步缺失PMT1基因,可以改善hac1?菌株在ERS反应条件下的生长状态;但缺失PMT1基因没有上调hac1?菌株UPR通路靶基因的转录水平。【结论】缺失PMT1基因可增强hac1?菌株对ERS诱导剂衣霉素的抗性,机制与已知的UPR通路不相关,提示可能存在其它途径参与ERS反应的调控。

[Objective] Endoplasmic reticulum stress（ERS） activates a cytoprotective signaling cascade, termed as unfolded protein response（UPR）. Recent advances have unveiled that UPR pathwaywas mainly mediated by Hac1p（transcription factor） and Ire1p（ERS sensor） in yeast. Our previous results suggest that protein-O-mannosyltransferase 1（PMT1） deficiency enhanced the basal activity of UPR, and extended the replicative lifespan of yeast. In this study, we attempted to further study the effect of PMT1-deficiency on ERS response induced by tunicamycin in Saccharomyces cerevisiae. [Methods] Colony-forming ability of PMT1/IRE1 and PMT1/HAC1 double-gene deletion strains（pmt1？ire1？ and pmt1？hac1？） was analyzed under ERS condition. Cell proliferation assay was performed using the Microbial Viability Assay Kit. Expression levels of canonical UPR target genes were determined by quantitative RT-PCR（q RT-PCR）. [Results] PMT1 deficiency strain（pmt1？） grew slowly under ERS condition, while both the IRE1- and HAC1- deletion strain（ire1？ and hac1？） were not viable under this condition, compared to the control strain. The double-gene deletion strain（pmt1？hac1？） exhibited enhanced growth ability under ERS condition, compared with the hac1？strain. Nonetheless, expression levels of UPR target genes showed no significant difference between pmt1？hac1？ and hac1？ strains. [Conclusion] PMT1 deficiency enables hac1？ strain to resist tunicamycin induced ERS, independent of the UPR activity.