内质网应激反应在PMT1基因调控酿酒酵母细胞寿命和自噬中的作用

Effects of endoplasmic reticulum stress on the regulation of the lifespan and autophagy of Saccharomyces cerevisiae by PMT1

【目的】研究内质网应激反应在酿酒酵母蛋白O-甘露糖基转移酶-1(protein O-mannosyltransferase-1,pmt1)基因调控细胞寿命和自噬中的作用。【方法】基于基因同源重组的原理,构建双基因缺失菌株(pho8Δ60 pmt1Δ)。在光学显微镜下计数PMT1基因缺失酵母菌株(pmt1Δ)在衣霉素(内质网应激诱导剂)培养条件下的母细胞产生子代细胞的数量,统计菌株的复制性寿命。在SD-N培养中(自噬诱导培养基),通过酶标仪检测pho8Δ60pmt1Δ菌株的碱性磷酸酶活性。在衣霉素培养条件下,通过Western blotting检测细胞自噬标签Atg8蛋白的表达水平;通过RT-qPCR检测pmt1Δ菌株中自噬相关基因ATG1和ATG8的表达情况。【结果】在衣霉素培养条件下,pmt1Δ菌株的复制性寿命缩短38.7%,差异有统计学意义;PMT1基因缺失酵母菌株的碱性磷酸酶活性上升;随衣霉素浓度增加,pmt1Δ菌株中融合蛋白GFP-Atg8和游离GFP蛋白的表达水平逐渐升高;在衣霉素培养条件下,pmt1Δ菌株中ATG1和ATG8基因表达上调。【结论】内质网应激反应减弱了PMT1基因缺失酵母细胞复制性寿命,增强了细胞自噬活性。

[Objective]To study the effects of endoplasmic reticulum stress on the regulation of the lifespan and autophagy of Saccharomyces cerevisiae by the protein O-mannosyltransferase 1(PMT1).[Methods]The double deletion strain(pho8Δ60 pmt1Δ)was constructed based on the genetic homologous recombination.The daughter cells produced by the mother cell of the PMT1-deleted yeast strain(pmt1Δ)treated with tunicamycin(inducing endoplasmic reticulum stress)were counted under a light microscope,and the replicative lifespan of the strain was examined.A microplate reader was used to measure the alkaline phosphatase activity of the pho8Δ60 pmt1Δstrain in the SD-N medium(for inducing autophagy).Western blotting was employed to determine the expression level of the autophagy marker Atg8 in the presence of tunicamycin.The transcript levels of autophagy-related genes ATG1 and ATG8 in the pmt1Δstrain treated with tunicamycin were determined by RT-qPCR.[Results]The replicative lifespan of the pmt1Δstrain was shortened by 38.7%,while the alkaline phosphatase activity of pmt1Δstrain was increased compared with those of the wild type in the presence of tunicamycin.The expression levels of GFP-Atg8 fusion protein and free GFP in the pmt1Δstrain were up-regulated with the increase in the concentration of tunicamycin.The transcript levels of ATG1 and ATG8 were up-regulated in the pmt1Δstrain treated with tunicamycin.[Conclusion]Endoplasmic reticulum stress impairs the replicative lifespan and enhances the autophagy of PMT1-deleted yeast cells.