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

摘要:

【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pho8Δ60 pmt1Δ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。

Abstract:

[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 pmt1Δ strain 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 pmt1Δ strain treated with tunicamycin were determined by RT-qPCR. [Results] The replicative lifespan of the pmt1Δ strain was shortened by 38.7%, while the alkaline phosphatase activity of pmt1Δ strain 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 pmt1Δ strain were up-regulated with the increase in the concentration of tunicamycin. The transcript levels of ATG1 and ATG8 were up-regulated in the pmt1Δ strain treated with tunicamycin. [Conclusion] Endoplasmic reticulum stress impairs the replicative lifespan and enhances the autophagy of PMT1-deleted yeast cells.