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目的:研究Elongator复合物在低浓度氨基酸条件下对TORC1活性的影响。方法:通过生长实验来观察Elongator突变体在低浓度氨基酸和低浓度亮氨酸条件下的生长程度;通过免疫印迹法检测核糖体蛋白S6的磷酸化水平来检测TORC1的活性;将GFP-ATG8质粒转入到Elongator突变体及野生型酵母菌株WT里,并通过免疫印迹法检测游离的GFP蛋白的水平来检测体内自噬。结果:生长实验显示Elongator突变体在低浓度氨基酸条件下显示过度生长的特性,并且这种过度生长可被TORC1抑制剂雷帕霉素所抑制;免疫印迹检测TORC1活性的实验以及检测体内自噬实验显示Elongator突变体在低浓度氨基酸条件下TORC1活性上升,而自噬进程被抑制;进一步实验证明Elongator突变体可特异性忽略低浓度亮氨酸而激活TORC1,抑制自噬,从而持续生长。结论:Elongator复合物在氨基酸信号下可抑制TORC1的活性,同时促进体内自噬进程。推测Elongator复合物可通过抑制TORC1信号通路确保细胞内蛋白翻译的准确性。
Objective: To study the effect of Elongator complex on TORC1 activity under low concentration of amino acids. Methods: The growth of Elongator mutants was observed under the conditions of low concentration of amino acids and low concentration of leucine by growth experiments. The phosphorylation of ribosomal protein S6 was detected by Western blot to detect the activity of TORC1. The GFP-ATG8 plasmid Transferred to Elongator mutant and wild-type yeast strain WT, and tested for autophagy in vivo by measuring the level of free GFP protein by immunoblotting. RESULTS: Growth experiments showed that Elongator mutants showed overgrowth at low concentrations of amino acids and this overgrowth could be inhibited by the TORC1 inhibitor rapamycin; Western blotting to detect TORC1 activity and to test autophagy in vivo The results showed that Elongator mutants increased TORC1 activity and inhibited the autophagy in low concentrations of amino acids. Further experiments showed that Elongator mutants could specifically inhibit low concentrations of leucine and activate TORC1 to inhibit autophagy and continue to grow. Conclusion: The Elongator complex can inhibit the activity of TORC1 under the amino acid signal and promote autophagy in vivo. It is speculated that the Elongator complex can ensure the accuracy of intracellular protein translation by inhibiting the TORC1 signaling pathway.