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Date:
2021-04-05
[Abstract] The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae, TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to ...
[摘要] [摘要]雷帕霉素激酶复合体I(TORC1)的靶标是真核生物中细胞生长和代谢的主要调节剂。在促生长激素和丰富营养素的存在下,TORC1活跃并通过磷酸化多种蛋白质来驱动蛋白质,脂质和核苷酸合成。相反,当氮和/或葡萄糖水平下降时,TORC1被抑制,导致细胞从合成代谢转换为分解代谢,并最终进入静止状态。在发芽酵母中酿酒酵母 抑制TORC1会触发TORC1从其在液泡周围的位置移动到液泡膜边缘的单个焦点/物体。这种重新定位取决于许多关键的TORC1调节子的活动,因此可以使用TORC1定位分析来跟踪通过TORC1途径发出的信号。在这里,我们提供了使用荧光显微镜测量TORC1(特别是Kog1-YFP)重新定位/信号传递的详细协议。重点放在确保以下方面的程序上:(1)尽管葡萄糖和氮饥饿期间它们相对较低的荧光和自发荧光灶的积累,但仍正确地识别(并计数)TORC1抗体;(2)在每次实验开始时,使细胞保持对数生长期,以便正确监测TORC1体形成的动力学;(3)使用适当的荧光标签来避免检查错误定位的TORC1。
[背景]雷帕霉素的靶激酶复合物I(TORC1 )是真核生物的细胞生长控制网络中的中央轮毂(Loewith和Hall,2011;冈萨雷斯和Hall,2017; Liu和萨巴蒂尼,2020 ...
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