State Transitions in the TORC1 Signaling Pathway and Information Processing in S. cerevisiae

TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases, Gtr1, Gtr2 and Rho1, bind TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to down-regulation of 450 Sch9-dependent protein and ribosome synthesis genes, and up-regulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP activated protein kinase (AMPK/Snf1) and at least one other factor, push the TORC1 pathway into an off state where Sch9- branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A off) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different Sch9 off, PP2A off state, where PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis or a stress response, depending on the needs of the cell. 2-color microarray dataset. Experiments interrogate the immediate response of wild-type (ACY044) and mutant strains (ACY142 and ACY509) to log growth; stress, including heatshock (42C), oxidative (0.4mM H2O2), and osmotic stress (0.375M KCl); and starvation conditions, including nitrogen, amino acid, and glucose starvation. Cells in mid-log growth were subjected to stress and/or starvation and/or the TOR inhibitor rapamycin for 20 minutes. Taken together, these microarrays support a model where the TORC1 pathway acts an information-processing hub. We find that condition dependent gene expression programs are activated hierarchically to allocate available energy for growth, amino acid synthesis, or a stress response, depending on the needs of the cell. All yeast strains are of W303 background, MatA. Mutant strains were made using standard techniques (homologous recombination).

雷帕霉素靶蛋白激酶复合物I（TOR kinase complex I, TORC1）是所有真核生物细胞生长与代谢的关键调控因子。此前在酵母中的研究表明，在氮源与氨基酸饥饿条件下，三种GTP酶（GTPases）Gtr1、Gtr2与Rho1会结合TORC1，阻断S6激酶（S6 kinase）Sch9的磷酸化并激活蛋白磷酸酶2A（PP2A）。这会导致450个依赖Sch9的蛋白质与核糖体合成基因表达下调，同时使100个依赖PP2A的氮同化与氨基酸合成基因表达上调。本研究通过凝胶迁移实验（bandshift assay）与基因芯片（microarray）检测证实，TORC1信号通路还可介导另外三种应激/饥饿状态。其一，在葡萄糖饥饿条件下，AMP激活的蛋白激酶（AMP-activated protein kinase, AMPK/Snf1）与至少一种其他因子会将TORC1通路置于失活状态，此时Sch9分支信号与PP2A分支信号均被抑制。值得注意的是，即便细胞同时处于氮源与葡萄糖饥饿状态，TORC1通路仍维持在葡萄糖饥饿（PP2A失活）状态。其二，在渗透应激条件下，丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）Hog1/p38会将TORC1通路导向另一种Sch9失活、PP2A失活的状态，此时PP2A分支信号仍可被氮源饥饿激活。其三，在氧化应激与热应激条件下，TORC1-Sch9信号通路被阻断，但PP2A分支信号仅呈现弱激活状态。综上，本研究数据证实，TORC1通路可作为信息处理枢纽，在不同条件下激活不同的基因程序，以根据细胞需求将可用能量分配用于细胞生长、氨基酸合成或应激应答。本数据集为双色基因芯片数据集。实验旨在检测野生型菌株（ACY044）与突变菌株（ACY142、ACY509）在对数生长期、各类应激（包括42℃热激、0.4mM过氧化氢氧化应激、0.375M氯化钾渗透应激）以及饥饿条件（包括氮源饥饿、氨基酸饥饿与葡萄糖饥饿）下的即时应答反应。将处于对数中期的细胞置于应激、饥饿条件，或用TOR抑制剂雷帕霉素（rapamycin）处理，处理时长均为20分钟。综上，这些基因芯片数据支持了TORC1通路作为信息处理枢纽的模型。本研究发现，条件依赖性基因表达程序会被层级激活，从而根据细胞需求将可用能量分配用于细胞生长、氨基酸合成或应激应答。所有酵母菌株均为W303背景，交配型为MatA。突变菌株通过标准技术（同源重组，homologous recombination）构建获得。