Dissection of the TORC1 Signaling Network in Yeast

摘要/摘要 雷帕霉素激酶复合体I靶标(TORC1)是细胞生长和代谢的关键调节因子。 真核生物。过去15年的研究揭示了荷尔蒙背后的机制 和氨基酸信号传递给TORC1，但仍不清楚其他关键信号，如葡萄糖饥饿，是如何 传播到这个高度保守的复合体。在上一个授权期，我们研究了TORC1信令 研究发现，葡萄糖饥饿会导致TORC1途径迅速失活。然后，在一个 反应较慢(τ=10分钟)，TORc1解体，关键调控成分Kog1/Raptor进入 液泡/溶酶体边缘的单一小体。后一事件(部分)由AMPK/SNF1依赖驱动 Kog1在Ser 491/494处的磷酸化，以及附近的两个类Pron基序。Kog1-身体然后服务于增加 TORC1激活阈值，并确保细胞在次优状态下保持静止状态 条件。最近，我们发现自我情结(EGOC；人类的RAG/Ragator)-已知的 在氨基酸存在的情况下激活TORC1-也进入Kog1-小体。对这些数据进行跟踪 我们现在将确定：(A)葡萄糖饥饿如何触发TORC1途径的抑制；(B)哪些蛋白质 是在Kog1/EGOC小体中；(C)是什么驱动Kog1/EGOC小体的组装；以及(D)什么角色 Kog1/EGOC-小体在细胞信号中发挥作用。为此，我们将：(1)剖析Kog1的结构和功能- 身体。在初步实验中，我们已经证明Kog1/EGOC小体在纯化后保持完好 空泡。利用这一点，我们现在将映射Kog1/eGOC-Body和其 装配路径。然后，使用在Kog/EGOC-Body组装中有缺陷的突变体(包括一些WE )，我们将检验我们的预测，即Kog1小体在(A)下注对冲、(B)噪音中发挥作用 (C)应激反应和饥饿反应的协调。我们还将：(2)鉴定蛋白质 调控TORC1途径和Kog1/EGOC小体的形成，并剖析其作用机制。在……里面 为此，我们将测试葡萄糖饥饿和AMPK/Snf1，触发EGOC变化的预测 灭活TORC1并驱动Kog1/EGOC-小体的形成。要做到这一点，我们将绘制 调节TORC1信号和Kog1小体的形成，然后研究关键调节因子(包括 我们最近确定的两个)进行了详细说明。我们的建议是创新的，因为我们研究了 使用最先进的系统、基因组和生化方法进行TORC1信号传递。建议数 这项研究具有重要意义，因为它有望阐明细胞生长控制的潜在机制 人类和/或低等真核生物--对于(A)理解与TORC1相关的疾病，例如 癌症、糖尿病和肥胖症，以及(B)开发选择性地阻止致病真菌生长的药物。 此外，我们的工作有望阐明由数百种蛋白质组成的蛋白体在 在压力和饥饿条件下，以及从酵母到人类在健康细胞中发挥作用的各种生物体中。

SUMMARY/ABSTRACT The Target of Rapamycin kinase Complex I (TORC1) is a key regulator of cell growth and metabolism in eukaryotes. Work carried out over the last 15 years has shed light on the mechanisms underlying hormone and amino acid signaling to TORC1, but it is still unclear how other key signals, such as glucose starvation, are transmitted to this highly conserved complex. In the last grant period, we examined TORC1 signaling in budding yeast and found that glucose starvation leads to rapid inactivation of the TORC1 pathway. Then, in a slower reaction (τ=10min), TORC1 falls apart and the key regulatory component Kog1/Raptor moves into a single body on the edge of the vacuole/lysosome. The latter event is driven (in part) by AMPK/Snf1 dependent phosphorylation of Kog1 at Ser 491/494, and two nearby prion-like motifs. Kog1-bodies then serve to increase the threshold of TORC1 activation and ensure that cells remain committed to a quiescent state in suboptimal conditions. More recently, we found that the EGO complex (EGOC; Rag/Ragulator in humans)—known to activate TORC1 in the presence of amino acids—also moves into Kog1-bodies. Following up on these data we will now determine: (A) How glucose starvation triggers inhibition of the TORC1 pathway; (B) What proteins are in Kog1/EGOC-bodies; (C) What drives assembly of the Kog1/EGOC-bodies; and, (D) What role Kog1/EGOC-bodies play in cell signaling. To do this we will: (1) Dissect the structure and function of Kog1- bodies. In preliminary experiments we have shown that the Kog1/EGOC bodies remain intact on purified vacuoles. Taking advantage of this, we will now map the composition of the Kog1/EGOC-body and its assembly pathway. Then, using mutants that have defects in Kog/EGOC-body assembly (including some we have already isolated), we will test our prediction that Kog1-bodies play a role in (a) bet hedging, (b) noise dampening, and (c) the coordination of stress and starvation responses. We will also: (2) Identify the proteins that regulate the TORC1 pathway and Kog1/EGOC-body formation, and dissect their mechanism of action. In this aim, we will test the predication that glucose starvation and AMPK/Snf1, trigger changes in EGOC to inactivate TORC1 and drive the formation of Kog1/EGOC–bodies. To do this we will map the circuit that regulates TORC1 signaling and Kog1-body formation, and then study the function of key regulators (including two we recently identified) in detail. Our proposal is innovative in that we study new and unexplored aspects of TORC1 signaling using state-of-the-art systems, genomic, and biochemical approaches. The proposed research is significant in that it promises to shed light on the mechanisms underlying cell growth control in humans and/or lower eukaryotes—with implications for (a) understanding TORC1 related diseases such as cancer, diabetes and obesity, and (b) developing drugs that selectively block the growth of pathogenic fungi. In addition, our work promises to shed light on the role that protein bodies—formed by hundreds of proteins in stress and starvation conditions, and in organisms ranging from yeast to man—play in a healthy cell.