Dissection of the TORC1 Signaling Network in Yeast

摘要/摘要 雷帕霉素激酶复合体I(TORC1)的靶点是细胞生长的主要调节因子和 真核生物的新陈代谢。过去20年来开展的研究揭示了这种机制 潜在的激素和氨基酸向TORC1发出信号，但目前仍不清楚其他关键信号是如何发出的， 例如葡萄糖饥饿，被传递到这个高度保守的复合体。在上一次拨款期间， 我们检测了发芽酵母中的TORC1信号，发现PKC、GCN2、Sit4和CK2 信号通路与GAP SEAC(人类的GATOR1/2)共同作用，通过 高度保守的GTP酶，GTR1/2(人类中的Rag A/B和C/D)。这进而将TORC1释放到 移入液泡/溶酶体边缘的单个不活跃小体--这一事件取决于 TORC1结合蛋白，Pib2。在这一框架的基础上，我们现在希望：(1)确定和描述 这些蛋白质和通路与GTR1/2并行工作来调节TORC1，以及(2)决定如何 保守的GCN2、PKC、Sit4、CK2通路，通过GTR1/2调节TORC1。 我们从暴露在各种应激和饥饿条件下的细胞中纯化了TORC1，并鉴定了 无数新的互动角色。最值得注意的是未特指的空泡/溶酶体膜。 Ydl180w、Ygr125w和Syg1蛋白，因为它们与TORC1紧密结合，是ITS所必需的 移入或移出不活跃的物体。我们现在建议研究这些TORC1的功能 详细地结合蛋白质，检验以下假设：(I)Ydl180w是TORC1的抑制子并竞争 用GTR1/2控制TORC1活性，(Ii)Ygr125w是TORC1的硫依赖激活剂，和(Iii) Syg1是一种磷酸依赖的TORC1激活剂。为了解决第二个问题，我们提纯了 GTR1/2的主要调控因子SEAC，并定位了其在葡萄糖和氮饥饿中的磷酸化 条件。这导致了150多个磷酸化位点的鉴定，其中许多是高度或 葡萄糖和/或氮饥饿期间的低磷酸化。这项赠款补充申请资金 将URM学生添加到实验室。学生将专注于剖析Ydl180w(现在)的功能 Ait1)，研究了饥饿依赖的Ait1磷酸化在TORC1中所起的作用 调节和剖析与Ait1结合的配体在调节细胞生长和 新陈代谢。我们的建议是创新的，因为我们研究了TORC1的新的和未探索的方面 使用最先进的系统、蛋白质组学和生化方法进行信号传递。建议数 这项研究意义重大，因为它有望阐明细胞生长控制的潜在机制， 和复杂的信号整合，在一个重要的模式生物体中--对(A) 了解与TORC1相关的疾病，如癌症、癫痫、糖尿病和肥胖，因为许多 正在研究的蛋白质和途径是保守的，并且(B)开发选择性地 阻断病原真菌的生长。

SUMMARY/ABSTRACT The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Work carried out over the last 20 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 the PKC, Gcn2, Sit4, and CK2 signaling pathways work together with the GAP SEAC (GATOR1/2 in humans) to inhibit TORC1 via the highly-conserved GTPases, Gtr1/2 (Rag A/B and C/D in humans). This in turn releases TORC1 to move into a single inactive body at the edge of the vacuole/lysosome—an event that depends on the TORC1 binding protein, Pib2. Building on this framework, we now wish to: (1) Identify and characterize the proteins and pathways work in parallel with Gtr1/2 to regulate TORC1, and (2) determine how the conserved Gcn2, PKC, Sit4, CK2 pathways, regulate TORC1 via Gtr1/2. To address the first question, we purified TORC1 from cells exposed to a variety of stress and starvation conditions, and identified numerous new interactors. The most notable are the uncharacterized vacuolar/lysosomal membrane proteins Ydl180w, Ygr125w and Syg1, since they bind tightly to TORC1 and are required for its movement into, or out of, the inactive bodies. We now propose to study the function of these TORC1 binding proteins in detail, testing the hypotheses that: (i) Ydl180w is repressor of TORC1 and competes with Gtr1/2 to control TORC1 activity, (ii) Ygr125w is a sulfur dependent activator of TORC1, and (iii) Syg1 is a phosphate dependent activator of TORC1. To address the second question, we purified the major Gtr1/2 regulator SEAC, and mapped its phosphorylation in glucose and nitrogen starvation conditions. This led to the identification of over 150 phosphorylation sites, many of which are hyper- or hypo-phosphorylated during glucose and/or nitrogen starvation. This grant supplement request funds to add a URM student to the lab. The student will focus on dissecting the function of Ydl180w (now called Ait1), examining the role that starvation dependent phosphorylation of Ait1 plays in TORC1 regulation and dissecting the role that ligand binding to Ait1 plays in regulating cell growth and metabolism. Our proposal is innovative in that we study new and unexplored aspects of TORC1 signaling using state-of-the-art systems, proteomic, and biochemical approaches. The proposed research is significant in that it promises to shed light on the mechanisms underlying cell growth control, and complex signal integration, in an important model organism—with implications for (a) understanding TORC1 related diseases such as cancer, epilepsy, diabetes and obesity, since many of the proteins and pathways under investigation are conserved and (b) developing drugs that selectively block the growth of pathogenic fungi.