Dissection of the TORC1 Signaling Network in Yeast

总结/摘要 雷帕霉素激酶复合物I（TORC 1）的靶标是细胞生长的主要调节剂， 真核生物的新陈代谢过去20年开展的工作阐明了这些机制， 潜在的激素和氨基酸信号传导到TORC 1，但目前还不清楚其他关键信号， 例如葡萄糖饥饿，被传递到这个高度保守的复合体。在最后一个赠款期间， 我们检测了芽殖酵母中的TORC 1信号，发现PKC、Gcn 2、Sit4和CK 2 信号通路与GAP SEAC（人类中的GATOR 1/2）一起工作，通过 高度保守的GTP酶，Gtr 1/2（人类中的Rag A/B和C/D）。这反过来又释放了TORC 1， 在液泡/溶酶体的边缘移动到一个单一的无活性体-这一事件取决于 TORC 1结合蛋白，Pib 2。在这个框架的基础上，我们现在希望：（1）确定和表征 蛋白质和途径与Gtr 1/2平行工作以调节TORC 1，以及（2）确定如何在细胞中表达TRC 1。 保守的Gcn2、PKC、Sit4、CK2通路通过Gtr1/2调节TORC 1。回答第一个问题， 我们从暴露于各种应激和饥饿条件的细胞中纯化了TORC 1，并鉴定了 许多新的互动。最值得注意的是未表征的空泡/溶酶体膜 蛋白质Ydl 180w、Ygr 125w和Syg 1，因为它们与TORC 1紧密结合，并且是其功能所必需的。 进入或离开不活动的身体。我们现在建议研究这些TORC 1的功能， 结合蛋白的详细，测试的假设：（i）Ydl180w是TORC 1的阻遏物，并竞争 与Gtr 1/2一起控制TORC 1活性，（ii）Ygr 125 w是TORC 1的硫依赖性激活剂，和（iii） Syg1是TORC 1的磷酸依赖性激活剂。为了解决第二个问题，我们纯化了 主要的Gtr1/2调节因子SEAC，并在葡萄糖和氮饥饿下定位其磷酸化 条件这导致了超过150个磷酸化位点的鉴定，其中许多是超或 在葡萄糖和/或氮饥饿期间低磷酸化。这笔赠款补充请求资金 给实验室增加一个URM的学生学生将重点剖析Ydl 180w（现在）的功能 称为Ait 1），研究饥饿依赖的Ait 1磷酸化在TORC 1中的作用。 调节和剖析与Ait 1结合的配体在调节细胞生长中的作用， 新陈代谢.我们的建议是创新的，因为我们研究新的和未开发的方面TORC 1 使用最先进的系统、蛋白质组学和生物化学方法进行信号传导。拟议 研究的重要性在于它有望揭示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-与影响（a） 了解TORC1相关疾病，如癌症，癫痫，糖尿病和肥胖，因为许多 所研究的蛋白质和途径是保守的，以及（B）开发选择性的药物 阻止病原真菌的生长。

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.