Dissection of the TORC1 Signaling Network in Yeast

摘要/摘要 雷帕霉素激酶复合体I(TORC1)的靶点是细胞生长和代谢的主要调节因子 真核生物。在过去的20年里进行的研究揭示了荷尔蒙和 氨基酸向TORC1传递信号，但目前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，我们研究了萌芽中的TORC1信号 发现PKC、GCN2、Sit4和CK2信号通路与GAP SEAC协同工作 (人类中的GATOR1/2)通过高度保守的GTP酶GTR1/2(Rag A/B和C/D在 人类)。这进而释放TORC1，进入液泡/溶酶体边缘的一个不活跃的小体- 一种依赖于TORC1结合蛋白Pib2的事件。在这个框架的基础上，我们现在希望：(1) 鉴定和鉴定与GTR1/2平行调节TORC1的蛋白质和通路，以及(2) 确定保守的GCN2、PKC、Sit4、CK2通路是如何通过GTR1/2调节TORC1的。 问题：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。最值得注意的是未鉴定的空泡/溶酶体膜蛋白。 Ydl180w、Ygr125w和Syg1，因为它们与TORC1紧密结合，并且是TORC1进入或移出TORC1所必需的。 不活跃的身体。我们现在建议详细研究这些TORC1结合蛋白的功能，测试 假设：(I)Ydl180w是TORC1的抑制子，并与GTR1/2竞争控制TORC1的活性，(Ii) Ygr125w是TORC1的硫磺依赖活化剂，以及(Iii)Syg1是TORC1的磷酸盐依赖活化剂。 为了解决第二个问题，我们纯化了主要的GTR1/2调节因子SEAC，并绘制了它的磷酸化图谱 在葡萄糖和氮气饥饿的条件下。这导致了150多个磷酸化位点的鉴定， 其中许多在葡萄糖和/或氮饥饿期间过度或低度磷酸化。建立在这些基础上 数据，我们现在希望检验保守的GCN2、PKC、Sit4、CK2和其他 激酶/磷酸酶通过(去)磷酸化和激活SEAC来抑制TORC1。我们还计划探索一种 我们发现了关键的丝氨酸合成酶Ser3/33(人类的PHGDH)和 TORC1调节器Pib2-测试Ser3/33在存在的情况下通过Pib2激活TORC1的假设 丝氨酸的缺失。我们的建议是创新的，因为我们研究了TORC1信号的新的和未被探索的方面 使用最先进的系统、蛋白质组学和生化方法。这项拟议的研究具有重要意义 它有望阐明细胞生长控制和复杂信号整合的潜在机制， 在一个重要的模式生物中--对(A)理解与TORC1相关的疾病，如 癌症、癫痫、糖尿病和肥胖症，因为许多正在研究的蛋白质和途径是 保护和(B)开发选择性地阻止病原真菌生长的药物。

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. Building on these data, we now wish to test the hypothesis that the conserved Gcn2, PKC, Sit4, CK2, and other kinases/phosphatases inhibit TORC1 by (de)phosphorylating and activating SEAC. We also plan to explore a new connection we identified between the key serine synthesis enzymes Ser3/33 (PHGDH in humans) and the TORC1 regulator Pib2—testing the hypothesis that Ser3/33 activate TORC1 via Pib2 in the presence, but not absence, of serine. 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.