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。过去20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 发芽酵母，发现PKC、GCN2、Sit4和CK2信号通路与GAP一起工作 SEAC(人类中的GATOR1/2)通过高度保守的GTP酶GTR1/2(Rag A/B和C/D)抑制TORC1 在人类中)。这进而释放TORC1以移动到位于 液泡/溶酶体--这一事件依赖于TORC1结合蛋白Pib2。在这个框架的基础上， 我们现在希望：(1)鉴定和表征与GTR1/2平行调节的蛋白质和通路 以及(2)确定保守的GCN2、PKC、Sit4、CK2通路如何通过GTR1/2调节TORC1。 为了解决第一个问题，我们从暴露在各种压力和饥饿下的细胞中提纯了TORC1 并确定了许多新的相互作用者。最值得注意的是那些没有个性的人 液泡/溶酶体膜蛋白Ydl180w、Ygr125w和Syg1，因为它们与TORC1紧密结合，并且 进入或移出不活跃的物体所需的。我们现在建议研究这些基因的功能 TORC1结合蛋白的详细研究，验证了以下假设：(I)Ydl180w是TORC1和TORC1的阻遏物 与GTR1/2竞争控制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。为了回答第二个问题，我们净化了少校 GTR1/2调节子SEAC，并绘制了其在葡萄糖和氮饥饿条件下的磷酸化图谱。这导致了 超过150个磷酸化位点的鉴定，其中许多是在 葡萄糖和/或氮饥饿。在这些数据的基础上，我们现在希望检验这一假设 保守的GCN2、PKC、Sit4、CK2和其他激酶/磷酸酶通过(去)磷酸化和 启动SEAC。我们还计划探索我们确定的关键丝氨酸合成之间的新联系 Ser3/33酶(人类中的PHGDH)和TORC1调节因子Pib2-检验Ser3/33的假设 在丝氨酸存在而不是不存在的情况下，通过Pib2激活TORc1。我们的建议是创新的，因为我们 使用最先进的系统、蛋白质组和蛋白质组学研究TORC1信号的新的和未探索的方面 生化方法。这项拟议的研究具有重要意义，因为它有望揭示 在一个重要的模式生物中，细胞生长控制和复杂信号整合的潜在机制- 有助于(A)了解与TORC1相关的疾病，如癌症、癫痫、糖尿病和 肥胖，因为研究中的许多蛋白质和途径是保守的；和(B)开发药物 选择性地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. 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.