Genetic dissection of Ragulator structure and function in amino acid-dependent regulation of mTORC1
Genetic dissection of Ragulator structure and function in amino acid-dependent regulation of mTORC1
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mTORC1 氨基酸依赖性调节中 Ragulator 结构和功能的遗传解析
DOI：
10.1093/jb/mvaa076
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Okada Masato
中科院分区：
文献类型：
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作者：
Nada Shigeyuki;Okada Masato
Ragulator是一种异五聚体蛋白复合物，由两个被膜锚p18/Lamtor 1包裹的路障异二聚体组成。Ragulator复合物作为Rag GTP酶的溶酶体膜支架发挥功能，以募集和激活雷帕霉素复合物1（mTORC 1）的机制靶标。然而，Ragulator结构在mTORC 1功能调节中的作用仍然难以捉摸。在这项研究中，我们通过将RagC直接锚定到溶酶体来破坏Ragulator结构，并监测对氨基酸依赖性mTORC 1激活的影响。在p18缺陷细胞中表达溶酶体锚定的RagC导致mTORC 1的组成性溶酶体定位和氨基酸非依赖性激活。Ragulator在该系统中的共表达恢复了mTORC 1激活的氨基酸依赖性。此外，Gator 1，一个抑制Rag GTP酶，消融诱导的mTORC 1的氨基酸非依赖性激活，即使在Ragulator的存在。这些结果表明，Ragulator结构是必不可少的氨基酸依赖性调控Rag GTP酶通过Gator 1。此外，我们的遗传分析揭示了氨基酸在mTORC 1调控中的新作用如下：氨基酸可以以Rheb非依赖性方式激活mTORC 1的一部分，并且还可以通过蛋白磷酸酶驱动mTORC 1信号传导的负反馈调节。这些有趣的发现有助于我们全面了解mTORC 1信号转导的调控机制。
Ragulator is a heteropentameric protein complex consisting of two roadblock heterodimers wrapped by the membrane anchor p18/Lamtor1. The Ragulator complex functions as a lysosomal membrane scaffold for Rag GTPases to recruit and activate mechanistic target of rapamycin complex 1 (mTORC1). However, the roles of Ragulator structure in the regulation of mTORC1 function remain elusive. In this study, we disrupted Ragulator structure by directly anchoring RagC to lysosomes and monitored the effect on amino acid-dependent mTORC1 activation. Expression of lysosome-anchored RagC in p18-deficient cells resulted in constitutive lysosomal localization and amino acid-independent activation of mTORC1. Co-expression of Ragulator in this system restored the amino acid dependency of mTORC1 activation. Furthermore, ablation of Gator1, a suppressor of Rag GTPases, induced amino acid-independent activation of mTORC1 even in the presence of Ragulator. These results demonstrate that Ragulator structure is essential for amino acid-dependent regulation of Rag GTPases via Gator1. In addition, our genetic analyses revealed new roles of amino acids in the regulation of mTORC1 as follows: amino acids could activate a fraction of mTORC1 in a Rheb-independent manner, and could also drive negative-feedback regulation of mTORC1 signalling via protein phosphatases. These intriguing findings contribute to our overall understanding of the regulatory mechanisms of mTORC1 signalling.