Abstract

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.

中文翻译：

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Ragulator结构和功能的遗传解剖在mTORC1的氨基酸依赖性调控中。

摘要

Ragulator是一种杂五聚体蛋白质复合物，由两个被膜锚p18 / Lamtor1包裹的路障异二聚体组成。Ragulator复合物充当Rag GTPases的溶酶体膜支架，以募集并激活雷帕霉素复合物1（mTORC1）的机制靶标。但是，Ragulator结构在调节mTORC1功能中的作用仍然难以捉摸。在这项研究中，我们通过直接将RagC锚定到溶酶体来破坏Ragulator的结构，并监测了对氨基酸依赖性mTORC1激活的影响。p18缺陷细胞中溶酶体锚定的RagC的表达导致mTORC1的组成型溶酶体定位和氨基酸非依赖性激活。Ragulator在此系统中的共表达恢复了mTORC1激活的氨基酸依赖性。此外，消融了Rag GTPases的抑制剂Gator1，甚至在存在Ragulator的情况下也诱导了mTORC1的氨基酸非依赖性激活。这些结果表明，Ragulator结构对于通过Gator1进行的Rag GTPases氨基酸依赖性调节至关重要。此外，我们的遗传分析揭示了氨基酸在mTORC1调控中的新作用，如下所示：氨基酸可以以Rheb独立的方式激活一部分mTORC1，并且还可以通过蛋白质磷酸酶驱动mTORC1信号的负反馈调控。这些有趣的发现有助于我们对mTORC1信号调节机制的整体理解。我们的遗传分析揭示了氨基酸在mTORC1调控中的新作用，如下所示：氨基酸可以以Rheb独立的方式激活一部分mTORC1，还可以通过蛋白质磷酸酶驱动mTORC1信号的负反馈调控。这些有趣的发现有助于我们对mTORC1信号调节机制的整体理解。我们的遗传分析揭示了氨基酸在mTORC1调控中的新作用，如下所示：氨基酸可以以Rheb独立的方式激活一部分mTORC1，还可以通过蛋白质磷酸酶驱动mTORC1信号的负反馈调控。这些有趣的发现有助于我们对mTORC1信号调节机制的整体理解。