Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan.
J Biochem. 2020 Dec 26;168(6):621-632. doi: 10.1093/jb/mvaa076.
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.
Ragulator 是一种由两个 roadblock 异二聚体组成的杂五聚体蛋白复合物，由膜锚定蛋白 p18/Lamtor1 包裹。Ragulator 复合物作为 Rag GTPases 的溶酶体膜支架，招募并激活雷帕霉素靶蛋白复合物 1（mTORC1）。然而，Ragulator 结构在调节 mTORC1 功能方面的作用仍不清楚。在这项研究中，我们通过直接将 RagC 锚定在溶酶体上来破坏 Ragulator 结构，并监测对氨基酸依赖性 mTORC1 激活的影响。在 p18 缺陷细胞中表达溶酶体锚定的 RagC 导致 RagC 持续定位于溶酶体，并且 mTORC1 激活不受氨基酸的影响。在这个系统中表达 Ragulator 恢复了 mTORC1 激活的氨基酸依赖性。此外，Rag GTPases 的抑制因子 Gator1 的缺失即使在存在 Ragulator 的情况下也会诱导 mTORC1 的非氨基酸依赖性激活。这些结果表明，Ragulator 结构对于通过 Gator1 进行 Rag GTPases 的氨基酸依赖性调节是必不可少的。此外，我们的遗传分析揭示了氨基酸在调节 mTORC1 方面的新作用，如下所述：氨基酸可以以 Rheb 独立的方式激活 mTORC1 的一部分，并且还可以通过蛋白磷酸酶驱动 mTORC1 信号的负反馈调节。这些有趣的发现有助于我们全面理解 mTORC1 信号的调节机制。
