Amino acid (AA) availability is a robust determinant of cell growth through controlling mechanistic/mammalian target of rapamycin complex 1 (mTORC1) activity. According to the predominant model in the field, AA sufficiency drives the recruitment and activation of mTORC1 on the lysosomal surface by the heterodimeric Rag GTPases, from where it coordinates the majority of cellular processes. Importantly, however, the teleonomy of the proposed lysosomal regulation of mTORC1 and where mTORC1 acts on its effector proteins remain enigmatic. Here, by using multiple pharmacological and genetic means to perturb the lysosomal AA-sensing and protein recycling machineries, we describe the spatial separation of mTORC1 regulation and downstream functions in mammalian cells, with lysosomal and non-lysosomal mTORC1 phosphorylating distinct substrates in response to different AA sources. Moreover, we reveal that a fraction of mTOR localizes at lysosomes owing to basal lysosomal proteolysis that locally supplies new AAs, even in cells grown in the presence of extracellular nutrients, whereas cytoplasmic mTORC1 is regulated by exogenous AAs. Overall, our study substantially expands our knowledge about the topology of mTORC1 regulation by AAs and hints at the existence of distinct, Rag- and lysosome-independent mechanisms that control its activity at other subcellular locations. Given the importance of mTORC1 signalling and AA sensing for human ageing and disease, our findings will probably pave the way towards the identification of function-specific mTORC1 regulators and thus highlight more effective targets for drug discovery against conditions with dysregulated mTORC1 activity in the future.

氨基酸 （AA） 可用性是通过控制雷帕霉素复合物 1 （mTORC1） 活性的机制/哺乳动物靶标来决定细胞生长的稳健决定因素。根据该领域的主要模型，AA 充足性驱动异二聚体 Rag GTP 酶在溶酶体表面募集和激活 mTORC1，从那里协调大多数细胞过程。然而，重要的是，拟议的 mTORC1 溶酶体调节以及 mTORC1 作用于其效应蛋白的位置仍然是一个谜。在这里，通过使用多种药理学和遗传学手段来扰乱溶酶体 AA 感应和蛋白质回收机制，我们描述了哺乳动物细胞中 mTORC1 调节和下游功能的空间分离，溶酶体和非溶酶体 mTORC1 响应不同的 AA 来源磷酸化不同的底物。此外，我们揭示了一小部分 mTOR 位于溶酶体，这是由于基础溶酶体蛋白水解局部提供新的 AA，即使在细胞外营养物质存在下生长的细胞中也是如此，而细胞质 mTORC1 受外源性 AA 调节。总体而言，我们的研究大大扩展了我们对 AA 调节 mTORC1 拓扑结构的了解，并暗示存在独特的、不依赖 Rag 和溶酶体的机制来控制其在其他亚细胞位置的活性。鉴于 mTORC1 信号传导和 AA 感应对人类衰老和疾病的重要性，我们的研究结果可能会为识别功能特异性 mTORC1 调节因子铺平道路，从而突出未来针对 mTORC1 活性失调的疾病的更有效药物发现靶点。