Cells need to coordinate nutrient availability with their growth and proliferation. In eukaryotic cells, this coordination is mediated by the mechanistic target of the rapamycin complex 1 (mTORC1) pathway. mTORC1 activation is regulated by two GTPase units, the Rag GTPase heterodimer and the Rheb GTPase. The RagA-RagC heterodimer controls the subcellular localization of mTORC1, and its nucleotide loading states are strictly controlled by upstream regulators including amino acid sensors. A critical negative regulator of the Rag GTPase heterodimer is GATOR1. In the absence of amino acids, GATOR1 stimulates GTP hydrolysis by the RagA subunit to turn off mTORC1 signaling. Despite the enzymatic specificity of GATOR1 to RagA, a recent cryo-EM structural model of the human GATOR1-Rag-Ragulator complex reveals an unexpected interface between Depdc5, a subunit of GATOR1, and RagC. Currently, there is no functional characterization of this interface, nor do we know its biological relevance. Here, combining structure-function analysis, enzymatic kinetic measurements, and cell-based signaling assays, we identified a critical electrostatic interaction between Depdc5 and RagC. This interaction is mediated by the positively charged Arg-1407 residue on Depdc5 and a patch of negatively charged residues on the lateral side of RagC. Abrogating this interaction impairs the GAP activity of GATOR1 and cellular response to amino acid withdrawal. Our results reveal how GATOR1 coordinates the nucleotide loading states of the Rag GTPase heterodimer, and thus precisely controls cellular behavior in the absence of amino acids.

细胞需要协调营养供应与其生长和增殖。在真核细胞中，这种协调是由雷帕霉素复合物 1 (mTORC1) 途径的机械靶标介导的。 mTORC1 激活由两个 GTPase 单元（Rag GTPase 异二聚体和 Rheb GTPase）调节。 RagA-RagC异二聚体控制mTORC1的亚细胞定位，其核苷酸负载状态受到包括氨基酸传感器在内的上游调节因子的严格控制。 Rag GTPase 异二聚体的一个关键负调节因子是 GATOR1。在缺乏氨基酸的情况下，GATOR1 通过 RagA 亚基刺激 GTP 水解，从而关闭 mTORC1 信号传导。尽管 GATOR1 对 RagA 具有酶特异性，但最近人类 GATOR1-Rag-Ragulator 复合物的冷冻电镜结构模型揭示了 GATOR1 亚基 Depdc5 和 RagC 之间的意外界面。目前，还没有该界面的功能表征，我们也不知道其生物学相关性。在这里，结合结构功能分析、酶动力学测量和基于细胞的信号传导测定，我们确定了 Depdc5 和 RagC 之间的关键静电相互作用。这种相互作用是由 Depdc5 上带正电的 Arg-1407 残基和 RagC 侧面的一片带负电的残基介导的。消除这种相互作用会损害 GATOR1 的 GAP 活性和细胞对氨基酸撤退的反应。我们的结果揭示了 GATOR1 如何协调 Rag GTPase 异二聚体的核苷酸加载状态，从而在氨基酸缺失的情况下精确控制细胞行为。