Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein–protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1’s role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

Rheb 是一种小 G 蛋白，作为雷帕霉素复合物 1 (mTORC1) 机制靶标的直接激活剂，协调信号级联反应以响应营养物质和生长因子。尽管进行了大量研究，但直接激活 Rheb 的鸟嘌呤核苷酸交换因子 (GEF) 仍不清楚，至少部分是由于作为信号转导标志的蛋白质-蛋白质相互作用 (PPI) 的动态和瞬时性质。在此，我们报告了一种快速而强大的邻近标记系统的开发，该系统名为火球菌生物素蛋白连接酶 ( Ph BPL) 辅助生物素识别 (PhastID)，并详细介绍了使用 PhastID 识别的 Rheb 邻近蛋白网络中胰岛素刺激的变化。特别是，我们发现溶酶体 V-ATP 酶亚基 ATP6AP1 可以与 Rheb 动态相互作用。ATP6AP1 可以通过其最后 12 个氨基酸直接与 Rheb 结合，并利用其高度保守的 C 尾中的三天冬氨酸基序来增强 Rheb GTP 负载。事实上，靶向 ATP6AP1 C 尾可以阻断 Rheb 激活并抑制癌细胞增殖和迁移。我们的研究结果强调了 PhastID 在绘制活细胞中瞬时 PPI 方面的多功能性，揭示了 ATP6AP1 作为 Rheb 非常规 GEF 的作用，并强调了 ATP6AP1 在通过 Rheb 整合 mTORC1 激活信号、填补 Rheb/mTORC1 激活中缺失的环节中的重要性。