Phase-separated condensates are membrane-less intracellular structures comprising dynamic protein interactions that organize essential biological processes. Understanding the composition and dynamics of these organelles advances our knowledge of cellular behaviors and disease pathologies related to granule dysregulation. In this study, we apply microenvironment mapping with a HaloTag-based platform (HaloMap) to characterize intracellular stress granule dynamics in living cells. After validating the robustness and sensitivity of this approach, we then profile the stress granule proteome throughout the formation and disassembly and under pharmacological perturbation. These experiments reveal several ubiquitin-related modulators, including the HECT (homologous to E6AP C terminus) E3 ligases ITCH and NEDD4L, as well as the ubiquitin receptor toll-interacting protein TOLLIP, as key mediators of granule disassembly. In addition, we identify an autophagy-related pathway that promotes granule clearance. Collectively, this work establishes a general photoproximity labeling approach for unraveling intracellular protein interactomes and uncovers previously unexplored regulatory mechanisms of stress granule dynamics.

相分离的凝聚物是无膜的细胞内结构，包含组织基本生物过程的动态蛋白质相互作用。了解这些细胞器的组成和动力学有助于深入了解与颗粒失调相关的细胞行为和疾病病理。在这项研究中，我们使用基于 HaloTag 的平台 （HaloMap） 应用微环境映射来表征活细胞中的细胞内应激颗粒动力学。在验证了这种方法的稳健性和敏感性之后，我们然后在整个形成和拆卸以及药理学扰动下分析应力颗粒蛋白质组。这些实验揭示了几种泛素相关的调节剂，包括 HECT （与 E6AP C 末端同源） E3 连接酶 ITCH 和 NEDD4L，以及泛素受体 toll 相互作用蛋白 TOLLIP，作为颗粒分解的关键介质。此外，我们确定了促进颗粒清除的自噬相关途径。总的来说，这项工作建立了一种通用的光邻近标记方法，用于解开细胞内蛋白质相互作用组，并揭示了以前未探索的应激颗粒动力学的调节机制。