Sun Yang, Yang Lan, Zhang Jingzi, Tian Pu, Chen Shue, Fang Lei, Hong Zhi
Centre for Cellular Biology and Signaling, Zhejiang University-University of Edinburgh Institute, Haining 314400, Zhejiang, China.
University of Edinburgh Medical School, Biomedical Sciences, College of Medicine & Veterinary Medicine, University of Edinburgh, Edinburgh, EH8 9JZ, United Kingdom.
Biophys Rep. 2025 Aug 31;11(4):219-231. doi: 10.52601/bpr.2025.240051.
Protein-protein interactions at organelle membranes bridge organelles in close proximity, facilitating regulated metabolite exchange and maintaining cellular homeostasis. Enzyme-catalyzed proximity labeling (PL) has been widely used to uncover the molecular composition of these interactions, but excessive labeling of irrelevant cytosolic proteins complicates data analysis. To address this, we developed a streamlined protocol that combines the TurboID system with digitonin-permeabilization to efficiently map protein interactions at organelle membranes in live mammalian cells. Digitonin selectively permeabilizes the plasma membrane, removing cytosolic proteins while preserving the integrity of inner membranes like the ER and mitochondria. This approach enhances spatial resolution in proteo-mic analysis, enabling a more precise map for protein interactome. Using this method, we successfully achieved proximal labeling of ER-localized proteins REEP1 and REEP6 to decipher their interaction networks, demonstrating its applicability for studying membrane-associated interactions with greater clarity and reduced contamination.
细胞器膜上的蛋白质-蛋白质相互作用将相邻的细胞器连接起来，促进有调控的代谢物交换并维持细胞内稳态。酶催化的邻近标记（PL）已被广泛用于揭示这些相互作用的分子组成，但无关胞质蛋白的过度标记使数据分析变得复杂。为了解决这个问题，我们开发了一种简化方案，将TurboID系统与洋地黄皂苷通透法相结合，以有效地绘制活哺乳动物细胞中细胞器膜上的蛋白质相互作用图谱。洋地黄皂苷选择性地使质膜通透，去除胞质蛋白，同时保持内质网和线粒体等内膜的完整性。这种方法提高了蛋白质组学分析的空间分辨率，能够为蛋白质相互作用组绘制更精确的图谱。使用这种方法，我们成功地实现了内质网定位蛋白REEP1和REEP6的近端标记，以破译它们的相互作用网络，证明了其在更清晰且减少污染地研究膜相关相互作用方面的适用性。
