TurboID-based mapping of organelle membrane protein interactomes with digitonin-permeabilization

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 select-ively 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.