Optically activated proximity labeling for spatially and temporally resolved proteomics.

Abstract

Membrane protein interactions are essential for the proliferation, differentiation and survival of cells. However, due to the lack of appropriate technologies, the interactomes of membrane proteins, at distinct spatial locations within the cell and at different stages of the cell cycle, are poorly understood. Here, we develop a light-activated proximity labeling method for high-precision mapping of membrane protein interactions with high spatial and temporal resolution. Our method combines a split version of the proximity biotinylation enzyme, TurboID, with the optically activated protein dimerization pair CRY2 and CIB1. As proof of principle, we engineer cells expressing CIB1 and an inactive TurboID fragment on the cell membrane along with the complementary TurboID fused to CRY2 in the cytoplasm. Using immunofluorescence imaging and mass spectrometry, we demonstrate that the split-TurboID fragments assemble, become functional and biotinylate proteins, only upon exposure to light. Next we tag E-cadherin, an essential cell-cell adhesion protein, with our optically activated proximity labeling system and validate it by comparing the measured interactome upon light exposure with known E-cadherin binding partners. Our results demonstrate that light-triggered proximity labeling can map the E-cadherin interactome with high precision. We anticipate that our optically activated technique will serve as a revolutionary tool to map junctional protein interactions within cells, with high fidelity, in a previously unprecedented, spatially and temporally resolved fashion.