Split-TurboID enables contact-dependent proximity labeling in cells

Most of the thousands of proteins that comprise a human cell have specific subcellular localization patterns essential for their function. “Proximity labeling” (PL) is a method for mapping the localization of endogenous cellular proteins on a proteome-wide scale. To improve the specificity and versatility of PL, we developed split-TurboID, a promiscuous biotinylating enzyme split into two inactive fragments. The fragments are coexpressed in cells and brought together by a drug, protein–protein interaction, or organelle contact to reconstitute TurboID enzymatic activity. We used split-TurboID to map the protein composition of endoplasmic reticulum–mitochondria contact sites, which are essential for mitochondrial fission, lipid biosynthesis, and calcium signaling. For conditional or higher-specificity PL, split-TurboID may be a valuable tool for biological discovery.