Determining Multivalent Interactomes of Intrinsically Disordered Proteins in Phase-Separated Condensates in Live Cells

Many intrinsically disordered protein regions (IDRs) can form liquid-liquid phase separation (LLPS) condensates or related biomolecular assemblies via multivalent interactions, but how these interaction behaviors contribute to specific cellular functions remains unclear due to the unknown composition of the IDR-mediated assemblies. Here, we report phase -separation-induced i nteractome d etection (PhaseID), a new method that determines the protein components within the LLPS condensates driven by any given IDR in live human cells. Using PhaseID, we demonstrated that different IDRs with transactivation functions have distinct multivalent interactomes and thereby execute transactivation through distinct pathways. In-depth analyses of PhaseID-determined IDR interactomes revealed that LLPS tunes the interaction selectivity of IDRs, enables new roles of IDRs in specific cellular processes, and involves heterotypic interactions encoded by protein sequence grammars.