PhaseID, a novel method combining chemical induction of LLPS with proximity labeling mass spectrometry, reveals that intrinsically disordered regions of transcription factors form condensates with distinct, sequence-encoded protein interactomes that execute gene activation through IDR-specific pathways, and that LLPS itself tunes IDR interaction selectivity to enable participation in diverse cellular processes beyond those accessible under non-LLPS conditions.

Many intrinsically disordered protein regions (IDRs) can form liquid-liquid phase separation (LLPS) condensates via multivalent interactions, but how the interaction behaviors contribute to specific cellular functions remains unclear due to the unknown composition of the condensates. Here, we report phase-separation-induced interactome detection (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 transactivation IDRs each have unique multivalent interactomes, thereby executing transactivation through distinct pathways. In-depth analyses of PhaseID-determined IDR interactomes revealed that LLPS tunes the interaction selectivity of IDRs, enabling them to play new roles in specific cellular processes, and different LLPS condensates recruit proteins with distinct physicochemical properties encoded in their sequences.