Three archetypical classes of macromolecular regulators of protein liquid–liquid phase separation

Significance The assembly/disassembly of membraneless organelles, crucial for cellular functions and linked to human diseases, is delicately regulated by macromolecular components and other signals. To what extent and how macromolecular regulators affect this phase separation process are largely unknown. Here, by measuring the effects on the phase boundary and the levels of partitioning into the condensates of 2 proteins (SH3 5 and PRM 5 ), we define 3 archetypical classes of macromolecular regulators. Depending on their strengths of attraction with condensate proteins, regulators either promote or suppress phase separation, and can generally be classified as volume-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors. This classification provides a much needed understanding, given the numerous macromolecular species in and around membraneless organelles for exerting regulatory effects.