Malady, B.; Sarkar, S.; Wang, L.; Kadlecova, Z.; Lafer, E. M.; Owen, D. J.; Stachowiak, J.

During endocytosis a dynamic protein network is responsible for assembly of the clathrin coat. Early in the process the Eps15 protein is thought to undergo liquid-liquid phase-separation to form biomolecular condensates. These condensates facilitate clathrin assembly, producing clathrin-coated vesicles that are ultimately excluded from the condensate as the vesicles depart from the plasma membrane. An array of distinct clathrin accessory proteins drive this process, yet their interactions with protein condensates have not been explored. Here we show that clathrin accessory proteins promote the assembly of the clathrin lattice by simultaneously destabilizing protein condensates. By adding diverse accessory proteins to Eps15 condensates, we observed that accessory proteins which cross-linked Eps15 proteins stabilized condensates, while accessory proteins that compete with Eps15-Eps15 interactions destabilized condensates. Interestingly, accessory proteins that destabilized condensates also promoted the assembly of clathrin and its exclusion from condensates. In contrast, accessory proteins that stabilized condensates opposed exclusion of clathrin. Finally, we demonstrated that decreasing condensate stability enhanced clathrin assembly and exclusion, even when no additional clathrin assembly motifs were introduced. Taken together, these results help to explain how condensates can catalyze the assembly of coated vesicles by providing both a substrate for their initiation and a driving force for their exclusion and ultimate departure.