Protein phase separation helps to explain exclusion of initiator proteins from clathrin-coated vesicles

Clathrin-mediated endocytosis, the major uptake pathway in eukaryotic cells, is essential for cell signaling and receptor recycling. The invagination of the plasma membrane is orchestrated by an interconnected network of initiator and adaptor proteins that ultimately recruit the clathrin coat. The initiator proteins, Eps15 and Fcho, arrive early at endocytic sites. These proteins catalyze endocytosis by recruiting critical adaptor proteins, yet are not engulfed by the developing coated vesicle. Instead, they remain at the plasma membrane, where they nucleate successive rounds of vesicle assembly. How do the initiator proteins avoid being incorporated into coated vesicles, despite their biochemical interactions with multiple adaptor proteins? Recently, our group and others have shown that endocytic initiator proteins, form a liquid-like network, which serves as a flexible catalytic network for vesicle assembly. Here we show that assembly of clathrin triskelia into solid-like lattices within this network leads to the physical separation of clathrin from the initiator proteins. Specifically, we find that a fluid-like network of Eps15 and Fcho is able to recuruit AP2 and other adaptor proteins. These proteins in turn recruit clathrin. As clathrin assembles, it spontaneously separates from the initiator proteins, forming solid-like lattices that are physically insoluble in the liquid-like initiator network. These results provide a simple biophysical explanation for the long-standing observation that initiator proteins are excluded from growing endocytic vesicles, helping to explain a critical step in the endocytic pathway.