Poly-ubiquitin catalyzes endocytosis by promoting liquid-like assembly of early endocytic proteins.

Abstract

Clathrin-mediated endocytosis is an essential cellular pathway that enables cell signaling and receptor recycling. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives trafficking vesicle assembly. Recently it has been reported that early endocytic proteins, Eps15 and Fcho1/2, form liquid-like droplets through weak multivalent interactions, which provide a dynamic, catalytic scaffold for assembly of endocytic vesicles. How do cells modulate the stability of this network? Many receptors and endocytic proteins are ubiquitinated, and early endocytic proteins, such as Eps15, contain ubiquitin-interacting motifs (UIMs). Therefore, we examined the influence of poly-ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of ubiquitin to Eps15 condensates dramatically increased the critical temperature for assembly of Eps15 networks, suggesting that ubiquitination may play an important role in initiating endocytosis. In live cell imaging experiments, we added a deubiquitinase (DUB) domain at the C terminus of Eps15 (Eps15-DUB) to remove ubiquitin from Eps15 and its interaction partners. Expression of Eps15-DUB drove a sharp increase in the frequency of non-productive endocytic events that failed to form vesicles. Interestingly, the fraction of non-productive events in cells expressing Eps15-DUB exceeded that for Eps15 knockout cells, demonstrating the importance of ubiquitination in endocytosis. Collectively, these results suggest that poly-ubiquitination of the endocytic machinery and its target receptors plays an essential role in stabilizing the flexible protein network responsible for catalyzing endocytic events.