Dependence of Early Endocytosis on Clathrin Adaptors and the Scaffold Protein Ede1

Michael Dores

doi:https://doi.org/10.21985/N2MX6P

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Dependence of Early Endocytosis on Clathrin Adaptors and the Scaffold Protein Ede1

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The formation of a primary endocytic vesicle bud is a dynamic process involving the transient organization of adaptor and scaffold proteins at the plasma membrane. The proteins involved in early endocytosis have modular protein interaction motifs and domains that are predicted to contribute to scaffold formation. These motifs are found in multiple constituents of the early endocytic scaffold, suggesting that there is a degree of redundancy between different adaptor and scaffold proteins. Although the interaction partners of these motifs have been defined, their role in endocytosis has not been elucidated. The yeast clathrin adaptors Ent1, Ent2, Yap1801, and Yap1802 share many of the same protein interaction motifs. Each of the adaptors has a region that binds to Eps15-Homology (EH) domain-proteins that contains multiple tripeptide NPF motifs as well as a clathrin-binding motif (CBM). Experiments involving combinations of motif mutants revealed that these protein interaction regions are able to function redundantly during receptor internalization despite having different binding targets. Ubiquitin and ubiquitin binding also contribute to stabilizing and regulating the network of endocytic proteins during vesicle formation. There are at least three characterized early endocytic ubiquitin-binding proteins: the epsins Ent1 and Ent2, as well as the scaffold protein Ede1. These proteins are thought to act as adaptors for ubiquitinated vesicle cargo; however, many components of the endocytic machinery are also modified by ubiquitin. The Ent2 UIMs are required for proper receptor internalization in the absence of Ede1 EH-NPF scaffolding interactions. This functional redundancy suggests that the Ent2 UIMs are involved in scaffold formation. Contrary to previous predictions, the Ede1 UBA domain is not functionally redundant with the Ent2 UIMs, as cells that lack Ent2 and Ede1 ubiquitin-binding domains are able to internalize activated receptors. This study has identified a novel role for the UBA domain in regulating Ede1. In the absence of EH-mediated scaffolding interactions, the UBA domain inhibits internalization of surface receptors. These results suggest that the ubiquitin-binding domains within the epsins and Ede1 contribute to the formation and regulation of the endocytic scaffold.

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