Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein–protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein–protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo.

Clathrin-mediated endocytosis is driven by a complex machinery of proteins, which assemble in a regular order at the plasma membrane. The assembly of the endocytic machinery is conventionally thought to be a continuous process of mechanistically dependent steps, starting from a defined initiation step. Indeed, several initiation mechanisms involving single proteins have been proposed in mammalian cells. Here, we demonstrate that the initiation mechanism of endocytosis is highly flexible. We disrupted the long early phase of endocytosis in yeast by deleting seven genes encoding early endocytic proteins. Surprisingly, membrane uptake and vesicle budding dynamics were largely normal in these mutant cells. Regulated cargo recruitment was, however, defective. In addition, different early endocytic proteins were able to initiate vesicle budding when anchored to a plasma membrane domain where endocytosis does not normally take place. Our results suggest that the cargo-recruiting early phase is not mechanistically required for vesicle budding, but early-arriving proteins can recruit the budding machinery into position at the plasma membrane. Separable early and late phases allow for a robust process of vesicle budding to follow from variable initiation mechanisms. Such a modular design could easily adapt and evolve to respond to different cellular requirements.