Quantification of cytosolic interactions identifies E de1 oligomers as key organizers of endocytosis

Abstract

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 E de1, homolog of mammalian E ps15. E de1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of E de1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of E de1 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 . image Cytoplasmic protein–protein interactions between endocytic components are quantitatively analyzed by fluorescence correlation spectroscopy in yeast. Cytoplasmic oligomerization of the scaffold protein E de1 is shown to be critical for its function in endocytosis. Cytoplasmic diffusion coefficients and concentrations of endocytic proteins are analyzed by fluorescence correlation spectroscopy. The endocytic protein–protein interaction network in the cytoplasm is characterized by fluorescence cross‐correlation spectroscopy. Oligomerization of E de1 is essential for its localization and function as an early endocytic scaffold protein.