Epsin but not AP‐2 supports reconstitution of endocytic clathrin‐coated vesicles

Formation of clathrin‐coated vesicles (CCVs) in receptor‐mediated endocytosis is a mechanistically well‐established process, in which clathrin, the adaptor protein complex AP‐2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)‐based in vitro CCV reconstitution system with chemically defined components and the full‐length recombinant proteins clathrin, AP‐2, epsin‐1, and dynamin‐2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin‐1 alone does not have the propensity for scission but is required for bud formation, whereas AP‐2 and clathrin are not sufficient. Thus, our study reveals that epsin‐1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.