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

Abstract

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.

Global Identification of ANTH Genes Involved in Rice Pollen Germination and Functional Characterization of a Key Member, OsANTH3

“…This suggests that NPF-rich OsANTH3 not only combine with α-adaptin to participate in CCV but also function as a clathrinbinding motif. The main endocytic clathrin adapter AP-2 alone is not sufficient to generate CCVs (Brod et al, 2020). CALM and AP180, the animals ANTH proteins, are abundant in CCVs similar to AP-2 (Prasad and Lippoldt, 1988;Skruzny et al, 2015).…”

Dynamic interplay between cell membrane tension and clathrin‐mediated endocytosis

“…Removal of mechanical hindrances restores the structural and dynamic features of clathrin‐coated structures [Ferguson et al., 2017, Willy et al., 2017, Saleem et al., 2015]. Overall, the current understanding of the mechanoregulation of CME dynamics is built on a multitude of approaches that utilise in vitro reconstitution systems [Saleem et al., 2015, Brod et al., 2020, Meinecke et al., 2013, Purushothaman and Ungermann, 2018, Steinem and Meinecke, 2020], computational simulations [Hassinger et al., 2017, Walani et al., 2015, Agrawal et al., 2010, Irajizad et al., 2017, Giani et al., 2017, Giani et al., 2016, Mahmood et al., 2019] and live‐cell imaging assays that incorporate tension manipulation on the membrane [Ferguson et al., 2016, Willy et al., 2017].…”

The role of ligand endocytosis in notch signalling

“…Hence, Epsin is essential for the maturation of the CCP, but although possessing curvature inducing abilities, it is not essential for pit formation. However, a recent in vitro reconstitution study shows that Epsin, but not AP‐2, is able to support the formation of Clathrin‐coated vesicles (Brod et al., 2020). A recent study indicates that Epsin is also involved in Clathrin‐independent endocytosis of the EGF receptor (Sigismund et al., 2005).…”