<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Reconstitution of clathrin coated vesicles"^^ . "Receptor-mediated endocytosis by clathrin coated vesicles (CCVs) is a well investigated process, in which clathrin, the adaptor AP-2 and the large GTPase dynamin play crucial roles. However, the minimal machinery for the formation of CCVs has not been defined in vitro. Specifically, scission of clathrin coated endocytic buds (CCBs), the last step in this process that results in the release of free vesicles, is not clearly defined mechanistically. Actually, five APcomplexes are described and except AP-2, all are recruited in an Arf-dependent manner to their donor membrane. Here, Arf is likely to play a distinct and GTPase activity-independent role in the scission mechanism via its myristoylated amphipathic helix, as has been shown for Arf’s role in COPI vesicle scission. In case of AP-2 no Arf is known to be involved, but it has been shown that epsin-1 with its N-terminal amphiphilic helix (ENTH-domain) is an endocytic accessory protein involved in the biogenesis of endocytic clathrin coated vesicles and was suggested to be implicated in scission. To identify the minimal machinery required for CCV formation and to investigate the molecular roles of dynamin and epsin-1 in this process, an in vitro reconstitution system for the biogenesis of CCVs was established using defined components in the form of synthetic membranes and recombinant, purified proteins. I observed that a combination of AP-2 and epsin-1 recruits clathrin to synthetic liposomes more efficiently than each single adaptor, indicating cooperativity between AP-2 and epsin-1. CCVreconstitution experiments with giant unilamellar vesicles showed that clathrin coated vesicles can be reconstituted dependent on clathrin adaptors, clathrin, dynamin and GTP. Moreover, blocking GTP-hydrolysis abolished the formation of CCVs, supporting the widely accepted hypothesis that hydrolysis of GTP by dynamin is mandatory for vesicle release. However, as hydrolysis of GTP was an absolute requirement of vesicle release, epsin-1 alone does not have the propensity for scission, but is required for bud formation, as AP-2 and clathrin alone are not sufficient. A role of the endocytic protein epsin-1 for the progression of endocytosis to CCBs would also be structurally supported as epsin-1 contains a membrane bending ENTH-domain that would facilitate such a function by introducing curvature to the membrane. In summary this work comprises the first in vitro reconstitution system of endocytic CCVs that uses full length proteins in a chemically defined environment. Using this system, I confirmed and refined the roles of known constituents of the minimal endocytic CCV biogenesis machinery, which is comprised of AP-2, epsin-1, clathrin, dynamin, and by GTP-hydrolysis in dynamin."^^ . "2018" . . . . . . . "Jan"^^ . "Brod"^^ . "Jan Brod"^^ . . . . . . "Reconstitution of clathrin coated vesicles (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Reconstitution of clathrin coated vesicles (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Reconstitution of clathrin coated vesicles (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Reconstitution of clathrin coated vesicles (Other)"^^ . . . . . . "small.jpg"^^ . . . "Reconstitution of clathrin coated vesicles (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Reconstitution of clathrin coated vesicles (PDF)"^^ . . . "PhD Thesis --- Jan Brod --- 24.07.2018.pdf"^^ . . "HTML Summary of #25165 \n\nReconstitution of clathrin coated vesicles\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .