title: Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating.
abstract: Clathrin-coated pits invaginate from specific membrane compartments and pinch off as coated vesicles. These vesicles then uncoat rapidly once released. The Hsc70 molecular chaperone effects the uncoating reaction, and is guided to appropriate locations on clathrin lattices by the J-domain-containing co-chaperone molecule auxilin. This raises the question of how a local event such as ATP hydrolysis by Hsc70 can catalyse a global disassembly. Here, we have used electron cryomicroscopy to determine 12-A-resolution structures of in-vitro-assembled clathrin coats in association with a carboxy-terminal fragment of auxilin that contains both the clathrin-binding region and the J domain. We have located the auxilin fragment by computing differences between these structures and those lacking auxilin (described in an accompanying paper). Auxilin binds within the clathrin lattice near contacts between an inward-projecting C-terminal helical tripod and the crossing of two 'ankle' segments; it also contacts the terminal domain of yet another clathrin 'leg'. It therefore recruits Hsc70 to the neighbourhood of a set of critical interactions. Auxilin binding produces a local change in heavy-chain contacts, creating a detectable global distortion of the clathrin coat. We propose a mechanism by which local destabilization of the lattice promotes general uncoating.
authors.affiliation: Biophysics Graduate Program, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Fotin
authors.forename: Yifan
authors.initials: Y
authors.lastname: Cheng
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Grigorieff
authors.forename: Thomas
authors.initials: T
authors.lastname: Walz
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Harrison
authors.forename: Tomas
authors.initials: T
authors.lastname: Kirchhausen
mesh_terms: D000818:Animals; D034021:Auxilins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002417:Cattle; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D033922:Clathrin Heavy Chains / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D034021:Auxilins; D002966:Clathrin; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D033922:Clathrin Heavy Chains