title: Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly.
abstract: The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin(401-910) and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly.
authors.affiliation: School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Young
authors.forename: Svetla
authors.initials: S
authors.lastname: Stoilova-McPhie
authors.forename: Alice
authors.initials: A
authors.lastname: Rothnie
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Vallis
authors.forename: Phillip
authors.initials: P
authors.lastname: Harvey-Smith
authors.forename: Neil
authors.initials: N
authors.lastname: Ranson
authors.forename: Helen
authors.initials: H
authors.lastname: Kent
authors.forename: Frances M
authors.initials: FM
authors.lastname: Brodsky
authors.forename: Barbara M F
authors.initials: BM
authors.lastname: Pearse
authors.forename: Alan
authors.initials: A
authors.lastname: Roseman
authors.forename: Corinne J
authors.initials: CJ
authors.lastname: Smith
mesh_terms: D000818:Animals; D034021:Auxilins / Q000737:chemistry* / Q000378:metabolism*; D033941:Clathrin Light Chains / Q000737:chemistry* / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013552:Swine / Q000378:metabolism
chemical_list: D034021:Auxilins; D033941:Clathrin Light Chains; D050883:HSC70 Heat-Shock Proteins; C494373:Hspa8 protein, rat; D018832:Molecular Chaperones
keywords: 3D structure; cryo-electron microscopy (cryo-EM); endocytosis; molecular chaperone; vesicle uncoating