A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin


An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.

Publication DOI: https://doi.org/10.1073/pnas.1018845108
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
Uncontrolled Keywords: clathrin,Hsc70,auxilin,light scattering,stopped-flow,kKinetics,molecular models,multiprotein complexes,clathrin-mediated endocytosis,kinetic mechanism,macromolecular assemblies,vesicle uncoating,mathematical model,General
Publication ISSN: 1091-6490
Last Modified: 24 Jan 2024 18:16
Date Deposited: 03 Aug 2012 13:09
Full Text Link: http://www.pnas ... ent/108/17/6927
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
PURE Output Type: Article
Published Date: 2011-04-26
Published Online Date: 2011-04-11
Authors: Rothnie, Alice (ORCID Profile 0000-0002-4259-7015)
Clarke, Anthony R.
Kuzmic, Petr
Cameron, Angus
Smith, Corinne J.



Version: Accepted Version

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