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

Alice Rothnie, Anthony R. Clarke, Petr Kuzmic, Angus Cameron, Corinne J. Smith

Research output: Contribution to journalArticle

Abstract

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.
Original languageEnglish
Pages (from-to)6927-6932
Number of pages6
JournalProceedings of the National Academy of Sciences
Volume108
Issue number17
Early online date11 Apr 2011
DOIs
Publication statusPublished - 26 Apr 2011

Keywords

  • clathrin
  • Hsc70
  • auxilin
  • light scattering
  • stopped-flow
  • kKinetics
  • molecular models
  • multiprotein complexes
  • clathrin-mediated endocytosis
  • kinetic mechanism
  • macromolecular assemblies
  • vesicle uncoating
  • mathematical model

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  • Activities

    • 1 Participation in conference

    UK Endocytosis Conference

    Alice Rothnie (Speaker)

    5 Apr 2011

    Activity: Participating in or organising an event typesParticipation in conference

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