Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70

Yuchen Ma, Tsvika Greener, Michael E. Pacold, Shivani Kaushal, Lois E. Greene, Evan Eisenberg

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.

Original languageEnglish (US)
Pages (from-to)49267-49274
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number51
DOIs
StatePublished - Dec 20 2002

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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