Epsin binds to clathrin by associating directly with the clathrin- terminal domain. Evidence for cooperative binding through two discrete sites

Matthew T. Drake, Maureen A. Downs, Linton M. Traub

Research output: Contribution to journalArticle

111 Scopus citations

Abstract

Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so- called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the α subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence 257LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor β subunits, amphiphysin, and β-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the β subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence 480LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin- like molecules suggests that the ability to establish multiple protein- protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.

Original languageEnglish (US)
Pages (from-to)6479-6489
Number of pages11
JournalJournal of Biological Chemistry
Volume275
Issue number9
DOIs
StatePublished - Mar 3 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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