Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain

J Biol Chem. 2005 May 20;280(20):19600-6. doi: 10.1074/jbc.M501510200. Epub 2005 Mar 7.

Abstract

Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Endosomal Sorting Complexes Required for Transport
  • Endosomes / metabolism
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • Membrane Transport Proteins / chemistry
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Multiprotein Complexes
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Ubiquitin / metabolism*
  • Vesicular Transport Proteins

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • Endosomal Sorting Complexes Required for Transport
  • Membrane Transport Proteins
  • Multiprotein Complexes
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Ubiquitin
  • Vesicular Transport Proteins
  • Vps36 protein, S cerevisiae