The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer

Biochem J. 1999 Mar 1;338 ( Pt 2)(Pt 2):539-43.

Abstract

EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein-protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a approximately 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the approximately 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autoantigens / immunology
  • Autoantigens / metabolism*
  • Base Sequence
  • Cytosol / metabolism
  • DNA Primers
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • HeLa Cells
  • Humans
  • Membrane Proteins / immunology
  • Membrane Proteins / metabolism*
  • Molecular Weight
  • Phosphatidylinositol Phosphates / metabolism
  • Rats
  • Vesicular Transport Proteins

Substances

  • Autoantigens
  • DNA Primers
  • Membrane Proteins
  • Phosphatidylinositol Phosphates
  • Vesicular Transport Proteins
  • early endosome antigen 1
  • phosphatidylinositol 3-phosphate