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

EEA1, an early-endosomal protein originally identified as an autoantigen, i s essential for endocytic membrane fusion. It interacts' with early endosom es via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdI ns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequen ce contains heptad repeats characteristic of proteins involved in coiled-co il protein-protein interactions. Here we have investigated the ability of E EA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 result ed in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong a ppearance of a similar to 350-kDa crosslinked product. Glycerol gradient ce ntrifugation experiments indicated that native EEA1 had the same hydrodynam ic properties as the similar to 350-kDa crosslinked complex. Two-hybrid ana lysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel co iled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correla tes with its ability to bind to Rab5 and early endosomes, whereas its bindi ng to PtdIns3P is independent of dimerization. These data enable us to prop ose a model for the quaternary structure of EEA1.