The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding - Critical role of this dual interaction for endosomal localization

Early endosome antigen 1 (EEA1) is 170-kDa polypeptide required for endosom e fusion. EEA1 binds to both phosphtidylinositol 3-phosphate (PtdIns3P) and to Rab5-GTP in vitro, but the functional role of this dual interaction at the endosomal membrane is unclear. Here we have determined the structural f eatures in EEA1 required for binding to these ligands, We have found that t he FYVE domain is critical for both PtdIns3P and Rab5 binding, Whereas PtdI ns3P binding only required the FYVE domain, Rab5 binding additionally requi red a 30-amino acid region directly adjacent to the FYVE domain. Microinjec tion of glutathione S-transferase fusion constructs into Cos cells revealed that the FYVE domain alone is insufficient for localization to cellular me mbranes; the upstream 30-amino acid region required for Rab5 binding must a lso be present for endosomal binding. The importance of Rab5 in membrane bi nding of EEA1 is underscored by the finding that the increased expression o f wild-type Rab5 increases endosomal binding of EEA1 and decreases its depe ndence on PtdIns3P, Thus, the levels of Rab5 are rate-limiting for the recr uitment of EEA1 to endosome membranes. PtdIns3P may play a role in modulati ng the Rab5 EEA1 interaction.