Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes - Role of conserved residues

FYVE zinc finger domains, which are conserved in multiple proteins from yea st to man, interact specifically with the membrane lipid phosphatidylinosit ol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requi rements for the interaction of the FYVE finger of the early endosome antige n EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn2+-dependent, and Zn2+ could not be replaced by any othe r bivalent cations tested. By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K-D of about 50 nM and a 1:1 stoichiometry. Mutagenesis of cysteines involved in Zn2+ coordin ation, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6- to > 100-fold decreased affinit y for PtdIns(3)P, A mutation in the putative PtdIns(3)P-binding pocket, R13 75A, may prove particularly informative, because it led to a strongly decre ased affinity for PtdIns(3)P without affecting the FYVE three-dimensional s tructure, as measured by fluorescence spectroscopy. Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be lar gely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C ter minus interferes with early endosome morphology, the point mutants were wit hout detectable effect. These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.