FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn 2؉ -dependent, and Zn 2؉could not be replaced by any other 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 Zn 2؉ coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6-to >100-fold decreased affinity for PtdIns(3)P. A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, 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 largely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without 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.Phosphatidylinositol 3-kinases are important regulators of vital cellular processes, including endocytic membrane trafficking, signal transduction, apoptosis, and cytoskeletal organization (1-3). The recent discovery of the conserved FYVE zinc finger (for Fab1p, YOTB, Vac1p, EEA1) 1 (4) as a phosphatidylinositol 3-phosphate (PtdIns (3)P) -specific domain has shed light on the function of this phosphatidylinositol 3-kinase product in cellular processes (5-8). FYVE finger proteins include the membrane trafficking regulators EEA1, Hrs, Vac1p, Vps27p, and Fab1p, the signal transducer SARA, the putative cytoskeletal regulator Fgd1, as well as a number of proteins with unknown function (5, 9 -15).The determination of the crystal structure of the ligand-free FYVE domain of the yeast vacuolar sorting protein Vps27p at high resolution has enabled the modeling of the FYVE-PtdIns (3)P interaction (16). According to this model, the basic residues present in the ␤1 strand ( Fig.

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