Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane traf®cking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind speci®cally, and with high af®nity, to PI(3)P both in vitro and in vivo. When expressed in ®broblasts, a tagged probe localized to endosomes, as detected by¯uorescence microscopy. Electron microscopy of untransfected ®broblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells de®cient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27D yeast cells, which have impaired endosome to vacuole traf®cking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in de®ning the early endosome and the subsequent steps leading to multivesicular endosome formation.

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

Nature © Macmillan Publishers Ltd 1998 8 F Fi ig gu ur re e 1 1 The endosomal localization of EEA1-CT and its Hrs FYVE-finger hybrid depends on PI(3)K activity and an intact FYVE finger. BHK21 cells were co-transfected with C215S (k, l) and then incubated for 20 minutes at 37 ᑻC in the absence (a, b, e-h, k, l) or presence (c, d and i, j) of 20 nM wortmannin. The cells were permeabilized with 0.05% saponin to wash out cytosolic protein, then fixed and finally stained with anti-Rab5 (a, c, e, g, i, k) or anti-Myc-epitope (b, d, f, h, j, l) antibodies and examined by confocal immunofluorescence microscopy 5 . a shows the same cell as b, c shows the same cell as d, and so on. Arrows indicate examples of co-localization of Rab5 with epitope-tagged protein. Scale bar, 10 Ȗm. Nature

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the transGolgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.

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.