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.

Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis postulates that pairing between a vesicular v-SNARE and a target membrane z-SNARE is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting. Moreover, other proteins have been shown to be important in membrane docking or tethering. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase Rab5. Here we show that Rab5-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity. Rab5 and other associated proteins seem to act upstream of EEA1, implying that Rab5 effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that EEA1 mediates endosome docking and, together with SNAREs, leads to membrane fusion.

Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.

hosphatidylinositol-3-OH kinases (PI(3)Ks) constitute a family of lipid-modifying enzymes that are involved in signal transduction, cytoskeletal organization and membrane transport 1 . Three different classes of PI(3)K have been described, which differ in their specificities for phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate substrates, as well as in their regulation. PI(3)Ks are needed for receptor trafficking in the endocytic pathway in mammalian cells and for transport from the Golgi to the vacuole in yeast (reviewed in ref. 1). The docking and fusion of early endosomes, which is regulated by the small GTPase Rab5 (ref. 2), requires PI(3)K activity 3 . In addition, PI(3)Ks regulate the recruitment to membranes of EEA1, an effector of the small GTPase Rab5 and a core component of the docking and fusion machinery 4,5 that binds phosphatidylinositol-3-phosphate (PtdIns(3)P) through a FYVEfinger motif 6 . We have recently identified over 20 cytosolic proteins that interact with the active form of Rab5 (ref. 5). Here we report the identification of two distinct PI(3)Ks, hVPS34 and p85α-p110β, among these cytosolic proteins. We suggest a new mechanism by which Rab5 can modify its membrane environment by coupling the local production of phosphoinositides to the selective recruitment of Rab5 effector proteins.We used matrix-assisted laser-desorption-ionization mass spectrometry (MALDI-MS) to identify the proteins purified on the basis of a specific interaction with the active form of Rab5 (ref. 5). Surprisingly, among these proteins we identified the regulatory subunit of class-I PI(3)Ks, p85α, and confirmed its identity by western blotting (Fig. 1a). We therefore performed PI(3)K enzymatic assays to demonstrate the presence of catalytic activity in the Rab5-GTPγS column eluate. PI(3)K activity was markedly detected in this eluate and was inhibited >90% by 100 nM wortmannin (Fig. 1b). The activity was more than 100 times greater than the activity in the Rab5-GDP column eluate, and 900-fold greater than the background activity (Fig. 1b). The enrichment was ~10-fold higher than the corresponding value reported for Ras and PI(3)K 7 . PI(3)K therefore efficiently interacts with, and is recruited by, Rab5.Given the presence of a class-I-PI(3)K regulatory subunit in the Rab5-GTPγS eluate, we would expect the catalytic subunit to be either p110α or p110β (ref. 1). By using isoform-specific antibodies we established that p110β, but not p110α, was present in the eluate P Figure 1 The PI(3)Ks p85α-p110β and p150-hVPS34 bind specifically to a Rab5-GTPγS affinity column. a, Identification of p85α in the Rab5-GTPγS column eluate. Eluates from GST-Rab5-GDP or GST-Rab5-GTPγS affinity columns were analysed by SDS-PAGE, silver staining and MALDI analysis (left) or immunoblotting (right). b, PI(3)K-activity assay on eluates from GST-Rab5-GDP or GST-Rab5-GTPγS affinity columns, using phosphatidylinositol as a substrate, in the absence or presence of 100 nM wortmannin (WM); and quantifica...