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...

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