H. Stenmark Krebs medal review: PtdIns3P in tumour suppression secretory and endosomal pathways and ⁄ or between endosomes. A key effector of PtdIns3P in endocytic trafﬁcking is the FYVE-domain-containing protein Vac1, which interacts genetically and physically with the small GTPase Vps21, and with Vps45, a member of the Sec1 ⁄ Munc18 family of proteins regulating the formation of SNARE complexes that mediate mem- brane fusion [44,45]. Indeed, studies of the mammalian homologues of Vac1, Vps21 and Vps45, termed Rabe- nosyn-5, RAB5 and VPS45, respectively, have revealed that these proteins function in tethering and fusion reactions in the early endocytic pathway [46]. More- over, studies in Drosophila have revealed that Rabeno- syn functions to bridge Rab5 with Vps45, thereby regulating the function of the SNARE protein Ava- lanche in endosomal fusion [47]. Interestingly, func- tional interference with Rabenosyn-5 and its interacting partners causes a loss of both epithelial and planar polarity [47,48]. The loss of epithelial polarity is a prevailing characteristic of carcinomas, and muta- tion of Rabenosyn indeed causes epithelial tumours in Drosophila. To date, the mechanisms by which Rabe- nosyn controls epithelial polarity have not been eluci- dated, whereas more detailed insight is available in the case of planar cell polarity. One consequence of inter- ferring with Rabenosyn function is that Flamingo, a determinant of planar cell polarity through the nonca- nonical Wnt signalling pathway, accumulates in the cytoplasm instead of translocating to polarized mem- brane domains [48]. Accumulating evidence suggests a link between improper development of planar cell polarity and cancer [49], and even though it is still not clear whether Rabenosyn-5 is a tumour suppressor in mammals, the epithelial and planar cell polarity main- tained by this RAB5 and PtdIns3P effector has to be considered when dissecting the tumour suppressor activities of PI3K-III. Early studies in mammalian cells have also revealed another important PtdIns3P effector in endosomal tethering and fusion, namely EEA1, a protein that contains a Rab5 binding domain and a FYVE domain at its C-terminus, and a distinct Rab5 binding domain at its N-terminus [50]. EEA1 forms rod-shaped dimers through parallel coiled-coil interactions, and is well suited for tethering two opposing Rab5-containing membranes [51]. The exquisite localization of EEA1 to early endosomes is probably conferred by the coinci- dent detection of PtdIns3P and GTP-bound Rab5 [50]. EEA1 is structurally related to Rabenosyn-5, and also interacts with SNARE molecules [52,53]. In a remark- able reconstitution of Rab5-mediated fusion using lipo- somes and recombinant SNAREs and Rab5 effectors, the inclusion of PtdIns3P could bypass the requirement for PI3K-III, as expected based on previous studies [54]. Importantly, the omission of either EEA1 or Rabeno- syn-5 was sufﬁcient to inhibit fusion strongly, indicating that these proteins, despite their structural relatedness, have nonredundant functions in endocytic membrane fusion. PtdIns3P and endosomal sorting to the degradative pathway Consistent with the fact that Vps34 was originally identiﬁed as a mediator of protein sorting, studies of both yeast and mammalian cells have shown that PI3K-III is required for proper sorting of certain mem- brane proteins from endosomes to vacuoles ⁄ lysosomes [8,55]. Moreover, interference with the function of VPS34 in mammalian cells results in dilated late endo- somes devoid of intraluminal vesicles (ILVs) [56,57]. Recent studies have revealed that not only VPS34, but also VPS15, Beclin 1, UVRAG and BIF-1 are required for proper degradation of endocytosed epidermal growth factor receptors in lysosomes, highlighting the involvement of an entire PI3K-III complex in endosomal sorting [15]. A mechanistic explanation for these ﬁndings has emerged with the discovery of the endosomal sorting complex required for transport (ESCRT) machinery [58,59]. This molecular machinery, which consists of at least four subcomplexes (ESCRT-0, -I, -II and -III), is recruited to endosome membranes where it recognizes ubiquitinated membrane proteins (e.g. activated growth factor receptors and membrane-anchored hydrolases) and sorts these into ILVs. Recent reconsti- tution studies employing giant unilamellar vesicles have revealed that ESCRT-0, which contains as many as ﬁve ubiquitin-binding domains, serves to sequester ubiquitinated cargoes, whereas ESCRT-I and -II, which also contain ubiquitin-binding domains, serve to form invaginations of the endosomal membrane [60]. Finally, ESCRT-III is recruited, cargo is deubiquitinat- ed by deubiquitinating enzymes recruited by ESCRT-III [61], and ESCRT-III serves to sever the neck of the forming invagination, thereby securing the inclusion of cargo proteins within ILVs [60]. The main link between PI3K-III and the ESCRT pathway is the fact that Vps27 ⁄ HRS, a core component of ESCRT-0, contains a FYVE domain that mediates its recruitment to endosomal membranes through binding PtdIns3P [62]. Vps27 ⁄ HRS in turn recruits ESCRT-I through interac- tion with the Vps23 ⁄ TSG101 subunit, so the initial recruitment of ESCRT-0 to endosomal membranes via FYVE-PtdIns3P interactions is crucial for the function of the ESCRT machinery. In addition, a subunit of FEBS Journal 277 (2010) 4837–4848 ª 2010 The Author Journal compilation ª 2010 FEBS 4841