Krebs medal review: PtdIns3P in tumour suppression ESCRT-II, Vps36 ⁄ EAP45, contains a PtdIns3P-binding GLUE domain that is predicted to contribute to the membrane recruitment of ESCRT-II [32,34]. The involvement of the ESCRT machinery in protein sort- ing and ILV biogenesis, as well as the notion that key subunits of this machinery require PtdIns3P for their membrane recruitment, readily explains why interfer- ence with PI3K-III functions results in impaired protein sorting and causes endosomes devoid of ILVs. PI3K-III and PtdIns3P binding proteins in autophagy Macroautophagy (referred to here as autophagy) is a process that involves the sequestration of cytoplasm by a double membrane called phagophore or isolation membrane, followed by fusion of the resulting auto- phagosome with endosomes and lysosomes [63] (Fig. 2B). The degradation of the sequestered cytosolic material in autolysosomes is beneﬁcial for the cell, both under starvation conditions (when it is crucial to recycle free amino acids by degrading cytosolic pro- teins that are not housekeeping), during infection with cytosolic parasites, and under various stress conditions (e.g. those that result in the accumulation of cytosolic protein aggregates that are not readily degraded by proteasomes). The exact origin of the phagophore membrane is still a matter of debate, although there are strong arguments in favour of a contribution from both ER and mitochondrial membranes [42,64,65]. At least in the case of ER membranes, there is evidence for the production of PtdIns3P during the early phase of autophagosome formation [42,66]. Several lines of evidence point to a crucial role for PI3K-III in auto- phagy [66], and immunoelectron microscopy of starved yeast cells using the 2xFYVE probe has revealed that PtdIns3P is enriched on inner side of the phagophore during autophagosome formation [41]. Studies in yeast have revealed that a complex con- sisting of Vps34, Vps15, Vps30 and Atg14 is required for autophagy [10], and subsequent work in mamma- lian cells has shown a similar requirement for the mammalian homologues of these proteins [11,12]. In addition, an involvement of the Vps38 homologue UVRAG has been reported [7], which is surprising in light of the strong evidence that Vps38 mediates endosomal trafﬁcking and not autophagy in yeast. A possible role of UVRAG in autophagy is supported by the independent identiﬁcation of BIF-1, an interactor of UVRAG, as a regulator of autophagy [5]. On the other hand, monoallelic UVRAG mutations associated with microsatellite unstable colon cancer have no effect on autophagy, and the depletion of UVRAG has an H. Stenmark undetectable effect on autophagy in HEK293 cells, whereas endosomal sorting is affected [67]. One expla- nation for the conﬂicting ﬁndings on UVRAG may stem from the involvement of UVRAG in fusion between autophagosomes (and late endosomes) and lysosomes through its interactions with the HOPS complex [68]. Except for the catalytic activity of VPS34, little is known about the speciﬁc functions of the various PI3K-III subunits in autophagy. However, recent evidence suggests that the speciﬁc function of ATG14 in autophagy may reﬂect the ability of this protein to target PI3K-III to ER membranes [69]. How does PtdIns3P mediate autophagy? The only known PtdIns3P effector in autophagy in yeast is the Proppin protein Atg18 [70], whose binding to PtdIns3P is required for autophagy [71]. Although the exact function of Atg18 in autophagy remains to be clariﬁed, current evidence suggests that this protein, in complex with Atg2, facilitates the recruitment of lipidated Atg8, a key effector in autophagosome formation to phago- phore membranes [71]. A mammalian Atg18 homo- logue, WIPI2, is recruited to phagophore membranes along with ULK1, a protein kinase that positively reg- ulates autophagy [72]. Interestingly, the depletion of WIPI2 leads to a strong accumulation of omegasomes, comprising ER-localized PtdIns3P-containing struc- tures positive for DFCP1 (double FYVE domain-con- taining protein 1) that are considered to act as platforms for autophagosome formation. This suggests a role for WIPI2 in the progression from omegasomes into autophagosomes. PI3K-III and PtdIns3P binding proteins in cytokinesis A surprising ﬁnding when using a green ﬂuorescent protein-tagged version of the 2xFYVE probe was that PtdIns3P accumulates in the bridge separating two dividing cells, the so-called midbody. PtdIns3P is fre- quently associated with the central, electron-dense part of the midbody, referred to as the midbody ring or the Flemming body, but can also be observed on small vesicles throughout the midbody region [73]. This localization of PtdIns3P raises the question of whether its formation is required during cytokinesis, the ﬁnal stage of cell division (Fig. 2C). Indeed, small interfer- ing RNA-mediated knockdown of VPS34, as well as of the accessory PI3K-III components VPS15, Beclin 1, UVRAG and BIF-1, causes an increased proportion of cells undergoing cytokinesis, suggesting a role for PI3K-III in the completion of cytokinesis [15,73]. Small interfering RNA screening identiﬁed the large FYVE domain-containing protein FYVE-CENT 4842 FEBS Journal 277 (2010) 4837–4848 ª 2010 The Author Journal compilation ª 2010 FEBS