Krebs medal review: PtdIns3P in tumour suppression recombinant probe that can be used directly on ﬁxed specimens, makes this probe very versatile for monitor- ing the distribution of PtdIns3P. Subsequently, other probes have been used for monitoring PtdIns3P, including the FYVE domain of SARA, which has an intrinsic ability to dimerize and therefore does not need to be expressed as tandem fusion [36], and certain PX domains [37]. In general, the various probes have yielded comparable results, although the 2xFYVE probe has been most rigorously tested for ligand speci- ﬁcity. The original studies using 2xFYVE by ﬂuores- cence and electron microscopy showed that the bulk of PtdIns3P is associated with the limiting and intralumi- nal membranes of endosomes at steady-state [35]. Sub- sequent studies have revealed that PtdIns3P can also be detected at the plasma membrane of cells stimulated with insulin or lysophosphatidic acid [38,39]. Because this pool of PtdIns3P is generated through the activity of PI3K-II [40], which has so far not been implicated in tumour suppression, it will not be considered further in the present review. Recent analyses of starved yeast H. Stenmark cells have revealed a strong localization of PtdIns3P on autophagosomes, especially on the inner mem- branes [41], and, during induction of autophagy in mammalian cells, PtdIns3P is formed on membranes of the endoplasmic reticulum (ER) [42]. The impor- tance of this is discussed below. PI3K-III and PtdIns3P binding proteins in endosomal trafﬁcking Because Vps34 and Vps15 were originally identiﬁed as mediators of vacular protein sorting in yeast, the ﬁrst characterized functions of PI3K-III and PtdIns3P were those associated with endosomal trafﬁcking (Fig. 2A). PtdIns3P and endosomal fusion Yeast cells devoid of functional Vps34 or Vps15 secrete several hydrolases that normally are trans- ported to the lysosome-like vacuole [8,43], suggesting that these proteins control trafﬁcking between the Fig. 2. PtdIns3P effectors in cell regulation. Endocytic downregulation of a mitogenic receptor (A), autophagy (B) and cytokinesis (C). PtdIns3P effectors involved in the various processes are shown in red. Rabenosyn-5 and EEA1 mediate membrane fusion in the early endo- cytic pathway. HRS and EAP45 are subunits of the ESCRT machinery that sorts ubiquitinated mitogenic receptors into the ILVs of multive- sicular endosomes. DFCP1 mediates phagophore biogenesis, whereas WIPI2 mediates autophagosome biogenesis. FYVE-CENT facilitates cytokinesis. Microtubules are indicated by dashed green lines. 4840 FEBS Journal 277 (2010) 4837–4848 ª 2010 The Author Journal compilation ª 2010 FEBS