H. Stenmark Krebs medal review: PtdIns3P in tumour suppression Fig. 1. The human PI3K-III complex. (A) The various subunits of PI3K-III are indicated. VPS34, VPS15 and Beclin 1 are considered to form a core complex onto which acces- sory subunits can assemble. HEAT repeats (H), WD40 repeats (WD), Bcl-2 binding domain (BD), zinc ﬁngers (ZF) and coiled-coil domains (CC) are indicated. Note that there exist alternative sequence variants for most of the subunits (not indicated). (B) Distinct PI3K-III subcomplexes regulate autophago- some formation and endosomal trafﬁc. demonstration that this domain binds PtdIns3P. The FYVE domain was originally identiﬁed as a zinc ﬁnger required for localization of the early endosomal autoan- tigen 1 (EEA1) to early endosomes [19]. The ﬁnding that wortmannin, a general PI3K inhibitor, prevents the localization of EEA1 to endosomes, provided a clue that EEA1 might be recruited by a 3-phosphorylated PI [20], and biochemical studies showed that the FYVE domains from yeast and mammalian proteins bind to PtdIns3P with high speciﬁcity [21–23]. Further progress in deciphering the downstream functions of PtdIns3P was obtained when the PX domain was identiﬁed as a second PtdIns3P binding domain [24–27]. Although a few mammalian PX domains bind to other 3-PIs than PtdIns3P, most of the PX domains bind speciﬁcally to PtdIns3P with afﬁnities comparable to those of FYVE domains [28]. The human genome encodes approxi- mately 30 FYVE domain-containing proteins and some 45 PX domain-containing proteins that presumably mediate most (but not all) of the downstream functions of PtdIns3P [29]. Additional PtdIns3P-binding proteins that do not contain FYVE or PX domains include the Proppin ⁄ WIPI proteins, which bind PtdIns3P [and to some extent the related PtdIns(3,5)P2] through a WD40-repeat-containing b-propeller structure [30,31], and certain variant pleckstrin homology domains such as the GLUE (GRAM-like ubiquitin-binding in EAP45) domain [32–34]. Intracellular localization of PtdIns3P The identiﬁcation of PtdIns3P-binding domains offered the possibility to design probes that reveal the intracellular distribution of this lipid. Early work revealed that a single FYVE domain from the endoso- mal protein HRS binds PtdIns3P with too low afﬁnity to be useful as a probe. Remarkably, however, when two FYVE domains from HRS were fused in tandem (2xFYVE), the resulting construct could be used for imaging of cellular PtdIns3P with high sensitivity and speciﬁcity, presumably as a result of an avidity effect [35]. The fact that 2xFYVE can be either transfected into cells as a fusion with green ﬂuorescent protein or another tag, or expressed in bacteria and puriﬁed as a FEBS Journal 277 (2010) 4837–4848 ª 2010 The Author Journal compilation ª 2010 FEBS 4839