Review

TRENDS in Biochemical Sciences

Vol.31 No.1 January 2006

Phosphatidylinositol 3,5-bisphosphate: metabolism and cellular functions

Robert H. Michell1, Victoria L. Heath1, Mark A. Lemmon2 and Stephen K. Dove1
1 2

School of Biosciences, University of Birmingham, Birmingham, UK, B15 2TT Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Polyphosphoinositides (PPIn) are low-abundance membrane phospholipids that each bind to a distinctive set of effector proteins and, thereby, regulate a characteristic suite of cellular processes. Major functions of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] are in membrane and protein trafcking, and in pH control in the endosomelysosome axis. Recently identied PtdIns(3,5)P2 effectors include a family of novel b-propeller proteins, for which we propose the name PROPPINs [for b-propeller(s) that binds PPIn], and possibly proteins of the epsin and CHMP (charged multi-vesicular body proteins) families. All eukaryotes, with the exception of some pathogenic protists and microsporidians, possess proteins needed for the formation, metabolism and functions of PtdIns(3,5)P 2. The importance of PtdIns(3,5)P2 for normal cell function is underscored by recent evidence for its involvement in mammalian cell responses to insulin and for PtdIns(3,5)P2 dysfunction in the human genetic conditions X-linked myotubular myopathy, Type-4B CharcotMarie-Tooth disease and eck corneal dystrophy.

Introduction The seven polyphosphoinositides (PPIn; see Box 1 for a discussion of the naming and abbreviation of inositolcontaining molecules) are essential regulators of many cell functions, including signalling through hormone and growth-factor receptors, cell survival versus apoptosis, motility and membrane trafcking. The myo-inositol headgroup of phosphatidylinositol (PtdIns) includes ve stereochemically unique hydroxyls, and PPIn are PtdIns derivatives modied with various permutations of 3-, 4- and 5-phosphate groups. In vivo, each kinase involved in PPIn biosynthesis phosphorylates one hydroxyl group in PtdIns or in one or more PPIn. PPIn kinases are tightly regulated, often by signal-induced translocation to membranes. PPIn phosphatases metabolize PPIn to less phosphorylated PPIn and/or to PtdIns. Mutations of some PPIn phosphatases, including those acting on phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], are causal in human diseases (see later). Metabolically, the monoester phosphates of PPIn are strikingly dynamic, with turnover times in vivo of no more
Corresponding author: Michell, R.H. (r.h.michell@bham.ac.uk). Available online 20 December 2005

than a few minutes, so individual PPIn concentrations change quickly following any change in kinase and/or phosphatase activity. Local PPIn concentrations will often uctuate rapidly and asynchronously in individual cell elements, for example, during vesicle trafcking between different cell compartments, even when the cell-wide concentrations are fairly stable. Displayed as minor constituents of target membranes, PPIn regulate an ever-growing number of cellular processes. They bind to specic effector proteins, modifying their activities and regulating the processes that these effectors inuence. Most effectors of PPIn interact with their cognate PPIn through well-dened PPIn-binding domains [e.g. of the PH (pleckstrin homology), FYVE (Fab1, YOTB, Vac1 and EEA1), Tubby, ENTH (epsin N-terminal homology) and PX (phox homology) families]. Single PPIn often use several effectors to inuence multiple cell functions simultaneously at more than one site. How these processes are correctly segregated in space and time is not fully understood, but the suggestion that effector proteins of PPIn are often co-incidence detectors provides at least a part of the explanation [1]. To function, such PPIn effectors must recognize both their cognate PPIn and a second ligand, usually a protein resident in the relevant cell compartment: for example, the endosomal regulator EEA1, with a phosphatidylinositol 3-phosphate (PtdIns3P)-binding FYVE domain, becomes active only when it interacts at the early endosome with PtdIns3P and GTP-loaded Rab5. PtdIns(3,5)P2 synthesis: enzymology, regulation and phenotypes PtdIns(3,5)P2, the most recently identied phosphatidylinositol-bisphosphate (PtdInsP2) isomer, makes up a small proportion of total cell PPIn commonly 0.1% or less. Present in all eukaryotic cells so far examined [210], it presumably contributes to widely conserved cell function(s). Here, we review current knowledge of PtdIns(3,5)P2 metabolism and function, particularly focusing on effectors through which PtdIns(3,5)P2 acts. Processes in which PtdIns(3,5)P2 has been implicated, and the proteins involved, are summarized in Figure 1 and Table S1 (see Supplementary Material). To examine the phylogenetic distributions of proteins of each implicated family, BLAST searches were run against appropriate databases, both with multiple sequences that encompassed the sequence variability amongst divergent

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Box 1. Inositol lipids and inositol phosphates: names and abbreviations Abbreviations for phosphoinositides
Some years ago, the International Union of Biochemistry and Molecular Biology (IUBMB; www.iubmb.org) specied concise and unambiguous names and abbreviations for phosphoinositides (see http://www.chem.qmul.ac.uk/iupac/misc/phos2t8.html#t4; http://www.chem.qmul.ac.uk/iupac/cyclitol/myo.html). Despite this, papers on phosphoinositides and inositol polyphosphates are often marred by inconsistent, and even wrong, names and abbreviations, and journals seem often to aim for neither correctness nor consistency. Examples of correct usage include PtdIns (phosphatidylinositol) and PtdIns(3,4,5)P 3 (phosphatidylinositol 3,4,5-trisphosphate). For instance, the IUBMB-approved depiction of PtdIns(3,5)P2 biosynthesis is: PtdIns/PtdIns3P [not PtdIns(3)P]/PtdIns(3,5)P2 This is better than commonly used alternatives such as PI/PI3P/ PI3,5P2, for the following reasons: An italic P distinguishes a phosphate group from a P atom. PtdIns has only one meaning. By contrast, and depending on the author and the phase of the moon, PI can have several meanings: (i) PtdIns; (ii) any phosphoinositide; or (iii) a broad term embracing all phosphoinositides. When it pops up in catch-all phrases such as PI signalling only the author can know what is meant scientic descriptions should not admit such ambiguity. And a typographic slip can easily cause confusion with pI (isoelectric point) or with Pi (inorganic orthophosphate). numbering of the 1D conguration. As a consequence, L-Ins1P or DIns3P is correctly abbreviated simply as Ins3P.

To prime or not to prime?

Erroneous primes have recently tended to infect the names of polyphosphoinositides, inositol polyphosphates and the enzymes that handle them. As a result, papers discuss non-existent lipids (e.g. PtdIns3 0 P and PI3 0 P) and ctitious enzymes (e.g. 5 0 -phosphatases, phosphoinositide 3 0 -kinases and PI-3 0 Ks). This is wrong numbers with primes designate substituents on the second ring of a multi-ring molecule (e.g. ATP is adenosine 5 0 triphosphate). Most inositol derivatives include only one ring and so need no primes. There are rare exceptions such as di-myo-inositol1,1 0 -phosphate (Figure I), a protective molecule synthesized in response to thermal stress by some Archaea and Bacteria [89].

A proposed abbreviation for polyphosphoinositide(s)

Polyphosphoinositides (not polyphosphatidylinositols) is a useful term that encompasses all of the seven phosphorylated PtdIns derivatives, but it has no International Union of Pure and Applied Chemistry (IUPAC)-approved abbreviation. None of the several abbreviations that have been widely used in this sense, such as PtdIns(s) or PIP(s), is sufciently unambiguous to be accepted (see earlier). In this article, we have abbreviated polyphosphoinositides to PPIn with this abbreviation meaning one or more of the seven polyphosphoinositide(s). (PPIn is unambiguous, whereas PPIns, an obvious alternative, already means preproinsulin.) We, therefore, suggest that PPIn might be adopted as an unambiguous generic abbreviation meaning polyphosphoinositide(s).

Numbering of inositol substituents

All six carbons become asymmetric centres as soon as the myoinositol ring is modied on position 1, 3, 4 or 6. It is symmetric about the 2/5 axis. Standard chemical rules demand that the rst-named substituent takes the lowest possible number (whether the ring is designated D- or L-). For example, inositol synthase (Ino1p in yeast) makes L-inositol 1phosphate (L-Ins1P). However, this is synonymous with D-Ins3P. In Biology, strict application of this lowest numbering rule would often stipulate confusing changes of numbering midway through pathways. To avoid this, discussions of inositol lipids and phosphates are allowed to ignore the lowest-numbering rule (see http://www. chem.qmul.ac.uk/iupac/cyclitol/myo.html). Rather, the simple abbreviation Ins is approved as meaning myo-inositol with the

OH HO HO HO OH
0

O P O

O HO O

OH OH OH OH

Figure I. Di-myoinositol-1,1 -phosphate, a protective cytosolic metabolite made by some thermophilic and other archaea and bacteria when they are stressed by conditions beyond those under which they normally grow.

eukaryotes and with consensus sequences derived from collections of PtdIns3P 5-kinases (type-III PIPkins or PIPkIIIs) and PROPPINs [for b-propeller(s) that binds PPIn] (Boxes 2 and 3). Table 1 illustrates the nearubiquitous occurrence amongst eukaryotes of proteins known or suspected to be implicated in PtdIns(3,5)P2 metabolism and function. PtdIns(3,5)P2 is made by the consecutive actions on PtdIns of a type-III PPIn 3-kinase (Vps34p in Saccharomyces cerevisiae) and a PIPkIII (Fab1p in S. cerevisiae) [6,1113]. The fact that PtdIns3P production by type-III PPln 3-kinases occurs mainly in the endosomal system [14] suggests that PtdIns(3,5)P2 might also function in the endosomal and lysosomal trafcking arena, and identication of the large protein Fab1p as the PIPkIII that makes PtdIns(3,5)P2 in yeast reinforced this idea. The following phenotypes are characteristic of yeasts that lack their PIPkIII, that lack a Fab1p activator (vac14D or vac7D), that express only a kinase-dead version of Fab1p (fab1KK) or that lack a PtdIns(3,5)P2 effector [8,9,15,16]: A grossly enlarged vacuole that lls much of the cell. This indicates that PtdIns(3,5)P2 is needed for membrane and
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protein recycling from the vacuole (lysosome) to earlier compartments [11,12,15,1720] (Figure 1); Schizosaccharomyces pombe that lack the PIPkIII Ste12p illustrate this especially well [18,21] (Figure 2). Often, vacuoles are also strikingly enlarged in cells that lack Vps34p, the kinase that supplies PtdIns3P for PtdIns(3,5)P2 synthesis [18,21] (Figure 2). Moreover, mouse 3T3-L1 cells that overexpress a kinase-dead version of the mouse PIPkIII display a similar vacuolation defect, even with the active kinase still present their extensive vacuolation seems to be caused by a loss of compartment identity in the late endosomal system [22]. A defect in the ubiquitin-dependent sorting of proteins into the internal vesicles of multivesicular bodies (MVBs) [23,24], for their subsequent trafcking to the vacuole (Figure 1). Failure to acidify the vacuole normally [11,15,17], for reasons that are still unexplained. Abnormal sensitivity to some stresses, such as heat or treatment with the ionophore monensin [15,25]. This phenotype is corrected by including a substantial concentration of a non-nutrient osmolyte such as sorbitol in the growth medium, as is an accompanying

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(a) Vps34p Vac7p + Vac14p

PtdIns Ymr1p or Inp53p PIPkII?

PtdIns3P PIPkIII Fig4p

PtdIns5P

MTM-like ptases PtdIns(3,5)P2

Ent3p,Ent5p,Vps24p MVBvacuole protein sorting

Atg18p Vacuole MVB membrane retrieval

Svp3p Cell integrity

Other effectors? Vacuole (and endosome?) acidification

(b)

Golgi

Vacuole

MVB

Ti BS

Figure 1. A summary of the likely metabolism, effectors and functions of PtdIns(3,5)P2. PtdIns(3,5)P2 regulates at least four cellular processes (a), two of which are summarized in the lower part of the gure (b). Svp3p is a tentatively identied effector protein for which we are currently gathering supporting information.

sporulation defect [26]. The defect underlying this sensitivity seems to be a loss of cell-wall integrity (Figure 1), possibly caused by inefcient delivery of cellwall constituents during assembly. Failure of hyphal growth on solid media (Candida albicans) [17]. Slowing of endocytic and exocytic membrane trafcking. This might also be the underlying cause of inefcient mating, meiosis or sporulation and especially in S. pombe a failure to respond appropriately to mating factors [13,18,20,2730]. Failure to display the rapid increase in PtdIns(3,5)P2 levels that normally follows hyper-osmotic stress. Following application of a hyper-osmotic stress, the PtdIns(3,5)P2 concentration increases up to 30-fold in yeast, and 26-fold in plant and some animal cells [3,5,12,31]. Cells that carry partially active FAB1 mutants (e.g. fab11), and so have a reduced PtdIns(3,5)P2 content, display only the vacuole enlargement and increased sensitivity to stress. Thus, it seems that the processes
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that are impaired in these circumstances are those that have the most stringent PtdIns(3,5)P2 requirements [20]. Mouse or S. pombe PIPkIII can functionally replace S. cerevisiae PIPkIII in yeast that lack the FAB1 gene [32]. Molecular characteristics of PIPkIIIs When incubated in vitro with dened PPIn substrates, S. cerevisiae, S. pombe and mouse PIPkIIIs are all specic PtdIns3P 5-kinases [32]. Among eukaryotes with sequenced genomes, a gene encoding a PIPkIII is apparent in all genomes other than those of a few pathogenic protists and microsporidians (Table 1). Box 2 summarizes the main molecular features of PIPkIIIs but little is understood of how their complex organization dictates their function. The PtdIns3P-binding FYVE domain might contribute to substrate recognition [33], but most Fab1p in yeast is found at the vacuole rather than associated with the endosomal structures that are thought to house much of the PtdIns3P of a cell. The DEP (Disheveled, Egl-10, Pleckstrin) domain, which is present in chordates and insects but not in

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Table 1. Species distribution of genes encoding proteins implicated in PtdIns(3,5)P2 functiona

Organisms: species or group Comments (e.g. genome size as a number of proteinencoding genes) PtdIns 3kinase (Vps34plike) C C C C C Degenerate parasitic fungi (w2000) C C PIPkIII (Fab1plike) 1 1 1 1 1 0 1 Vac14p and homologues C C C C C K C PROPPIN Vps24p or other CHMPs C ? C ? ? K C Epsins 3 and 5 Fig4p and homologues C C C C C C C

Mammals, birds, sh and amphibia Lower chordate (Ciona intestinalis) Nematodes (Caenorhabditis elegans and C. briggsae) Insects (Drosophila, Apis and Anopheles) Fungi (e.g. Saccharomyces cerevisiae) Microsporidia (Encephalitozoon cuniculi and Nosema locustae) Amoebozoa (Dictyostelium discoideum and Entamoeba histolytica) Plants (Arabidopsis thaliana, Oryza sativa and Chlamydomonas reinhardtii) Apicomplexa (Cryptosporidium hominis and C. parvum) (Theileria parva and annulata) (Plasmodium spp.) Trypanosomatids (Trypanosoma brucei and T. cruzi) (Leishmania major and infantum) Diplomonad (Giardia intestinalis)
a

4 4 3 3 3 0 2

C C C C C/C K C

4/6/1

15

Pathogens (w4000) Pathogens (w4000) Malaria (w14,000) (w930012000) Sleeping sickness, Chagas disease Sandy-transmitted pathogens (w10,000) Earlydiverged eukaryote

C K? K? C K? C

0 0 0 1 1 K?

K C C ? ? K

0 0 1 C K 2

K C C K K C

C C C C C C

C? C C ? ? C

For PIPkIIIs and PROPPINs, the number of putative proteins found by BLAST searches and validated by ClustalW and Dialign alignments is given. For other proteins, a simpler indication is given of their presence (C) or absence (K).

nematode worms or in non-metazoan eukaryotes, is of unknown function. The CCT [chaperone containing TCP1 (T-complex protein 1)]-like domain seems to be important in the regulation of PIPkIII, probably through specic protein protein interactions. In particular, the fab11 mutant, which is not activated by hyper-osmotic stress, carries a mutation (G864E) in a glycine residue that is highly conserved in the CCT domains of PIPkIIIs and TriC/CCT complex/thermosome chaperone proteins (Box 2). These Group-II chaperonins are barrel-shaped hexadecameric (2!8) complexes that catalyse the efcient folding of actins, tubulins and some other cytosolic proteins, especially those that are b-propellers constructed of multiple WD40 domains. The CCT-like region of PIPkIIIs (see Box 2) corresponds to the hinged intermediate plus apical elements of a thermosome/CCT subunit, which is the part of the chaperone that interacts with substrate proteins. PtdIns3P 5-kinase activity can be abolished by mutating the ATP-binding site in the kinase domain [11,12,23,32,34]. Mouse PIPkIII (MmPIPkIII, also known as PIKfyve) also exhibits protein-serine kinase activity, which is catalysed by the same active site as the PtdIns3P 5-kinase. It has been suggested that protein phosphorylation of this type might be involved in the auto-inhibition of PIPkIIIs (see later) [34]. Whether other PIPkIIIs display protein kinase activity is not known. We failed to conrm the claim that MmPIPkIII directly 5-phosphorylates PtdIns [35,36]: in our hands it did not, even when studied in vivo in S. cerevisiae.
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Regulation of PIPkIII activity The control of PIPkIII activity is not clearly understood. In S. cerevisiae, two activator proteins (Vac7p and Vac14p) participate in the hyper-osmotic activation of Fab1p (Figure 1). If PIPkIII is to exhibit its basal PtdIns3P kinase activity and to be fully activated by hyper-osmotic stress, yeast must possess both Vac7p and Vac14p. Vac14p has homologues in many eukaryotes, and a mouse Vac14p (also termed ArPIKfyve) activates mouse PIPkIII [19,24,3739], but Vac7p is a protein restricted to fungi [11,19,24,37,40]. The level of PtdIns(3,5)P2 in unstressed yeast is low, even when wild-type Fab1p is greatly overexpressed. Fab1p, therefore, seems normally to be auto-inhibited in vivo, and this auto-inhibition is lost in the constitutively active mutant FAB15 [19]. FAB15 has multiple mutations in its C-terminal half and remains active even in the presence of wild-type Fab1p. This suggests that proteinprotein interactions involving the C-terminal half of Fab1p might be involved in auto-inhibition of wild-type Fab1p. Furthermore, lack of activation of the fab11 mutant during hyper-osmotic stress suggests that interactions between other proteins and the CCT domain might be important in this process. Cells that lack the PtdIns(3,5)P2 effector Atg18p (also known as Svp1p; see Supplementary Table S1) accumulate large amounts of PtdIns(3,5)P2 [41]. Whether this is due to faster synthesis rather than slower degradation is not yet clear. However, yeast two-hybrid studies have identied a robust physical interaction between Atg18p and parts of the Fab1p molecule that are mutated in FAB15 [42],

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S. pombe

Nomarski

FM4-64

Wild type

Lacking PtdIns 3-kinase (vps34)

Lacking PIPkIII (ste12)

Figure 2. Vacuole enlargement in Schizosaccharomyces pombe mutants that lack PtdIns 3-kinase or PIPkIII. In the right-hand images, vacuoles are labelled with the endocytosed dye FM464. Reproduced, with permission, from Ref. [21].

suggesting that Atg18p might be a direct negative regulator of Fab1p. The following might represent a simple model of the normal control of PIPkIII activity, exemplied by Fab1p. In response to a stress, activated Vac14p (and maybe also Vac7p) binds to the CCT domain of auto-inhibited Fab1p. Thereby, Fab1p becomes active and PtdIns(3,5)P2 accumulates at the vacuole. Atg18p associates with the PtdIns(3,5)P2 that, in turn, accumulates in the vacuole membrane; the PtdIns(3,5)P2-ligated Atg18p binds to active Fab1p; and this provokes Fab1p to revert to its auto-inhibited state. It remains to be determined to what degree Atg18p, Vac7p and Vac14p interact with overlapping regions of the Fab1p molecule, and whether they interact directly with one another. PtdIns(3,5)P2 dephosphorylation Evidence from S. cerevisiae suggests that the main route of PtdIns(3,5)P2 turnover is dephosphorylation by PPIn 5-phosphatase(s) (Figure 1); yeast contain no phosphatidylinositol 5-phosphate (PtdIns5P), the product of 3-dephosphorylation, even when they have abnormally elevated PtdIns(3,5)P2 levels [41]. The key enzyme for conversion of PtdIns(3,5)P2 to PtdIns3P is Fig4p [19,40], a PtdIns(3,5)P2selective member of the Sac domain family of PPIn 5-phosphatases. Assays in vitro suggest that Fig4p hydrolyses PtdIns(3,5)P2 specically [19]; and Fig4p homologues in other species probably do the same.
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Moreover, Fig4p and the PIPkIII activator Vac14p form a vacuole-associated complex that regulates Fab1p activity [19], which further emphasizes the spatial and temporal coordination of PtdIns(3,5)P2 synthesis and degradation. In contrast to the situation in yeast, recent evidence from mammalian cells has highlighted a role for myotubularin and related PPIn 3-phosphatases (collectively termed MTMs) in the 3-dephosphorylation of PtdIns(3,5)P2 to PtdIns5P. There are at least 12 mammalian MTM proteins, only some of which are catalytically active phosphatases [4345]. Mutations in MTM1 cause X-linked myotubular myopathy (a muscle degenerative disease) and mutations in MTMR2 and in MTMR13/SBF2 (a catalytically inactive MTM) cause CharcotMarie-Tooth disease types 4B1 and 4B2 (neurodegenerative conditions involving defective myelination) [44,46]. Originally regarded as PtdIns3Pspecic phosphatases, all of the catalytically active MTMs that have been tested readily dephosphorylate PtdIns(3,5)P2 or PtdIns3P [47,48], and one of the pathogenic MTMR2 mutations occurs in the PH-like GRAM (glucosyltransferases, Rab-like GTPase activators and myotubularins) domain, which binds PtdIns(3,5)P2 or other PPIn [49]. In Caenorhabditis elegans, RNAi knockout of three MTMs impairs the endocytic functions of coelomocytes [50,51]. It is obviously important to clarify which PPIn are substrates of the various MTM phosphatases in vivo. One observation that points to PtdIns(3,5)P2 hydrolysis is an increase in PtdIns5P accumulation in myotubes that overexpress MTM1 [52]. Whether failure to metabolize PtdIns(3,5)P2 and/or PtdIns3P correctly is central to the causation of the progressive diseases associated with MTM mutations is not known. Yeast has one MTM-like protein, Ymr1p, which seems to contribute to the regulation of PtdIns3P levels in vivo [53], but its detailed properties are yet to be determined.

Multiple PtdIns(3,5)P2 effectors Cells that lack a normal PIPkIII and PtdIns(3,5)P2 complement display multiple phenotypes, suggesting that PtdIns(3,5)P2 regulates several processes and, therefore, that cells probably possess several PtdIns(3,5)P2 effector proteins. This view is supported by the recent identication of several disparate, and widely conserved, proteins as possible PtdIns(3,5)P2 effectors in retrograde membrane trafcking from the vacuole and in anterograde protein trafcking through the MVB pathway (see Figure 1, Table 1 and Supplementary Table S1). Effectors that mediate other PtdIns(3,5)P2-dependent functions, such as vacuolar or lysosomal acidication and cell integrity, remain to be identied. Additional proteins that have been reported to bind to PtdIns(3,5)P2, but so far without evidence that the interactions are of biological importance, include the mammalian a-tocopherol-binding protein ATTP [54], two Sec14 domain-containing proteins from plants [55], the PXdomain-containing mammalian sorting nexin SNX1 [56] and the trafcking protein Ivy1p [57].

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Box 2. PtdIns3P 5-kinases

PtdIns3P 5-kinases (type-III PIPkins or PIPkIIIs; organized as depicted in Figure I), are found in almost all eukaryotes. They vary in length between 1494 (Caenorhabditis briggsae) and 2853 (Ustilago maydis) amino acids. Three of the domains, namely CCT (chaperone containing TCP1), PIPkIII-unique and PtdInsP kinase (PIPkinase), are ubiquitous and are always present at approximately the same positions. These, plus the near-ubiquitous FYVE domain, make up w3065% of the sequence. A group-3 DEP (Disheveled, Egl-10, Pleckstrin) domain [90] is present only in the PIPkIIIs of chordate and arthropod metazoans. Consensus sequences for the conserved domains of PIPkIIIs are shown in Figure II. Fungal and metazoan genomes usually encode one PIPkIII but plants can have several maybe as many as six in rice [8]. A few eukaryotic genomes, including those of the microsporidian intracellular parasitic fungus Encephalitozoon cuniculi and of some apicomplexan pathogens, lack a recognizable PIPkIII. PIPkins or PIPkIs) and of PtdIns5P 4-kinases (type-II PIPkins or PIPkIIs). The exception is the activation loop, which at least in PIPkIs and PIPkIIs has a major role in determining substrate specicity [95]. The consensus PIPkIII activation loop is alone sufcient to nd most PIPkIIIs when used in a BLAST search.

FYVE DEP

CCT-like

PIPkIII-unique

PIPkinase
Ti BS

Figure I. A schematic depiction of the distribution of domains in PIPkIIIs: the DEP domain is present in a minority of PIPkIIIs.

(a)

FYVE domain
This domain, which starts between the N terminus and up to w400 residues in, seems to be absent from some plant PIPkIIIs. When a consensus FYVE domain sequence derived from many PIPkIIIs (Figure IIa) is used to search genome databases, it identies the FYVE domains of PIPkIIIs with higher probability than FYVE domains from other proteins, so these FYVE domains include sequence elements that are unique to PIPkIIIs.

KqyWMPDsxx keCydCxxkF TtFRRrHHCR lCGQIFCsxC CxxxIg(4-8 insert) gdLRVCxYCx xiaxxyxxss d

(b)

TriC/CCT/Thermosome-like domain
The TriC/CCT/Thermosome-like domain (termed Cpn60_TCP1 in the PFAM database of protein domains; www.sanger.ac.uk/Software/ Pfam) is the central domain of w250 residues and aligns well with the central w50% of the sequence of any chaperone subunit of the TriC/CCT/thermosome family [9193]. Most similar to this domain are metazoan Tcp1g, its yeast homologue Cct3p and Archaeal thermosomes. PIPkIIIs are the only proteins outside the CCT/TriC/thermosome family that include this sequence element, but what functions PIPkIIIs and TriC/CCT/thermosome proteins have in common remains to be determined. If a consensus CCT-like sequence derived from the CCT-like domains of PIPkIIIs (Figure IIb) is submitted to genome databases, PIPkIIIs score above genuine chaperone subunits, so the CCT-like domains of PIPkIIIs constitute a distinct subgroup within the TriC/CCT/thermosome sequence family.

HxxALLxQLL gdxMDptqYV xsxixnPRIL xvxRIvsxrP xVLERISRCT KTlMFFEGCP AyhlxLExSF

(c)

xxxxlsxxWx KiKkixGGxx LLxgxLEYxR dliLVeksVS gadIisSidx KxLGcTIlLR LxDefaxpp

diixxLxxxa xdSxvvxGVV xexfxsidpi xyAqdxfxxx LxtxkLGxCs GgxxxeLkkV

xxxvrPdxxx csKnvahKxM vxQEkeylKn gItLvlnvKx xFxv(4-17 insert) Kxvlxfmvfa

Sxxsxxxpxp Cxxpxlvxid xxxCpSCexP mxxHirryvH xxgxxxIwMw(0-37 insert) SFaKyLELxF xghxxxxr xxyFgFxnxV axfxYspIxN
(d)

FYGEnDxtLG xFLexxcfxx gqGxvxivlk Eldspvpxxx CkxCxxxTxx vamSdxtwxl (4-24) pCgHslhhDx YevxvPxxki E

PIPkIII-unique domain
This newly identied PIPkIII-unique region (Figure IIc) was found by multiple alignment of PIPkIII sequences using the gap-tolerant alignment tool Dialign [94]. This region of the PIPkIII sequence has no recognizable homology with other proteins; it includes several well-conserved cysteine and histidine residues that were previously noted by Cooke [8]. This PIPkIII-unique consensus sequence reliably retrieves PIPkIIIs, and nothing else, from BLAST searches of genome databases.

HlxlqFedgx LSRCxkWdar xFAPxYFxYi exkxDlLVME eVLLDENLvE xnVMDYSLlV KsxgilGGxx

akfsCkiyYA GGKSgsTFxk xxsxSxxSPT NLFfxrkixr xiYxsPlfvx GvDdxxxeLv KxPTIVsPkq

eeFdalRxxi TlDDRFIiKq cLaKLIGvYq ifDLKGSlRn ShsKxLLrxS VGIIDyIRTF YrxRFreAMe

cpGEedFIrS msrlElesFl VsiKnxxxgk RxvxxtNgxn vWnDTAFLAR TWDKkLEMVV xYIlxPDxW

PIPkIII kinase domain

Most of the C-terminal PtdInsP kinase domain (Figure IId) of PIPkIIIs is similar in sequence to the kinase domains of PtdIns4P 5-kinases (type-I

Figure II. Consensus sequences for commonly occurring domains in PIPkIIIs. (Variable residue, x; basic, red; nonpolar, brown; uncharged, turquoise; acidic, blue; aromatic, yellow-green; O40% conserved, lower case; O60%, upper case; O80%, underlined; invariant, double underlined.) These consensuses are derived from up to w43 PIPkIII sequences (15 fungal, 14 metazoan, ten plant and four other). (a) FYVE domain. (b) TriC/CCT/Thermosome-like domain. Shading highlights the glycine residue that is mutated to glutamic acid in fab11. (c) PIPkIII-unique domain. (d) PIPkIII kinase domain (shaded, activation loop). (The Dialign implementation at www.genomatix.de/cgi-bin/dialign/dialign.pl gives these colour-coded alignments.)

PROPPINs: a family of PtdIns(3,5)P2 effectors that mediate trafcking between late endosomes, MVB and vacuole For cells to maintain normal vacuole morphology, a dynamic balance must be maintained between membrane delivery (anterograde trafc) and recycling of membrane to the late endosome compartment (retrograde trafc) [16,58] (Figure 1). Vacuole enlargement of the type seen in mutants such as fab1D or vac14D occurs, at least partly, because membrane retrieval (or degradation) does not counterbalance membrane delivery, suggesting that PtdIns(3,5)P2 is necessary for this retrograde trafc.
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Demonstration that Atg18p (see Supplementary Table S1 for its several synonyms) is a specic and high-afnity PtdIns(3,5)P2-binding protein that participates in retrograde membrane trafc from the vacuole was a major step towards understanding this PtdIns(3,5)P2 requirement [41]. A deletion screen for genes whose loss causes both a fab1D/fab1K--like vacuole enlargement and deranged PtdIns(3,5)P2 metabolism identied Atg18p (this screen previously identied Vac14p as a PIPkIII activator [24]). Cells lacking Atg18p contain abnormally large quantities of PtdIns(3,5)P2, even when unstressed.

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Atg18p contains no previously known PPIn-binding domain, but it binds to PtdIns(3,5)P2 with high afnity and specicity [as determined by quantitative surface plasmon resonance (SPR) analysis of Atg18p binding to PPIn-doped phospholipid surfaces]. Most of Atg18p almost certainly folds as a WD40-repeat-based seven-bladed b-propeller, and a conserved sequence that spans blades ve and six includes a basic Glu/GlnjArg-Arg-Gly motif that is essential for Atg18p to bind to PtdIns(3,5)P2 with high afnity (for details, see Box 3 and Supplementary Figure S1). Mutated Atg18p that no longer binds to PtdIns(3,5)P2 does not support retrograde vacuole-to-endosome trafcking [41]. Atg18p is the functional prototype of a novel family of proteins that includes several subfamilies, for which we propose the name PROPPINs. For details, see Box 3 and Supplementary Figure S1 [41,42,59,60]. Sequence conservation is greatest in a characteristic PROPPIN tract of w74 residues that includes the Glu/GlnjArg-Arg-Gly motif. A consensus sequence from that region is an efcient search tool for identifying proteins in this family (see Box 3 and Supplementary Figure S1). None of the three S. cerevisiae PROPPINs (Atg18p, Atg21p and Hsv2p; see Box 3, Supplementary Table S1 and Supplementary Figure S1) is essential for cell survival, and each has independent functions. atg21D and hsv2D
Box 3. PROPPINs, a novel family of PtdIns(3,5)P2 effectors
Atg18p and closely related proteins, for which we propose the generic name PROPPINs, include an approximately central tract of w75 highly conserved residues (Figure I). This characteristic tract includes the PtdIns(3,5)P2-interacting Glu/Glnj Arg-Arg-Gly motif that is characteristic of all PROPPINs. Almost all eukaryote genomes encode one or more PROPPINs. The few genomes from which they seem to be absent include Encephalitozoon cuniculi and some apicomplexan pathogens. The SMART and INTERPRO databases note 23 WD40 domains in most PROPPINs, and repetitive WD40-like character through much of the sequences. Fold prediction or threading programs invariably suggest that PROPPINs fold as seven-bladed b-propellers, with the bstrands occupying matching positions in the various PROPPINs (see Ref. [41]). The positioning of b-strands was found by submitting PROPPINS to 3D-PSSM [96] (http://www.sbg.bio.ic.ac.uk/w3dpssm/), FUGUE [97] (http://www-cryst.bioc.cam.ac.uk/wfugue/) and other fold-recognition servers. The closest structural matches always included the multi-WD40 b-propellers of Tup1 (PDB code: c1erja), transducin-b1 (d1 gotb), a bTrcp1-Skp1-b-catenin complex (c1p22a) and an actin-interacting protein-1 homologue (c1nr0). As noted by others, the amino acid sequences of PROPPINs are divided into two major groups, and each group is further divided into at least two subgroups. Fungi usually have three PROPPINs (typied by Saccharomyces cerevisiae Atg18p, Atg21p and Hsv2p), which are often located in syntenic positions in different fungi. Metazoans, from mammals to insects, have up to four conserved PROPPINs, in two pairs [60]. The metazoan 1 and 2 pair is more similar to the fungal Atg18p- and Atg21p-like and the metazoan 3 and 4 pair is closer in sequence to the fungal Hsv2p-like group; see Supplementary Figure S1 for examples of PROPPINs in these groups. Dictyostelium has one representative of each major group. Arabidopsis has ve PROPPINs, four of which are allied to the Hsv2p-centred group. A claim that Arabidopsis has eight genes encoding PROPPINs, for which the names ATG18a-h were proposed [98], was inappropriate for two reasons. First, the genes tentatively named ATG18f-g (encoded by At1g54710, At5g54730 and At1g03380) do not match the PROPPIN consensus, so are functionally unrelated WD-40 proteins. Second, only one of the ve bona de Arabidopsis
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mutants show neither the retrograde-trafcking defect nor the supra-normal accumulation of PtdIns(3,5)P2 that characterizes atg18D cells, and Atg21p and Hsv2p localize differently from Atg18p in cells. Atg21p is also implicated in autophagic processes (Supplementary Box S1), particularly those that degrade peroxisomes [6163], and Hsv2p contributes to cell-wall formation during sporulation [30]. Recognition of the PtdIns(3,5)P2 effector role of Atg18p/Svp1p raised the possibility that many, or all, PROPPINs might be effectors for PtdIns(3,5)P2, or maybe for other PPIn. The three S. cerevisiae PROPPINs all bind to PtdIns(3,5)P2 with high specicity and selectivity, as do several other PROPPINs, from mammals, C. elegans and Drosophila melanogaster. One D. melanogaster PROPPIN3 (CG11975; see Box 3 for the denition of PROPPIN-3) binds to PtdIns(3,5)P2 or PtdIns3P with similar afnities (KDZ80100 nM), but not to other PPIn, so PtdIns3P binding might be relevant for a few PROPPINs. Other studies have conrmed that Atg21p (a yeast PROPPIN-1/2) and WIPI49/WIPI-1 (human PROPPIN-1) bind to PPIn, but suggested less selectivity and lower afnity than we observed, even with the same proteins [62,64]. From lipid-overlay studies, it has been concluded that PtdIns3P might be the preferred ligand. However, this technique tends to bias the apparent selectivity towards phosphatidylinositol-phosphates (PtdInsPs) because

PROPPINs is allied to the Atg18p- or Atg21p-related group 1 and 2 PROPPINs.

PROPPINs: a generic name for Atg18p/Atg21p/ Hsv2p-related PPIn effectors

This group of proteins has only recently become a focus of attention but it has already garnered diverse names [41,42,59,60,64,98,99]. Based on the naming of a human protein as WIPI49 (WD40-repeat protein interacting with phosphoinositides of 49 kDa [64]), one suggested nomenclature would term each protein WIPI-n, where n is a numerical identier for an individual protein [60]. However, WIPI has a previous usage, in speech and hearing analysis. As mentioned, another paper suggests allying the ve Arabidopsis PROPPINs members to Atg18p (as AtAtg18a, AtAtg18b and so on), even though most of them are not most closely related to it. As an unambiguous and euphonious name for these proteins that suggests their function, we suggest PROPPIN(s) [for b-propeller(s) that binds PPIn]. The consensus sequence presented in Figure I, as the PROPPIN consensus, would be used to identify new family members and to eliminate inappropriate candidates. Following a previously proposed numbering scheme, we suggest that the numbering of PROPPINs would designate Homo sapiens protein FLJ10055 as HsPROPPIN-1, DKFZP434J154 as HsPROPPIN-2, WDR45L as HsPROPPIN-3 and WDRX1 as HsPROPPIN-4. A more detailed analysis of molecular relationships within the PROPPIN family will be necessary before each of the PROPPINs of non-metazoan organisms can condently be assigned to these (or possibly additional) PROPPIN groupings.

AHxspLAcLA lsxDGTxlAT ASEKGTlIRv Fstxxgxkly EFRRGxxxax IYSlnFSxDS xflCasSdtg TVHIF

Figure I. Consensus sequence for PROPPINs. The consensus sequence is derived from w150 PROPPIN sequences from diverse eukaryotes. (Variable residue, x; basic, red; nonpolar, brown; uncharged, turquoise; acidic, blue; aromatic, yellow-green; O40% conserved, lower case; O60%, upper case; O80%, underlined; invariant, double underlined.)

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PtdInsP2s and PtdIns(3,4,5)P3 wash off the nitrocellulose support more readily than PtdInsPs [65]. In line with this, our initial lipid-overlay analyses hinted at stronger Atg18p binding to PtdIns3P than to PtdIns(3,5)P2, but quantitative SPR and other experimental approaches then made it clear that Atg18p and most other PROPPINs show clear preference for PtdIns(3,5)P2 over other PPIn [41]. Other cell functions in which PROPPINs undoubtedly play a major part include autophagic processes: Supplementary Box S1 discusses whether PtdIns(3,5)P2 might be implicated in these functions. Epsins and Vps24p, putative PtdIns(3,5)P2 effectors in protein trafcking to the vacuole lumen Protein sorting via MVBs (Figure 1) is of crucial importance in trafcking some proteins to lysosomal or vacuolar compartments, in the trafcking of endocytosed cell-surface receptors and in the packaging of enveloped viruses for release from cells. This process normally delivers some protein cargoes to the late endosome or MVB: these cargoes include some lysosome and vacuole-resident hydrolases, and receptors internalized from the cell surface and destined for destruction. Most proteins destined for MVB sorting are tagged with a single ubiquitin molecule, in a process that involves the FYVE domain protein Hrs/ Vps27p and the protein complexes ESCRT (endosomal sorting complexes required for protein transport)-1, ESCRT-2 and ESCRT-3 [66]. Following protein sorting, the cargo-containing vesicles bud into the lumen of the forming MVB. At least two mechanisms concentrate protein cargo into the intra-lumenal MVB vesicles. For one of these, which fails in cells that cannot make PtdIns(3,5)P2, the sorted protein must carry a monoubiquitin tag. Constitutive N-terminal tagging of these cargoes with ubiquitin enables them to be sorted in a PtdIns(3,5)P2-independent manner, so the PtdIns(3,5)P2 requirement must precede ubiquitination. Three proteins, the epsins Ent3p and Ent5p (found in prevacuolar and endosomal structures) and the charged multivesicular body protein (CHMP) Vps24p (a component of the ESCRT-III complex), have so far been proposed as PtdIns(3,5)P2 effectors involved in MVB sorting [6769]. Other, better-characterized epsins have a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-binding ENTH domain and help to regulate the actin network beneath the plasma membrane [7072]. Ent3p and Ent5p lack some of the residues needed for PtdIns(4,5)P2 to interact with the ENTH domain, but they might interact with other PPIn. Deletion of both ENT3 and ENT5, which seem to function redundantly in S. cerevisiae, causes an MVB sorting defect similar to that of yeast lacking PIPkIII, and qualitative lipid-overlay studies have indicated that specic binding to PtdIns(3,5)P2 might underlie this role of Ent3p and/or Ent5p [68,69]. We have compared the binding of Ent3p and its isolated ENTH domain to PPIn by lipid overlay and quantitative SPR analysis on PPIn-doped phospholipid surfaces. We saw weak and similar binding to each of the three PtdInsP2 isomers (KDO3 mM), with no clear specicity for PtdIns(3,5)P2 (K. Narayan, M. Lemmon and G Payne, unpublished). It has been suggested that Ent3p and Ent5p
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contribute to inward budding of MVB vesicles in much the same way as other epsins participate in endocytosis [69]. However, this would place the participation of Ent3p and Ent5p beyond the ubiquitination-dependent step and at a stage that is different from that requiring PtdIns(3,5)P2. Further work is needed to clarify whether Ent3p and/or Ent5p do indeed contribute to MVB protein sorting in a PtdIns(3,5)P2-dependent manner. Recently, it has been suggested that Huntingtininteracting protein 1 (HIP1) and HIP1-related protein (HIP1r), two other proteins with ENTH domains, might need to interact with PPIn, possibly PtdIns(3,5)P2, when they mediate the cycling of receptors for growth or survival factors in and out of broblasts. If the PPIn-binding ENTH domain of HIP1r is excised, this cycling of receptors fails and the cells undergo apoptosis [73]. Small coiled-coil proteins of the CHMP family, including Vps24p, are components of the ESCRT-III complex [66,74], so it was intriguing when a phage display-based screen for proteins that bind PtdIns(3,5)P2 picked out a rat Vps24p (RnVps24p) homologue that has no recognized PPInbinding domain [67]. It was reported that RnVps24p interacts with PtdIns(3,5)P2 (and/or phosphatidylinositol 3,4-bisphosphate) through a binding site that includes a lysine residue that is present in many CHMPs. In rat cells, overexpression of the N-terminal half of RnVps24p, which includes the PtdIns(3,5)P2-binding motif, interferes with normal membrane trafcking and causes endosomal vacuolation that seems similar to that seen in 3T3-L1 cells overexpressing a kinase-dead version of mouse PIPkIII (see later) [67]. We have used SPR measurements to examine PPIn binding by RnVps24p and other CHMPs, and have not seen specic or high-afnity recognition of PtdIns(3,5)P2 (M. Baumeister and M. Lemmon, unpublished). Rather, most of the tested CHMPs, including Vps24p from yeast and rat, bound all phosphoinositides with low afnity (KDO50 mM). Whether Vps24p (or other CHMPs) are bona de effector(s) for PtdIns(3,5)P2, a particularly low-abundance PPIn, therefore remains unclear. PIPkIII, PtdIns(3,5)P2 and PtdIns(3,5)P2 effectors in diverse eukaryotes Eubacterial and Archaeal genomes lack the protein machinery for PtdIns(3,5)P2 formation and function, but all eukaryotes other than a few microsporidian diplomonad and apicomplexan species with reduced genomes have these proteins (Table 1). These proteins, therefore, fall into an enigmatic group of proteins categorized by Hartman and Federov [75] as eukaryote signature proteins (ESPs). ESPs are close to ubiquitous in eukaryotes, so are likely to have been recruited early into the eukaryotic lineage, but they do not seem to have originated from the acknowledged Eubacterial or Archaeal progenitors of eukaryotes. The source of this intriguing grouping of mainly cytosolic and plasma membrane components during the emergence of eukaryotes remains unresolved. Soon after the discovery of PtdIns(3,5)P2, it was reported that hyper-osmotic stress provokes accumulation of this lipid in somatic cells and pollen tubes of plants [5,76], and that this might be implicated in hyper-osmotically driven

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vacuole fragmentation. Moreover, both interleukin-2 and UV exposure stimulate PtdIns(3,5)P2 formation in T-lymphocytes [4], and brinogen has the same effect in platelets [77]. In addition to the involvement of MTMs in human diseases, mutations in the only human PIPkIII (HsPIPkIII) seem to cause FrancoisNeetens eck corneal dystrophy, an autosomal dominant and largely asymptomatic human condition in which refractile ecks are present in the cells of the corneal stroma [78]. Provocatively, the majority of the relevant mutations occur in and around the potentially regulatory CCT domain of HsPIPkIII [78]. Indications of PtdIns(3,5)P2 function in mammalian cells have come mostly from studies of the mouse PIPkIII (MmPIPkIII; also known as PIKfyve) in 3T3-L1 cells [7]. Cloned as a protein that shows increasing expression as 3T3-L1 pre-adipocytes differentiate [35,79], MmPIPkIII has mainly been interrogated for involvement in cell regulation by insulin. The ability of MmPIPkIII to make PtdIns(3,5)P2 in fab1D yeast established that it functions as a PIPkIII in vivo [32]. Contrary to our statement in 1999 [32], we nd that, when expressed effectively in yeast, MmPIPkIII fully rescues the phenotypes caused by loss of Fab1p (e.g. the ability of yeast to display an accumulation of PtdIns(3,5)P2 in response to hyper-osmotic shock). MmPIPkIII is necessary for formation of PtdIns5P in adipocytedifferentiated 3T3-L1 broblasts, but the available information does not distinguish between direct PtdIns5P synthesis and formation via PtdIns(3,5)P2 [80]. Treating differentiated 3T3-LI adipocytes with insulin (or with pervanadate as an insulin mimic) increases the quantity and activity of membrane-associated MmPIPkIII, which is probably mainly in trans-Golgi elements and/or in MVB membrane elements that do not contain the insulin-responsive glucose transporter GLUT4 [81,82]. However, some PIKfyve is in the same vesicles as insulin-responsive aminopeptidase (IRAP), a protein with behaviour that mimics GLUT4 [83]. Procedures that inhibit PIPkIII activity in vivo delay adipocytic differentiation and also slow the emergence of the ability of the cell to respond to insulin with Akt/PKB phosphorylation, mobilization of glucose transporters and enhanced DNA synthesis [79]. Moreover, Ser318 of MmPIPkIII undergoes Akt-catalysed phosphorylation in insulin-treated cells; this somehow facilitates the insulin-stimulated appearance of IRAP, and presumably also that of GLUT4, at the cell surface [83]. In intact cells, some of the non-cytosolic fraction of tagged and overexpressed MmPIPkIII seems to be associated with endosomal and MVB compartments that contain the recycling mannose phosphate receptor (MPR) [81] and are the destination of horseradish peroxidase that entered cells by uid-phase endocytosis [84]. This PIPkIII-positive compartment is not entirely coincident with any welldened endosomal structure, so its identity remains less than clear. Some MmPIPkIII is present on the intracellular vacuoles that accumulate in cells intoxicated with VacA (Helicobacter pylori vacuolating toxin). Over-expressing active MmPIPkIII counteracts this vacuolation, but not the vacuolation caused by an ATPase-dead version of the endosomal AAA-ATPase suppressor of KC-transport
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growth defect-1 (SKD1; homologous to yeast Vps4p). VacA is not a direct MmPIPkIII inhibitor, at least in vitro [85]. Over-expressing kinase-dead MmPIPkIII alongside the active enzyme provokes the formation of prominent vacuolated structures; at least some of these are MVBs containing abnormally few internal vesicles [22]. This treatment also impedes the trafcking of endocytosed horseradish peroxidase, but not of endocytic cargoes that enter by receptor-mediated mechanisms [86]. Unexpectedly, overexpression of inactive MmPIPkIII does not seem to decrease the cellular PtdIns(3,5)P2 complement, which leaves the mechanism of this dominant negative effect unclear. Finally, during phagocytosis of Salmonella typhimurium expressing the virulence factor SigD (h SopB), a PPIn 5-phosphatase with high activity against PtdIns(3,5)P2, cells form abnormally extended phagocytic vacuoles, with an accumulation of PtdIns3P in their bounding membranes [87]. Might this accumulation be driven, at least in part, by conversion of PtdIns(3,5)P2 to PtdIns3P in the phagosome membranes?

Future perspectives The eight years since PtdIns(3,5)P2 was rst described have seen identication of enzymes that make and degrade it, recognition of PIPkIII- and PtdIns(3,5)P2-regulated cell functions, identication of some novel PPIn-binding proteins as PtdIns(3,5)P2 effectors and the description of diseases involving impaired PtdIns(3,5)P2-dependent functions. But many questions remain unanswered. How are PtdIns(3,5)P2 synthesis and degradation coordinated in time and space? How does a single PIPkIII (in most species) make several functional pools of PtdIns(3,5)P2 at different places in the cell for different purposes? Which MTM phosphatase or Fig4p homologue targets each pool, and how does loss of MTM phosphatases that hydrolyse PtdIns(3,5)P2 and PtdIns3P cause multiple diseases? How is PtdIns(3,5)P2 involved in the sorting of proteins going to and from the vacuole or lysosome via MVB, and are PtdIns(3,5)P2 effector proteins other than PROPPINs, epsins and Vps24p involved? How does PtdIns(3,5)P2 inuence lumenal acidication of the vacuole and lysosome (and maybe also of some endosomes)? How does PtdIns(3,5)P2 contribute to cell responses to insulin, particularly adipocytic differentiation and transporter trafc to and from the plasma membrane? Are other PtdIns(3,5)P2-dependent cellular processes still to be found? How does enhanced PtdIns(3,5)P2 synthesis contribute to cellular stress responses? And what role, if any, does PtdIns(3,5)P2 have in autophagy? The next few years should be very exciting.

Note added in proof A recent study has shown that RNAi suppression of PIPkIII expression in a human HeLa-cell-derived line inhibits HIV1 replication, probably as a consequence of blocking a membrane-trafcking step between late endosomes and the trans-Golgi network [88].

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Supplementary data Supplementary material associated with this article, which can be found at doi: 10.1016/j.tibs.2005.11.013, consists of: an alignment of the conserved core region of PROPPINs from diverse eukaryotes a table of the proteins involved in PtdIns(3,5)P2 function including, for several proteins, the multiple synonyms under which they have been studied a discussion of the possible involvement of PROPPINs and/or PtdIns(3,5)P2 in autophagy

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