pi 3,5 p2 metabolism and function

Phosphatidylinositol 3,5-bisphosphate:metabolism and cellular functionsRobert H. Michell1, Victoria L. Heath1, Mark A. Lemmon2 and Stephen K. Dove1

1School of Biosciences, University of Birmingham, Birmingham, UK, B15 2TT2Department 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 trafficking,

and in pH control in the endosome–lysosome axis.

Recently identified 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)P2. 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 Charcot–Marie-Tooth disease and fleck corneal

dystrophy.

Introduction

The seven polyphosphoinositides (PPIn; see Box 1 for adiscussion of the naming and abbreviation of inositol-containing molecules) are essential regulators of many cellfunctions, including signalling through hormone andgrowth-factor receptors, cell survival versus apoptosis,motility and membrane trafficking. The myo-inositolheadgroup of phosphatidylinositol (PtdIns) includes fivestereochemically unique hydroxyls, and PPIn arePtdIns derivatives modified with various permutationsof 3-, 4- and 5-phosphate groups. In vivo, each kinaseinvolved in PPIn biosynthesis phosphorylates onehydroxyl group in PtdIns or in one or more PPIn. PPInkinases are tightly regulated, often by signal-inducedtranslocation to membranes. PPIn phosphatases metab-olize PPIn to less phosphorylated PPIn and/or to PtdIns.Mutations of some PPIn phosphatases, including thoseacting on phosphatidylinositol 3,5-bisphosphate[PtdIns(3,5)P2], are causal in human diseases (see later).

Metabolically, the monoester phosphates of PPIn arestrikingly dynamic, with turnover times in vivo of no more

Corresponding author: Michell, R.H. ([email protected]).Available online 20 December 2005

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than a few minutes, so individual PPIn concentrationschange quickly following any change in kinase and/orphosphatase activity. Local PPIn concentrations will oftenfluctuate rapidly and asynchronously in individual cellelements, for example, during vesicle trafficking betweendifferent cell compartments, even when the cell-wideconcentrations are fairly stable.

Displayed as minor constituents of target membranes,PPIn regulate an ever-growing number of cellularprocesses. They bind to specific effector proteins, modify-ing their activities and regulating the processes that theseeffectors influence. Most effectors of PPIn interact withtheir cognate PPIn through well-defined PPIn-bindingdomains [e.g. of the PH (pleckstrin homology), FYVE(Fab1, YOTB, Vac1 and EEA1), Tubby, ENTH (epsinN-terminal homology) and PX (phox homology) families].Single PPIn often use several effectors to influencemultiple cell functions simultaneously at more than onesite. How these processes are correctly segregated in spaceand time is not fully understood, but the suggestion thateffector proteins of PPIn are often co-incidence detectorsprovides at least a part of the explanation [1]. To function,such PPIn effectors must recognize both their cognatePPIn and a second ligand, usually a protein resident in therelevant cell compartment: for example, the endosomalregulator EEA1, with a phosphatidylinositol 3-phosphate(PtdIns3P)-binding FYVE domain, becomes active onlywhen it interacts at the early endosome with PtdIns3Pand GTP-loaded Rab5.

PtdIns(3,5)P2 synthesis: enzymology, regulation

and phenotypes

PtdIns(3,5)P2, the most recently identified phosphatidyl-inositol-bisphosphate (PtdInsP2) isomer, makes up a smallproportion of total cell PPIn – commonly 0.1% or less.Present in all eukaryotic cells so far examined [2–10], itpresumably contributes to widely conserved cell func-tion(s). Here, we review current knowledge ofPtdIns(3,5)P2 metabolism and function, particularlyfocusing on effectors through which PtdIns(3,5)P2 acts.Processes in which PtdIns(3,5)P2 has been implicated, andthe proteins involved, are summarized in Figure 1 andTable S1 (see Supplementary Material). To examine thephylogenetic distributions of proteins of each implicatedfamily, BLAST searches were run against appropriatedatabases, both with multiple sequences that encom-passed the sequence variability amongst divergent

Review TRENDS in Biochemical Sciences Vol.31 No.1 January 2006

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Box 1. Inositol lipids and inositol phosphates: names and abbreviations

Abbreviations for phosphoinositidesSome years ago, the International Union of Biochemistry and

Molecular Biology (IUBMB; www.iubmb.org) specified 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)P3 (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 – scientific

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 inositol substituentsAll six carbons become asymmetric centres as soon as the myo-

inositol ring is modified on position 1, 3, 4 or 6. It is symmetric about

the 2/5 axis.

Standard chemical rules demand that the first-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 1-

phosphate (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

numbering of the 1D configuration’. As a consequence, L-Ins1P or D-

Ins3P 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 0P and PI3 0P) and fictitious enzymes (e.g. 5 0-phosphatases,

phosphoinositide 3 0-kinases and PI-3 0Ks).

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-inositol-

1,1 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 sufficiently 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 polypho-

sphoinositide(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 polyphosphoinosi-

tide(s).

HOHO

OH

HOOH

OP

OO

OHO

OHOH

OH

OH_

Figure I. Di-myoinositol-1,1 0-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.

Review TRENDS in Biochemical Sciences Vol.31 No.1 January 2006 53

eukaryotes and with consensus sequences derived fromcollections of PtdIns3P 5-kinases (type-III PIPkins orPIPkIIIs) and PROPPINs [for b-propeller(s) that bindsPPIn] (Boxes 2 and 3). Table 1 illustrates the near-ubiquitous occurrence amongst eukaryotes of proteinsknown 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 Sacchar-omyces cerevisiae) and a PIPkIII (Fab1p in S. cerevisiae)[6,11–13]. The fact that PtdIns3P production by type-IIIPPln 3-kinases occurs mainly in the endosomal system[14] suggests that PtdIns(3,5)P2 might also function in theendosomal and lysosomal trafficking arena, and identifi-cation of the large protein Fab1p as the PIPkIII thatmakes PtdIns(3,5)P2 in yeast reinforced this idea.

The following phenotypes are characteristic of yeaststhat 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 fills much of the cell. Thisindicates that PtdIns(3,5)P2 is needed for membrane and

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protein recycling from the vacuole (lysosome) to earliercompartments [11,12,15,17–20] (Figure 1); Schizosac-charomyces pombe that lack the PIPkIII Ste12p illus-trate this especially well [18,21] (Figure 2). Often,vacuoles are also strikingly enlarged in cells that lackVps34p, the kinase that supplies PtdIns3P forPtdIns(3,5)P2 synthesis [18,21] (Figure 2). Moreover,mouse 3T3-L1 cells that overexpress a kinase-deadversion of the mouse PIPkIII display a similar vacuo-lation defect, even with the active kinase still present –their extensive vacuolation seems to be caused by a loss ofcompartment identity in the late endosomal system [22].

† A defect in the ubiquitin-dependent sorting of proteinsinto the internal vesicles of multivesicular bodies (MVBs)[23,24], for their subsequent trafficking to the vacuole(Figure 1).

† Failure to acidify the vacuole normally [11,15,17], forreasons that are still unexplained.

† Abnormal sensitivity to some stresses, such as heat ortreatment with the ionophore monensin [15,25]. Thisphenotype is corrected by including a substantialconcentration of a non-nutrient osmolyte such as sorbitolin the growth medium, as is an accompanying

http://www.iubmb.org

http://www.chem.qmul.ac.uk/iupac/misc/phos2t8.html#t4

http://www.chem.qmul.ac.uk/iupac/cyclitol/myo.html




PtdIns

PIPkIII Fig4p

Vps34pPIPkII?

MTM-like p’tases

Ent3p,Ent5p,Vps24p Other effectors?Atg18p Svp3p

‘Cell integrity’

Vac7p

Vac14p+

MVB

PtdIns(3,5)P2

PtdIns5PPtdIns3P

Ymr1por Inp53p

Vacuole(and endosome?)

acidification

Vacuole → MVB membrane

retrieval

MVB→vacuoleprotein sorting

(a)

(b)

GolgiVacuole

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 figure (b). Svp3p is a tentatively identified effector protein for which we are currently gathering supporting information.


sporulation defect [26]. The defect underlying thissensitivity seems to be a loss of cell-wall integrity(Figure 1), possibly caused by inefficient delivery of cell-wall constituents during assembly.

† Failure of hyphal growth on solid media (Candidaalbicans) [17].

† Slowing of endocytic and exocytic membrane trafficking.This might also be the underlying cause of inefficientmating, meiosis or sporulation and – especially in S.pombe – a failure to respond appropriately to matingfactors [13,18,20,27–30].

† 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, thePtdIns(3,5)P2 concentration increases up to 30-fold inyeast, and 2–6-fold in plant and some animal cells[3,5,12,31].Cells that carry partially active FAB1 mutants (e.g.

fab1–1), and so have a reduced PtdIns(3,5)P2 content,display only the vacuole enlargement and increasedsensitivity to stress. Thus, it seems that the processes


that are impaired in these circumstances are those thathave 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 defined PPIn substrates, S.cerevisiae, S. pombe and mouse PIPkIIIs are all specificPtdIns3P 5-kinases [32]. Among eukaryotes withsequenced genomes, a gene encoding a PIPkIII is apparentin all genomes other than those of a few pathogenic protistsand microsporidians (Table 1). Box 2 summarizes the mainmolecular features of PIPkIIIs but little is understood ofhow their complex organization dictates their function.

The PtdIns3P-binding FYVE domain might contributeto substrate recognition [33], but most Fab1p in yeast isfound at the vacuole rather than associated with theendosomal structures that are thought to house much ofthe PtdIns3P of a cell.

The DEP (Disheveled, Egl-10, Pleckstrin) domain,which is present in chordates and insects but not in


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 protein-

encoding genes)

PtdIns 3-

kinase

(Vps34p-

like)

PIPkIII

(Fab1p-

like)

Vac14p

and

homol-

ogues

PROPPIN Vps24p

or other

CHMPs

Epsins 3

and 5

Fig4p and

homol-

ogues

Mammals, birds, fish and amphibia C 1 C 4 C C C

Lower chordate (Ciona intestinalis) C 1 C 4 ? C C

Nematodes (Caenorhabditis elegans

and C. briggsae)

C 1 C 3 C C C

Insects (Drosophila, Apis and

Anopheles)

C 1 C 3 ? C C

Fungi (e.g. Saccharomyces

cerevisiae)

C 1 C 3 ? C/C C

Microsporidia (Encephalitozoon

cuniculi and Nosema locustae)

‘Degenerate’ para-

sitic fungi (w2000)

C 0 K 0 K K C

Amoebozoa (Dictyostelium

discoideum and Entamoeba

histolytica)

C 1 C 2 C C C

Plants (Arabidopsis thaliana, Oryza

sativa and Chlamydomonas

reinhardtii)

C 4/6/1 C 1–5 C C C

Apicomplexa

(Cryptosporidium hominis and

C. parvum)

Pathogens (w4000) C 0 K 0 K C C?

(Theileria parva and annulata) Pathogens (w4000) K? 0 C 0 C C C

(Plasmodium spp.) Malaria (w14,000) K? 0 C 1 C C C

Trypanosomatids (w9300–12000)

(Trypanosoma

brucei and T. cruzi)

Sleeping sickness,

Chagas’ disease

C 1 ? C K C ?

(Leishmania major and infantum) Sandfly-transmitted

pathogens

K? 1 ? K K C ?

Diplomonad (Giardia intestinalis) (w10,000) Early-

diverged eukaryote

C K? K 2 C C C

aFor 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 ofunknown function.

The CCT [chaperone containing TCP1 (T-complexprotein 1)]-like domain seems to be important in theregulation of PIPkIII, probably through specific protein–protein interactions. In particular, the fab1–1 mutant,which is not activated by hyper-osmotic stress, carries amutation (G864E) in a glycine residue that is highlyconserved in the CCT domains of PIPkIIIs and TriC/CCTcomplex/thermosome chaperone proteins (Box 2). TheseGroup-II chaperonins are barrel-shaped hexadecameric(2!8) complexes that catalyse the efficient folding of actins,tubulins and some other cytosolic proteins, especially thosethat are b-propellers constructed of multiple WD40domains. 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 partof the chaperone that interacts with ‘substrate’ proteins.

PtdIns3P 5-kinase activity can be abolished by mutatingthe ATP-binding site in the kinase domain [11,12,23,32,34].Mouse PIPkIII (MmPIPkIII, also known as PIKfyve) alsoexhibits protein-serine kinase activity, which is catalysedby the same active site as the PtdIns3P 5-kinase. It hasbeen suggested that protein phosphorylation of this typemight be involved in the auto-inhibition of PIPkIIIs (seelater) [34]. Whether other PIPkIIIs display protein kinaseactivity is not known. We failed to confirm the claim thatMmPIPkIII directly 5-phosphorylates PtdIns [35,36]: inour hands it did not, even when studied in vivo in S.cerevisiae.


Regulation of PIPkIII activity

The control of PIPkIII activity is not clearly understood. InS. 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 kinaseactivity and to be fully activated by hyper-osmotic stress,yeast must possess both Vac7p and Vac14p. Vac14p hashomologues in many eukaryotes, and a mouse Vac14p (alsotermed ArPIKfyve) activates mouse PIPkIII [19,24,37–39],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-inhibitedin vivo, and this auto-inhibition is lost in the constitutivelyactive mutant FAB1–5 [19]. FAB1–5 has multiplemutations in its C-terminal half and remains active evenin the presence of wild-type Fab1p. This suggests thatprotein–protein interactions involving the C-terminal halfof Fab1p might be involved in auto-inhibition of wild-typeFab1p. Furthermore, lack of activation of the fab1–1mutant during hyper-osmotic stress suggests that inter-actions between other proteins and the CCT domain mightbe important in this process.

Cells that lack the PtdIns(3,5)P2 effector Atg18p (alsoknown as Svp1p; see Supplementary Table S1) accumulatelarge amounts of PtdIns(3,5)P2 [41]. Whether this is due tofaster synthesis rather than slower degradation is not yetclear. However, yeast two-hybrid studies have identified arobust physical interaction between Atg18p and parts ofthe Fab1p molecule that are mutated in FAB1–5 [42],


Nomarski FM4-64S. pombe

Wild type

LackingPtdIns3-kinase(∆vps34)

LackingPIPkIII(∆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 FM4–64. Reproduced, with permission, from Ref. [21].


suggesting that Atg18p might be a direct negativeregulator of Fab1p.

The following might represent a simple model of thenormal control of PIPkIII activity, exemplified by Fab1p. Inresponse to a stress, activated Vac14p (and maybe alsoVac7p) binds to the CCT domain of auto-inhibited Fab1p.Thereby, Fab1p becomes active and PtdIns(3,5)P2 accumu-lates at the vacuole. Atg18p associates with thePtdIns(3,5)P2 that, in turn, accumulates in the vacuolemembrane; the PtdIns(3,5)P2-ligated Atg18p binds toactive Fab1p; and this provokes Fab1p to revert to itsauto-inhibited state. It remains to be determined to whatdegree Atg18p, Vac7p and Vac14p interact with overlap-ping regions of the Fab1p molecule, and whether theyinteract directly with one another.

PtdIns(3,5)P2 dephosphorylation

Evidence from S. cerevisiae suggests that the main route ofPtdIns(3,5)P2 turnover is dephosphorylation by PPIn5-phosphatase(s) (Figure 1); yeast contain no phosphatidyl-inositol 5-phosphate (PtdIns5P), the product of 3-depho-sphorylation, even when they have abnormally elevatedPtdIns(3,5)P2 levels [41]. The key enzyme for conversion ofPtdIns(3,5)P2 to PtdIns3P is Fig4p [19,40], a PtdIns(3,5)P2-selective member of the Sac domain family of PPIn5-phosphatases. Assays in vitro suggest that Fig4phydrolyses PtdIns(3,5)P2 specifically [19]; and Fig4phomologues in other species probably do the same.


Moreover, Fig4p and the PIPkIII activator Vac14p form avacuole-associated complex that regulates Fab1p activity[19], which further emphasizes the spatial and temporalcoordination of PtdIns(3,5)P2 synthesis and degradation.

In contrast to the situation in yeast, recent evidencefrom mammalian cells has highlighted a role for myotubu-larin and related PPIn 3-phosphatases (collectively termedMTMs) in the 3-dephosphorylation of PtdIns(3,5)P2 toPtdIns5P. There are at least 12 mammalian MTM proteins,only some of which are catalytically active phosphatases[43–45]. Mutations in MTM1 cause X-linked myotubularmyopathy (a muscle degenerative disease) and mutationsin MTMR2 and in MTMR13/SBF2 (a catalytically inactiveMTM) cause Charcot–Marie-Tooth disease types 4B1 and4B2 (neurodegenerative conditions involving defectivemyelination) [44,46]. Originally regarded as PtdIns3P-specific phosphatases, all of the catalytically active MTMsthat have been tested readily dephosphorylatePtdIns(3,5)P2 or PtdIns3P [47,48], and one of the patho-genic MTMR2 mutations occurs in the PH-like GRAM(glucosyltransferases, Rab-like GTPase activators andmyotubularins) domain, which binds PtdIns(3,5)P2 orother PPIn [49]. In Caenorhabditis elegans, RNAi‘knockout’ of three MTMs impairs the endocytic functionsof coelomocytes [50,51].

It is obviously important to clarify which PPIn aresubstrates of the various MTM phosphatases in vivo. Oneobservation that points to PtdIns(3,5)P2 hydrolysis is anincrease in PtdIns5P accumulation in myotubes thatoverexpress MTM1 [52]. Whether failure to metabolizePtdIns(3,5)P2 and/or PtdIns3P correctly is central to thecausation of the progressive diseases associated with MTMmutations is not known. Yeast has one MTM-like protein,Ymr1p, which seems to contribute to the regulation ofPtdIns3P levels in vivo [53], but its detailed properties areyet 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 thatPtdIns(3,5)P2 regulates several processes and, therefore,that cells probably possess several PtdIns(3,5)P2 effectorproteins. This view is supported by the recent identificationof several disparate, and widely conserved, proteins aspossible PtdIns(3,5)P2 effectors in retrograde membranetrafficking from the vacuole and in anterograde proteintrafficking through the MVB pathway (see Figure 1, Table 1and Supplementary Table S1). Effectors that mediate otherPtdIns(3,5)P2-dependent functions, such as vacuolar orlysosomal acidification and ‘cell integrity’, remain tobe identified.

Additional proteins that have been reported to bind toPtdIns(3,5)P2, but so far without evidence that theinteractions are of biological importance, include themammalian a-tocopherol-binding protein ATTP [54], twoSec14 domain-containing proteins from plants [55], the PX-domain-containing mammalian sorting nexin SNX1 [56]and the trafficking protein Ivy1p [57].


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 contain-

ing TCP1), PIPkIII-unique and PtdInsP kinase (PIPkinase), are ubiqui-

tous and are always present at approximately the same positions.

These, plus the near-ubiquitous FYVE domain, make up w30–65% 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 patho-

gens, lack a recognizable PIPkIII.

FYVE domainThis 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 identifies 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.

TriC/CCT/Thermosome-like domainThe 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 [91–93]. Most similar to this domain are

metazoan Tcp1g, its yeast homologue Cct3p and Archaeal thermo-

somes. PIPkIIIs are the only proteins outside the CCT/TriC/thermo-

some 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.

PIPkIII-unique domain

This newly identified ‘PIPkIII-unique’ region (Figure IIc) was found by

multiplealignmentofPIPkIII sequencesusingthe gap-tolerantalignment

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.

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

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 specificity [95]. The

consensus PIPkIII activation loop is alone sufficient to find most

PIPkIIIs when used in a BLAST search.

HlxlqFedgx akfsCkiyYA eeFdalRxxi cpGEedFIrSLSRCxkWdar GGKSgsTFxk TlDDRFIiKq msrlElesFl xFAPxYFxYi xxsxSxxSPT cLaKLIGvYq VsiKnxxxgkexkxDlLVME NLFfxrkixr ifDLKGSlRn RxvxxtNgxneVLLDENLvE xiYxsPlfvx ShsKxLLrxS vWnDTAFLAR xnVMDYSLlV GvDdxxxeLv VGIIDyIRTF TWDKkLEMVVKsxgilGGxx KxPTIVsPkq YrxRFreAMe xYIlxPDxW

Sxxsxxxpxp Cxxpxlvxid FYGEnDxtLG xFLexxcfxx xxxCpSCexP mxxHirryvH gqGxvxivlk Eldspvpxxx xxgxxxIwMw(0-37 insert) CkxCxxxTxx vamSdxtwxl SFaKyLELxF xghxxxxr (4-24) pCgHslhhDx xxyFgFxnxV axfxYspIxN YevxvPxxki E

HxxALLxQLL xxxxlsxxWx diixxLxxxa xxxvrPdxxx gdxMDptqYV KiKkixGGxx xdSxvvxGVV csKnvahKxM xsxixnPRIL LLxgxLEYxR xexfxsidpi vxQEkeylKn xvxRIvsxrP dliLVeksVS xyAqdxfxxx gItLvlnvKx xVLERISRCT gadIisSidx LxtxkLGxCs xFxv(4-17 insert)KTlMFFEGCP KxLGcTIlLR GgxxxeLkkV Kxvlxfmvfa AyhlxLExSF LxDefaxpp

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

(a)

(b)

(c)

(d)

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 fab1–1. (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.)

DEPFYVE 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.


PROPPINs: a family of PtdIns(3,5)P2 effectors that mediate

trafficking between late endosomes, MVB and vacuole

For cells to maintain normal vacuole morphology, adynamic balance must be maintained between membranedelivery (anterograde traffic) and recycling of membrane tothe late endosome compartment (retrograde traffic) [16,58](Figure 1). Vacuole enlargement of the type seen in mutantssuch as fab1D or vac14D occurs, at least partly, becausemembrane retrieval (or degradation) does not counter-balance membrane delivery, suggesting that PtdIns(3,5)P2

is necessary for this retrograde traffic.


Demonstration that Atg18p (see Supplementary TableS1 for its several synonyms) is a specific and high-affinityPtdIns(3,5)P2-binding protein that participates in retro-grade membrane traffic from the vacuole was a major steptowards understanding this PtdIns(3,5)P2 requirement[41]. A deletion screen for genes whose loss causes both afab1D/fab1K--like vacuole enlargement and derangedPtdIns(3,5)P2 metabolism identified Atg18p (this screenpreviously identified Vac14p as a PIPkIII activator [24]).Cells lacking Atg18p contain abnormally large quantities ofPtdIns(3,5)P2, even when unstressed.

http://www.sanger.ac.uk/Software/Pfam

http://www.sanger.ac.uk/Software/Pfam

http://www.genomatix.de/cgi-bin/dialign/dialign.pl



Atg18p contains no previously known PPIn-bindingdomain, but it binds to PtdIns(3,5)P2 with high affinityand specificity [as determined by quantitative surfaceplasmon resonance (SPR) analysis of Atg18p binding toPPIn-doped phospholipid surfaces]. Most of Atg18p almostcertainly folds as a WD40-repeat-based seven-bladedb-propeller, and a conserved sequence that spans bladesfive and six includes a basic Glu/GlnjArg-Arg-Gly motif thatis essential for Atg18p to bind to PtdIns(3,5)P2 with highaffinity (for details, see Box 3 and Supplementary FigureS1). Mutated Atg18p that no longer binds to PtdIns(3,5)P2

does not support retrograde vacuole-to-endosometrafficking [41].

Atg18p is the functional prototype of a novel family ofproteins that includes several subfamilies, for which wepropose the name PROPPINs. For details, see Box 3 andSupplementary Figure S1 [41,42,59,60]. Sequence con-servation is greatest in a characteristic PROPPIN tract ofw74 residues that includes the Glu/GlnjArg-Arg-Gly motif.A consensus sequence from that region is an efficient searchtool for identifying proteins in this family (see Box 3 andSupplementary Figure S1).

None of the three S. cerevisiae PROPPINs (Atg18p,Atg21p and Hsv2p; see Box 3, Supplementary Table S1 andSupplementary Figure S1) is essential for cell survival, andeach 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 character-

istic of all PROPPINs.

Almost all eukaryote genomes encode one or more PROPPINs. The

few genomes from which they seem to be absent include Encepha-

litozoon cuniculi and some apicomplexan pathogens.

The SMART and INTERPRO databases note 2–3 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 b-

strands 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 (typified

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 five 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 five bona fide Arabidopsis


mutants show neither the retrograde-trafficking defectnor the supra-normal accumulation of PtdIns(3,5)P2 thatcharacterizes atg18D cells, and Atg21p and Hsv2p localizedifferently from Atg18p in cells. Atg21p is also implicated inautophagic processes (Supplementary Box S1), particularlythose that degrade peroxisomes [61–63], and Hsv2pcontributes to cell-wall formation during sporulation [30].

Recognition of the PtdIns(3,5)P2 effector role ofAtg18p/Svp1p raised the possibility that many, or all,PROPPINs might be effectors for PtdIns(3,5)P2, or maybefor other PPIn. The three S. cerevisiae PROPPINs all bindto PtdIns(3,5)P2 with high specificity and selectivity, as doseveral other PROPPINs, from mammals, C. elegans andDrosophila melanogaster. One D. melanogaster PROPPIN-3 (CG11975; see Box 3 for the definition of PROPPIN-3)binds to PtdIns(3,5)P2 or PtdIns3P with similar affinities(KDZ80–100 nM), but not to other PPIn, so PtdIns3Pbinding might be relevant for a few PROPPINs.

Other studies have confirmed that Atg21p (a yeastPROPPIN-1/2) and WIPI49/WIPI-1 (human PROPPIN-1)bind to PPIn, but suggested less selectivity and loweraffinity than we observed, even with the same proteins[62,64]. From lipid-overlay studies, it has been concludedthat PtdIns3P might be the preferred ligand. However, thistechnique tends to bias the apparent selectivity towardsphosphatidylinositol-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 effectorsThis 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 identifier for an individual protein [60]. However, WIPI has

a previous usage, in speech and hearing analysis. As mentioned,

another paper suggests allying the five 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 HsPROP-

PIN-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

confidently 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%, under-

lined; invariant, double underlined.)

http://www.sbg.bio.ic.ac.uk/~3dpssm/

http://www-cryst.bioc.cam.ac.uk/~fugue/



PtdInsP2s and PtdIns(3,4,5)P3 wash off the nitrocellulosesupport more readily than PtdInsPs [65]. In line with this,our initial lipid-overlay analyses hinted at stronger Atg18pbinding to PtdIns3P than to PtdIns(3,5)P2, but quantitativeSPR and other experimental approaches then made it clearthat Atg18p and most other PROPPINs show clearpreference for PtdIns(3,5)P2 over other PPIn [41].

Other cell functions in which PROPPINs undoubtedlyplay a major part include autophagic processes: Sup-plementary 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 trafficking to the vacuole lumen

Protein sorting via MVBs (Figure 1) is of crucial importancein trafficking some proteins to lysosomal or vacuolarcompartments, in the trafficking of endocytosed cell-surfacereceptors and in the packaging of enveloped viruses forrelease from cells. This process normally delivers someprotein cargoes to the late endosome or MVB: these cargoesinclude some lysosome and vacuole-resident hydrolases,and receptors internalized from the cell surface anddestined for destruction. Most proteins destined for MVBsorting are tagged with a single ubiquitin molecule, in aprocess that involves the FYVE domain protein Hrs/Vps27p and the protein complexes ESCRT (endosomalsorting complexes required for protein transport)-1,ESCRT-2 and ESCRT-3 [66]. Following protein sorting,the cargo-containing vesicles bud into the lumen of theforming MVB. At least two mechanisms concentrateprotein cargo into the intra-lumenal MVB vesicles. Forone of these, which fails in cells that cannot makePtdIns(3,5)P2, the sorted protein must carry a mono-ubiquitin tag. Constitutive N-terminal tagging of thesecargoes with ubiquitin enables them to be sorted in aPtdIns(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 chargedmultivesicular body protein (CHMP) Vps24p (a componentof the ESCRT-III complex), have so far been proposed asPtdIns(3,5)P2 effectors involved in MVB sorting [67–69].

Other, better-characterized epsins have a phosphatidyl-inositol 4,5-bisphosphate [PtdIns(4,5)P2]-binding ENTHdomain and help to regulate the actin network beneaththe plasma membrane [70–72]. Ent3p and Ent5p lack someof the residues needed for PtdIns(4,5)P2 to interact with theENTH domain, but they might interact with other PPIn.Deletion of both ENT3 and ENT5, which seem to functionredundantly in S. cerevisiae, causes an MVB sorting defectsimilar to that of yeast lacking PIPkIII, and qualitativelipid-overlay studies have indicated that specific binding toPtdIns(3,5)P2 might underlie this role of Ent3p and/orEnt5p [68,69].

We have compared the binding of Ent3p and its isolatedENTH domain to PPIn by lipid overlay and quantitativeSPR analysis on PPIn-doped phospholipid surfaces. We sawweak and similar binding to each of the three PtdInsP2

isomers (KDO3 mM), with no clear specificity forPtdIns(3,5)P2 (K. Narayan, M. Lemmon and G Payne,unpublished). It has been suggested that Ent3p and Ent5p


contribute to inward budding of MVB vesicles in much thesame way as other epsins participate in endocytosis [69].However, this would place the participation of Ent3p andEnt5p beyond the ubiquitination-dependent step and at astage that is different from that requiring PtdIns(3,5)P2.Further work is needed to clarify whether Ent3p and/orEnt5p do indeed contribute to MVB protein sorting in aPtdIns(3,5)P2-dependent manner.

Recently, it has been suggested that Huntingtin-interacting protein 1 (HIP1) and HIP1-related protein(HIP1r), two other proteins with ENTH domains, mightneed to interact with PPIn, possibly PtdIns(3,5)P2, whenthey mediate the cycling of receptors for growth or survivalfactors in and out of fibroblasts. If the PPIn-binding ENTHdomain of HIP1r is excised, this cycling of receptors failsand the cells undergo apoptosis [73].

Small coiled-coil proteins of the CHMP family, includingVps24p, are components of the ESCRT-III complex [66,74],so it was intriguing when a phage display-based screen forproteins that bind PtdIns(3,5)P2 picked out a rat Vps24p(RnVps24p) homologue that has no recognized PPIn-binding domain [67]. It was reported that RnVps24pinteracts with PtdIns(3,5)P2 (and/or phosphatidylinositol3,4-bisphosphate) through a binding site that includes alysine residue that is present in many CHMPs. In rat cells,overexpression of the N-terminal half of RnVps24p, whichincludes the PtdIns(3,5)P2-binding motif, interferes withnormal membrane trafficking and causes endosomalvacuolation that seems similar to that seen in 3T3-L1cells overexpressing a kinase-dead version of mousePIPkIII (see later) [67].

We have used SPR measurements to examine PPInbinding by RnVps24p and other CHMPs, and have not seenspecific or high-affinity recognition of PtdIns(3,5)P2 (M.Baumeister and M. Lemmon, unpublished). Rather, most ofthe tested CHMPs, including Vps24p from yeast and rat,bound all phosphoinositides with low affinity (KDO50 mM).Whether Vps24p (or other CHMPs) are bona fide 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 proteinmachinery for PtdIns(3,5)P2 formation and function, butall eukaryotes other than a few microsporidian diplomonadand apicomplexan species with ‘reduced’ genomes havethese proteins (Table 1). These proteins, therefore, fall intoan enigmatic group of proteins categorized by Hartman andFederov [75] as ‘eukaryote signature proteins’ (ESPs).ESPs are close to ubiquitous in eukaryotes, so are likelyto have been recruited early into the eukaryotic lineage, butthey do not seem to have originated from the acknowledgedEubacterial or Archaeal progenitors of eukaryotes. Thesource of this intriguing grouping of mainly cytosolic andplasma membrane components during the emergence ofeukaryotes remains unresolved.

Soon after the discovery of PtdIns(3,5)P2, it was reportedthat hyper-osmotic stress provokes accumulation of thislipid in somatic cells and pollen tubes of plants [5,76], andthat this might be implicated in hyper-osmotically driven



vacuole fragmentation. Moreover, both interleukin-2 andUV exposure stimulate PtdIns(3,5)P2 formation in T-lym-phocytes [4], and fibrinogen has the same effect in platelets[77]. In addition to the involvement of MTMs in humandiseases, mutations in the only human PIPkIII (HsPIPkIII)seem to cause Francois–Neetens fleck corneal dystrophy,an autosomal dominant and largely asymptomatic humancondition in which refractile flecks are present in the cellsof the corneal stroma [78]. Provocatively, the majority of therelevant mutations occur in and around the potentiallyregulatory CCT domain of HsPIPkIII [78].

Indications of PtdIns(3,5)P2 function in mammaliancells 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 as3T3-L1 pre-adipocytes differentiate [35,79], MmPIPkIIIhas mainly been interrogated for involvement in cellregulation by insulin. The ability of MmPIPkIII to makePtdIns(3,5)P2 in fab1D yeast established that it functionsas a PIPkIII in vivo [32]. Contrary to our statement in 1999[32], we find that, when expressed effectively in yeast,MmPIPkIII fully rescues the phenotypes caused by loss ofFab1p (e.g. the ability of yeast to display an accumulation ofPtdIns(3,5)P2 in response to hyper-osmotic shock). MmPIP-kIII is necessary for formation of PtdIns5P in adipocyte-differentiated 3T3-L1 fibroblasts, but the available infor-mation does not distinguish between direct PtdIns5Psynthesis and formation via PtdIns(3,5)P2 [80].

Treating differentiated 3T3-LI adipocytes with insulin(or with pervanadate as an insulin mimic) increases thequantity and activity of membrane-associated MmPIP-kIII, which is probably mainly in trans-Golgi elementsand/or in MVB membrane elements that do not containthe insulin-responsive glucose transporter GLUT4[81,82]. However, some PIKfyve is in the same vesiclesas insulin-responsive aminopeptidase (IRAP), a proteinwith behaviour that mimics GLUT4 [83]. Procedures thatinhibit PIPkIII activity in vivo delay adipocytic differen-tiation and also slow the emergence of the ability of thecell to respond to insulin with Akt/PKB phosphorylation,mobilization of glucose transporters and enhanced DNAsynthesis [79]. Moreover, Ser318 of MmPIPkIII undergoesAkt-catalysed phosphorylation in insulin-treated cells;this somehow facilitates the insulin-stimulated appear-ance of IRAP, and presumably also that of GLUT4, at thecell surface [83].

In intact cells, some of the non-cytosolic fraction oftagged and overexpressed MmPIPkIII seems to be associ-ated with endosomal and MVB compartments that containthe recycling mannose phosphate receptor (MPR) [81] andare the destination of horseradish peroxidase that enteredcells by fluid-phase endocytosis [84]. This PIPkIII-positivecompartment is not entirely coincident with any well-defined endosomal structure, so its identity remains lessthan clear. Some MmPIPkIII is present on the intracellularvacuoles that accumulate in cells intoxicated with VacA(Helicobacter pylori vacuolating toxin). Over-expressingactive MmPIPkIII counteracts this vacuolation, but not thevacuolation caused by an ATPase-dead version of theendosomal AAA-ATPase suppressor of KC-transport


growth defect-1 (SKD1; homologous to yeast Vps4p). VacAis not a direct MmPIPkIII inhibitor, at least in vitro [85].

Over-expressing kinase-dead MmPIPkIII alongside theactive enzyme provokes the formation of prominentvacuolated structures; at least some of these are MVBscontaining abnormally few internal vesicles [22]. Thistreatment also impedes the trafficking of endocytosedhorseradish peroxidase, but not of endocytic cargoes thatenter by receptor-mediated mechanisms [86]. Unexpect-edly, overexpression of inactive MmPIPkIII does not seemto decrease the cellular PtdIns(3,5)P2 complement, whichleaves the mechanism of this ‘dominant negative’effect unclear.

Finally, during phagocytosis of Salmonella typhimuriumexpressing the virulence factor SigD (h SopB), a PPIn5-phosphatase with high activity against PtdIns(3,5)P2,cells form abnormally extended phagocytic vacuoles, withan accumulation of PtdIns3P in their bounding membranes[87]. Might this accumulation be driven, at least in part, byconversion of PtdIns(3,5)P2 to PtdIns3P in the phagosomemembranes?

Future perspectives

The eight years since PtdIns(3,5)P2 was first describedhave seen identification of enzymes that make and degradeit, recognition of PIPkIII- and PtdIns(3,5)P2-regulated cellfunctions, identification of some novel PPIn-bindingproteins as PtdIns(3,5)P2 effectors and the description ofdiseases involving impaired PtdIns(3,5)P2-dependent func-tions. But many questions remain unanswered. How arePtdIns(3,5)P2 synthesis and degradation coordinated intime and space? How does a single PIPkIII (in most species)make several functional pools of PtdIns(3,5)P2 at differentplaces in the cell for different purposes? Which MTMphosphatase or Fig4p homologue targets each pool, andhow does loss of MTM phosphatases that hydrolysePtdIns(3,5)P2 and PtdIns3P cause multiple diseases? Howis PtdIns(3,5)P2 involved in the sorting of proteins going toand from the vacuole or lysosome via MVB, and arePtdIns(3,5)P2 effector proteins other than PROPPINs,epsins and Vps24p involved? How does PtdIns(3,5)P2

influence lumenal acidification of the vacuole and lysosome(and maybe also of some endosomes)? How doesPtdIns(3,5)P2 contribute to cell responses to insulin,particularly adipocytic differentiation and transportertraffic to and from the plasma membrane? Are otherPtdIns(3,5)P2-dependent cellular processes still to befound? How does enhanced PtdIns(3,5)P2 synthesis con-tribute to cellular stress responses? And what role, if any,does PtdIns(3,5)P2 have in autophagy? The next few yearsshould be very exciting.

Note added in proof

A recent study has shown that RNAi suppression of PIPkIIIexpression in a human HeLa-cell-derived line inhibits HIV-1 replication, probably as a consequence of blocking amembrane-trafficking step between late endosomes and thetrans-Golgi network [88].



Supplementary data

Supplementary material associated with this article, whichcan be found at doi: 10.1016/j.tibs.2005.11.013, consists of:

† an alignment of the conserved core region of PROPPINsfrom diverse eukaryotes

† a table of the proteins involved in PtdIns(3,5)P2 function– including, for several proteins, the multiple synonymsunder which they have been studied

† a discussion of the possible involvement of PROPPINsand/or PtdIns(3,5)P2 in autophagy

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