Multiple pools of PtdIns 4-phosphate detected using the pleckstrin

Multiple pools of PtdIns 4-phosphate detected using the

pleckstrin homology domain of Osh2p

Anjana Roy and Timothy P Levine

Division of Cell Biology, Institute of Ophthalmology, University College London, Bath

Street, London EC1V 9EL

Address correspondence to Tim Levine:

phone (+44)-20 7608 4027/8, fax (+44)-20 7608 4034

email [email protected]

Running Title:

A PH domain that binds multiple pools of PtdIns(4)P

JBC Papers in Press. Published on July 22, 2004 as Manuscript M401583200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Roy and Levine, 2004

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SUMMARY

Phosphatidylinositol (PtdIns) phosphate (PtdInsP) lipids are used as intracellular

signposts for the recruitment and activation of peripheral membrane proteins. While

the distribution of most PtdInsPs is restricted to a single organelle, PtdIns(4)P is

unique in that it exists in several discrete pools, and so proteins that bind

PtdIns(4)P must use extra receptors to achieve a restricted localization. Here we

compare the two highly related pleckstrin homology (PH) domains from Osh1p and

Osh2p, yeast homologues of oxysterol binding protein (OSBP), that target

membranes using PtdIns(4)P, and in vitro bind both PtdIns(4)P and PtdIns(4,5)P2.

We show that Golgi targeting is specified by an additional site on PHOsh1, that lies on

a face of the domain not previously known to interact with receptors. In contrast,

PHOsh2 does not have a demonstrable second site, and targets multiple pools of

PtdInsPs, each dependent on a different PtdIns 4-kinase. This lack of a second site

in PHOsh2 allows it to be used as an unbiased reporter for altered distribution of

4–phosphorylated PtdIns. For example, in cells with excess PtdIns(4)P caused by

inactivation of the phosphatase Sac1p, PHOsh2 indicates that PtdIns(4)P accumulates

on the plasma membrane, while other Golgi-targeted PH domains fail to detect this

change.


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INTRODUCTION

Membrane recruitment of peripheral membrane proteins is determined by one or

more targeting domains that bind other proteins or membrane lipids. An important

class of membrane lipid used to recruit proteins are the PtdInsPs, phosphates being

added at positions 3, 4 or 5 of the inositol sugar in any one of 7 combinations. Most

PtdInsPs have quite restricted distributions, and can be considered as intracellular

signposts or signals that recruit proteins to specific compartments (1). These

distributions are created by the targeting of lipid modifying enzymes. For example,

PtdIns(3)P is largely restricted to endosomes: it is synthesized by a PtdIns 3-kinase

lying on the trans-Golgi network to endosome pathway, and it is prevented from

reaching other compartments by degradation in the vacuole/lysosome (2).

PtdIns(4)P differs from other PtdInsPs as it performs multiple roles. Not only is it a

signpost for protein recruitment, but it is also an essential intermediate in the

production of PtdIns(4,5)P2. Further, PtdIns(4)P is synthesized by multiple,

separate PtdIns 4-kinases and is therefore likely to have multiple intracellular pools.

There are two families of PtdIns 4-kinases: types II and III (type I are now known

to be PtdIns 3-kinases (3)). In mammalian cells, the predominant PtdIns 4-kinases

are type II, but have only recently been cloned and are still partially characterized

(4-7). In addition, there are two mammalian type III kinases: α and β (8). Analysis

of the relative importance of the different kinases in membrane traffic has been

carried out in the model organism S. cerevisiae, which also has type II and III

kinases, but with a different emphasis, since here the type III kinases are dominant


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(9). The yeast type III α PtdIns 4-kinase homologue, Stt4p, is an essential protein

that has been localized to the plasma membrane (10), and has been shown to

synthesize the bulk of PtdIns(4)P for cellular requirements. This pool acts as a

precursor for PtdIns(4,5)P2 (11), which is perhaps exclusively on the plasma

membrane (12,13), and which is made by a single PtdIns(4)P 5-kinase, Mss4p, that

is active on the plasma membrane (14,15). However, there are important roles for

PtdIns(4)P beyond being an intermediate in PtdIns(4,5)P2 production, in particular

in budding from the late Golgi. This is the location of Pik1p, another essential PtdIns

4-kinase that is homologous to the human type III β kinase (16,17). A number of

peripheral Golgi proteins are now known to be recruited by PtdIns(4)P. Pleckstrin

homology (PH) domains from three families of lipid binding proteins target the Golgi

(12,13,18-21). In addition, clathrin adaptor AP1 complex was also shown to

interact with PtdIns(4)P on the Golgi (22). Yeast also harbor a third PtdIns 4-

kinase, Lsb6p, which is in the type II family, and has no known function (23).

The question posed by the presence of these different PtdIns 4-kinases is how

might proteins detect one specific pool of PtdIns(4)P among the multiple organelles

that have active PtdIns 4-kinases? One plausible explanation supported by previous

observations with the PH domain of OSBP is that proteins cannot target a specific

compartment by virtue of PtdIns(4)P alone, but achieve restricted targeting by

binding a membrane lipid in combination with other membrane receptors (12,24).

OSBP is a peripheral membrane protein that targets the late Golgi solely by virtue

of a PH domain that binds to PtdIns(4)P and PtdIns(4,5)P2 with equal affinity

(12,18). In addition, PHOSBP binds a Golgi-restricted receptor dependent on the


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small GTPase Arf1p (12), and has recently been shown to bind weakly to ARF1

(21).

Here, we focus on two PH domains from yeast homologues of OSBP: Osh1 and

Osh2. While PHOsh1 targets Golgi membranes, PHOsh2, a closely related domain (71%

identical and 91% homologous) targets membranes weakly (25). We have

identified the site within PHOsh1 that binds a second membrane receptor, and

developed a tool based on PHOsh2 that lacks the second site and so detects pools of

4-phosphorylated PtdIns in an unbiased manner. Using PHOsh2, we demonstrate the

physical separation of the two pools of PtdIns(4)P dependent on Stt4p and Pik1p,

on the plasma membrane and Golgi respectively. We then compare PHOsh2 to other

PH domains that bind PtdIns(4)P and PtdIns(4,5)P2 as a probe for excess

PtdIns(4)P in ∆sac1 cells.


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Experimental Procedures

Plasmids

PH domains were defined as follows (alternative gene names together with

accession numbers given in brackets, followed by starting and ending residues,

identified using single letter amino acid code): Osh1p (YAR042W/YAR044W,

AY241177) E280-R384; Osh2p (YDL019C, NP_010265) long form: 256P-424P;

short form: S286-K391; phospholipase C δ1 (PLCδ1), ceramide transfer protein

(CERT, previously called Goodpasture’s antigen binding protein – GPBP) and

phosphoinositol 4-phosphate adaptor protein 1 (FAPP1) as before (12). For

chimeras between Osh1p and Osh2p PH domains, 4 restriction sites that do not

alter the translated sequence were introduced at roughly equal spacing in both PH

domains, to produce 5 segments that shared the same boundary residues in both

two domains. Plasmids containing PH domains were based on either pRS406

(integrating at URA3) or pRS416 (CEN URA3) and contained the constitutive portion

of the PHO5 promoter, followed by GFP, a linker with the Myc epitope

(GSMEQKLISEEDLRS), then the PH domain and a carboxy-terminal extension of

XNS (where X is encoded by the last two nucleotides from the source DNA and a

guanine) as previously (12,24,25). Plasmids are listed in Table 1.

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Yeast strains For localization in wild-type yeast, plasmids were routinely

transformed into strain RS453B. Strains harboring the temperature-sensitive alleles

pik1-83, stt4-4 and mss4-2 together with their wild type controls and ∆vps34 were

as described previously (12). A ∆sac1 strain (TLY218) was derived from SEY6210

by replacing SAC1 with the heterologous marker gene S. pombe HIS5 using the

PCR method (26), and deletion of SAC1 was confirmed by PCR and inositol

auxotrophy.

Liposome centrifugation assays His6-tagged fluorescent PH domains were

expressed in bacteria and purified as previously (12). Small unilamellar liposomes

were prepared, incubated with GFP-PH domain and removed from suspension by

centrifugation as described previously (12). The GFP content remaining in the

supernatant was measured in a LS50 spectrophotometer (excitation 485 nm,

emission 515 nm, slit widths 5 nm). Phosphoinositides were assumed to be equally

distributed between the two leaflets of the liposome bilayer.

Cell imaging GFP-PH domain fusion proteins were visualized in yeast cells growing

at mid-log phase (OD 600 nm = 1) and in COS cells as previously (12,24). To label

yeast with FM4-64, the dye was added at final concentration of 30 µM 15 minutes

prior to imaging endosomal/late Golgi compartments.


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RESULTS

Identification of the second site in PHOsh1 for Golgi targeting. PHOsh1 targets

Golgi membranes in yeast (Fig. 1A) and mammalian fibroblasts (24,25), while

PHOsh2 shows barely discernible membrane targeting in yeast (Fig. 1B) and

fibroblasts (data not shown). This differs from a recent study showing stronger

Golgi targeting by monomeric PHOsh2, although in that case a basic patch adjacent

to the core PH domain sequence was also included, which may alter targeting (27).

The portion of primary sequence responsible for the difference in targeting between

PHOsh1 and PHOsh2 was mapped using a series of chimeras in which portions of the

Osh1p sequence were exchanged for the homologous Osh2p sequence. For this

purpose, the PH domain (105 amino acids in Osh1p) was divided into five

approximately equal segments, and chimeras made that contained mixtures of

Osh1p and Osh2p sequence (see Materials and Methods). Substitution of the N-

terminal three fifths of PHOsh1 with the equivalent region of PHOsh2 had no effect on

targeting (Fig. 1C), despite the almost universal location of the binding site in PH

domains for PtdInsPs within this N-terminal region (28,29). In contrast, inclusion of

the next (i.e. fourth) segment of PHOsh2 inhibited membrane localization (Fig. 1D).

The same requirement for the fourth segment of PHOsh1 was seen for Golgi targeting

in mammalian COS cell fibroblasts (data not show).

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Within the critical fourth segment of 22 residues, PHOsh1 differs from PHOsh2 at 8

positions. By further dissection of this segment, a single residue, histidine at

position 79 in PHOsh1 compared to arginine at position 80 in PHOsh2, was shown to be

critical for targeting (Table 2). To confirm the role of this single residue, arginine

was introduced at position 79 of PHOsh1, in which it strongly inhibited Golgi targeting

(Fig. 1E). Introduction of histidine at the same site in PHOsh2 induced a striking gain

of Golgi targeting (Fig. 1F).

Modeling of the sequence of PHOsh1 on known PH domain structures (28,29) shows

that this residue lies in the middle of the β7 strand. In all known structures, this

residue points away from the core of the domain on a side of the molecule at some

distance from the membrane interface, assuming that this is defined by the site

that binds PtdInsPs (Fig. 2). These results therefore indicate that the second site is

unlikely to interact with a membrane lipid receptor, but is more likely to be a

protein.

PHOsh2 binds both PtdIns(4)P and PtdIns(4,5)P2. If PHOsh2 is fully capable of

membrane targeting, but just lacks a second site, it should bind PtdInsPs similarly

to PHOsh1. To test this, we compared the binding of GFP-tagged PH domains to

liposomes containing 2.5% PtdIns(4)P or PtdIns(4,5)P2 (12) (Fig. 3). As controls,

PHPlcδ1 showed its well-documented absolute preference for PtdIns(4,5)P2 over

PtdIns(4)P, and PHFAPP1 bound both lipids, with a slight preference for PtdIns(4)P

(Fig. 3). PHOsh1 and PHOsh2 bound PtdIns(4,5)P2 with almost identical affinity, greater



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than that of PHFAPP1 (Fig. 3A); PHOsh1 bound PtdIns(4)P more strongly than PHOsh2

(approximately 2-fold more) (Fig. 3B). Overall, these experiments show that PHOsh2

binds PtdIns(4)P and PtdIns(4,5)P2 with affinity similar to other PH domains that

target membranes efficiently, despite its very weak membrane targeting in vivo.

A PHOsh2 dimer targets two pools of 4 phosphorylated PtdInsPs in growing

cells. The above data show that PHOsh2 binds 4-phosphorylated PtdIns, but it differs

from closely related PH domains by the absence of a second site. Therefore, we

reasoned that if a reporter construct could be made to target membranes

dependent purely on PHOsh2, it would reveal the overall distribution of PtdIns(4)P

and PtdIns(4,5)P2. To achieve membrane localization, we expressed a tandem

dimer of PHOsh2, as has been done for PHPLCδ1 and FYVE domains previously. The

presence of two membrane binding sites vastly increases avidity without altering

specificity (12,30). GFP-PHOsh2-dimer showed tight dual localisation to punctate

internal structures and the plasma membrane, with little cytoplasmic background

(Fig. 4A). GFP-PHOsh2-dimer expressed from either a relatively weak promoter

(OSH1), or a rapidly inducible promoter (GAL1) produced the same dual localisation

(data not shown), indicating that it is independent of duration and level of

expression. Although the distribution of GFP-PHOsh2-dimer became diffuse after

fixation (data not shown), partial co-localisation was found in live cells with punctae

stained by FM4-64, which traverses the endocytic pathway and also enters late

Golgi compartments prior to being sorted back to the cell surface (Fig. 4B) (31).

Therefore, we conclude that the punctate structures are late Golgi and/or early



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endosomes. In addition, the dual targeting of PHOsh2 implies either that it interacts

with two different ligands, or alternatively that a single ligand is present in two

sites. An indication that both localisations of GFP-PHOsh2-dimer are PtdInsP-

dependent was gained using a dimer in which two conserved positive residues of

both PH domain sequences contained mutations that are known to prevent PtdInsP

binding site of the PH domains (12), which had an entirely diffuse localisation (Fig.

4C).

To test whether Pik1p plays a role in the localisation of PHOsh2, as it does for PHOSBP

(12), GFP-PHOsh2-dimer was expressed in a strain carrying a conditional pik1 allele.

Shift to the non-permissive temperature caused a rapid and almost complete loss of

internal punctate localisation in the mutant cells, with only a slight effect seen in

wild-type cells undergoing the same temperature shift (Fig. 5A). By comparison,

PHOSBP is only incompletely delocalized under these conditions (12). Therefore,

PtdIns(4)P produced by Pik1p is likely to be the Golgi ligand for PHOsh2. Next, we

sought to identify the plasma membrane ligand for PHOsh2. Initially we looked at the

role of PtdIns(4,5)P2 by expressing GFP-PHOsh2-dimer in cells carrying the mss4-2ts

allele. Little effect on the plasma membrane localisation was detected at the non-

permissive temperature under conditions where PtdIns(4,5)P2 levels are known to

fall (Fig. 5B) (12). This result does not rule out a role for PtdIns(4,5)P2 at the

plasma membrane, but indicates that there must be an additional ligand present

when PtdIns(4,5)P2 production is blocked.



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We next tested whether the other essential PtdIns(4)-kinase, Stt4p, was

responsible for synthesising a pool of PtdInsP on the plasma membrane that is

recognised by PHOsh2. In temperature sensitive strains carrying the mutant allele

stt4-4ts, the localisation of PHOsh2-dimer was altered, with much reduced plasma

membrane localization even at the permissive temperature (data not shown).

Plasma membrane targeting was almost completely lost at the non-permissive

temperature, while punctate localisation was preserved (Fig. 5C, 37˚C), and these

effects were not seen in wild-type controls (Fig. 5C), indicating that Stt4p

synthesizes the 4-phosphorylated PtdIns that PHOsh2 binds on the plasma

membrane. We also examined the role of the single PtdIns 3-kinase in yeast,

VPS34, since it is possible that PHOsh2 binds either PtdIns(3)P or PtdIns(3,5)P2.

However, the localisation of GFP-PHOsh2-dimer was unaffected in ∆vps34 cells (data

not shown). The combination of this result and the complete loss of punctate

localization with pik1 mutation indicates that the punctate localization of PHOsh2 is

not mediated by PtdIns(3)P. Overall, these results show that each of the dual

localisations of this PH domain is sensitive to inactivation of a different essential

PtdIns 4-kinase, demonstrating that PHOsh2 interacts with two separate pools of 4-

phosphorylated PtdIns.

PHOsh2 as a reporter for altered PtdIns(4)P levels in ∆sac1 cells.

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The results above indicate that PHOsh2 is an unbiased reporter of PtdIns(4)P and

PtdIns(4,5)P2, so we next examined how its distribution was affected by a well-

characterised mutation that alters PtdInsP levels. Sac1p is one of five PtdInsP

phosphatases in yeast, and its inactivation leads to a large increase in cellular

PtdIns(4)P, up to ten-fold, together with a fall in PtdIns(4,5)P2 (16,32). To

determine the effect of these changes in 4-phospholrylated PtdIns, we expressed

tagged PH domains in a ∆sac1 strain. The GFP-PHOsh2-dimer showed much

increased plasma membrane targeting, and an apparent decrease in punctate

localization (Fig. 6A, compare with Fig. 4A). This change might be caused by either

a reduction in ligand concentration on Golgi/endosomes or an increase in ligand on

the plasma membrane. To distinguish between these possibilities, we expressed

monomeric GFP-PHOsh2 in the ∆sac1 strain, and found that a single copy of PHOsh2

now localized appreciably to the plasma membrane (Fig. 6B, compare with Fig. 1B).

This result indicates that the ∆sac1 mutation leads to an increase in PtdIns(4)P at

the plasma membrane, which is likely, from the results above, to be synthesized by

Stt4p. Therefore, our findings agree with previous results that the excess

PtdIns(4)P that accumulates in ∆sac1 cells is synthesized by Stt4p rather than by

Pik1p (32).

We next examined the effect of the ∆sac1 mutation on other PH domains that bind

PtdIns(4)P. GFP-PHOsh1 did not target the enlarged pool of PtdIns(4)P at the plasma

membrane, but remained largely punctate (Fig. 6C). This is further evidence that

PHOsh1 does not target membranes solely because they are rich in PtdIns(4)P. We



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next examined the effect of ∆sac1 on the distribution of other Golgi-targeted PH

domains, using sequences from OSBP, FAPP1 and ceramide transfer protein (CERT,

previously called Goodpasture’s antigen binding protein – GPBP). Like PHOsh1, these

target the Golgi by interacting with both PtsIns(4)P and a second site (12). The

distribution of GFP-PHOSBP and GFP-PHFAPP1 remained punctate, as seen for GFP-

PHOsh1 (Fig. 6D and E). However, GFP-PHCERT was notably different, being targeted

to the nuclear envelope and to peripheral patches, characteristic of the yeast ER

(Fig. 6F). To check whether this targeting was caused by binding to PtdInsPs, we

next expressed a mutated version of this construct, in which the critical basic

residue for PtdInsP binding was mutated to an arginine. GFP-PHCERT(R43E) was

entirely diffuse, with no ER or punctate localization (Fig. 6G). On close inspection,

all three of PHOsh1, PHOSBP and PHFAPP1showed much weaker targeting to the nuclear

envelope and periphery (Fig. 6). These results suggest that some PtdIns(4)P

accumulates on the ER in ∆sac1 cells, and that this pool is specifically targeted by

PHCERT, possibly because of a unique second site interaction.


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Discussion

PH domains are sequences usually 100–120 amino acids in length that have a

characteristic fold of a seven-stranded β sandwich closed off at one side by an

amphipathic alpha helix (28,29,33). PH domains have been found to interact with a

variety of different ligands, using sites that are somewhat conserved for each

purpose. The alpha helix can interact with WD40 proteins including Gβγ (34), and

differing parts of the alpha helix of PH domains in Guanine nucleotide exchange

factors bind the neighbouring catalytic Dbl homology domain (35). Finally, the best

characterized interaction of PH domains arises from the formation of a pocket by

loops β1-β2 and β3-β4 to bind PtdInsPs. Lipid specificity is determined by a

combination of basic residues lining the pocket and a generally positive electrostatic

charge on this face of the domain. While a few PH domains, in particular those that

bind PtdIns(3,4,5)P3, show high affinity binding for just a single PtdInsP, most PH

domains, including PHOSBP and related sequences, bind PtdInsPs relatively

promiscuously (28,36-38). This lack of specificity need not preclude specific overall

targeting if the domain also interacts with a second receptor (12).

We compared two highly related PH domains with different Golgi targeting. Both PH

domains bound PtdInsPs with moderate strength, similar to the recent findings of

Lemmon and co-workers (38). Indeed, out of the 33 PH domains in the entire yeast

genome, these two are among only seven that demonstrably bind PtdInsPs at all

(38). Given a similar binding affinity for PtdInsPs, what determines the difference in

targeting? This was mapped to the third residue of strand β7, which in PHOsh1 (tight


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binding) consists of RWHLKG, compared to RWRLKG in PHOsh2 (weak binding). The

histidine is conserved in Osh1p homologues in humans (OSBP, ORP4 and ORP9),

flies, worms, plants and in other fungi (C. albicans, A. nidulans but not S. pombe –

serine, or M. Grisea and N. crassa – threonine), whereas the arginine is unique to

Osh2p in Saccharomyces. Different PH domain structures all indicate that this

residue lies at the bottom of a shallow groove on a face of the domain adjacent to

but not overlapping with the PtdInsP binding site (Fig. 2) (28,29,37). The finding

that this histidine can specify Golgi localization identifies this side of PH domains as

a site in this sub-family of PH domains that binds a second ligand. Given the

location of the site away from the membrane, it would appear that the ligand is a

protein, rather than a membrane lipid. Following our initial studies where we found

that this second site depends on Arf1p in yeast (12), De Matteis and co-workers

have now demonstrated a direct interaction between both PHFAPP1 and PHOSBP with

ARF1 in mammalian cells (the equivalent of yeast Arf1p) (21). On this basis, we

therefore predict that ARF1/Arf1p interacts with histidine 79 in PHOsh1 and PHOSBP

(12).

Although Golgi targeting can be achieved by combining two binding interactions,

without a second site monomeric PHOsh2 does not target the Golgi. This reduced

affinity for the Golgi may be crucial to targeting of full-length Osh2p, which has

diverged in multiple ways from Osh1p (25). Because PHOsh2 interacts only with

PtdInsPs, we have been able to develop a tool based on dimeric PHOsh2 that detects

PtdIns(4)P and PtdIns(4,5)P2 alone (Fig. 3). Binding to other PtdInsPs was shown to

be irrelevant by the lack of a role for Vps34p. The dual localization of GFP-PHOsh2-


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dimer in log-phase cells was consistent with previous studies of the two essential

PtdIns 4-kinases (9), and their localizations (10,17). While previous studies have

shown that Pik1p produces PtdIns(4)P at the Golgi (12,13), this is the first

demonstration that Stt4p synthesizes PtdIns(4)P on the plasma membrane. Since

the Stt4-dependent pools of PtdIns(4)P and PtdIns(4,5)P2 are of roughly equivalent

size (11), it is difficult to determine which lipid is being detected by the GFP-PHOsh2-

dimer, even using cells in which Mss4p can be inactivated.

If GFP-PHOsh2-dimer is indeed a probe for 4-phosphorylated PtdIns, its distribution

should change in response to changes in PtdInsP metabolism. We used cells in

which the SAC1 gene was disrupted, since in the absence of this PtdInsP

phosphatase cells accumulate up to ten times the normal levels of PtdIns(4)P, while

PtdIns(4,5)P2 decreases by 50% (16,32). Results with PHOsh2 in these cells showed

that excess PtdIns(4)P accumulated mainly on the plasma membrane. However,

other PH domains that bind PtdIns(4)P, such as those from Osh1p, OSBP and FAPP1

remained largely localized to the Golgi in ∆sac1 cells, similar to the recent findings

of Lemmon and co-workers (38). This is further evidence that PH domains that

target the Golgi as monomers localize using a second receptor in addition to

PtdInsP. Interestingly, PHCERT behaved differently in cells carrying ∆sac1 compared

to the other sequences tested in targeting the ER in a PtdInsP-dependent manner.

This suggests that PHCERT recognizes a second Golgi receptor that is relocated to the

ER in a ∆sac1 strain. Since this receptor differs from that recognized by PHOsh1p,

PHOSBP and PHFAPP1, it may be that PHCERT does not bind ARF1. ER relocalization of a

Golgi protein in ∆sac1 cells has also been reported for the short yeast OSBP


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homologue Kes1p, which also binds PtdIns(4)P and PtdIns(4,5)P2 (39). CERT was

recently shown to transfer ceramide in mammalian cells from its site of synthesis

(the ER) to the site of its conversion to complex sphingolipid (late Golgi) (19), a

process which also occurs in yeast (40). Identical targeting by PHCERT and Kes1p

suggests the possibility that they both access a sub-domain of the late Golgi that is

specialized for non-vesicular ceramide import.

It is clear from all studies of PH domains targeted to Golgi membranes that they

bind PtdIns(4)P. However, these PH domains can also show fairly indiscriminate

binding to a broad range of PtdInsPs (12,24,36,38), i.e. there is no intrinsic

specificity for PtdIns(4)P. This is sometimes over-looked (41) because different

techniques for measuring protein-lipid interaction yield differing results. By binding

protein to lipids immobilized on nitrocellulose (so-called “Fat Western”) originally

showed that PHFAPP1 specifically binds PtdIns(4)P (20), however this specificity was

not seen with an amended technique (38). Although it is not yet known if other

proteins that target PtdIns(4)P on the Golgi are equally promiscuous (22), it is

tempting to speculate why such lack of specificity exists for PtdIns(4)P binding

proteins. PtdIns(4)P exists in multiple discrete pools, not only in yeast, but also in

mammalian cells where multiple organelles can recruit PtdIns 4-kinases (4-

8,22,42). Thus, proteins that target PtdIns(4)P can only achieve a narrow

distribution among the multiple pools of PtdIns(4)P by combining this interaction

with others. Therefore, there may be very little evolutionary disadvantage to

binding the combination of PtdIns(4)P and PtdIns(4,5)P2, and it is possible that a

truly PtdIns(4)P-specific binding protein may not exist.


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In summary, PHOsh2 appears to detect two independent pools of 4-phosphorylated

PtdIns without the bias introduced by a second site that is otherwise found in PH

domains that bind PtdIns(4)P. The potential use of the probe is complicated by its

interaction with another lipid, PtdIns(4,5)P2. Now that the micro-heterogeneity of

all intracellular organelles has become evident (43-45), it will become increasingly

important to study how multiple targeting signals integrate to specify a protein’s

overall distribution.

Acknowledgements We would like to acknowledge the support of Sean Munro, in

whose lab this work was initiated, and who has given much useful advice. We would

also like to thank Scott Emr for providing yeast strains and communicating results

prior to publication, and Anjon Audhya, Olga Perisic, Howard Riezman, Steve Moss

and Chris Loewen for helpful discussions. Funded by the Wellcome Trust (Grant no.

060537) and Fight For Sight.

Abbreviations:

CERT – ceramide transfer protein, GPBP - Goodpasture’s antigen binding protein,

NLS - nuclear localization signal, OSBP - oxysterol binding protein, PH – pleckstrin

homology, PtdIns – phosphatidylinositol, PtdInsP - phosphatidylinositol phosphate

(PtdInsP), FAPP1 - phosphoinositol 4-phosphate adaptor protein 1, PLCδ1 -

phospholipase C δ1.


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Figure Legends:

Figure 1. Identification of second site within PHOsh1 that determines Golgi targeting.

PHOsh1, PHOsh2 and the indicated chimeras of these two homologous sequences were

expressed as GFP fusion proteins as indicated in RS453, a wild-type yeast strain,

and images taken of cells growing in log-phase. The different PH domains were: A.

PHOsh1 (105 residues); B. PHOsh2 (106 residues); C. residues 1-61 of PHOsh2 followed

by residues 61-105 of PHOsh1; D. PHOsh1 with residues 61-82 replaced with 62-83 of

PHOsh2. The region of 61-82 near the C-terminus of PHOsh1 was critical for Golgi

targeting. Fine dissection of this region then indicated that a single residue is

critical (see Table 2). E. PHOsh1 with mutation H79R; F. PHOsh2 with mutation R80H. ,

Substitution of arginine into PHOsh1 inhibited targeting, whereas histidine 79 brought

about Golgi targeting when inserted into PHOsh2. Plasmids used in A-F were pTL331,

pTL502, pTL503, pTL504, pTL508 and pTL509 respectively.

Fig. 2. Separation of the site that determines Golgi localization from the site that

binds PtdIns.

Ribbon diagram of the structure of PH domain from the kinase Akt (PHAkt) together

with Ins(1,3,4,5)-tetrakisphosphate ligand, the soluble headgroup of PtdIns(4,5)P2

(red and grey) drawn by CHIMERA software using known co-ordinates (46).

Features indicated are: loop β1-β2 (1), and loop β3-β4 (2) that encompass the

binding site of the ligand, leading to the predicted orientation of the domain with

respect to membrane lipids, which have been drawn to approximate scale (PH


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domain diameter = approx 4 nm, Golgi membrane thickness = 7.3 nm (47),

phospholipid diameter 0.7 nm) ; and T77 of PHAkt (blue, arrowed), the residue

equivalent to H79 of PHOsh1 and R80 of PHOsh2, positioned on a side of the domain at

one side away from the known ligand binding site. Out of 14 solved PH domain

structures, the predicted secondary structure of the core β sheets and α helix in

PHOsh1 is most similar to PHAkt. The extra helix (asterisk) in loop β3-β4 of PHAkt is a

unique feature absent from PHOsh1/2 that is unlikely to affect the core structure.

Fig. 3. Binding of PHOsh2 to PtdIns(4)P and PtdIns(4,5)P2 in vitro.

GFP-tagged PH domains from PLCδ1, FAPP1, Osh1p and Osh2p (final concentration

0.5–0.8 µM) were incubated with increasing amounts of small unilamellar liposomes

made up from DOPC containing A. 2.5% PtdIns(4,5)P2 B. 2.5% PtdIns(4)P, or C.

neither (DOPC alone). Binding was detected as the decrease in the proportion of

GFP fluorescence remaining in the supernatant after centrifugation to remove

liposomes and bound GFP-PH domain. PHPlcδ1 specifically bound PtdIns(4,5)P2, and

the other PH domains bound both this and PtdIns(4)P, PHOsh2 alone showing a slight

preference for PtdIns(4,5)P2. None of the PH domains interacted with DOPC alone.

Fig. 4. Dual localisation of PHOsh2 dimer.

A. PHOsh2 was expressed as a tandemly repeated dimer of PHOsh2 extended

approximately 30 amino acids at each end (total of 169 amino acids) bracketed by

GFP (using pTL511). The dimer showed a dual localisation: at the plasma

membrane particularly enriched in small buds; and in numerous punctate structures


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often seen at the bud-neck in large budded cells, which is the site of secretion

(arrows). The same dual localization was seen with another dimeric PHOsh2 construct

with only a single GFP, and much narrower definition of PHOsh2 (106 residues,

pTL512, data not shown).

B. Cells expressing GFP-PHOsh2-dimer as in A, co-stained with the endocytic tracer

FM4-64 (15 mins uptake), which has entered late Golgi compartments. In the

bottom panel, each image has been falsely coloured: GFP in green, FM4-64 in

magenta, with co-localization indicated in white. The very brightest punctae all

showed good co-localization, as do a proportion of the less bright punctae

(arrowhead). Using these settings, cells containing single markers showed complete

separation of the fluorophores (data not shown).

C. GFP-PHOsh2 dimer as in A, but both PH domains of the dimer carry mutations in

two basic residues implicated in PtdInsP binding (K307E R309E, pTL513). The

construct was entirely cytoplasmic, indicating loss of both of the localisations of

wild-type PHOsh2.

Fig. 5. Different PtdIns 4-kinases synthesize the two pools of PtdIns(4)P.

All images are of the GFP-PHOsh2-dimer expressed (from pTL511) in the indicated

mutant strains and the corresponding wild-type parental strains, grown to mid-log

phase at the permissive temperature (25˚C) and shifted to a strongly non-


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permissive temperature (39˚C) for 15 minutes prior to imaging. A. Inactivation of

Pik1p completely inhibited the punctate localization of PHOsh2. B. Targeting to both

sites was unaffected by inactivation of Mss4p, indicating no requirement for

PtdIns(4,5)P2 for localization. C. Plasma membrane localisation was lost selectively

on inactivation of Stt4p, but not in the wild-type control. Plasma membrane

targeting was also reduced in the stt4-4 mutant strain at a temperature permissive

for growth (25˚C, data not shown).

Fig. 6. PHOsh2 and other Golgi-targeted PH domains reveal an altered distribution of

PtdIns(4)P in cells lacking Sac1p.

Images of GFP-tagged PH domains expressed in a ∆sac1 strain, grown to mid-log

phase at 30˚C. A. GFP-PHOsh2-dimer, B. GFP-PHOsh2-monomer, C. GFP-PHOsh1, D.

GFP-PHOSBP, E. GFP-PHFAPP, F. GFP-PHCERT and G. GFP-PHCERT* carrying the mutation

R43E . The constructs used are indicated diagrammatically above each image (2–2,

2, 1, O, F, C, C*), and were expressed from pTL511, pTL342, pTL331, pTL332,

pTL334, pTL333 and pTL514 respectively. Deletion of ∆sac1 shifted dimeric PHOsh2

from punctate to plasma membrane targeting. Monomeric PHOsh2 showed enhanced

targeting to the plasma membrane (compare with Fig 1A). In comparison, PHOsh1,

PHOSBP and PHFAPP1 targeted punctae predominantly in ∆sac1 cells. In addition, these

sequences showed minor targeting to the nuclear envelope (arrows) and cell

periphery (arrowheads). PHCERT was unique in targeting the ER (nuclear envelope

and peripheral patches). This was dependent on binding to PtdInsPs, as is was not

abolished by mutating the critical basic residue in PHCERT (R43) to an acid (E).


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Table 1. Plasmids used.

pRS406 Integrates at URA3 ref (48)

pRS416 CEN URA3 (low copy) ref (48)

pTL331 pRS406 PHO5 prom., GFP, myc, PHOsh1 (105 aa = 280–122 aa) ref (25)

pTL332 pRS406 PHO5 prom., GFP, myc, PHOSBP (99 aa = 87-185 aa) ref (12)

pTL333 pRS406 PHO5 prom., GFP, myc, PHCERT (=GPBP) (122 aa = 1-122 aa) ref (12)

pTL334 pRS406 PHO5 prom., GFP, myc, PHFAPP1 (98 aa = 1-98 aa) ref (12)

pTL342 pRS416 PHO5 prom., GFP, PHOsh2 (256–424 aa), GFP ref (25)

pTL502 pRS406 PHO5 prom., GFP, myc, PHOsh2 (106 aa = 286-391 aa)

391K391

this study

pTL503 pRS406 PHO5 prom., GFP, myc, PHOsh2 (aa 1-61) – PHOsh1 (aa 61-105) this study

pTL504 pRS406 PHO5 prom., GFP, myc, PHOsh1 (aa 61-82 62-83 of PHOsh2) this study




pTL508 pRS406 PHO5 prom., GFP, myc, PHOsh1 as pTL331 (with mutation H79R) this study

pTL509 pRS406 PHO5 prom., GFP, myc, PHOsh2 as pTL502 (with mutation R80H) this study

pTL510 pTrcHisA His6, GFP, myc, PHOsh2 (106 aa)

391K391

this study

pTL511 pRS416 PHO5 prom., GFP, PHOsh2 (169 aa = 256-424 aa) – VNSKL linker –

PHOsh2 repeat - GFP

391K391

this study

pTL512 pRS406 PHO5 prom., GFP, PHOsh2

(106 aa) – KGAGAGAGS linker – PHOsh2 repeat

this study

pTL513 pTL511 carrying mutations K307E R309E in both copies of PHOsh2 this study

pTL514 pTL333 carrying mutation R43E in PHCERT this study


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Table 2. Analysis of the section of PHOsh1 that determines Golgi targeting.

GFP-tagged PHOsh1 constructs which varied only in the sequences in the fourth out of

five segments of PHOsh1 (residues 61-82) were scored for their punctate localization.

Introduction of the Osh2p sequence (construct 1) made 8 changes and rendered

membrane targeting only weakly detectable (w). While the N-terminal 3 variant

residues had little effect on targeting (construct 2), the C-terminal 5 variant

residues were responsible for the majority of targeting (construct 3). Introduction

of the first 4 of these 5 changes had no effect on targeting (construct 4), indicating

an important role for the last variant residue: H79 in PHOsh1 (R80 in PHOsh2).

construct plasmid Sequence from residues 61-82

in PHOsh1

# changes

cf Osh1p

punctae

Osh1p pTL331 SEKLKFEIIGGNNGVIRWHLKG 0 ++

1 pTL504 SEKLNFELLGGITGTTRWRLKG 8 w

2 pTL505 SEKLNFELLGGNNGVIRWHLKG 3 +

3 pTL506 SEKLKFEIIGGITGTTRWRLKG 5 w

4 pTL507 SEKLKFEIIGGITGTTRWHLKG 4 ++


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A B

C

E

D

FR80HH79R

Roy and Levine Figure 1

G 2G 1

G 1 G 2

G G


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*1 2


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7035

B

0

C100

60

200

80

40

70350

PtdInsP added (µM)


0 8040

A100

60

200

80

40

% u

nb

ou

nd

GF

P-P

H

PtdIns(4,5)P2 PtdIns(4)P

DOPC only

100

60

200

80

40


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A BG 2 G2 GFP

FM4-64

mergeG G

C2* *2


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A

B

C

pik1-83wild-type

stt4-4wild-type

mss4-2wild-type

G 2 G2


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F


G 2 G2 G 2 G

all in ∆sac1 cells

G OG 1

G CG F

G *C

A B

C

E

G

D


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Anjana Roy and Timothy P. Levineof Osh2p

Multiple pools of PtdIns 4-phosphate detected using the pleckstrin homology domain

published online July 22, 2004J. Biol. Chem.

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Multiple pools of PtdIns 4-phosphate detected using the pleckstrin