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Originally published In Press as doi:10.1074/jbc.M405589200 on July 20, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39604-39610, September 17, 2004
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Negative Regulation of Phosphatidylinositol 4,5-Bisphosphate Levels by the INP51-associated Proteins TAX4 and IRS4*

Helena Morales-Johansson{ddagger}, Paul Jenoe{ddagger}, Frank T. Cooke§, and Michael N. Hall{ddagger}

From the {ddagger}Division of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland and the §Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom

Received for publication, May 19, 2004 , and in revised form, July 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an important second messenger in signaling pathways in organisms ranging from yeast to mammals, but the regulation of PI(4,5)P2 levels remains unclear. Here we present evidence that PI(4,5)P2 levels in Saccharomyces cerevisiae are down-regulated by the homologous and functionally redundant proteins TAX4 and IRS4. The EPS15 homology domain-containing proteins TAX4 and IRS4 bind and activate the PI(4,5)P 5-phosphatase INP51 via an Asn-Pro-Phe motif in INP51. Furthermore, the INP51-TAX4/IRS4 complex negatively regulates the cell integrity pathway. Thus, TAX4 and IRS4 are novel regulators of PI(4,5)P2 and PI(4,5)P2-dependent signaling. The interaction between TAX4/IRS4 and INP51 is analogous to the association of EPS15 with the 5-phosphatase synaptojanin 1 in mammalian cells, suggesting that EPS15 is an activator of synaptojanin 1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphoinositides are conserved from yeast to mammals as second messengers. They mediate the signal transduction involved in many different cellular processes and thereby comprise a complex signaling system (1). One of the more thoroughly studied phosphoinositides is phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2).1 PI(4,5)P2 was originally shown to be cleaved by phospholipase C to generate the two second messengers inositol-1,4,5-phosphate and diacylglycerol (2). More recently, uncleaved PI(4,5)P2 has also been shown to act as a second messenger (3). Uncleaved PI(4,5)P2 acts by binding conserved domains in target proteins, such as the pleckstrin homology domain (4–6). In mammalian cells, PI(4,5)P2 signaling is important in regulating vesicular transport, the organization of the actin cytoskeleton, and the regulation of ion channels (7–9). In the model organism Saccharomyces cerevisiae, PI(4,5)P2 is also essential and is produced by MSS4, the sole phosphatidylinositol 4-phosphate 5-kinase in yeast (10, 11).

One target of PI(4,5)P2 signaling in S. cerevisiae is the GDP/GTP exchange factor ROM2. ROM2 contains a pleckstrin homology domain that binds PI(4,5)P2 in the plasma membrane, thus mediating the localization of ROM2 (12, 13). ROM2 has two homologs, ROM1 and TUS1, which also harbor putative pleckstrin homology domains (12, 14). ROM2 and its homologs are components of the cell integrity pathway necessary for the cellular response to cell wall damage induced by stress such as heat shock (14, 15). The response to cell wall damage includes a reorganization of the actin cytoskeleton and an up-regulation of cell wall synthesis (16–18). Activation of the cell integrity pathway is mediated by its most upstream component, the cell wall sensor WSC1, which signals to ROM2 (19, 20). Subsequently, ROM2 stimulates the exchange of GDP to GTP in the Rho GTPase RHO1, thereby activating RHO1 (12). The GTPase-activating protein (GAP) SAC7 converts RHO1-GTP into the inactive GDP-bound form (21). RHO1 in its active (GTP-bound) form has several effectors such as the glucan synthase FKS1 and the PKC1-MAP kinase cascade (22–25). Activation of the MAP kinase MPK1 induces transcription of genes involved in cell wall biosynthesis such as CHS3 encoding chitin synthase III, which is important for cell wall repair (16, 18, 26).

The ROM2-MAP kinase-signaling pathway is also an effector branch of the TOR signaling network. The TOR signaling network contains two structurally and functionally distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2) (27). TORC2 regulates the polarization of the actin cytoskeleton, and this regulation is via the ROM2-MAP kinase pathway (21, 27–29). Interestingly, the sole phosphatidylinositol 4-phosphate 5-kinase MSS4 is required for the polarization of the actin cytoskeleton, and overexpression of MSS4 restores growth in conditional TORC2 mutants (10, 11, 27, 30). However, the link between PI(4,5)P2 and TORC2 in the regulation of ROM2 and, ultimately, the actin cytoskeleton is not understood.

The phosphoinositide 5-phosphatases INP51, INP52, and INP53 (also known as SJL1–3) mediate the turnover of PI(4,5)P2 and are implicated in several cellular processes such as cell wall biosynthesis and the organization of the actin cytoskeleton (31, 32). Heat shock has been observed to induce an increase in PI(4,5)P2 levels, suggesting the existence of a mechanism regulating the levels of this phosphoinositide (10). However, the nature of this regulatory mechanism and, more specifically, the possible regulation of the phosphoinositide 5-phosphatases are not well understood. In mammalian cells, the INP family orthologue synaptojanin 1 interacts with several proteins involved in endocytosis in nerve terminals (33–35). To investigate the regulation of PI(4,5)P2 turnover in S. cerevisiae, we focused on INP51, INP52, and INP53 and their role in PI(4,5)P2 signaling linked to the cell integrity pathway and the TORC2 pathway.

Here we present evidence for two novel positive regulators of the PI(4,5)P2 5-phosphatase INP51, the redundant EPS15 homology (EH) domain-containing proteins TAX4 and IRS4. INP51 associates with TAX4 or IRS4 to form a complex important for the turnover of PI(4,5)P2 linked to the cell integrity pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Media—The S. cerevisiae strains used in this study are listed in Table I. All strains were isogenic derivatives of TB50. Rich medium (yeast extract, peptone, and dextrose (YPD)) and minimal medium (synthetic dextrose (SD)) were as described previously (36). PCR cassettes were used to generate gene deletions and for tagging with HA, Myc, or the tandem affinity purification tag, as described (37–39). Yeast transformation was performed by the lithium acetate procedure (40). Correct integration was verified by PCR, and tagged proteins were tested for their functionality.


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  		TABLE I
Strains used in this study

 
Spot Assay—Logarithmically growing cells were harvested and resuspended in 10 mM Tris-HCl (pH 7.4). The resuspended cells were diluted in a 10-fold dilution series. 3 µl of each dilution (10x, 100x, 1,000x and 10,000x diluted) were spotted on a YPD plate. Growth was scored after 2 days at 30 or 37 °C.

INP51 Purification—Cells expressing a tagged or untagged (mock purification) version of INP51 were grown in 5 liters of YPD to an A600 of 0.8 at 30 °C, harvested by centrifugation, and washed with cold water before resuspension in lysis buffer. The lysis buffer used to prepare cell extracts contained phosphate-buffered saline, 5% glycerol. 0.5% Tween 20, phosphatase inhibitors (10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM sodium pyrophosphate, 10 mM {beta}-glycerophosphate, and 1 mM phenylmethylsulfonyl fluoride), and protease inhibitor mixture tablets (Roche Applied Science). Cell lysate was obtained by glass bead lysis. Cell lysates containing ~700 mg of protein were cleared with a 5-min, 500 x g spin, diluted with lysis buffer to 10 mg/ml, and subsequently passed over an ion exchange resin (SP-Sepharose). The resin was washed twice with lysis buffer and twice with lysis buffer containing 50 mM potassium acetate. Bound proteins were eluted with lysis buffer containing 600 mM potassium acetate. The eluate was precleared over a protein A-Sepharose (Amersham Biosciences) column prior to the addition of anti-HA (12CA5) cross-linked to protein ASepharose beads. After 5 times washing with lysis buffer, immunoprecipitated proteins were visualized by silver staining (41). Analysis of protein bands by mass spectrometry was performed as described (27).

Immunoprecipitation—Yeast extracts from cells (500-ml culture in YPD grown to A600 of 0.8 at 30 °C) expressing tagged proteins of interest were prepared as described above. An aliquot of extract containing 10 mg of protein was adjusted to 1 ml with lysis buffer plus inhibitors. For immunoprecipitations, 20 µl of anti-HA (12CA5) or anti-Myc (9E10) cross-linked to protein A-Sepharose beads were added and mixed for 4 h at 4 °C. Beads were collected by centrifugation, washed five times with 1 ml of lysis buffer, and resuspended in 5x SDS-polyacrylamide gele sample buffer for electrophoresis. After SDS-PAGE, the proteins were blotted onto nitrocellulose membranes, blocked in 5% dry milk powder in 1x phosphate-buffered saline and 0.1% Nonidet P-40, and incubated with primary antibody anti-HA (clone 12CA5) or anti-Myc (clone 9E10) (1:10000 in blocking solution). Subsequently tagged proteins were detected using horseradish peroxidase-conjugated goat anti-mouse secondary antibodies and ECL reagents (Amersham Biosciences).

Phosphoinositide Analysis—Yeast cells were diluted 5 x 104/ml in SD medium lacking inositol containing 10 µC/ml [3H]inositol and grown to a density of 2–4 x 106/ml (12–16 h). 2-ml aliquots were taken and mixed with 2 ml of MeOH. Cells were pelleted by centrifugation and processed as described previously (42).

Differential Centrifugation—Differential centrifugation was performed as described previously (43).

Indirect Immunofluorescence—Logarithmically growing cells containing INP51-HA, TAX4-Myc, or IRS4-Myc were fixed for 2 h in the growth medium supplemented with formaldehyde (3.7% final) and potassium phosphate buffer (100 mM final, pH 6.5). Cells were washed and resuspended in sorbitol buffer (1.2 M sorbitol and 100 mM potassium phosphate, pH 6.5). Cell walls were digested for 45 min at 37 °C in sorbitol buffer supplemented with {beta}-mercaptoethanol (20 mM final) and zymolyase 20T (12.5 mg/ml; Seigagaku Corporation). Spheroblasts were fixed on poly-L-lysine-coated glass slides and permeabilized with PBT (53 mM Na2HPO4, 13 mM NaH2PO4, 75 mM NaCl, 1% bovine serum albumin, and 0.1% Triton X-100). Immunofluorescence directed against the HA epitope was performed by application of a primary antibody anti-HA (clone 12CA5) or anti-Myc (clone 9E10) at a dilution of 1:1,000 in PBT for 2 h and, subsequently, the application of a Cy3-conjugated rabbit anti-mouse IgG (Molecular Probes) diluted 1:1,000 in PBT for 90 min. Washed cells were examined with a Zeiss Axiophot microscope (100x objective). Immunofluorescent detection of FKS1 was performed as described previously (17).

MAP Kinase Activation Assay—YPD cultures of logarithmically growing cells at 24 or 39 °C were harvested, and cell extracts were prepared as described previously (44). Protein concentrations of extracts were determined by using a Bradford assay (Bio-Rad). Samples were denatured by the addition of 5x SDS-PAGE sample loading buffer and heating at 95 °C for 5 min. A total of 25 µg of protein (for MPK1 protein detection) or 40 µg of protein (for phosphorylated MAP kinase detection) was loaded for standard SDS-PAGE (10% acrylamide) and Western blot. For immunodetection, a goat anti-MPK1 antibody (clone yN-19, 1:1000 dilution; Santa Cruz Biotechnology) and a rabbit antiphospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (1:1000; Cell Signaling) were used. The anti-MPK1 and anti-phospho-MAP kinase were verified to specifically recognize activated MPK1 under heat stress conditions (data not shown). Secondary antibodies were horseradish peroxidase-conjugated anti-goat (anti-MPK1) or anti-rabbit (anti-phospho-MAP kinase) secondary antibody and detection by ECL reagents (Amersham Biosciences). The nitrocellulose membrane used for the Western blot was stained with Coomassie Blue for the visualization of total protein.

Actin Staining—Logarithmically growing cells were fixed in formaldehyde (3.7%) and potassium phosphate buffer (100 mM, pH 6.5) and stained with TRITC-phalloidin (Sigma) to visualize actin, as described previously (45).

Chitin Staining—1 ml of cells at an A600 of 0.5 were collected by centrifugation and washed with water. Cells were then incubated in a solution with 0.5 mg/ml calcofluor white (Sigma) for 5 min to visualize chitin. To remove residual calcofluor white, cells were washed twice with water. Cells were examined with a Zeiss Axiophot microscope (100x objective).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Disruption of INP51 Restores Growth of a tor2 Mutant—To understand further how the turnover of PI(4,5)P2 is important for PI(4,5)P2 signaling, we tested whether the phosphoinositide 5-phosphatase INP51, INP52, or INP53 antagonizes MSS4 signaling. We asked if the lack of any INP gene mimics MSS4 overexpression in suppressing a TOR2 signaling defect. A deletion of each INP gene was introduced into a temperaturesensitive tor2 (tor2ts) mutant defective in the organization of the actin cytoskeleton. The double mutants were grown at permissive (30 °C) and restrictive (37 °C) temperatures to determine whether an INP mutation can suppress the tor2ts mutation. Deletion of only INP51, INP52, or INP53 conferred no growth defect at any temperature (data not shown). Interestingly, the deletion of INP51, but not the deletion of INP52 or INP53, suppressed the growth defect of tor2ts cells (data not shown). The inp51 tor2ts cells grew almost as well as wild-type cells and as well as tor2ts cells overexpressing MSS4 (Fig. 1). This result suggests that INP51, but not INP52 or INP53, antagonizes the role of MSS4 in the TORC2 signaling pathway. Curiously, although inp51 suppressed the growth defect of tor2ts, it did not appear to suppress the actin defect of the tor2ts mutant (data not shown).



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  		FIG. 1.
Disruption of INP51 restores growth of a tor2 temperature-sensitive mutant (tor2ts). Wild-type (TB50a), inp51, tor2ts, and tor2ts inp51 cells and tor2ts cells transformed with plasmid overexpressing MSS4 were spotted on to YPD medium and incubated at 30 or 37 °C. tor2ts cells are not viable at 37 °C.

 
INP51 Synthetically Interacts with Mutations Affecting the Cell Integrity Pathway—The finding that an inp51 deletion suppresses the growth defect of a tor2ts mutant is similar to the previous observation that activation of the cell integrity pathway suppresses the growth defect of a tor2ts mutant (15) and suggests that INP51 may antagonize the cell integrity pathway. Thus, we investigated whether an inp51 mutation interacts with mutations affecting components of the cell integrity pathway. Specifically, we asked if an inp51 deletion suppresses or enhances the effect of mutations in WSC1, ROM2, SAC7, or MPK1. We observed that the deletion of INP51 suppresses the growth defects of wsc1 and rom2 cells at 37 and 30 °C, the restrictive temperatures of these mutants, respectively (Fig. 2, A and B). Conversely, the combination of inp51 with sac7 conferred a synthetic growth defect (Fig. 2C). Finally, the growth defect of an mpk1 mutant grown at restrictive temperature (38 °C) was not suppressed by INP51 deletion (data not shown). The above results show that INP51 genetically interacts with at least some components of the cell integrity pathway. Deletion of INP51 suppresses mutations in positive components of the pathway (WSC1 and ROM2) and causes a synthetic defect when combined with a mutation in a negative element of the pathway (SAC7). The nature of these interactions suggests that the phosphoinositide phosphatase INP51 negatively regulates signaling through the cell integrity pathway. This conclusion is consistent with the previous conclusions (13, 27, 30, 46) that the phosphoinositide kinase MSS4 and PI(4,5)P2 act positively on the TORC2 and cell integrity pathways.



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  		FIG. 2.
INP51 synthetically interacts with mutations affecting the cell integrity pathway. A, wild-type, inp51, wsc1, and wsc1 inp51 cells were spotted on to YPD medium and incubated at 30 or 37 °C. wsc1 has a growth defect at 37 °C. B, wild-type, inp51, rom2, and rom2 inp51 cells were spotted onto YPD medium and incubated at 30 °C. The rom2 mutant has a growth defect at 30 °C. C, wild-type, inp51, sac7, and sac7 inp51 cells were spotted onto YPD medium and incubated at 30 °C.

 
INP51 Associates with the EH Domain-containing Proteins TAX4 and IRS4 —In mammalian cells, the kinases and phosphatases that determine PI(4,5)P2 levels are regulated by interacting proteins (33, 47–51). An indication that the level of PI(4,5)P2 is regulated in S. cerevisiae has been suggested previously by the observation that PI(4,5)P2 levels increase upon heat shock (10). However, proteins regulating such kinases and phosphatases in S. cerevisiae remain to be identified. To identify potential INP51 regulatory proteins, we opted to isolate INP51-interacting proteins by using a biochemical approach described previously (27). A functional, epitope-tagged version of INP51 (INP51-HA) was constructed and purified from yeast cell extracts as described under "Experimental Procedures." Two homologous proteins were co-purified with INP51, IRS4, and an uncharacterized protein encoded by the open reading frameYJL083w that we named TAX4 (Fig. 3). The interaction between INP51 and TAX4 or IRS4 was confirmed by coimmunoprecipitation using epitope-tagged versions of TAX4 (TAX4Myc) and IRS4 (IRS4-Myc), as described under "Experimental Procedures" (Fig. 4). We did not observe interaction between TAX4 and IRS4 by coimmunoprecipitation of the heterologously tagged versions (TAX4-TAP and IRS4-Myc) of these proteins (data not shown). IRS4 was identified previously in a screen for mutants defective in rDNA silencing (52). Untagged TAX4 has an apparent molecular mass of 77 kDa and a predicted size of 68.7 kDa. Untagged IRS4 is observed as two bands between 70 and 75 kDa, slightly larger than the predicted size of 68.8 kDa. TAX4 and IRS4 have an overall identity of 31% and contain a C-terminal EH domain. The EH domains of TAX4 and IRS4 are 64% identical. The EH domain, conserved from yeast to human, is a protein-protein interaction domain of ~100 amino acids that interacts specifically with short motifs containing an asparagine-proline-phenylalanine (NPF) core (53, 54). INP51 has a C-terminal NPF motif (55), amino acids 932–934, which is important for the interaction between INP51 and TAX4 or IRS4. The absence of the NPF motif and amino acids C-terminal to NPF in INP51 (i.e. the deletion of amino acids 932–946) abolishes the interaction between INP51 and TAX4 or IRS4, as assayed by coimmunoprecipitation (Fig. 4). Our results show that INP51, via its NPFmotif, interacts separately with the two EH domain-containing proteins TAX4 and IRS4, suggesting that INP51 forms separate INP51-TAX4 and INP51-IRS4 complexes.



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  		FIG. 3.
INP51 co-purifies with TAX4 and IRS4. A silver-stained gel of INP51 purification (see "Experimental Procedures") from HM47-1C cells (INP51-HA) and TB50 cells (mock purification) is shown. Protein bands unique to the INP51 purification are indicated. These bands were excised and identified by mass spectrometry (see "Experimental Procedures"). Equivalent regions of the mock purification were also excised to confirm the co-purification with INP51.

 



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  		FIG. 4.
INP51 coimmunoprecipitates with TAX4 and IRS4. Lysates from cells expressing INP51-HA (HM47-1C), TAX4-Myc (HM92-1A), IRS4-Myc (HM104-2D), or INP51{Delta}NPF-HA (HM102-3B) or co-expressing INP51-HA and TAX4-Myc (HM96-1B), INP51-HA and IRS4-Myc (HM119-2B), INP51{Delta}NPF-HA and TAX4-Myc (HM127-3B), or INP51{Delta}NPF-HA and IRS4-Myc (HM132-2D) were used to analyze the interaction between INP51 and TAX4 and IRS4. IP, immunoprecipitation.

 
There are additional EH-containing proteins in S. cerevisiae, including PAN1, END3, and EDE1 (45, 56, 57). Interestingly, a conditional pan1 mutation shows synthetic interaction with an inp51 mutation (58). Because PAN1 and END3 belong to the same protein complex, we tested whether PAN1 and/or END3 interact with INP51. We could not detect, by coimmunoprecipitation, an interaction between INP51 and PAN1 or END3, suggesting that INP51 interacts specifically with TAX4 and IRS4.

TAX4 and IRS4 Positively Regulate INP51—The interaction of INP51 with TAX4 and IRS4 led us to examine a possible involvement of TAX4 and IRS4 in the function of INP51. TAX4 and IRS4 are non-essential genes, as shown previously (59). Furthermore, cells lacking both TAX4 and IRS4 (a tax4 irs4 double mutant) did not display a detectable growth defect on standard rich or minimal media at various temperatures (Fig. 6 and data not shown).



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  		FIG. 6.
Double deletion of TAX4 and IRS4 restores growth of tor2ts. Wild-type, inp51, tax4 irs4, tor2ts, tor2ts inp51, and tor2ts tax4 irs4 cells and tor2ts cells transformed with plasmid overexpressing MSS4 were spotted onto YPD medium and incubated at 30 or 37 °C. tor2ts cells are not viable at 37 °C.

 
To determine the function of TAX4 and IRS4, we measured the levels of all detectable S. cerevisiae phosphoinositides, namely phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, phosphatidylinositol 3,5-bisphosphate, and PI(4,5)P2, in inp51, tax4, irs4, and tax4 irs4 mutant cells. The double mutant tax4 irs4 was examined in anticipation of the likelihood that the structurally homologous TAX4 and IRS4 proteins are also functionally homologous. We observed that an inp51 mutant has higher levels of PI(4,5)P2 than does wild-type, as observed previously (60), but is not altered in the levels of other phosphoinositides (Fig. 5). The deletion of TAX4 or IRS4 did not affect the level of any phosphoinositide. However, the double deletion mutant tax4 irs4, like the inp51 mutant, exhibited a significant and specific increase in the level of PI(4,5)P2. Our data suggest that TAX4 and IRS4 indeed act redundantly as positive regulators of INP51 activity and, thereby, PI(4,5)P2 turnover.



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  		FIG. 5.
PI(4,5)P2 is increased in inp51 and tax4 irs4 cells. Wild-type, inp51, tax4, irs4, and tax4 irs4 cells were labeled at 30 °C with [3H]inositol, and [3H]inositol-containing lipids from the cell extracts were deacylated, separated by high pressure liquid chromatography, and quantified. PI(3)P, phosphatidylinositol 3-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate.

 
Because TAX4 or IRS4 appears to be required for INP51 activity, we asked if the tax4 irs4 double mutation, like inp51, genetically interacts with TOR2 and components of the cell integrity pathway. We combined the tax4 irs4 double mutation with tor2ts, rom2, or sac7. In agreement with the positive role of TAX4 and IRS4 on INP51 activity, tax4 irs4 suppressed the growth defect of a tor2ts mutant at a non-permissive temperature to the same extent as did the deletion of INP51 or the overexpression of MSS4 (Fig. 6). The growth defect of a rom2 mutant was also suppressed by the tax4 irs4 mutation (data not shown). Finally, the combination of tax4 irs4 with a sac7 mutation produced a synthetic growth defect similar to the combination of inp51 and sac7 (Fig. 7). Deletion of TAX4 or IRS4 alone showed no synthetic interaction with components of the cell integrity pathway (data not shown). Our results suggest that TAX4 or IRS4, like INP51, is important for PI(4,5)P2 turnover linked to TORC2 signaling and the cell integrity pathway.



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  		FIG. 7.
tax4 irs4 double mutation interact with mutations in the cell integrity pathway. Wild-type, sac7, inp51, sac7 inp51, tax4 irs4, and sac7 tax4 irs4 cells were spotted onto YPD medium and incubated at 30 °C.

 
To investigate the mechanism by which TAX4 and IRS4 regulate INP51, we examined the cellular localization of TAX4, IRS4, and INP51 by indirect immunofluorescence on strains expressing epitope-tagged versions of these proteins. We observed an intracellular punctate staining pattern for both TAX4 and IRS4 (data not shown). However, for unknown reasons, we were unable to detect a signal for INP51 and were thus unable to determine by this assay if TAX4 or IRS4 is required for the correct localization of INP51. INP51 has previously been shown to be membrane-associated (31). However, by differential centrifugation (see "Experimental Procedures") we observed that INP51 is a cytosolic protein and that this localization did not change in a tax4 irs4 double mutant (data not shown).

INP51, TAX4, and IRS4 Negatively Regulate MPK1—The genetic interactions described above suggest that INP51, TAX4, and IRS4 negatively regulate signaling through the cell integrity pathway. However, the above experiments (and data not shown for TAX4 and IRS4) did not reveal a genetic interaction between MPK1 and the INP51 complexes. To investigate further a possible functional interaction between the MPK1 and the INP51 complexes, we examined MPK1 phosphorylation in wild-type cells and mutant cells containing rom2, sac7, inp51, tax4, irs4, tax4 irs4, or any possible combination of rom2 or sac7 with inp51, tax4, irs4, or tax4 irs4. Upon stimulation of the cell integrity pathway, MPK1 is phosphorylated and thereby activated (61). MPK1 phosphorylation was analyzed with an anti-phospho-p44/p42 MAP kinase antibody that also specifically recognizes the phosphorylated form of MPK1. The amount of total MPK1 was assayed with a separate antibody that recognizes all forms of MPK1. As shown previously, MPK1 phosphorylation is almost undetectable in wild-type cells grown at 23 °C (non-inducing condition) but increases upon shift to 39 °C (61). We examined the phosphorylation state of MPK1 in wild-type cells and mutant cells grown at low temperature (24 °C) and at high temperature (39 °C). The level of MPK1 phosphorylation in heat-shocked cells (shift to 39 °C for 30 and 60 min) was not altered in any of the mutants examined (data not shown). However, the sac7, sac7 inp51, and sac7 tax4 irs4 mutations affected the basal level (24 °C) of MPK1, as follows. A sac7 mutation resulted in a slight increase in MPK1 phosphorylation but also a similar increase in the amount of the MPK1 protein as compared with wild-type (Fig. 8). The sac7 inp51 and sac7 tax4 irs4 cells exhibited a 10-fold increase in MPK1 phosphorylation and a 2.5-fold increase in MPK1 protein level compared with the sac7 cells. Thus, inp51 and tax4 irs4 mutations cause constitutive MPK1 activity, although this effect is detectable only when inp51 and tax4 irs4 mutations are combined with a sac7 mutation. Furthermore, as suggested by some of the genetic interactions described above, these findings confirm that INP51 complexes indeed negatively regulate signaling through MPK1 and the cell integrity pathway.



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  		FIG. 8.
MPK1 activity increases in sac7 inp51 and sac7 tax4 irs4 cells. MPK1 activation and protein levels were analyzed in wild-type, sac7, inp51, sac7 inp51, tax4 irs4, and sac7 tax4 irs4 cells at 24 °C. Analysis of activated MPK1 with antibodies recognizing specific Thr/Tyr phosphorylation (top section) and MPK1 protein level with antibodies recognizing MPK1 (middle section) was performed. The bottom section (total extract) shows a Coomassie-stained version of the gel from the middle section. Equal amounts of protein were loaded in each lane, i.e. 40 µg for the gel of top section and 25 µg for the gel of the middle and bottom sections.

 
The Distribution of Chitin Is Negatively Regulated by INP51, TAX4, and IRS4 —To further investigate the regulation of MPK1 signaling by the INP51 complexes, we examined MPK1 readouts in sac7 inp51 and sac7 tax4 irs4 cells. In particular, we examined the actin cytoskeleton, glucan synthase (FKS1) and chitin (see the Introduction) in sac7, inp51, tax4 irs4, sac7 inp51, and sac7 tax4 irs4 cells grown at low (24 °C) and high temperatures (39 °C). Heat stress transiently depolarizes both the actin cytoskeleton and glucan synthase (17). The above mutant cells behaved like wild-type cells with regard to the actin cytoskeleton and FKS1 (data not shown), suggesting that INP51, TAX4, and IRS4 do not regulate signaling of the cell integrity pathway to these targets. However, the amount and distribution of chitin was changed in some mutants. In wild-type cells, chitin is most visible at the neck and at a future bud site (Fig. 9). In the sac7 and tax4 irs4 cells the chitin signal was slightly depolarized, whereas in the sac7 inp51 and sac7 tax4 irs4 cells the chitin signal and depolarization were strikingly increased. In sac7 inp51 and sac7 tax4 irs4 cells, the chitin was clearly distributed over the entire cell surface in unbudded cells, the mother cell surface in small budded cells, and both mother and daughter cell surfaces in large budded cells (Fig. 9). The changes in chitin amounts and distribution suggest that INP51, TAX4, and IRS4 regulate a specific branch of the cell integrity pathway affecting chitin.



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  		FIG. 9.
Chitin distribution is delocalized in sac7 inp51 and sac7 tax4 irs4 cells. Cells from logarithmically grown cultures were fixed with formaldehyde and stained with calcofluor white. The cells were examined with Nomarski optics and fluorescence to visualize the chitin.

 
To investigate if the affect on chitin is the cause of the observed growth defect of sac7 inp51 and sac7 tax4 irs4 cells (Fig. 7), CHS3 was deleted in these mutants. CHS3 encodes chitin synthase III that is responsible for 90% of the chitin produced in the cell, although deletion of CHS3 does not cause a growth defect (62–64). A deletion of CHS3 in sac7 inp51 and sac7 tax4 irs4 mutants reduced the amount of chitin to wild-type levels but did not suppress the growth defect (data not shown). This suggests that targets of the cell integrity pathway other than chitin could be affected (hyperactivated), thus causing the growth defect observed in sac7 inp51 and sac7 tax4 irs4 cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we present two proteins, TAX4 and IRS4, that are novel partners of the phosphoinositide 5-phosphatase INP51. The structurally homologous and functionally redundant TAX4 and IRS4 proteins positively regulate INP51 activity and thereby negatively regulate PI(4,5)P2 levels (Fig. 10). Furthermore, the INP51 complexes (INP51-TAX4 and INP51-IRS4) negatively regulate signaling through the cell integrity pathway, including the MAP kinase MPK1.



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  		FIG. 10.
Model showing PI(4,5)P2 regulation linked to the cell integrity pathway. PI(4,5)P2 synthesis and turnover is regulated by MSS4 and INP51, respectively. INP51 activity is controlled by TAX4 and IRS4. PI(4,5)P2 signaling impinges on a PKC1 pathway controlled by two independent pathways, the TORC2 signaling pathway and the cell integrity pathway. PI(4,5)P2 could signal to ROM2 or through an unknown parallel pathway.

 
TAX4 and IRS4 contain an EH domain. EH domains are found mainly in proteins involved in endocytosis and the organization of the actin cytoskeleton, and they interact specifically with short motifs containing an NPF core (54, 56, 57, 65, 66). INP51 contains an NPF motif (55) that is important for the interaction with TAX4 or IRS4 and is not found in any other phosphoinositide phosphatase in S. cerevisiae. Thus, the interaction between INP51 and TAX4 or IRS4 is similar to the interaction between the mammalian phosphoinositide 5-phosphatase synaptojanin 1 (NPF-containing) and EPS15, suggesting a conserved regulatory mechanism (33). The functional consequence of EPS15 binding to synaptojanin 1 is not known, but our findings suggest that this binding may result in the activation of synaptojanin 1 phosphatase activity.

How does TAX4 or IRS4 activate INP51? The EH domain has been suggested to play an important role in recruiting proteins to a specific cellular location (67). Our studies did not reveal a change in INP51 localization in the absence of TAX4 and IRS4; however, TAX4 and IRS4 might control a small pool of INP51 whose change in localization cannot be detected in the fractionation experiment. Furthermore, we did not detect a change in the binding of TAX4 or IRS4 to INP51 in response to several different types of cell wall stress, including heat shock and detergent treatment (data not shown). Future studies will be required to determine the mechanism by which TAX4 or IRS4 activates INP51.

We also present evidence that INP51, TAX4, and IRS4 genetically interact with two signaling pathways, the TORC2 signaling pathway and the cell integrity pathway (Fig. 10). The mechanism by which inp51 and tax4 irs4 mutations suppress the growth defect of a tor2 mutant remains unclear but could be through the activation of the cell integrity pathway. The inp51 or tax4 irs4 mutations activate MPK1, and the cell integrity pathway is a downstream effector pathway of TORC2. The activation of MPK1, which is detected only when inp51 or tax4 irs4 is combined with a sac7 mutation, could be a result of increased RHO1 activity (balance toward the active, GTP-bound form of RHO1). The sac7 mutation eliminates a RHO1 GAP and thereby prevents down-regulation of RHO1, whereas inp51 or tax4 irs4 causes an increase in the level of PI(4,5)P2 that could then activate RHO1 via the pleckstrin homology domain in the RHO1 GDP/GTP exchange factors.

The combined action of a GAP (SAC7) and a phosphoinositide phosphatase (INP51) could be a conserved mechanism for achieving proper spatial and temporal regulation of a signaling pathway, in this particular case signaling through the cell integrity pathway. The GAP and phosphoinositide phosphatase combination has been observed in other organisms such as Dictyostelium discoideum, where the phosphoinositide phosphatase Dd5P4 contains both a PI(4,5)P2 5-phosphatase catalytic domain and a GAP catalytic domain (68). This domain combination is also present in the human PI(4,5)P2 5-phosphatase OCRL1 that, when mutated, causes mental retardation and kidney and eye failure (oculocerebrorenal Lowe syndrome) (69–71).


   FOOTNOTES
 
* This work was supported by grants from the Wellcome Trust (to F. C.) and the Canton of Basel and the Swiss National Science Foundation (to M. N. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 41-61-267-2150; E-mail: m.hall{at}unibas.ch.

1 The abbreviations used are: PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; EH, EPS115 homology; GAP, GTPase-activating protein; HA, hemagglutinin A; MAP, mitogen-activated protein; TOR, target of rapamycin; TORC, TOR complex; TRITC, tetramethylrhodamine isothiocyanate; ts, temperature sensitive. Back


   ACKNOWLEDGMENTS
 
We thank Thierry Mini and Wolfgang Oppliger for technical assistance and Anja Lorberg and Dietmar Martin for comments on the manuscript.



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