PtdIns(3)P accumulation in triple lipid-phosphatase-deletion mutants triggers lethal hyperactivation of the Rho1p/Pkc1p cell-integrity MAP kinase pathway

William R. Parrish*, Christopher J. Stefan and Scott D. Emr{ddagger}

Department of Cellular and Molecular Medicine and the Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, California 92093-0668, USA

{ddagger} Author for correspondence (e-mail: semr{at}ucsd.edu)

Accepted 16 August 2005


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the budding yeast Saccharomyces cerevisiae, the regulation of phosphatidylinositol 3-phosphate [PtdIns(3)P] is an essential function shared by the myotubularin-related phosphatase Ymr1p and the synaptojanin-like phosphatases Sjl2p and Sjl3p. The aim of this study was to gain further insight into the mechanisms underlying the toxicity of PtdIns(3)P accumulation in ymr1{Delta} sjl2{Delta} sjl3{Delta} mutant cells. We conducted a genetic screen to isolate genes that, when overexpressed, would rescue the conditional lethality of ymr1{Delta} sjl2{Delta} sjl3{Delta} triple-mutant cells expressing YMR1 from the dextrose-repressible GAL1 promoter. This approach identified 17 genes that promoted growth of the triple mutant on media containing dextrose. Interestingly, the most frequently isolated gene product was a truncated form of PKC1 (Pkc1-T615) that lacked the C-terminal kinase domain. This Pkc1-T615 fragment also rescued the lethality of ymr1ts sjl2{Delta} sjl3{Delta} cells at restrictive temperature, and further mapping of the rescuing activity showed that the N-terminal Rho1-GTP-interacting HR1 domains (Pkc1-T242) were sufficient. This indicated that the PKC1 fragments might act by interfering with Rho1-GTP signal propagation. Consistent with this, deletion of the ROM2 gene, which encodes a major Rho1p guanine-nucleotide exchange factor, bypassed the lethal effect of PtdIns(3)P accumulation in ymr1{Delta} sjl2{Delta} sjl3{Delta} triple-mutant cells. Furthermore, cells deficient in phosphoinositide 3-phosphatase (PI 3-phosphatase) activity were defective for Rho1p/Pkc1p pathway regulation, which included an inability of these cells to adapt to heat stress. Taken together, the results of this study indicated that aberrant Rho1p/Pkc1p signaling contributes to the lethal effects of PtdIns(3)P accumulation in cells deficient in PI 3-phosphatase activity.

Key words: Rho1p, PtdIns 3-kinase, Myotubularin, Cell-integrity MAP kinase pathway, Pkc1p


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phosphoinositides are phosphorylated derivatives of the membrane phospholipid phosphatidylinositol (PtdIns) and have emerged as key regulators of many aspects of cellular physiology (reviewed by Martin, 2001Go; Rameh and Cantley, 1999Go). In particular, phosphoinositides modified on the D-3 position of the inositol ring play integral roles in controlling signaling pathways including those that result in the coordinated regulation of membrane trafficking within the endomembrane system of eukaryotic cells (Odorizzi et al., 2000Go; Rameh and Cantley, 1999Go; Simonsen et al., 2001Go). The seminal discovery that Vps34p, the sole phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity in Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi system to the vacuole of yeast (Schu et al., 1993Go; Stack et al., 1993Go) initiated studies that yielded much information regarding the role of phosphatidylinositol 3-phosphate [PtdIns(3)P] in driving endomembrane sorting reactions. For example, it is now recognized that a set of proteins that contain PtdIns(3)P-binding motifs such as the FYVE (for Fab1p, YGL023, Vps27p, EEA1p) domain serve as downstream effectors that regulate the nucleation of sorting reactions (Katzmann et al., 2003Go; Pelham, 2002Go; Sato et al., 2001Go; Wurmser et al., 1999Go). In yeast, specific examples include key roles for the FYVE-domain-containing proteins Vac1p and Vps27p in the coordination of endosome fusion (Burd et al., 1997Go), and in assembly of ESCRT (for `endosomal sorting complex required for transport') machinery required for multi-vesicular body (MVB) formation, respectively (Katzmann et al., 2003Go).

In addition to the FYVE domain, PtdIns(3)P effectors containing PX (for `phagocyte oxidase') domains, such as sorting nexins, also function in vesicle-mediated transport of membrane proteins. As such, sorting nexins contribute to the maintenance of endosomal system organization (Pelham, 2002Go). For example, the well-characterized retromer sorting complex components Vps5p and Vps17p are prototypical sorting nexins that are thought to assemble into a multivalent vesicle coat that selects cargos such as Vps10p [the carboxypeptidase Y (CPY) receptor] from late endosome compartments for recycling to the Golgi apparatus (Seaman, 2004Go; Seaman et al., 1998Go). Another set of sorting nexins, Snx4p, Snx41p and Snx42p, appear to function independently of the retromer complex to drive recycling of cargoes such as the plasma membrane v-SNARE Snc1p from earlier endocytic compartments (Hettema et al., 2003Go).

Recent studies in budding yeast have indicated that phosphoinositide phosphatase-mediated regulation of the signaling events that are initiated by the binding of various effectors to phosphoinositides is also required for the maintenance of cellular homeostasis (Foti et al., 2001Go; Gary et al., 2002Go; Parrish et al., 2004Go; Rudge et al., 2004Go; Stolz et al., 1998Go). In particular, yeast has seven well-characterized phosphoinositide phosphatases: three contain a promiscuous SAC1 domain that can hydrolyze PtdIns(3)P, PtdIns(4)P, PtdIns(5)P and PtdIns(3,5)P2 (Sac1p, Sjl2p and Sjl3p); two phosphatases that are specific for the D-5 position of PtdIns(4,5)P2 [Sjl1p and Inp54p (reviewed by Hughes et al., 2000Go)]; one that is specific for PtdIns(3,5)P2 (Rudge et al., 2004Go); and one that is specific for PtdIns(3)P [Ymr1p (Parrish et al., 2004Go; Taylor et al., 2000Go)]. In addition, Sjl2p and Sjl3p contain a separate PI 5-phosphatase domain that renders them uniquely capable of converting all major phosphoinositides to PtdIns (Hughes et al., 2000Go). Consistent with their in vitro activities, it is not surprising that studies on Sjl2p and Sjl3p have indicated largely redundant in vivo roles for these phosphatases with others that are dedicated to the hydrolysis of specific phosphoinositides. For example, an essential role for phosphatase-mediated control of PtdIns(4)P in the maintenance of Golgi structure and in driving secretion was elucidated through the construction of a mutant lacking Sac1p, Sjl2p and Sjl3p (Foti et al., 2001Go). Similarly, simultaneous deletion of SJL1, SJL2 and SJL3 is lethal, and the temperature-sensitive sjl1{Delta} sjl2ts sjl3{Delta} mutant exemplifies an essential role for PtdIns(4,5)P2 regulation in the control of endocytosis and actin dynamics (Stefan et al., 2002Go; Stolz et al., 1998Go).

Interestingly, many of the cellular defects that are seen in sjl1{Delta} sjl2ts sjl3{Delta} temperature-sensitive phosphatase mutant cells at restrictive temperature (Stefan et al., 2002Go) phenocopy cellular defects associated with either overexpression of hyperactivated Rho1p (Delley and Hall, 1999Go) or with a temperature-sensitive PtdIns(4)P 5-kinase (mss4ts) mutant at restrictive temperature (Desrivieres et al., 1998Go). Further studies have implicated a mechanism that can in part account for these observations (Audhya and Emr, 2002Go). Mss4p PtdIns(4)P 5-kinase activity synthesizes a specific pool of PtdIns(4,5)P2 that functions in the activation of the Rho1p guanine-nucleotide exchange factor Rom2p (Audhya and Emr, 2002Go). Coupled with the cell-wall sensors Wsc1p and Mid2p (Philip and Levin, 2001Go), this lipid kinase pathway is thought to represent a principal means by which yeast initiate and subsequently control (perhaps through phosphoinositide phosphatases) cellular responses to environmental stresses (Audhya and Emr, 2002Go; Stefan et al., 2002Go). Collectively, the results of these studies emphasize the general importance of a dynamic equilibrium between the activity of phosphoinositide kinases and phosphatases for the establishment and maintenance of the proper cellular compartmentalization of phosphoinositides.

The concept of establishment and maintenance of phosphoinositide compartmentalization seems to be a general theme, as an essential role for the regulation of PtdIns(3)P was also recently characterized (Parrish et al., 2004Go). This was particularly surprising in that, unlike PtdIns(4)P and PtdIns(4,5)P2, which are essential phosphoinositides (Audhya et al., 2000Go; Desrivieres et al., 1998Go), PtdIns(3)P is not essential under normal growth conditions (Herman and Emr, 1990Go). Mutants lacking the PI 3-phosphatase activity of Ymr1p and Sjl3p were shown to have significant defects in controlling the cellular levels and subcellular distribution of PtdIns(3)P, which caused the loss of endosome sorting system integrity (Parrish et al., 2004Go). Interestingly, further loss of Sjl2p SAC domain phosphatase activity in ymr1{Delta} sjl3{Delta} mutant cells caused lethality, suggesting that accumulation of PtdIns(3)P lead to toxic effects that resulted in cell death.

In this study, we sought to gain an understanding of the cellular basis for the requirement of phosphatase-mediated PtdIns(3)P regulation. Our results were consistent with PtdIns(3)P accumulation-dependent lethality in ymr1{Delta} sjl2{Delta} sjl3{Delta} cells. In addition, we isolated a truncation mutant of PKC1 (PKC1-T615) that lacked coding DNA for the C-terminal kinase domain required to propagate signals downstream of Rho1-GTP (reviewed by Heinisch et al., 1999Go); this mutant was found to be a multi-copy suppressor of PtdIns(3)P-mediated lethality. Furthermore, we found that the Rho1p guanine-nucleotide exchange factor Rom2p was required for the lethal effect of PtdIns(3)P accumulation in cells deficient in PI 3-phosphatase, indicating a link between PtdIns(3)P metabolism and regulation of Rho1p signaling. Collectively, the data argued that aberrant regulation of Rho1p/Pkc1p signaling participates in the lethal effect of PtdIns(3)P accumulation.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reagents and media
Enzymes used for recombinant DNA techniques were purchased from commercial sources and used according to the manufacturer's recommendations. Methods were performed essentially as described (Sambrook et al., 1989Go). Sources for growth media for yeast and bacterial strains have been described elsewhere (Gaynor et al., 1994Go). Standard yeast genetic methods were used throughout the study (Sherman et al., 1979Go). S. cerevisiae strains used in this study are listed in Table 1. Where pertinent, strain construction is described below. All oligonucleotides used in this study are available upon request.


View this table:
[in this window]
[in a new window]
 
Table 1. Strains used in this study

 

Yeast strains
The ymr1{Delta} sjl2{Delta} sjl3{Delta} strain carrying the GAL1-6HIS-YMR1-13xmyc allele was generated by cotransforming ymr1{Delta} sjl2ts sjl3{Delta} (BPY13) with pRS415-GAL3 and pBP69 (pRS415-GAL1-6HIS-YMR1-13xmyc), and selecting for growth on media containing galactose at 38°C. The GAL3-containing plasmid was necessary to complement the defect that SEY6210 has in galactose metabolism. These cotransformants were then plated for growth on 5-fluoro-orotic acid (FOA) galactose plates to select against cells harboring the pRS416-sjl2ts plasmid, but retaining both LEU2 plasmids. Resulting colonies were then picked and tested by western blot using monoclonal antibodies directed against the Myc epitope (Boehringer Mannheim Biochemicals) for galactose-dependent expression of YMR1-13xmyc, and subsequently for dextrose-dependent depletion. One isolate that fulfilled all of these criteria was saved for further studies (BPY141). The ymr1{Delta} sjl2{Delta} sjl3{Delta} vps30{Delta} quadruple mutant was created by crossing BPY13 to SEY6210-vps30{Delta} and following each allele in the progeny by PCR analysis. From this initial cross, ymr1{Delta} sjl2ts vps30{Delta} and ymr1{Delta} sjl3{Delta} vps30{Delta} triple mutants were isolated and subsequently crossed to generate a ymr1{Delta} sjl2ts sjl3{Delta} vps30{Delta} quadruple mutant (BPY63), which was subjected to two rounds of selection on media containing 5-FOA, creating a ymr1{Delta} sjl2{Delta} sjl3{Delta} vps30{Delta} quadruple mutant.

Plasmids
The S. cerevisiae genomic library used for isolation of 2 µ suppressors of Ymr1p depletion was previously described (Stagljar et al., 1994Go). 6-HIS-YMR1-13xmyc cloning: oligo-directed mutagenesis (Quick-change mutagenesis kit, Stratagene) was used to engineer a hexa-histidine tag downstream of a NdeI restriction site in plasmid pBP02 to generate plasmid pBP08 (pRS415-6-HIS-YMR1). Subsequently, the GAL1 promoter sequence was amplified from the genome of SEY6210 using oligos that added a 5' NaeI restriction site and a 3' NdeI restriction site. This fragment was then subcloned into plasmid pBP08, replacing the endogenous YMR1 promoter with the GAL1 promoter. Finally, the 13xmyc sequence was chromosomally integrated in frame with the C-terminus of the YMR1 gene (YJR110W) using the method of Longtine (Longtine et al., 1998Go). The C-terminal end of this modified YMR1 gene was then amplified from the genome by PCR using the Pfu Turbo DNA polymerase (Stratagene) with oligos that added a PacI restriction site to the 5' end of the fragment, and a SacI site to the 3' end in the ADH1 terminator sequence. The resulting fragment was then ligated into plasmid pBP08 to generate plasmid pBP69 (pRS415-GAL1-6HIS-YMR1-13xmyc), which was confirmed by DNA sequence analysis. VPS30 cloning: a pRS414-derived minimal genomic library clone of VPS30 (Seaman et al., 1997Go) was digested with ApaI and SacI, and the resulting ~2.5 kb fragment was subcloned into the LEU2-marked plasmid pRS415 at the same sites to generate the VPS30-containing plasmid pBP19. The ROM2-containing plasmid was a generous gift from M. Hall (University of Basel, Basel, Switzerland) (Bickle et al., 1998Go).

Serial dilution spot assays
In each case, log-phase cultures were harvested and resuspended at a final concentration of 1x107 cells/ml, and 3 µl of each tenfold serial dilution was spotted onto the appropriate selective media. Plates were then incubated at the indicated temperature for 3 days.

In vivo phosphoinositide analysis
Analysis of in vivo phosphoinositides was carried out as previously described (Rudge et al., 2004Go).

Slt2p activation assays
Slt2p phosphorylation assays were performed on log-phase cultures (OD600 ~0.4) that were shifted from growth at 26°C to 38°C, and 1 OD equivalent of cells was harvested per indicated time point into ice-cold trichloroacetic acid (TCA) at a final concentration of 10% and processed for analysis essentially as previously described (Gaynor et al., 1994Go). Rabbit polyclonal antisera directed against dual phosphorylated active p42/p44 mitogen-activated protein (MAP) kinase was purchased from commercial sources and used according to the manufacturer's suggestions.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PtdIns(3)P accumulation is toxic in ymr1{Delta} sjl2{Delta} sjl3{Delta} triple-mutant cells
Our previous results indicated that the lipid phosphatases Ymr1p, Sjl2p and Sjl3p share an essential function in the regulation of PtdIns(3)P (Parrish et al., 2004Go). To test if the lethal phenotype observed in ymr1{Delta} sjl2{Delta} sjl3{Delta} triple-mutant cells resulted from PtdIns(3)P accumulation, the ability of ymr1{Delta} sjl2ts sjl3{Delta} cells to synthesize PtdIns(3)P was compromised by deletion of VPS30, a subunit of Vps34p PtdIns 3-kinase complexes (Kihara et al., 2001Go) that is required for full enzymatic activity (Burda et al., 2002Go). Vps30p was chosen for this analysis because vps30{Delta} mutants show a 4-5-fold decrease in cellular PtdIns(3)P levels when compared with wild-type SEY6210 cells [see below and (Burda et al., 2002Go)]. Moreover, in contrast to cells deleted for VPS15 or VPS34 that do not synthesize PtdIns(3)P and are inviable at elevated temperature (Herman and Emr, 1990Go), vps30{Delta} cells display no obvious growth defects on standard media (Burda et al., 2002Go; Kihara et al., 2001Go). Interestingly, ymr1{Delta} sjl2ts sjl3{Delta} vps30{Delta} quadruple-mutant cells grew on plates containing 5-FOA, which selects against cells that maintain the URA3-marked sjl2ts plasmid (Fig. 1A). The quadruple-mutant cells transformed with a LEU2-marked VPS30 plasmid were not able to grow on this media, indicating that the VPS30 plasmid restored the requirement for the Sjl2p activity provided by the URA3-marked plasmid. This demonstrated that the loss of Vps30p was sufficient to rescue the lethal phenotype of ymr1{Delta} sjl2{Delta} sjl3{Delta} triple-mutant cells (Fig. 1A). As expected, this rescue was directly correlated to decreased cellular levels of PtdIns(3)P as ymr1{Delta} sjl2ts sjl3{Delta} vps30{Delta} quadruple-mutant cells showed a roughly sevenfold reduction when compared with PtdIns(3)P levels in ymr1{Delta} sjl2ts sjl3{Delta} parent cells at 38°C (Fig. 1B). Taken together with our previous studies (Parrish et al., 2004Go), these results argued that the lethal phenotype of ymr1{Delta} sjl2{Delta} sjl3{Delta} triple-deletion mutant cells was dependent on PtdIns(3)P accumulation.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 1. PtdIns(3)P accumulation is lethal in PI 3-phosphatase-deficient cells. (A) Deletion of the Vps34p PI 3-kinase complex component VPS30 rescues the lethal phenotype of ymr1{Delta} sjl2{Delta} sjl3{Delta} cells. ymr1{Delta} sjl2ts sjl3{Delta} vps30{Delta} cells were transformed with either pRS415-VPS30 or an empty vector. Transformants were then streaked to 5-FOA plates lacking the amino acid leucine, and growth was scored after 5 days at 26°C. (B) Quantitative comparison of in vivo PtdIns(3)P levels in wild-type, ymr1{Delta} sjl2ts sjl3{Delta} triple-mutant, ymr1{Delta} sjl2ts sjl3{Delta} vps30{Delta} quadruple-mutant, and vps30{Delta} cells at 38°C. Levels of deacylated product corresponding to PtdIns(3)P are shown. Data represent the mean±s.d. of three independent experiments.

 
Repression of Ymr1p expression in ymr1{Delta} sjl2{Delta} sjl3{Delta} cells results in inviability
In order to understand better the mechanism(s) underlying the toxic effect of PtdIns(3)P accumulation in ymr1{Delta} sjl2{Delta} sjl3{Delta} cells, a 6HIS-YMR1-13xmyc fusion was expressed in these cells from the dextrose-repressible GAL1 promoter. GAL1-6HIS-YMR1-13xmyc sjl2{Delta} sjl3{Delta} cells (henceforth referred to as YMR1-myc) grew on media containing galactose, which induces expression from the GAL1 promoter. When YMR1-myc cells were plated on media containing dextrose, which represses GAL1 expression, they failed to form colonies (Fig. 2A). Western blot analysis on lysates from YMR1-myc cells maintained in log phase in media containing dextrose showed that the inability of these cells to grow on dextrosecontaining medium was directly correlated with the reduction of Ymr1-myc protein (Fig. 2B). The change in OD600 over time of these cultures suggested that YMR1-myc cells were able to undergo an average of eight division cycles prior to arrest (our unpublished observations). Under the conditions of these experiments, the Ymr1-myc protein had a half-life of approximately 4 hours, which roughly corresponded to the average division time of YMR1-myc cells when grown in YP-dextrose liquid media (Fig. 2B and our unpublished observations). Consistent with our previous studies (Parrish et al., 2004Go), depletion of Ymr1-myc by growth on dextrose in the ymr1{Delta} sjl2{Delta} sjl3{Delta} mutant also resulted in increased cellular levels of PtdIns(3)P, whereas the levels of other phosphoinositides remained relatively constant (Fig. 2C). Therefore, these results reaffirmed our previous conclusions that phosphatase-mediated regulation/turnover of PtdIns(3)P is essential for maintaining cell viability (Parrish et al., 2004Go).



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 2. Depletion of GAL1-driven Ymr1-13xmyc in ymr1{Delta} sjl2{Delta} sjl3{Delta} cells on dextrose leads to inviability. (A) GAL1-YMR1-13xmyc sjl2{Delta} sjl3{Delta} cells were transformed either with pRS416-YMR1 or an empty vector, and transformants were grown overnight in selective media containing either galactose to stimulate Ymr1-myc expression, or dextrose to suppress expression. Aliquots of these cultures were subsequently streaked to selective plates containing the same sugar, and growth was scored after 3 days at 26°C. (B) Growth on dextrose causes depletion of GAL1-driven Ymr1-myc. GAL1-YMR1-13xmyc sjl2{Delta} sjl3{Delta} cells were maintained in log phase in YP dextrose media, and 1 OD600 equivalent of cells was harvested at each indicated time point into ice-cold TCA. Samples were processed as described in Materials and Methods. Results are representative of three independent experiments. The estimated half-life of Ymr1-myc was approximately 4 hours, which roughly corresponded to the doubling time of the cultures when grown under these conditions. (C) Depletion of Ymr1-myc in GAL1-YMR1-13xmyc sjl2{Delta} sjl3{Delta} cells on dextrose specifically affects cellular PtdIns(3)P levels. Cells were shifted into YP dextrose medium at the indicated time prior to labeling and were maintained in early log phase. Cells measuring 5OD600 units were harvested per time point, and labeling was carried out as in Fig. 1 except that there was no pre-incubation at 38°C. Results are representative of two independent experiments conducted in duplicate.

 

Suppressors of YMR1 depletion implicate cell-integrity pathway signaling in PtdIns(3)P toxicity
In order to gain further insight into the toxicity of accumulated PtdIns(3)P, we performed a genetic screen to identify yeast genomic fragments that when overexpressed would rescue the inability of YMR1-myc cells to grow on media containing dextrose. This strategy uncovered 26 suppressor of Ymr1p depletion (SYD) plasmids. Following restriction endonuclease mapping, 21 of these plasmids were found to contain unique inserts (Fig. 3A; Table 2; our unpublished observations). DNA sequence analysis identified 15 plasmids that were predicted to express a single open-reading frame (ORF), and three plasmids that could potentially express multiple ORFs (Fig. 3A; Table 2; our unpublished observations). In addition, there were three plasmids where different regions of the same ORF (SJL2, SJL3 and PKC1) were isolated independently (Table 2). Because it was possible that suppressors isolated in this fashion could act indirectly, for example by promoting leaky expression from the GAL1 promoter, or by altering the kinetics of Ymr1-myc depletion, each of the SYD plasmids that could express only a single ORF was subsequently tested for its ability to rescue ymr1ts sjl2{Delta} sjl3{Delta} cells at restrictive temperature. Only the SYD plasmids that promoted growth of both PI 3-phosphatase-deficient strains under restrictive conditions were considered for further investigation (see Table 2). Twelve of the SYD plasmids met these criteria, and fell into four groups based on the known cellular function of the genes they encoded: (1) complementing phosphatase domains of Sjl2p, Sjl3p and Ymr1p; (2) regulatory components of the cell-integrity MAP kinase pathway (Sac7p, Msg5p and truncated Pkc1p); (3) an oxysterol-binding protein (Osh7p); and (4) proteins involved in vesicle transport (Ypt1p and Vam6p; Fig. 3A and Table 2). Of these suppressors, only the lipid phosphatases significantly reduced cellular levels of PtdIns(3)P (Table 2). Likewise, only Sjl3-T893 (SYD4) corrected the fragmented vacuole phenotype that is associated with ymr1{Delta} sjl3{Delta} or sjl2{Delta} sjl3{Delta} double-mutant strains (our unpublished observations) (Parrish et al., 2004Go; Stolz et al., 1998Go), and only full-length SJL2 (SYD28) complemented defects in actin polarization that are associated with sjl2{Delta} sjl3{Delta} double-mutant strains (our unpublished observations) (Stolz et al., 1998Go). Therefore, despite their ability to promote growth of these PI 3-phosphatase-deficient mutants under restrictive conditions, most of the SYDs did not obviously alleviate other mutant phenotypes. One simple explanation is that the SYDs that do not decrease the levels of PtdIns(3)P might bypass its toxic effects by promoting adaptation to aberrant PtdIns(3)P regulation/localization, or by interfering with the ability of PtdIns(3)P to trigger detrimental signaling events.



View larger version (93K):
[in this window]
[in a new window]
 
Fig. 3. Gene-dosage-dependent suppressors of YMR1-13xmyc depletion. (A) GAL1-YMR1-13xmyc sjl2{Delta} sjl3{Delta} cells were grown overnight in YP dextrose medium to initiate depletion of Ymr1-myc and transformed with yEP352 high-copy-number S. cerevisiae genomic library plasmids (see Materials and Methods) and subsequently plated to selective media in the presence of dextrose. Colonies were picked 4 days later and re-streaked to selective media containing dextrose. Plates were incubated for 4 days at 26°C. A representative transformant for each unique SYD plasmid is shown. Isolates that also rescued ymr1ts sjl2{Delta} sjl3{Delta} cells at 35°C are underlined and set in bold print (also refer to Table 2). (B) Osmotic support rescues the lethal phenotype of ymr1{Delta} sjl2ts sjl3{Delta} cells at 38°C (upper panels). ymr1{Delta} sjl2ts sjl3{Delta} cells were transformed with either pRS415-YMR1 or an empty vector. Transformants were grown under selection to log phase and tenfold serial dilutions were plated to selective plates with or without 1M sorbitol and the plates were incubated for 3 days at the indicated temperature. Osmotic support rescues the lethal phenotype of sjl2{Delta} sjl3{Delta} depleted of Ymr1 (lower panels). ymr1{Delta} sjl2{Delta} sjl3{Delta} cells carrying pGAL1-YMR1-myc were grown on galactose and then changed to media containing glucose to deplete Ymr1. Tenfold serial dilutions were plated to selective plates (+dextrose) with or without 1M sorbitol and the plates were incubated for 3 days.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Gene-dosage-dependent suppressors of PI 3-phosphatase-deficient mutant inviability

 

The most frequently isolated suppressor (5 isolates) identified a portion of the PKC1 gene (YBL105C), an integral component of the Rho1p/Pkc1p-mediated cell-integrity MAP kinase pathway that is responsible for controlling cell wall homeostasis in response to cell stress (Heinisch et al., 1999Go). Interestingly, all of these PKC1 isolates were truncated, and lacked the C-terminal kinase domain that is required for the ability of Pkc1p to stimulate downstream signaling events (Heinisch et al., 1999Go) (Table 2). In addition to these truncated versions of Pkc1p, two negative regulatory components of the cell-integrity pathway [SAC7 and MSG5 (Martin et al., 2000Go)] were also isolated from the screen (Table 2 and see below). Together, these suppressors suggested that mis-regulation of the Pkc1p-activated cell-integrity signaling pathway might play a key role in triggering the toxic effects of PtdIns(3)P accumulation in PI 3-phosphatase-deficient cells. Consistent with this hypothesis, media containing 1 M sorbitol, which can prevent lysis due to defective cell wall maintenance, was sufficient to rescue the lethal phenotype of ymr1{Delta} sjl2ts sjl3{Delta} cells at the restrictive temperature (Fig. 3B). Taken together, these results indicate that defects in the maintenance of cell integrity are a probable cause of the lethal phenotype of PI 3-phosphatase-deficient mutants.

Proper regulation of cell-integrity pathway signaling depends on PtdIns(3)P metabolism
In light of the results described above, we reasoned that signaling in the cell-integrity pathway should be hyperactivated in PI 3-phosphatase-deficient cells. Therefore, to test this hypothesis directly, we took advantage of commercially available antibodies directed against the phosphorylated active form of p44/42 (Erk1/2-like) MAP kinases (Cell Signaling Technology) that recognize the yeast cell-integrity pathway p42-like MAP kinase Slt2p, to assay the activity of this pathway (see Fig. 4A for schematic). To do this, we used heat shock, a potent activator of the Pkc1p-mediated cell-integrity pathway, to stimulate either wild-type or ymr1ts sjl2{Delta} sjl3{Delta} cells and followed their ability to regulate the activity of Slt2p. Although most strains where this has been tested maintain high levels of Slt2p activation while heat stress is maintained (Flandez et al., 2004Go; Kamada et al., 1995Go; Martin et al., 2000Go), wild-type SEY6210 cells behave differently, and displayed Slt2p activation kinetics that were more consistent with results from Mattison et al., in which Slt2p is rapidly activated in response to heat stress, but over time the level of active Slt2p drops back to the original base line even though the stress is maintained [(Mattison et al., 1999Go); Fig. 4]. Maximal Slt2p phosphorylation occurred approximately 20-25 minutes after the cultures were shifted to 38°C (Fig. 4B,C, lane 3, top panel). Wild-type cells recovered quickly from this stimulus, and the level of phosphorylated Slt2p returned to basal level within 60 minutes (Fig. 4B,C, lane 5, top panel). At later time points, active Slt2p levels fell below the original basal level (Fig. 4B,C, lane 6, top panel), possibly due to an adaptation pathway involving the activity of dual-specificity phosphatases such as Msg5p (Fig. 4A) (Flandez et al., 2004Go; Martin et al., 2000Go). In sharp contrast to wild-type cells, ymr1ts sjl2{Delta} sjl3{Delta} mutant cells showed a significant defect in the regulation of the cell-integrity pathway (Fig. 4B, bottom panel). The ymr1ts sjl2{Delta} sjl3{Delta} mutant cells exhibited near peak levels of active Slt2p within 10 minutes of temperature shift, and this high level of active Slt2p was maintained through the time course of the experiment (Fig. 4B, lane 2, bottom panel). This indicated that ymr1ts sjl2{Delta} sjl3{Delta} cells display a potentiated response to heat stress in that they prematurely trigger maximal activation of the pathway, and in addition, since Slt2p phosphorylation did not decrease over time in these cells, there was apparently also a strong defect in adaptation (Fig. 4B, compare lanes 4, 5 and 6). Interestingly, the basal level of Slt2p phosphorylation in ymr1ts sjl2{Delta} sjl3{Delta} mutant cells was much greater than the heat-induced maximal activation seen for wild-type cells (see the legend for Fig. 4). Similar results were obtained for ymr1{Delta} sjl2ts sjl3{Delta} mutant cells (see below), indicating that constitutively activated signaling in the Pkc1p-mediated cell-integrity pathway might contribute to the lethal effects of PtdIns(3)P accumulation in PI 3-phosphatase-deficient cells.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 4. Proper PtdIns(3)P metabolism is important for appropriate regulation of Rho1p/Pkc1p-mediated cell-integrity pathway signaling. (A) Schematic representation of the Rho1p/Pkc1p-mediated cell-integrity pathway. Figure is adapted from Audhya and Emr (Audhya and Emr, 2002Go), and shows the level at which SYDs are expected to act on the cell-integrity pathway based on their known cellular functions (also refer to Table 2). (B) Slt2p activity is severely mis-regulated in ymr1ts sjl2{Delta} sjl3{Delta} cells. Wild-type and ymr1ts sjl2{Delta} sjl3{Delta} cells were grown to log phase at 26°C in YP dextrose media and subsequently shifted to 38°C. Cells were harvested at 1 OD600 unit for each time point into TCA at a final concentration of 10%, and samples were processed for western blot analysis as described in Materials and Methods. The equivalent of 0.2 OD600 units was loaded per lane. Note that results represent a 2-minute exposure using a super signal ECL detection system (Pierce) for the wild-type cells. Phospho-Slt2p signals are significantly stronger in ymr1ts sjl2{Delta} sjl3{Delta} samples, and represent a 10-second exposure under the same incubation conditions as the wild-type sample. Accordingly, Slt2p is constitutively activated in the basal state, and further hyperactivated upon heat stress in ymr1ts sjl2{Delta} sjl3{Delta} cells compared with wild-type cells. Blots were stripped and re-probed with antisera directed against the soluble glucose-6-phosphate dehydrogenase (G6PDH) enzyme as a control for protein loading. (C) vps34{Delta} cells are defective for regulation of heat-shock-induced cell-integrity pathway signaling. Wild-type and vps34{Delta} samples were resolved together by SDS-PAGE, transferred to the same nitrocellulose filter, which was probed as in Panel B. As an additional control, there was no detectable difference in the stability of 3xHA-tagged Slt2p between the wild-type and either ymr1ts sjl2{Delta} sjl3{Delta} or vps34{Delta} cells (our unpublished observations). (D) Slt2p is required to promote viability of ymr1{Delta} sjl2ts sjl3{Delta} cells. Cells were grown to log phase in YP dextrose media and tenfold serial dilutions were plated as in Fig. 1. Plates were incubated at the indicated temperature and growth was scored after 3 days. Synergism with slt2{Delta} implicates the Rho1p/Pkc1p signaling interface in PtdIns(3)P accumulation-mediated toxicity.

 
Because these results indicated the possibility of a link between PtdIns(3)P regulation and the control of Pkc1p-mediated signaling events in PI 3-phosphatase-deficient cells, we asked whether PtdIns(3)P might play a normal role in the regulation of the cell-integrity pathway. To address this, we took advantage of cells deleted for VPS34, which lack the only PtdIns 3-kinase in yeast and do not synthesize PtdIns(3)P or its derivatives (Schu et al., 1993Go), and assayed their response to heat shock by following the phosphorylation state of Slt2p as described above. Interestingly, vps34{Delta} cells also showed strong defects in the regulation of cell-integrity pathway signaling (Fig. 4C, bottom panel). Compared with wild-type cells, vps34{Delta} cells displayed a delay in the activation of Slt2p in response to heat shock (Fig. 4C, compare lanes 2 and 3 between top and bottom panels). In addition to this delay in cell-integrity pathway activation, vps34{Delta} cells failed to adapt to elevated temperature. This was demonstrated by the observation that the level of activated Slt2p never decreased in these cells at later time points, but rather increased through the time course of the experiment (Fig. 4C, compare lanes 4, 5 and 6). Therefore, it is noteworthy that vps34{Delta} cells show a temperature-sensitive lethal phenotype and lose viability within 180 minutes of shift to non-permissive temperature (Herman and Emr, 1990Go). Thus, it is likely that a failure in the proper regulation of cell-integrity pathway signaling plays a role in the death of vps34{Delta} cells at elevated temperatures. Importantly, these results indicated that normal PtdIns(3)P metabolism might play a novel key role in the regulation of cell-integrity pathway signaling.

Slt2p is required for viability of PI 3-phosphatase-deficient cells at elevated temperatures
In light of the results described above, we set out to test whether Slt2p hyperactivation itself might cause inviability of ymr1{Delta} sjl2{Delta} sjl3{Delta} mutant cells. To do this, SLT2 was deleted in ymr1{Delta} sjl2ts sjl3{Delta} cells and the resulting quadruple mutant was tested for its ability to grow on 5-FOA, which selects against cells maintaining the URA3-marked sjl2ts plasmid. This ymr1{Delta} sjl2ts sjl3{Delta} slt2{Delta} quadruple mutant failed to grow on 5-FOA (our unpublished observation), indicating that it required the phosphatase activity provided by the sjl2ts plasmid. Interestingly, as shown in Fig. 4D, when ymr1{Delta} sjl2ts sjl3{Delta} slt2{Delta} quadruple-mutant cells were tested for growth by serial dilution spot assays, it became apparent that Slt2p was required to promote viability of these cells at the normally permissive temperature of 34°C (Fig. 4D). Although they grew more slowly than the wild-type cells, slt2{Delta} cells did not display any significant loss in viability when grown at 38°C (Fig. 4D). These results indicated that deletion of SLT2 cannot bypass the lethal effects of PtdIns(3)P accumulation. Therefore, the additive effect of the loss of Slt2p in ymr1{Delta} sjl2ts sjl3{Delta} cells demonstrated that Slt2p hyperactivation is not sufficient to explain the failure of ymr1{Delta} sjl2{Delta} sjl3{Delta} mutant cells to grow.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5. Pkc1p fragments that contain the HR1 domains but lack the protein kinase domain interfere with Rho1p/Pkc1p signal propagation. (A) Overexpression of the Pkc1-T615 fragment quenches hyperactivation of Slt2p in ymr1ts sjl2{Delta} sjl3{Delta} cells. ymr1ts sjl2{Delta} sjl3{Delta} cells were transformed with either an empty vector, or the SYD22 Pkc1-T615-containing plasmid, and samples were prepared as in Fig. 4. Samples were resolved together by SDS-PAGE, transferred to the same nitrocellulose filter, which was probed as in Fig. 4B. Results are representative of two independent transformants. (B) Overexpression of the Rho1p-interacting HR1 domains of Pkc1p is sufficient to promote viability of ymr1ts sjl2{Delta} sjl3{Delta} cells at restrictive temperature. The sequences corresponding to the domain architecture of Pkc1p as predicted by the SMART database is shown for each Pkc1p construct. ymr1ts sjl2{Delta} sjl3{Delta} cells were transformed with the indicated high-copy-number plasmid and transformants were grown to log phase under the appropriate selection. Tenfold serial dilutions were then spotted to selective media at the indicated temperature and growth was scored after 3 days. Note that overexpression of the full-length PKC1 is growth inhibitory in these cells.

 
The rescuing activity of PKC1-T615 maps to the N-terminal Rho1-GTP interaction domain
On the basis of the results described above, the ability of the Pkc1p fragment to rescue ymr1ts sjl2{Delta} sjl3{Delta} cells at restrictive temperature could imply that defects in signaling events at or upstream of the level of Pkc1p function cause the lethality of PI 3-phosphatase-deficient cells. This raised the interesting possibility that the Pkc1-T615 fragment might act by interfering with the ability of endogenous Pkc1p to propagate signals to downstream effectors. To test this hypothesis, we assayed the ability of ymr1ts sjl2{Delta} sjl3{Delta} mutant cells carrying the SYD22 plasmid (PKC1-T615) to control signaling in the cell-integrity pathway by following the phosphorylation state of Slt2p. Consistent with interference with Pkc1p signaling, PKC1-T615 expression significantly reduced the levels of heat-stress-induced phospho-Slt2p compared with ymr1ts sjl2{Delta} sjl3{Delta} mutant cells (Fig. 5A). Given that Pkc1p, and signaling to the downstream cell-integrity pathway, is largely controlled by Rho1p activity [(reviewed by Heinisch et al., 1999Go); Fig. 4A], we next tested if the Pkc1-T615 fragment might compete with endogenous Pkc1p for upstream activating component(s) such as Rho1p. Pkc1 contains two N-terminal Rho1-GTP interacting domains (HR1), a calcium-dependent lipid-binding domain (C2), two zinc-dependent diacylglycerol-binding domains (C1), a predicted coiled-coil, and a C-terminal serine/threonine kinase domain (Schmitz et al., 2002Go). As depicted in Fig. 5B, the Pkc1-T615 fragment lacked sequences C-terminal to the C1 domains including the coiled-coil and the kinase domain. The combination of targeting and effector domains contained within this fragment suggested two simple models for how overexpression of this truncation might rescue PI 3-phosphatase-deficient cells when grown under restrictive conditions. First, since this Pkc1p fragment maintained the sequences that facilitate interaction with GTP-bound Rho1 but lacked the kinase domain that is required for signal transduction (see Fig. 4A and Fig. 5B for schematic), it could interfere with the propagation of upstream signals to endogenous Pkc1p by active Rho1p. Alternatively, this Pkc1p fragment could bind to intracellular membranes through its C1 and C2 membrane-targeting domains and, as such, could simply mask phospholipids and effectively block the toxic effects of PtdIns(3)P accumulation.

To distinguish between these models, the HR1 domains (Pkc1-T242) were genetically separated from the C2 and C1 domains (Pkc1-243-615; see Fig. 5B), and each sub-fragment was expressed at high levels in ymr1ts sjl2{Delta} sjl3{Delta} mutant cells as a GFP-fusion protein to facilitate detection. Cells carrying these high-copy-number plasmids were then assayed for their ability to grow at restrictive temperature. The ymr1ts sjl2{Delta} sjl3{Delta} strain was used for this experiment because its restrictive temperature of 34°C is not sufficient to trigger potent Slt2p activation in wild-type cells (our unpublished observations). Therefore, the effect of expression of various Pkc1p fragments could be assayed without inducing the heat shock response.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6. Rom2p is required for the lethal effects of PtdIns(3)P accumulation in ymr1{Delta} sjl2{Delta} sjl3{Delta} cells. (A) ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells were transformed with a pRS415-derived plasmid carrying either wild-type ROM2, YMR1 or no insert. Transformants were then streaked to 5-FOA plates lacking the amino acid leucine to select for maintenance of the pRS415 vector. Growth was scored after 5 days at 26°C. Results are representative of two independent transformants. (B) Cellular PtdIns(3)P levels are not significantly reduced in ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells. Cells were grown to early log phase under appropriate selection, then pre-incubated at either 26°C or 38°C for 20 minutes, and deacylated [3H]-glycero-phosphoinositols were analyzed by HPLC as in Fig. 1. Data represent the mean±s.d. of two experiments performed in duplicate. (C) Deletion of ROM2 attenuates hyperactivation of the Rho1p/Pkc1p-mediated Slt2p MAPK pathway. Samples were resolved together by SDS-PAGE and the nitrocellulose filter was probed as in Fig. 4. Results represent a 20-second exposure under the same conditions described for Fig. 4A. Note that the basal level of active Slt2p in ymr1{Delta} sjl2ts sjl3{Delta} cells is greater than the maximal heat-stress-induced signal in wild-type cells, indicating constitutive Rho1p/Pkc1p signaling in PI 3-phosphatase-deficient cells.

 
Western blot analysis using antibodies directed against GFP showed that each of these Pkc1p fragments (Pkc1-T615, -T242 and -243-615) was stable in vivo, and each was expressed to similar levels (our unpublished observations). Consistent with interference with Rho1p-stimulated Pkc1p signaling, in serial dilution spot assays the Pkc1-T242 fragment (isolated Rho1-GTP-interacting HR1 domains) promoted growth of ymr1ts sjl2{Delta} sjl3{Delta} cells at the restrictive temperature, whereas the Pkc1-243-615 fragment (C2 and C1 lipid-targeting domains) did not (Fig. 5B). In these assays, the complete Pkc1-T615 fragment was more effective at promoting growth than the isolated HR1 domains (Fig. 5B). Thus, it is likely that there is cooperativity between the targeting domains and the HR1 domains that improved the suppression efficiency, potentially by aiding in the membrane localization and/or targeting of the Pkc1p fragment to Rho1p. Surprisingly, overexpression of these Pkc1p truncations in wild-type cells did not cause a dominant phenotype in that there were no readily detectable effects on Slt2p phosphorylation in heat stress activation assays (our unpublished observations). Importantly, overexpression of full-length Pkc1p was toxic to ymr1ts sjl2{Delta} sjl3{Delta} cells as it inhibited growth at 30°C (our unpublished observations) and caused slow growth at 26°C (Fig. 5B). Taken together, these results lend support to a model where Pkc1p fragments containing the HR1 domains, but lacking the kinase domain, interfere with Rho1-GTP signal propagation and, as such, promote viability of PI 3-phosphatase-deficient mutants.

The Rho1p exchange factor ROM2 is required for the toxicity of PtdIns(3)P accumulation in ymr1{Delta} sjl2{Delta} sjl3{Delta} cells
Since interference with Rho1p signal propagation appeared to rescue the lethal phenotype displayed by ymr1ts sjl2{Delta} sjl3{Delta} cells, we asked whether mutants defective for Rho1p activity would bypass the lethality caused by PtdIns(3)P accumulation. Since RHO1 is an essential gene in yeast, to test this hypothesis we deleted ROM2, the gene encoding the principal upstream Rho1p guanine-nucleotide exchange factor (see Fig. 4A), in ymr1{Delta} sjl2ts sjl3{Delta} cells. We then tested ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} quadruple-mutant cells for their ability to grow on media containing 5-FOA, which selects against cells maintaining the URA3-marked sjl2ts plasmid. Therefore, the ability of these cells to grow on 5-FOA would indicate a bypass of the essential requirement for PI 3-phosphatase activity supplied by Ymr1p, Sjl2p or Sjl3p. To do this, ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells were transformed with LEU2-marked plasmids carrying either no insert, YMR1 or ROM2, and transformants were streaked to 5-FOA plates lacking leucine. Interestingly, ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells carrying the empty pRS415 vector grew on 5-FOA comparable with cells complemented with the pRS415-YMR1 plasmid (Fig. 6A). By contrast, ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells transformed with the pRS415-ROM2 plasmid failed to grow on 5-FOA, which reconstituted the PI 3-phosphatase requirement of the ymr1{Delta} sjl2ts sjl3{Delta} parent strain (Fig. 6A). Thus, deletion of ROM2 bypassed the lethality of ymr1{Delta} sjl2{Delta} sjl3{Delta} cells. These results demonstrated that Rom2p was required for the lethal effect of PtdIns(3)P accumulation, and further implicated aberrant regulation of Rho1p/Pkc1p signaling in PtdIns(3)P toxicity.

There were two possible simple explanations for how the loss of Rom2p could alleviate the requirement for PI 3-phosphatase activity in ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells. First, it could have been possible that Rom2p was required for ymr1{Delta} sjl2ts sjl3{Delta} triple-mutant cells to accumulate toxic levels of PtdIns(3)P. If this were true, then the loss of Rom2p should lead to significantly decreased cellular PtdIns(3)P levels in ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} quadruple-mutant cells. Alternatively, the loss of Rom2p guanine-nucleotide exchange factor activity might reduce the ability of these cells to activate Rho1p and subsequently Pkc1p and other downstream signaling events (see Fig. 4A), thus providing for bypass of the toxicity of PtdIns(3)P accumulation. Therefore, we metabolically labeled cells with myo-[2-3H]inositol, and measured the levels of PtdIns(3)P generated in ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells. Consistent with our previous studies (Parrish et al., 2004Go), ymr1{Delta} sjl2ts sjl3{Delta} cells showed a roughly 3-fold increase in PtdIns(3)P levels when compared with wild-type cells at permissive temperature, which was comparable with the levels of PtdIns(3)P detected in ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells (Fig. 6B). Upon shift to the restrictive temperature, PtdIns(3)P further accumulated to similar levels in both mutant strains (Fig. 6B). Thus, further deletion of ROM2 did not significantly affect cellular PtdIns(3)P levels in ymr1{Delta} sjl2ts sjl3{Delta} cells at either 26°C or at 38°C (Fig. 6B). Therefore, these results are consistent with a bypass of the toxicity of PtdIns(3)P accumulation, and indicated that aberrant regulation of Rho1p/Pkc1p signaling is a probable cause of the lethal effect of PtdIns(3)P accumulation in PI 3-phosphatase-deficient cells.

Next, we tested whether the deletion of ROM2 directly affected the activity of the Rho1p/Pkc1p-mediated cell-integrity pathway (see Fig. 4A) in the PI 3-phosphatase-deficient cells by comparing the phosphorylation state of Slt2p in ymr1{Delta} sjl2ts sjl3{Delta} cells and in ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells (Fig. 6C). Strong constitutive activation of cell-integrity pathway signaling was evident in ymr1{Delta} sjl2ts sjl3{Delta} cells, as the basal level of active Slt2p was greater than the heat-shock-stimulated level of active Slt2p in wild-type cells (Fig. 6C). Surprisingly, ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells also showed a strong constitutive activation of Slt2p (Fig. 6C), suggesting that Rom1p, the Rom2p homolog in yeast, might be sufficient to support the basal constitutive activation of cell-integrity pathway signaling in these quadruple-mutant cells. By contrast, the loss of Rom2p was very effective at blocking hyperactivation of Slt2p upon heat shock in ymr1{Delta} sjl2ts sjl3{Delta} rom2{Delta} cells when compared with ymr1{Delta} sjl2ts sjl3{Delta} parent cells (Fig. 6C). Therefore, taken together with our previous results, these data collectively argued that Rom2p-dependent hyperactivation of Rho1p/Pkc1p signaling mediated the lethal effects of PtdIns(3)P accumulation. Overall, these results suggested a novel essential function for proper PtdIns(3)P metabolism in the regulation of cell-integrity pathway signaling.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously demonstrated that phosphatase-mediated PtdIns(3)P regulation is an essential cellular function that requires the yeast myotubularin-related phosphatase Ymr1p and the synaptojanin-like phosphatases Sjl2p and Sjl3p (Parrish et al., 2004Go). The ymr1{Delta} sjl2ts sjl3{Delta} triple mutant redistributed PtdIns(3)P to aberrant subcellular compartments, and these new pools of PtdIns(3)P also lead to the mis-localization of various PtdIns(3)P effectors that caused the dysfunction of multiple PtdIns(3)P-dependent pathways such as the MVB, the CPY and the cytoplasm-to-vacuole (CVT) sorting pathways (Parrish et al., 2004Go). Here, we have taken a genetic approach to probe the molecular mechanisms that contribute to the lethal effects of PtdIns(3)P accumulation in yeast. The results of this study have indicated that the lethal phenotype of PI 3-phosphatase-deficient ymr1{Delta} sjl2{Delta} sjl3{Delta} cells is due in part to aberrant activation of Rho1p, a member of the RAS superfamily of small G proteins that regulates the activity of Pkc1p and the cell-integrity Slt2p MAP kinase pathway (see Fig. 4A for schematic). Taken together, the data presented here imply that inappropriate PtdIns(3)P metabolism alters the regulation of a Rho1p-mediated signaling pathway.

SYDs
It is unlikely that endomembrane sorting defects present in PI 3-phosphatase-deficient mutants are responsible for the lethal effects of PtdIns(3)P accumulation (Parrish et al., 2004Go). In support of this, we observed that deletion of VPS30, a subunit of the Vps34p PtdIns 3-kinase, in ymr1{Delta} sjl2ts sjl3{Delta} mutant cells rescued lethality by impeding PtdIns(3)P accumulation (Fig. 1). Thus, the lethal phenotype of PI 3-phosphatase-deficient cells was not directly correlated to endomembrane sorting defects since Vps30p function is required for proper sorting between compartments of the endomembrane system as well as for the CVT/autophagy pathways (Kihara et al., 2001Go). Consequently, defects in sorting of the soluble vacuolar hydrolase CPY and in the maturation of the cytoplasmic CVT cargo protein aminopeptidase-1 were exacerbated in ymr1{Delta} sjl2ts sjl3{Delta} vps30{Delta} quadruple-mutant cells compared with ymr1{Delta} sjl2ts sjl3{Delta} triple-mutant cells (Parrish et al., 2004Go) (our unpublished observations). Additionally, vps34{Delta} cells do not synthesize PtdIns(3)P, and although they display significant growth defects under stress conditions (for example heat stress, oxidative stress and hypo-osmotic stress), they are viable under standard growth conditions (Herman and Emr, 1990Go). This indicates that PtdIns(3)P itself, and therefore PtdIns(3)P-dependent trafficking pathways, are not essential for viability. Consistent with this, none of the vacuole protein sorting components or PtdIns(3)P effectors that have been identified is an essential protein. In light of this, it was somewhat surprising that proteins such as the Rab GTPase Ypt1p and Vam6p (Vps39p), the guanine-nucleotide exchange factor for the Rab GTPase Ypt7p, which are involved in regulating secretion and vacuolar fusion, respectively, came through our screen as suppressors of PtdIns(3)P-dependent lethality. Since neither of these suppressors lowered PtdIns(3)P levels when overexpressed in ymr1ts sjl2{Delta} sjl3{Delta} cells (Table 2), one possibility for explaining this is that they could potentially improve the trafficking and/or the subcellular distribution of various essential cell-surface proteins. Experiments to explore this hypothesis will be the focus of future work.

Hyperactivation of Rho1p/Pkc1p signaling mediates the lethality of PI 3-phosphatase-deficient cells
Our studies uncovered several lines of evidence that suggest PtdIns(3)P accumulation leads to lethal hyperactivation of Rho1p/Pkc1p signaling in ymr1{Delta} sjl2{Delta} sjl3{Delta} cells. First, our screen for suppressors of Ymr1p depletion identified negative regulatory components of the cell-integrity pathway such as SAC7 and MSG5. Sac7p is known to function as a GTPase-activating protein (GAP) for Rho1p, and its activity is important for antagonizing Rom2p-mediated Rho1p activation [(Martin et al., 2000Go); see Fig. 4A for schematic]. Similarly, Msg5p is a dual-specificity phosphatase that, in conjunction with other dual-specificity phosphatases (Mattison et al., 1999Go), plays a key role in maintaining the basal activity, as well as restricting the maximal activation of Slt2p (Flandez et al., 2004Go; Martin et al., 2000Go). Although a role for MSG5 in downregulating Slt2p activity has been proposed (Flandez et al., 2004Go), a definitive role for MSG5 in adaptation to heat stress has not yet been characterized. Additionally, our data suggest that the Pkc1p fragments lacking the kinase domain that were isolated in our screen interfere with Rho1-GTP effector activation. Furthermore, osmotic support was sufficient to promote viability of cells lacking Ymr1, Sjl2 and Sjl3 activity (Fig. 3), implying that defects in the regulation of Rho1p/Pkc1p-mediated signaling pathways lead to cell lysis in PI 3-phosphatase-deficient mutants where PtdIns(3)P accumulation is toxic. Consistent with this, the activity of the cell-integrity pathway MAP kinase Slt2p is mis-regulated in these mutants when compared with wild-type cells (Fig. 4B and Fig. 6C). Finally, deletion of the Rho1p guanine-nucleotide exchange factor ROM2 bypassed the requirement for PI 3-phosphatase activity supplied by Ymr1p, Sjl2p or Sjl3p, indicating that Rom2p activity is necessary for the lethal effects of PtdIns(3)P accumulation. Somewhat surprising was the observation that none of the suppressor plasmids (except the full-length Sjl2p-encoding plasmid), or deletion mutations that rescued the lethality of ymr1{Delta} sjl2{Delta} sjl3{Delta} cells, appreciably improved defects in actin organization. This potentially argues that the actin polarity defects presented by ymr1{Delta} sjl2{Delta} sjl3{Delta} cells are more closely associated with diminished PtdIns(4,5)P2 metabolism as a result of the lack of Sjl2p or Sjl3p activity and do not directly contribute to the inviability of PI 3-phosphatase-deficient mutant cells. On the basis of these observations, we conclude that aberrant regulation of Rho1p signaling activity is an important underlying cause of the PtdIns(3)P-mediated lethality of ymr1{Delta} sjl2{Delta} sjl3{Delta} cells.

Previous studies have highlighted the importance for PtdIns(4,5)P2 metabolism in controlling the signaling activity of Rom2p, and the subsequent Rho1p/Pkc1p-mediated cell-integrity pathway [(Audhya and Emr, 2002Go; Bickle et al., 1998Go); see Fig. 4A for schematic]. However, although neither PtdIns(3)P nor its known cellular effectors have been previously implicated in the control of Rho1p signaling activity, our current study has now shown that proper PtdIns(3)P metabolism is also required for the appropriate regulation of cell-integrity pathway signaling. In addition to the results discussed above, further support for this comes from our finding that vps34{Delta} cells display strong defects in the regulation of Rho1p/Pkc1p pathway signaling. In contrast to the PI 3-phosphatase-deficient mutants where Slt2p is constitutively activated (Fig. 6C), basal levels of phospho-Slt2p in vps34{Delta} cells are not significantly different from wild-type cells (Fig. 4B, lane 1). Rather, the absence of Vps34p PtdIns 3-kinase activity in vps34{Delta} cells apparently leads to a delay in the kinetics of Slt2p activation. Interestingly, like the ymr1ts sjl2{Delta} sjl3{Delta} triple-mutant cells, vps34{Delta} cells also display an inability to adapt (by inactivation of Slt2p) to heat stress in these assays, and this impediment to adaptation correlates with the inviability of these mutants (Herman and Emr, 1990Go) (our unpublished observations). Thus, Vps34p, and presumably PtdIns(3)P, is likely to have a role in mediating an adaptation pathway to heat stress. This is further supported by our observation that overexpression of the Pkc1-T615 fragment quenched Slt2p activation in ymr1ts sjl2{Delta} sjl3{Delta} cells (Fig. 5A), which was sufficient to promote viability (Fig. 5B). However, this was not the case for vps34{Delta} cells where overexpression of the Pkc1-T615 fragment, or deletion of ROM2, did not promote growth at elevated temperatures (our unpublished observations). This suggests that the presence of PtdIns(3)P may be important for the ability of cells to initiate a program of adaptation to heat stress, and argues that although PtdIns(3)P accumulation is toxic as a result of detrimental effects on the regulation of Rho1p/Pkc1p signaling, it is equally disadvantageous for cells to have no PtdIns(3)P.

A possible explanation for this phenomenon is that cells might have an intrinsic mechanism for monitoring the subcellular distribution of PtdIns(3)P and potentially its derivatives, as a means for determining endomembrane organelle integrity. Of the known PtdIns(3)P effector-domain-contain proteins in yeast, the PX-domain-containing Bem1p [a positive regulator of Cdc24p guanine-nucleotide exchange factor activity towards Cdc42p (Zheng et al., 1995Go)] and Bem3p [a GTPase-activating protein for Cdc42p (Zheng et al., 1994Go)] proteins could be attractive candidates for performing such a function. In line with this, it is noteworthy that a recent report from Molina and coworkers indicated that Cdc42p signaling can influence Slt2p activity (Rodriguez-Pachon et al., 2002Go). Therefore, it is intriguing that overexpression of BEM3 in ymr1{Delta} sjl2ts sjl3{Delta} cells prevented growth at the normally permissive temperature of 34°C (our unpublished observations). Accordingly, future studies will be geared towards the examination of potential roles for Bem1p and Bem3p in the lethality of PI 3-phosphatase-deficient mutants.

In addition to its well-characterized roles in directing intracellular membrane traffic, we have now shown that PtdIns(3)P metabolism plays an important role in the proper regulation of Rho1p-mediated signaling events. This is a particularly important finding since the etiology of severe human genetic disorders such as myotubular myopathy and Marie-Charcot tooth syndrome that are caused by the lack of proper PtdIns(3)P metabolism is poorly understood, and the full range of signaling pathways that are governed by PtdIns(3)P remains largely unexplored. It is likely that the equilibrium that is established and maintained by the concerted action of the Vps34p PtdIns 3-kinase and the lipid phosphatases might set threshold levels within which normal cellular functions occur unabatedly, whereas perturbations in this balance could trigger cellular stress responses and/or adaptation pathways. One possibility is that multiple PtdIns(3)P effectors are involved in the regulation of Rho1p/Pkc1p signaling. Consequently, a key question that remains open is whether the observed effects of PtdIns(3)P accumulation on the regulation of cell-integrity pathway signaling are direct, as the primary effectors that mediate this hyperactive response/failure in adaptation remain elusive. Alternatively, the fact that Rom2p activity is necessary for the lethal effect of PtdIns(3)P accumulation in PI 3-phosphatase-deficient cells suggests that Rom2p could either be a key integration point for the influence of PtdIns(3)P signaling on Rho1p activation, or that Rom2p might be directly triggered to activate Rho1p by binding to PtdIns(3)P or its derivatives. However there is no evidence in vitro that the Rom2p PH domain can interact with 3-OH-phosphorylated phosphoinositides (Audhya and Emr, 2002Go). Finally, it remains possible that PtdIns(3)P itself can alter the signaling properties of intracellular membranes where it is not normally represented. The mis-localization of PtdIns(3)P and/or PtdIns(3)P effectors to aberrant membranes might be sensed by the cell as a loss in the integrity of intracellular compartmentalization, which could be sufficient to trigger signaling pathways that lead to the activation of Rho1p and downstream stress-activated responses. Further work will be required to elucidate the molecular mechanisms that connect PtdIns(3)P metabolism to the regulation of Rho1p activity and the downstream Slt2p cell-integrity MAP kinase signaling pathway.


    Acknowledgments
 
We are grateful for excellent technical support through the course of the study from Steven Padilla, Perla Arcaira and Joshua Kaufman. We thank Mitsuaki Tabuchi, Roberto Botelho, Sara Rue and SoniaTerrillon as well as Jem Efe and Alex Rusnak for advice and critical comments on the work. We also thank members of the Emr laboratory, both past and present, especially Simon Rudge, Takeshi Noda and Anjon Audhya, for their generosity with strains and reagents, and invaluable suggestions regarding the manuscript. C.J.S. is a postdoctoral research associate of the Howard Hughes Medical Institute. W.R.P. was supported by National Research Service Award postdoctoral research training grant 5 T32 AI07036-25 from the NIH/NIAID. S.D.E. is an investigator of the Howard Hughes Medical Institute.


    Footnotes
 
* Present address: Laboratory of Biomedical Sciences, North Shore University Hospital, Long Island Jewish Health System, Manhasset, NY 11030, USA Back


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Audhya, A. and Emr, S. D. (2002). Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade. Dev. Cell 2, 593-605.[CrossRef][Medline]

Audhya, A., Foti, M. and Emr, S. D. (2000). Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics. Mol. Biol. Cell 11, 2673-2689.[Abstract/Free Full Text]

Bickle, M., Delley, P.-A., Schmidt, A. and Hall, M. N. (1998). Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO J. 17, 2235-2245.[Abstract/Free Full Text]

Burd, C. G., Peterson, M., Cowles, C. R. and Emr, S. D. (1997). A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast. Mol. Biol. Cell 8, 1089-1104.[Abstract]

Burda, P., Padilla, S. M., Sarkar, S. and Emr, S. D. (2002). Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase. J. Cell Sci. 115, 3889-3900.[Abstract/Free Full Text]

Delley, P.-A. and Hall, M. N. (1999). Cell wall stress depolarizes cell growth via hyperactivation of RHO1. J. Cell Biol. 147, 163-174.[Abstract/Free Full Text]

Desrivieres, S., Cooke, F. T., Parker, P. J. and Hall, M. N. (1998). MSS4, a Phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae. J. Biol. Chem. 273, 15787-15793.[Abstract/Free Full Text]

Flandez, M., Cosano, I. C., Nombela, C., Martin, H. and Molina, M. (2004). Reciprocal regulation between Slt2 MAPK and isoforms of Msg5 dual-specificity protein phosphatase modulates the yeast cell integrity pathway. J. Biol. Chem. 279, 11027-11034.[Abstract/Free Full Text]

Foti, M., Audhya, A. and Emr, S. D. (2001). Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology. Mol. Biol. Cell 12, 2396-2411.[Abstract/Free Full Text]

Gary, J. D., Sato, T. K., Stefan, C. J., Bonangelino, C. J., Weisman, L. S. and Emr, S. D. (2002). Regulation of Fab1 phosphatidylinositol 3-phosphate 5-kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide phosphatase family member. Mol. Biol. Cell 13, 1238-1251.[Abstract/Free Full Text]

Gaynor, E. C., te Heesen, S., Graham, T. R., Aebi, M. and Emr, S. D. (1994). Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J. Cell Biol. 127, 653-665.[Abstract]

Heinisch, J. J., Lorberg, A., Schmitz, H.-P. and Jacoby, J. J. (1999). The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae. Mol. Microbiol. 32, 671-680.[CrossRef][Medline]

Herman, P. K. and Emr, S. D. (1990). Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol. Cell. Biol. 10, 6742-6754.[Medline]

Hettema, E. H., Lewis, M. J., Black, M. W. and Pelham, H. R. B. (2003). Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes. EMBO J. 22, 548-557.[Abstract/Free Full Text]

Hughes, W. E., Cooke, F. T. and Parker, P. J. (2000). Sac phosphatase domain proteins. Biochem. J. 350, 337-352.[CrossRef][Medline]

Kamada, Y., Jung, U., Piotrowski, J. and Levin, D. (1995). The protein kinase C-activated MAP kinase pathway of Saccharyomyces cerevisiae mediates a novel aspect of the heat shock response. Genes Dev. 9, 1559-1571.[Abstract]

Katzmann, D. J., Stefan, C. J., Babst, M. and Emr, S. D. (2003). Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J. Cell Biol. 162, 413-423.[Abstract/Free Full Text]

Kihara, A., Noda, T., Ishihara, N. and Ohsumi, Y. (2001). Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J. Cell Biol. 152, 519-530.[Abstract/Free Full Text]

Longtine, M., McKenzie, A. R., Demarini, D., Shah, N., Wach, A., Brachat, A., Philippsen, P. and and Pringle, J. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953-961.[CrossRef][Medline]

Martin, H., Rodriguez-Pachon, J. M., Ruiz, C., Nombela, C. and Molina, M. (2000). Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae. J. Biol. Chem. 275, 1511-1519.[Abstract/Free Full Text]

Martin, T. (2001). PI(4,5)P2 regulation of surface membrane traffic. Curr. Opin. Cell Biol. 13, 493-499.[CrossRef][Medline]

Mattison, C. P., Spencer, S. S., Kresge, K. A., Lee, J. and Ota, I. M. (1999). Differential regulation of the cell wall integrity mitogen-activated protein kinase pathway in budding yeast by the protein tyrosine phosphatases Ptp2 and Ptp3. Mol. Cell. Biol. 19, 7651-7660.[Abstract/Free Full Text]

Odorizzi, G., Babst, M. and Emr, S. D. (2000). Phosphoinositide signaling and the regulation of membrane trafficking in yeast. Trends Biochem. Sci. 25, 229-235.[CrossRef][Medline]

Parrish, W. R., Stefan, C. J. and Emr, S. D. (2004). Essential role for the myotubularin-related phosphatase Ymr1p and the synaptojanin-like phosphatases Sjl2p and Sjl3p in regulation of phosphatidylinositol 3-phosphate in yeast. Mol. Biol. Cell 15, 3567-3579.[Abstract/Free Full Text]

Pelham, H. (2002). Insights from yeast endosomes. Curr. Opin. Cell Biol. 14, 454-462.[CrossRef][Medline]

Philip, B. and Levin, D. E. (2001). Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol. Cell. Biol. 21, 271-280.[Abstract/Free Full Text]

Rameh, L. E. and Cantley, L. C. (1999). The role of phosphoinositide 3-kinase lipid products in cell function. J. Biol. Chem. 274, 8347-8350.[Free Full Text]

Rodriguez-Pachon, J. M., Martin, H., North, G., Rotger, R., Nombela, C. and Molina, M. (2002). A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators. J. Biol. Chem. 277, 27094-27102.[Abstract/Free Full Text]

Rudge, S. A., Anderson, D. M. and Emr, S. D. (2004). Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase. Mol. Biol. Cell 15, 24-36.[Abstract/Free Full Text]

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning, a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.

Sato, T. K., Overduin, M. and Emr, S. D. (2001). Location, location, location: membrane targeting directed by PX domains. Science 294, 1881-1885.[Abstract/Free Full Text]

Schmitz, H.-P., Lorberg, A. and Heinisch, J. J. (2002). Regulation of yeast protein kinase C activity by interaction with the small GTPase Rho1p through its amino-terminal HR1 domain. Mol. Microbiol. 44, 829-840.[CrossRef][Medline]

Schu, P. V., Takegawa, K., Fry, M. J., Stack, J. H., Waterfield, M. D. and Emr, S. D. (1993). Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260, 88-91.[Medline]

Seaman, M. N. J. (2004). Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J. Cell Biol. 165, 111-122.[Abstract/Free Full Text]

Seaman, M. N. J., Marcusson, E. G., Cereghino, J. L. and Emr, S. D. (1997). Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products. J. Cell Biol. 137, 79-92.[Abstract/Free Full Text]

Seaman, M. N. J., Michael McCaffery, J. and Emr, S. D. (1998). A membrane coat complex essential for endosome-to-golgi retrograde transport in yeast. J. Cell Biol. 142, 665-681.[Abstract/Free Full Text]

Sherman, F., Fink, G. R. and Lawrence, L. W. (1979). Methods in Yeast Genetics: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.

Simonsen, A., Wurmser, A., Emr, S. and Stenmark, H. (2001). The role of phosphoinositides in membrane transport. Curr. Opin. Cell Biol. 13, 485-492.[CrossRef][Medline]

Stack, J. H., Herman, P. K., Schu, P. V. and Emr, S. D. (1993). A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J. 12, 2195-2204.[Abstract]

Stagljar, I., te Heesen, S. and Aebi, M. (1994). New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus. PNAS 91, 5977-5981.[Abstract/Free Full Text]

Stefan, C. J., Audhya, A. and Emr, S. D. (2002). The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate. Mol. Biol. Cell 13, 542-557.[Abstract/Free Full Text]

Stolz, L. E., Huynh, C. V., Thorner, J. and York, J. D. (1998). Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae. Genetics 148, 1715-1729.[Abstract/Free Full Text]

Taylor, G. S., Maehama, T. and Dixon, J. E. (2000). Inaugural article: Myotubularin, a protein tyrosine phosphatase mutated in myotubular myopathy, dephosphorylates the lipid second messenger, phosphatidylinositol 3-phosphate. PNAS 97, 8910-8915.[Abstract/Free Full Text]

Wurmser, A. E., Gary, J. D. and Emr, S. D. (1999). Phosphoinositide 3-kinases and their FYVE domain-containing effectors as regulators of vacuolar/lysosomal membrane trafficking pathways. J. Biol. Chem. 274, 9129-9132.[Free Full Text]

Zheng, Y., Cerione, R. and Bender, A. (1994). Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. J. Biol. Chem. 269, 2369-2372.[Abstract/Free Full Text]

Zheng, Y., Bender, A. and Cerione, R. A. (1995). Interactions among proteins involved in bud-site selection and bud-site assembly in Saccharomyces cerevisiae. J. Biol. Chem. 270, 626-630.[Abstract/Free Full Text]





This Article
Summary
Figures Only
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Parrish, W. R.
Articles by Emr, S. D.
PubMed
PubMed Citation
Articles by Parrish, W. R.
Articles by Emr, S. D.