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
Author for correspondence (e-mail: semr{at}ucsd.edu)
Accepted 16 August 2005
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Summary |
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Key words: Rho1p, PtdIns 3-kinase, Myotubularin, Cell-integrity MAP kinase pathway, Pkc1p
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Introduction |
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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, 2002). 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, 2004
; Seaman et al., 1998
). 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., 2003
).
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., 2001; Gary et al., 2002
; Parrish et al., 2004
; Rudge et al., 2004
; Stolz et al., 1998
). 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., 2000
)]; one that is specific for PtdIns(3,5)P2 (Rudge et al., 2004
); and one that is specific for PtdIns(3)P [Ymr1p (Parrish et al., 2004
; Taylor et al., 2000
)]. 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., 2000
). 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., 2001
). Similarly, simultaneous deletion of SJL1, SJL2 and SJL3 is lethal, and the temperature-sensitive sjl1
sjl2ts sjl3
mutant exemplifies an essential role for PtdIns(4,5)P2 regulation in the control of endocytosis and actin dynamics (Stefan et al., 2002
; Stolz et al., 1998
).
Interestingly, many of the cellular defects that are seen in sjl1 sjl2ts sjl3
temperature-sensitive phosphatase mutant cells at restrictive temperature (Stefan et al., 2002
) phenocopy cellular defects associated with either overexpression of hyperactivated Rho1p (Delley and Hall, 1999
) or with a temperature-sensitive PtdIns(4)P 5-kinase (mss4ts) mutant at restrictive temperature (Desrivieres et al., 1998
). Further studies have implicated a mechanism that can in part account for these observations (Audhya and Emr, 2002
). 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, 2002
). Coupled with the cell-wall sensors Wsc1p and Mid2p (Philip and Levin, 2001
), 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, 2002
; Stefan et al., 2002
). 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., 2004). This was particularly surprising in that, unlike PtdIns(4)P and PtdIns(4,5)P2, which are essential phosphoinositides (Audhya et al., 2000
; Desrivieres et al., 1998
), PtdIns(3)P is not essential under normal growth conditions (Herman and Emr, 1990
). 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., 2004
). Interestingly, further loss of Sjl2p SAC domain phosphatase activity in ymr1
sjl3
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 sjl2
sjl3
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., 1999
); 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.
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Materials and Methods |
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Yeast strains
The ymr1 sjl2
sjl3
strain carrying the GAL1-6HIS-YMR1-13xmyc allele was generated by cotransforming ymr1
sjl2ts sjl3
(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
sjl2
sjl3
vps30
quadruple mutant was created by crossing BPY13 to SEY6210-vps30
and following each allele in the progeny by PCR analysis. From this initial cross, ymr1
sjl2ts vps30
and ymr1
sjl3
vps30
triple mutants were isolated and subsequently crossed to generate a ymr1
sjl2ts sjl3
vps30
quadruple mutant (BPY63), which was subjected to two rounds of selection on media containing 5-FOA, creating a ymr1
sjl2
sjl3
vps30
quadruple mutant.
Plasmids
The S. cerevisiae genomic library used for isolation of 2 µ suppressors of Ymr1p depletion was previously described (Stagljar et al., 1994). 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., 1998
). 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., 1997
) 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., 1998
).
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., 2004).
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., 1994
). 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.
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Results |
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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 sjl3
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
sjl3
or sjl2
sjl3
double-mutant strains (our unpublished observations) (Parrish et al., 2004
; Stolz et al., 1998
), and only full-length SJL2 (SYD28) complemented defects in actin polarization that are associated with sjl2
sjl3
double-mutant strains (our unpublished observations) (Stolz et al., 1998
). 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.
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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., 1999). 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., 1999
) (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., 2000
)] 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
sjl2ts sjl3
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 sjl3
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., 2004
; Kamada et al., 1995
; Martin et al., 2000
), 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., 1999
); 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., 2004
; Martin et al., 2000
). In sharp contrast to wild-type cells, ymr1ts sjl2
sjl3
mutant cells showed a significant defect in the regulation of the cell-integrity pathway (Fig. 4B, bottom panel). The ymr1ts sjl2
sjl3
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
sjl3
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
sjl3
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
sjl2ts sjl3
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.
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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 sjl2
sjl3
mutant cells. To do this, SLT2 was deleted in ymr1
sjl2ts sjl3
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
sjl2ts sjl3
slt2
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
sjl2ts sjl3
slt2
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
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
sjl2ts sjl3
cells demonstrated that Slt2p hyperactivation is not sufficient to explain the failure of ymr1
sjl2
sjl3
mutant cells to grow.
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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 sjl3
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
sjl3
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.
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The Rho1p exchange factor ROM2 is required for the toxicity of PtdIns(3)P accumulation in ymr1 sjl2
sjl3
cells
Since interference with Rho1p signal propagation appeared to rescue the lethal phenotype displayed by ymr1ts sjl2 sjl3
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
sjl2ts sjl3
cells. We then tested ymr1
sjl2ts sjl3
rom2
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
sjl2ts sjl3
rom2
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
sjl2ts sjl3
rom2
cells carrying the empty pRS415 vector grew on 5-FOA comparable with cells complemented with the pRS415-YMR1 plasmid (Fig. 6A). By contrast, ymr1
sjl2ts sjl3
rom2
cells transformed with the pRS415-ROM2 plasmid failed to grow on 5-FOA, which reconstituted the PI 3-phosphatase requirement of the ymr1
sjl2ts sjl3
parent strain (Fig. 6A). Thus, deletion of ROM2 bypassed the lethality of ymr1
sjl2
sjl3
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 sjl2ts sjl3
rom2
cells. First, it could have been possible that Rom2p was required for ymr1
sjl2ts sjl3
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
sjl2ts sjl3
rom2
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
sjl2ts sjl3
rom2
cells. Consistent with our previous studies (Parrish et al., 2004
), ymr1
sjl2ts sjl3
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
sjl2ts sjl3
rom2
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
sjl2ts sjl3
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 sjl2ts sjl3
cells and in ymr1
sjl2ts sjl3
rom2
cells (Fig. 6C). Strong constitutive activation of cell-integrity pathway signaling was evident in ymr1
sjl2ts sjl3
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
sjl2ts sjl3
rom2
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
sjl2ts sjl3
rom2
cells when compared with ymr1
sjl2ts sjl3
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.
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Discussion |
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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., 2004). In support of this, we observed that deletion of VPS30, a subunit of the Vps34p PtdIns 3-kinase, in ymr1
sjl2ts sjl3
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., 2001
). 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
sjl2ts sjl3
vps30
quadruple-mutant cells compared with ymr1
sjl2ts sjl3
triple-mutant cells (Parrish et al., 2004
) (our unpublished observations). Additionally, vps34
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, 1990
). 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
sjl3
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 sjl2
sjl3
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., 2000
); see Fig. 4A for schematic]. Similarly, Msg5p is a dual-specificity phosphatase that, in conjunction with other dual-specificity phosphatases (Mattison et al., 1999
), plays a key role in maintaining the basal activity, as well as restricting the maximal activation of Slt2p (Flandez et al., 2004
; Martin et al., 2000
). Although a role for MSG5 in downregulating Slt2p activity has been proposed (Flandez et al., 2004
), 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
sjl2
sjl3
cells, appreciably improved defects in actin organization. This potentially argues that the actin polarity defects presented by ymr1
sjl2
sjl3
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
sjl2
sjl3
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, 2002; Bickle et al., 1998
); 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
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
cells are not significantly different from wild-type cells (Fig. 4B, lane 1). Rather, the absence of Vps34p PtdIns 3-kinase activity in vps34
cells apparently leads to a delay in the kinetics of Slt2p activation. Interestingly, like the ymr1ts sjl2
sjl3
triple-mutant cells, vps34
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, 1990
) (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
sjl3
cells (Fig. 5A), which was sufficient to promote viability (Fig. 5B). However, this was not the case for vps34
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., 1995)] and Bem3p [a GTPase-activating protein for Cdc42p (Zheng et al., 1994
)] 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., 2002
). Therefore, it is intriguing that overexpression of BEM3 in ymr1
sjl2ts sjl3
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, 2002). 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.
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