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Originally published In Press as doi:10.1074/jbc.M701055200 on April 9, 2007

J. Biol. Chem., Vol. 282, Issue 22, 15946-15953, June 1, 2007
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Up-regulation of the Cell Integrity Pathway in Saccharomyces cerevisiae Suppresses Temperature Sensitivity of the pgs1{Delta} Mutant*

Quan Zhong12, Guiling Li1, Jelena Gvozdenovic-Jeremic, and Miriam L. Greenberg3

From the Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202

Received for publication, February 5, 2007 , and in revised form, March 19, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously shown that mutants in the cardiolipin (CL) pathway exhibit temperature-sensitive growth defects that are not associated with mitochondrial dysfunction. The pgs1{Delta} mutant, lacking the first enzyme of the CL pathway, phosphatidylglycerolphosphate synthase (Pgs1p), has a defective cell wall due to decreased beta-1,3-glucan (Zhong, Q., Gvozdenovic-Jeremic, J., Webster, P., Zhou, J., and Greenberg, M. L. (2005) Mol. Biol. Cell 16, 665–675). Disruption of KRE5, a gene involved in cell wall biogenesis, restores beta-1,3-glucan synthesis and suppresses pgs1{Delta} temperature sensitivity. To gain insight into the mechanisms underlying the cell wall defect in pgs1{Delta}, we show in the current report that pgs1{Delta} cells have reduced glucan synthase activity and diminished levels of Fks1p, the glucan synthase catalytic subunit. In addition, activation of Slt2p, the downstream effector of the protein kinase C (PKC)-activated cell integrity pathway, was defective in pgs1{Delta}. The kre5W1166X suppressor restored Slt2p activation and dramatically increased (>10-fold) mRNA levels of FKS2, the alternate catalytic subunit of glucan synthase, partially restoring glucan synthase activity. Consistent with these results, up-regulation of PKC-Slt2 signaling and overexpression of FKS1 or FKS2 alleviated sensitivity of pgs1{Delta} to cell wall-perturbing agents and restored growth at elevated temperature. These findings demonstrate that functional Pgs1p is essential for cell wall biogenesis and activation of the PKC-Slt2 signaling pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiolipin (CL)4 is a unique anionic phospholipid that is ubiquitous in eukaryotes. It is primarily found in the mitochondrial inner membrane, where it interacts with and is required for the optimal activities of a large number of proteins. The importance of CL in mitochondrial function is underscored by the discovery that defective CL remodeling is associated with the severe X-linked genetic disorder Barth syndrome, which is characterized by cardiomyopathy, neutropenia, skeletal myopathy, and respiratory chain defects (2). Barth syndrome is due to mutations in a single gene (G4.5) encoding the protein tafazzin, a transacylase that remodels CL (3). The clinical presentation of Barth syndrome varies widely, even among patients who have the identical tafazzin mutation, suggesting that the phenotype is dependent upon multiple factors that are not well understood (4, 5). Elucidation of the cellular functions of CL and tafazzin will help to clarify the abnormalities associated with this disorder.

The identification of yeast genes encoding CL biosynthetic enzymes and subsequent construction of deletion mutants deficient in CL biosynthesis facilitated in vivo studies to elucidate the role of CL in mitochondrial function and cell viability. Disruption of PGS1 encoding the first enzyme of the CL pathway, phosphatidylglycerolphosphate synthase (Pgs1p) (6), results in the complete loss of both phosphatidylglycerol (PG) and CL (6, 7). Mutants lacking the CRD1 gene encoding CL synthase (8–10) contain no detectable CL but accumulate the upstream precursor PG (8–13). In vivo comparisons of cell functions in wild type and mutants deficient in the CL pathway confirmed the importance of CL in mitochondrial bioenergetics (11, 12, 14–16) and biogenesis (12, 17). Interestingly, these mutants also exhibit cellular defects that are not attributed to mitochondrial bioenergetics. The most severe growth defects are exhibited by the pgs1{Delta} mutant, which cannot grow on any medium at 37 °C (6, 18). The crd1{Delta} mutant loses viability after prolonged growth on both fermentable and nonfermentable media and does not form colonies from single cells at elevated temperature even on glucose (11, 13, 19). These growth defects suggest that the CL pathway plays an essential role in maintaining cell viability in response to the stress of increased temperature.

In order to understand the essential functions of the CL pathway, we took the genetic approach of isolating spontaneous mutants that suppress defective growth of pgs1{Delta} at elevated temperatures. One such suppressor mutant contained a loss of function mutation in KRE5, a gene involved in cell wall biogenesis (1). Subsequent biochemical analysis of the pgs1{Delta} cell wall revealed a decrease in beta-1,3-glucan levels, which were restored in the presence of the kre5W1166X suppressor. These findings were consistent with a large scale screen by Lussier et al. (20) to identify genes involved in cell wall biogenesis, in which it was reported that disruption of the promoter of PGS1 leads to hypersensitivity to cell wall-perturbing agents. Stabilization of the cell wall with osmotic support suppressed the temperature sensitivity of pgs1{Delta}, suggesting that the growth defects of pgs1{Delta} are associated with cell wall biogenesis.


Figure 1
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  		FIGURE 1.
Glucan synthesis and cell integrity signaling pathways.

 
The decreased beta-1,3-glucan levels in pgs1{Delta} suggested an intriguing link between the CL pathway, which is localized in the mitochondria, and beta-glucan synthesis in the plasma membrane. The current study was undertaken to elucidate the mechanisms whereby these pathways are linked. Yeast beta-1,3-glucan is synthesized by glucan synthase (21), which consists of a catalytic subunit encoded by the two homologous genes FKS1 and FKS2 (22, 23) and a regulatory subunit, Rho1p, a small GTPase (21, 24). As shown in Fig. 1, Rho1p alternates between GDP-bound inactive and GTP-bound active states (25, 26). Rho1p is regulated by a variety of effectors. The putative cell surface sensor protein Wsc1p activates Rho1p by stimulating nucleotide exchange through the GDP/GTP exchange factor, Rom2p (27). Lrg1p, a GTPase-activating protein, promotes formation of the inactive GDP-bound Rho1p (28). Posttranslational modification of Rho1p by the geranylgeranyl group is required for binding and activation of glucan synthase (29). The catalytic subunit of glucan synthase is also subject to regulation. Movement of Fks1p, which is required for the construction of a uniform and solid cell wall, is driven by actin (30). Transcription of FKS2 is activated in response to cell wall stress induced by heat, cell wall mutations, and cell wall-perturbing agents (31–34). A striking example of FKS2 activation is the 114-fold increase in FKS2 expression in the kre5 mutant (35).

Cell wall biogenesis is also controlled by the cell integrity pathway, which consists of a family of cell surface sensor proteins, Rho1p, protein kinase C (PKC), and a downstream mitogen-activated protein kinase (MAPK) cascade (36, 37) (Fig. 1). Activation of the cell integrity pathway is initiated by signals transmitted from cell wall sensor proteins to Rom2p, which stimulates formation of the GTP-bound activated form of Rho1p and the subsequent activation of Pkc1p. Pkc1p activates a cascade of phosphorylation reactions involving the MAPK kinase kinase Bck1p, the redundant MAPK kinases Mkk1p and Mkk2p, and the MAPK Slt2p. Signaling through the MAPK cascade results in dual phosphorylation and activation of Slt2p, which exerts its effects on cell wall biogenesis via two transcription factors, Rlm1p (38, 39) and the Swi4-Swi6 cell cycle box-binding factor complex (40). These transcription factors activate many genes involved in cell wall synthesis (39, 41–45), among which is FKS2. A constitutively activated PKC-Slt2 cell integrity pathway in cell wall mutants fks1{Delta} and gas1{Delta} leads to increased FKS2 expression and an Slt2p-dependent increase in thermal resistance (32).

To gain insight into the mechanism linking the CL pathway to cell wall biogenesis, we examined the possibility that glucan synthase and/or the PKC-Slt2 pathway are defective in the pgs1{Delta} mutant. Here we report that glucan synthase activity is decreased in pgs1{Delta}, accompanied by impaired cell integrity signaling in response to heat shock, both of which are restored in the presence of the kre5W1166X suppressor mutation. Overexpression of the glucan synthase catalytic subunits and up-regulation of PKC-Slt2 cell integrity signaling alleviate cell wall defects and restore growth at elevated temperatures. These data indicate that the CL pathway is required for glucan synthase activity and for activation of the PKC-Slt2 cell integrity pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Growth Media—The S. cerevisiae strains and plasmids used in this work are listed in Table 1. Synthetic complete medium contained adenine (20.25 mg/liter), arginine (20 mg/liter), histidine (20 mg/liter), leucine (60 mg/liter), lysine (200 mg/liter), methionine (20 mg/liter), threonine (300 mg/liter), tryptophan (20 mg/liter), and uracil (20 mg/liter), vitamins, salts (essentially components of Difco Vitamin Free Yeast Base without amino acids), inositol (75 µM), and glucose (2%). Synthetic drop out medium (Ura–) contained all of the above ingredients except uracil. Sporulation medium contained potassium acetate (1%), glucose (0.05%), and the essential amino acids. Complex medium contained yeast extract (1%), peptone (2%), and glucose (2%) (YPD).


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  		TABLE 1
Plasmids and yeast strains used in this study

 
Glucan Synthase Activity—Glucan synthase activity was determined as outlined by Shematek et al. (46) with minor modifications. Logarithmic phase cells resuspended in 1 mM EDTA (pH 8) were subjected to mechanical breakage with glass beads. Cell debris and unbroken cells were removed by low speed centrifugation (5,000 x g for 5 min at 4 °C). The resulting supernatant was centrifuged at 48,000 x g for 60 min, and pellets were washed once with 50 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and 1 mM beta-mercaptoethanol. Final pellets were resuspended in the same buffered solution containing 33% glycerol and stored at –20 °C. To determine glucan synthase activity, the standard reaction mixture contained 5.3 mM UDP-D-[14C]glucose (4 x 104 cpm/mmol), 80 µM GTP{gamma}S (Sigma), 0.8% bovine serum albumin, 2.2 mM EDTA, 75 mM Tris-HCl (pH 8), 8.25% (v/v) glycerol, and enzyme (100–200 µg of protein) in a total volume of 40 µl. The reaction was carried out at 30 °C for 30 min and stopped by adding 1 ml of 10% trichloroacetic acid. The reaction mixture was transferred onto a fiberglass filter mat (Whatman GF/C), washed three times with 1 ml of 10% trichloroacetic acid and twice with 1 ml of 95% ethanol, and then counted in a liquid scintillation counter. One unit of enzyme was defined as the amount that catalyzes the incorporation of 1 nmol of substrate (glucose) into glucan/min at 30 °C.

Northern Blot Analysis—The nonhomologous regions of the FKS1 and FKS2 coding sequences were amplified using the following primers as described previously (22): FKS1, 5'-CAGAACACTACAGCTGTTTTAACCG-3' (sense) and 5'-CCATATTGGTCATAGTCTTGTTCC-3' (antisense); FKS2, 5'-GGCATATTAAAGAAGTTACAAAAGG-3' (sense) and 5'-CCAGTTGGTTTTGTGTATAGATTGG-3' (antisense). The purified PCR fragments were cloned into pGEM-T-EASY vector for riboprobe synthesis. RNA was isolated by hot phenol extraction (47) from cells grown to the midlogarithmic phase in YPD, fractionated on an agarose gel, and transferred to a nylon membrane. The blots were hybridized with 32P-labeled FKS1 or FKS2 riboprobes, followed by the riboprobe against the constitutively expressed actin filament protein gene ACT1 to normalize for loading variation. RNA probes for Northern blot analysis were synthesized using the Promega Riboprobe System and from plasmids linearized with restriction enzymes as follows (gene, restriction enzyme, RNA polymerase): FKS1, PstI, T7; FKS2, ApaI, SP6; ACT1, BamHI, SP6 (17). The results were quantitated by phosphorimaging.

Western Blot Analysis of Fks1p—Aliquots of crude extract (100 µg of protein) from cells grown to midlogarithmic phase in YPD were subjected to 6% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was incubated in 5% nonfat milk in a solution containing 50 mM Tris-HCl (pH 7.4), 0.5 M NaCl, and 0.1% Tween 20 (TBS-T) for 1 h at room temperature and then overnight at 4 °C in the same buffer containing antibody against Fks1p (yN-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membrane was washed with TBS-T and incubated with alkaline phosphatase-conjugated donkey anti-goat IgG antibody (Promega) at room temperature for 1.5 h. The color was developed with an alkaline phosphatase substrate kit (Bio-Rad).

Detection of Dually Phosphorylated Slt2p—Dual phosphorylation of Slt2p was determined as previously described with minor modifications (32, 49). Midlog phase cells were diluted to A550 = 0.3 and grown at 30 or 37 °C for 2 h (1 generation). Cells were collected on ice by adding 20 ml of the culture to an equal volume of ice in a Falcon centrifuge tube and pelleted in a refrigerated centrifuge. Cells were then transferred with 1 ml of ice-cold water to an Eppendorf tube, pelleted, and immediately frozen on dry ice for 15 min. Cells were subsequently lysed in 120 µl of cold lysis buffer (50 mM Tris/HCl, pH 7.5, 10% glycerol, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 50 mM NaF, 1 mM sodium orthovanadate, 50 mM beta-glycerol phosphate, 5 mM sodium pyrophosphate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and the 1x protease inhibitor mixture (Roche Applied Science) by vigorous shaking with an equal amount of 0.45-mm glass beads. Cell extracts were separated from glass beads and cell debris, collected in a new Eppendorf tube by centrifugation, and further clarified by centrifuge at 13,000 x g for 15 min at 4 °C. Protein samples (50 µg) were boiled for 5 min, 2x loading buffer was added, and samples were fractionated by SDS-polyacrylamide gel electrophoresis using 8% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were probed with either anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cell signaling Technology rabbit IgG) at a 1:1000 dilution to detect dually phosphorylated Slt2p or with anti-Slt2p goat IgG (Santa Cruz Biotechnology) at a 1:100 dilution to detect Slt2p in the presence of 5% nonfat milk overnight at 4 °C. The primary antibody was detected using the alkaline phosphatase-conjugated anti-rabbit/goat antibody (Promega) with an alkaline phosphatase substrate kit (Bio-Rad).

Determination of Cell Wall beta-Glucan—Alkaline soluble and insoluble beta-glucan levels in the cell wall were measured as described previously (1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The major cell wall defect observed in the pgs1{Delta} mutant was markedly reduced beta-1,3-glucan, which was restored in the pgs1{Delta}kre5W1166X double mutant (1). One likely explanation for reduced beta-1,3-glucan is that glucan synthase activity is decreased in the pgs1{Delta} mutant. To address this possibility, glucan synthase activity was assayed in isogenic wild type, {rho}0, pgs1{Delta}, and pgs1{Delta}kre5W1166X cells. As seen in Fig. 2, glucan synthase activity was reduced to less than 30% of wild-type levels in the pgs1{Delta} mutant. Activity was partially restored by the kre5W1166X suppressor mutation. Wild type levels of glucan synthase activity were observed in petite mutants completely devoid of mitochondrial DNA ({rho}0 strain), indicating that defects in respiration per se are not the cause of decreased glucan synthase activity in the pgs1{Delta} mutant.

To understand the mechanism underlying reduced glucan synthase activity in the pgs1{Delta} mutant, expression of the glucan synthase catalytic subunits FKS1 and FKS2 was characterized in isogenic wild type and pgs1{Delta} mutant cells by Northern blot analysis. As shown in Fig. 3, A and B, FKS1 mRNA was only slightly reduced in pgs1{Delta} mutant cells to ~75% of the wild type level. Expression was increased 4-fold in the presence of the kre5W1166X suppressor. The small decrease in FKS1 mRNA levels in pgs1{Delta} was unlikely to account for the reduction in glucan synthase activity. However, in contrast to the minor decrease in FKS1 mRNA, Fks1 protein was barely detectable in pgs1{Delta} or in pgs1{Delta}kre5W1166X (Fig. 3C). Remarkably, expression of FKS2, encoding the alternate subunit of glucan synthase, was induced more than 10-fold in the pgs1{Delta}kre5W1166X suppressor (Fig. 3, A and B). These results suggest that decreased beta-1,3-glucan in the pgs1{Delta} mutant is at least partly due to decreased synthesis or stability of Fks1p. Antibodies to Fks2p are not available. However, in view of the absence of detectable Fks1p and the >10-fold increase in FKS2 expression, the most likely explanation for partial restoration of glucan synthase activity in the pgs1{Delta}kre5W1166X suppressor is that activity increased due to increased expression of FKS2.


Figure 2
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  		FIGURE 2.
Reduced glucan synthase activity in the pgs1{Delta} mutant. Isogenic wild type (FGY3), {rho}0, pgs1{Delta} (QZY24B), and suppressor mutant pgs1{Delta} kre5W1166X (QZY11A) cells were grown in YPD to midlog phase. Glucan synthase activity was determined as described under "Experimental Procedures." Data represent the average of three independent experiments. The error bars represent S.D. values (*, p < 0.02). WT, wild type.

 
Previous studies have shown that mutations in genes for cell wall synthesis, including FKS1 and GAS1, lead to stimulation of the cell integrity pathway, resulting in constitutive activation of Slt2p and increased expression of FKS2 mRNA (32). In this manner, fks1{Delta} and gas1{Delta} mutant cells, which have decreased beta-1,3-glucan, acquire SLT2-dependent thermotolerance and resistance to glucanase digestion (32). In this light, we wished to determine if suppression of pgs1{Delta} temperature sensitivity and increased expression of FKS1 and FKS2 in the kre5W1166X suppressor could be attributed to activation of the PKC-Slt2 cell integrity pathway. To this end, we assayed activation (dual phosphorylation) of Slt2p in response to heat shock in isogenic wild type, {rho}0, pgs1{Delta}, and pgs1{Delta}kre5W1166X cells (Fig. 4). Consistent with previous studies, activation of Slt2p was observed 120 min after shifting to 37 °C in wild type and {rho}0 cells. Surprisingly, phosphorylated Slt2p was not detectable in the pgs1{Delta} mutant. In contrast, phosphorylation of Slt2p was constitutive in the presence of the kre5W1166X suppressor. These results indicate that loss of PGS1 led to decreased activation of the Slt2p signaling pathway, which was restored in the pgs1{Delta}kre5W1166X suppressor.

The experiments described above suggested that kre5W1166X suppresses the temperature-sensitive growth defect of pgs1{Delta} by activating the PKC-Slt2 cell integrity pathway and increasing expression of structural genes (especially FKS2) encoding the glucan synthase catalytic subunit. To address this possibility, we asked if up-regulation of the PKC-Slt2 pathway and/or overexpression of genes encoding the glucan synthase catalytic subunits suppressed the growth and cell wall defects of pgs1{Delta}. Activation of the cell integrity pathway can be achieved by overexpression of the PKC-Slt2 pathway components or by expression of constitutively activated forms of PKC1 or BCK1 (50, 51). Overexpression of plasmids bearing ROM2, RHO1, MKK1, SLT2, and constitutively active alleles of PKC1 and BCK1 in pgs1{Delta} restored Slt2p phosphorylation to the pgs1{Delta} mutant (Fig. 5). Overexpression also significantly improved growth of pgs1{Delta} in the presence of the cell wall-perturbing agents calcofluor white (CFW) and caffeine (Fig. 6). Similarly, overexpression of both FKS1 and FKS2 enabled growth of pgs1{Delta} on CFW and caffeine. Consistent with a causative role of decreased Slt2p activation and decreased expression of FKS1 and FKS2 in pgs1{Delta} growth defects, overexpression also enabled growth of pgs1{Delta} at 36 °C (Fig. 7). Overexpression of the upstream components of the Slt2 pathway exerted somewhat stronger effects than components downstream of PKC. Thus, overexpression of ROM2, RHO1, and PKC1 enabled growth of pgs1{Delta} at 37 °C, whereas the constitutively activated BCK1 (BCK1-20) or overexpression of MKK1 or SLT2 did not enable growth at this temperature. SLT2 was also less effective than the other genes in rescuing sensitivity to cell wall-perturbing agents. In summary, restoring activation of Slt2p or increasing expression of glucan synthase genes suppressed the growth defect of the pgs1{Delta} mutant.


Figure 3
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  		FIGURE 3.
Reduced glucan synthase expression in the pgs1{Delta} mutant. Cells were grown in YPD to midlog phase. Total mRNA was isolated and analyzed by Northern blot hybridizing with 32P-labeled FKS1, FKS2, and ACT1 riboprobes as described under "Experimental Procedures." A representative membrane is shown in A. 32P was quantified by phosphorimaging analysis using ImageQuant software. Expression of FKS1 and FKS2 was normalized by comparison with ACT1 (B). Data represent the average of four independent experiments. C, Western blot analysis of Fks1p in cell extracts. A representative membrane is shown for four independent experiments. WT, wild type.

 


Figure 4
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  		FIGURE 4.
Impaired heat-induced Slt2p phosphorylation in the pgs1{Delta} mutant. Cells were grown in YPD to midlog phase and shifted to 37 °C for 2 h. Dual phosphorylation of Slt2p was determined by Western analysis with antibody to total Slt2p or to the dual phosphorylated protein (Phospho-Slt2p). A representative membrane is shown for three independent experiments. WT, wild type.

 


Figure 5
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  		FIGURE 5.
Up-regulation of Slt2p phosphorylation in the pgs1{Delta} mutant. Cells from the pgs1{Delta} mutant were transformed with empty vector pYES2/CT (vec), with plasmids bearing the wild type PGS1, ROM2, RHO1, MKK1, or SLT2 genes, or with the constitutively active alleles of PKC1 (PKC1*) or BCK1 (BCK1-20); were grown in YPD to midlog phase; and were shifted to 37 °C for 2 h. Dual phosphorylation of Slt2p was determined by Western blot analysis. A representative membrane is shown for three independent experiments.

 


Figure 6
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  		FIGURE 6.
Effects of up-regulation of the PKC-Slt2 cell integrity pathway and overexpression of GS cat-subunits on CFW and caffeine sensitivity of pgs1{Delta}. Serial diluted pgs1{Delta} cells transformed with plasmids as described in the legend to Fig. 5, as well as with FKS1 or FKS2 were spotted on YPD plates with the indicated concentration of CFW or caffeine and incubated at 30 °C. A representative image is shown for three independent experiments. WT, wild type.

 


Figure 7
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  		FIGURE 7.
Effects of up-regulation of the PKC-Slt2 cell integrity pathway and overexpression of GS catalytic subunits on temperature sensitivity of pgs1{Delta}. Serial diluted pgs1{Delta} cells carrying vectors described in Fig. 6 were spotted on YPD plates and incubated at the indicated temperatures. A representative image is shown for three independent experiments. WT, wild type.

 
To further elucidate the primary cause of cell wall defects in mutant cells lacking Pgs1p, we examined cell wall properties in the crd1{Delta} mutant, which lacks CL but accumulates PG. In contrast to the severe cell wall defects observed in pgs1{Delta}, including hypersensitivity to cell wall-perturbing agents and dramatically reduced beta-1,3-glucan (1), the crd1{Delta} mutant did not exhibit increased sensitivity to cell wall-perturbing agents CFW or caffeine (Fig. 8) and contained only slightly reduced levels of beta-1,3-glucan (Table 2). These findings indicate that the cell wall structure of the crd1{Delta} mutant is largely intact and that PG satisfies the CL pathway requirement for cell wall biogenesis.


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  		TABLE 2
Glucan composition in the crd1{Delta} mutant

Isogenic wild type (FGY3) and crd1{Delta} (FGY2) mutant cells were grown in YPD at 30 °C. Cells were harvested at the early stationary phase, and glucan levels were determined as described under "Experimental Procedures." Alkaline-insoluble glucan is expressed as micrograms per milligram of cell dry weight. Alkaline-soluble beta-1,3-glucan is expressed relative to wild-type cells. Data represent three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The pgs1{Delta} mutant, which lacks both PG and CL, exhibits severely defective growth at elevated temperature even on fermentable carbon sources (6, 18), suggesting that one or both of these anionic phospholipids is required for essential cellular functions. We have shown previously that this mutant has a defective cell wall due to reduced beta-1,3-glucan levels (1). In the current report, we further demonstrate that the cell wall defect is attributed to both decreased glucan synthase activity (Fig. 2) and to a defective PKC-activated cell integrity pathway (Fig. 4). When the glucan synthase and PKC pathway defects were alleviated in the pgs1{Delta} mutant, either by overexpression of the glucan synthase catalytic subunits or components of the cell integrity pathway (Fig. 5) or by genetic suppression of these defects in the presence of kre5W1166X (Figs. 2 and 4), cell integrity and growth at elevated temperature were restored (1) (Figs. 6 and 7). These findings indicate that PG and/or CL is required for glucan synthase activity and PKC signaling and that loss of both mitochondrial anionic phospholipids in pgs1{Delta} leads to defective growth at elevated temperature.

In contrast to pgs1{Delta}, the crd1{Delta} mutant did not exhibit increased sensitivity to cell wall-perturbing agents (Fig. 8) and contained only slightly reduced beta-1,3-glucan levels (Table 2). Furthermore, the crd1{Delta} mutant exhibits milder growth defects at elevated temperature than does pgs1{Delta} (1, 11, 13, 19). These findings suggest that PG satisfies the anionic phospholipid requirement for cell wall biogenesis. Although PG appears to be sufficient for cell wall biogenesis, the current study did not ascertain which phospholipid, PG or CL, plays a more direct role in this process. This question is currently under investigation in our laboratory.


Figure 8
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  		FIGURE 8.
The crd1{Delta} mutant does not exhibit increased sensitivity to cell wall-perturbing agents. Serial diluted wild type (WT) and crd1{Delta} mutant cells were spotted on YPD plates supplemented with CFW or caffeine at the indicated concentrations and incubated at 30 °C. A representative image is shown for three independent experiments.

 
One possible mechanism linking decreased glucan synthase activity and impaired PKC-Slt2 signaling to disruption of PGS1, suggested by our current findings, is dysfunctional Rho1p. Inactive Rho1p could explain the decrease in Fks1p protein levels in pgs1{Delta}, since loss of Rho1p activity has been associated with decreased Fks1p. Deficient prenylation of Rho1p in the cal1–1 mutant (which lacks a functional type I geranylgeranyl transferase) leads to defective targeting of Rho1p to the membrane and decreased binding to Fks1p (29). This results in dramatically reduced Fks1p levels, similar to what we observed in Fig. 3, presumably due to loss of protein stability (29). Dysfunctional Rho1p in the pgs1{Delta} mutant would also be consistent with the observed decrease in glucan synthesis and PKC-Slt2 signaling and with suppression of these defects by overexpression of ROM2.

How could mitochondrial anionic phospholipids affect Rho1p function? Membrane association is critical for the function of Rho family proteins, which appear to shuttle rapidly between membrane and cytosolic compartments (52). The mechanism of specific targeting of these GTPases, however, is largely unknown. Interestingly, Yeung et al. (53) have recently demonstrated that the electrostatic surface potential of a membrane can influence protein targeting. During phagosome formation, remodeling of anionic phospholipid composition, including phosphatidylserine (PS) and phosphoinositides, modulates the focal surface potential of the inner leaflet of the plasma membrane, thus regulating membrane anchoring of several proteins, including the Rho family protein Rac1p (53). In addition, Finkielstein et al. (54) investigated the mechanism of Rac1p recruitment to the plasma membrane using liposome binding and NMR spectroscopy. Rac1p was found preferentially associated with PS-containing bilayers via its C-terminal polybasic region. Subsequent functional analysis revealed that selective Rac1p membrane targeting via PS plays an important role in cytoskeletal rearrangement and cell migration. Interestingly, Cdc42p, another Rho family protein, only associates with PS when prenylated under the same condition, whereas other Rho GTPases, such as Rac2p, Rac3p, and RhoA, do not bind to PS even when prenylated, presumably due to their highly divergent polybasic C termini (54). Thus, the specific interaction between PS and Rac1p provides an additional layer of control in Rac1p-mediated signaling and functional specificity among other GTPase proteins. Evidence has emerged from several independent laboratories in the past few years that Rho1p is associated with mitochondria (55–57). The membrane potential-independent import of Rho1p to the mitochondrion was demonstrated in vitro (57). Twelve GTP-binding proteins were found to localize in the mitochondrial outer membrane, suggesting mitochondrial involvement in various regulatory processes (57). Two members of the Rho family, Ypt7p and Gem1p, were found to have mitochondria-associated functions. The mammalian homologue of Ypt7p, Rab32p, was previously demonstrated to be specifically associated with mitochondria (58), and the mitochondrial outer membrane localization of Gem1p and its regulatory function in mitochondrial morphology was also reported previously (59). We do not yet know if the presence of Rho1p at the mitochondrial outer membrane is physiologically relevant. However, it is tempting to speculate that PG and/or CL could potentially play a role in regulating the association of Rho1p with the mitochondrial membrane and that such an association affects Rho1p-dependent regulatory pathways, including glucan synthesis and PKC-Slt2 signaling.

Other mechanisms leading to decreased Fks1p level and impaired PKC-Slt2 signaling in the pgs1{Delta} mutant may be involved, including misregulation of nuclear gene translation and/or defective activation of PKC. Diminished levels of Fks1p in pgs1{Delta} and the pgs1{Delta}kre5W1166X suppressor strain despite normal or elevated FKS1 mRNA levels suggest that the defect is downstream of FKS1 transcription. In addition to the possibility of decreased protein stability as observed in cells with dysfunctional Rho1p (29), deficiency may also occur at the translational level. Recent studies have shown that lack of both PG and CL in the mitochondrial membrane leads to translational inhibition of the nuclear encoded gene COX4 (60). Such inhibition is mediated by the stem-loop structure in the COX4 5'-untranslated region and a putative protein trans-acting factor (61). Although to date, similar regulation of nuclear genes other than COX4 has not been reported, it is possible that this novel cross-talk pathway may be a general mechanism mediating translational regulation of nuclear genes in response to signals triggered by the loss of mitochondrial anionic phospholipids PG and CL.

Misregulation of cell integrity signaling in pgs1{Delta} may also result from defective activation of PKC1. In human promyelocytic (HL60) leukemia cells, PG was shown to specifically stimulate the PKC betaII isoform, leading to subsequent activation and phosphorylation of nuclear substrates by this enzyme (62). In addition, pkc1{Delta} and pgs1{Delta} mutants exhibit similar cell wall defects, including a significant reduction in both beta-1,3- and beta-1,6-glucan (1). These findings are consistent with defective activation of PKC in the pgs1{Delta} mutant.

In summary, we show for the first time that the synthesis of mitochondrial phospholipids PG and/or CL is required for glucan synthase activity and for activation of the PKC/Slt2 cell integrity pathway. Future studies will focus on the relative roles of PG and CL in Rho1p localization and membrane association, in the translation and stability of Fks1p, and in Pkc1p activation. These studies will shed light on the importance of mitochondrial lipids in functions that are essential for viability.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL62263 and by a grant from the Barth Syndrome Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These two authors contributed equally to this work. Back

2 Present address: Dept. of Cancer Biology, Dana Farber Cancer Institute, Harvard Medical School, 44 Binney St., Boston, MA. Back

3 To whom correspondence should be addressed: Dept. of Biological Sciences, Wayne State University, Detroit, MI 48202. Tel.: 313-577-5202; Fax: 313-577-6891; E-mail: MLGREEN{at}sun.science.wayne.edu.

4 The abbreviations used are: CL, cardiolipin; PG, phosphatidylglycerol; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; CFW, calcofluor white; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate; PS, phosphatidylserine. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. N. Hall for providing the plasmids that up-regulate the cell integrity pathway and Dr. John Lopes for providing the plasmid pPLG SP6.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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