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The cell wall of yeast cells preserves osmotic integrity and determines the morphology of the cells during budding growth and the processes of mating, sporulation, and pseudohyphal growth. Its constituents, 1,3-/1,6-glucans, mannoproteins, and chitin, are normally present in proportions of about 50 to 60%, 40%, and 1 to 2% of the cell wall dry weight, respectively (15). Several proteins are involved in cell wall biogenesis. Fks1p and Fks2p are the putative catalytic subunits of the plasma membrane 1,3-glucan synthase; Chs1, Chs2, and Chs3 are responsible for the synthesis of chitin; and several proteins are involved in the synthesis and elaboration of 1,6-glucan and cell wall mannoproteins (CWPs) (15). The assembly of the polymers occurs outside the cell. Several pieces of evidence indicate that the glycosylphosphatidylinositol-CWPs (GPI-CWPs) are convalently linked to 1,3-glucan through a 1,6-glucan chain, whereas proteins with internal repeats (PIR-CWPs) can be directly linked to it (22). The 1,3-glucan molecules can be also linked to some chitin molecules (13). Moreover, a fraction of proteins corresponding to about 2% of total CWPs are bound to chitin through short branches of the 1,6-glucan that are resistant to 1,3-glucanase (11, 14). One putative cell wall polymer cross-linker is Gas1p, a GPI-containing glycoprotein (17). In contrast with GPI-CWPs that at the cell surface undergo a probable transglycosylation reaction that ends with the loss of part of GPI, Gas1p retains the glycolipid and remains anchored to the plasma membrane. The increased alkali solubility of the glucan and the release in the medium of 1,3-glucan and mannoproteins shown by the gas1 mutant have suggested for Gas1p a cross-linker function (16, 20). The gas1 cells appear to respond to the weakening of the cell wall by activating a complex set of reactions, among which are a 10-fold increase in the chitin level, a 3-fold increase in the CWP1 mRNA level, a 20-fold increase in the 1,3-glucanase-resistant cross-links between GPI-CWPs and chitin, and the triggering of Fks2p expression (11, 16, 20). Some of these responses are common to other cell wall mutants and could be part of a rescue mechanism activated by cell wall stresses.

The rationale behind the experiments described here is that if gasI cells trigger a compensatory response to protect cell integrity, the osmolarity of the growth medium would affect this response. The genetic interactions between the GAS1 gene and the pathway that governs cell integrity have also been analyzed.

The growth kinetics parameters of the parental Saccharomyces cerevisiae W303-1B (MAT ade2-1 his3-11,15 trp1-1 ura3-1 leu2-3,112 can1-100) strain and the derived WB2d (gas1::LEU2) strain grown in YNB-glucose minimal medium (6.7 g of Difco yeast nitrogen base [without amino acids] per liter and 2% glucose supplemented with 50 mg of the appropriate amino acids and uracil and 100 mg of adenine per liter) at 30°C in the presence of an osmotic stabilizer are shown in Table 1. No significant effect on the duplication time (T_d) of the parental strain was observed with 0.5 M KCl and other osmotic supports (data not shown). In contrast, the T_d of the gas1-null mutant was about 40 min shorter in the presence of an osmotic support, and the length of the lag phase elapsing from the inoculum to the initiation of the exponential growth was reduced (data not shown). In addition, the percentage of pluribudded cells at the stationary phase decreased from 40% to about 4 to 6%, although a high percentage of budded cells were still present (last two columns in Table 1). Since the effects are present as well in 1 M sorbitol, they are due to the increase in osmolarity and not to the action of specific cations.

A microscopic examination indicated that gas1 cells in 0.5 M KCl lose the typical swollen aspect and are slightly smaller, and cells carrying two or more buds (pluribudded cells), characteristic of the "aggregated" phenotype of gas1, are almost absent (Fig. 1A and B). These results indicate that the increase in osmolarity of the growth medium ameliorates the growth kinetics parameters and the morphological defects of the gas1 mutant.

View larger version (69K): [in this window] [in a new window]   FIG. 1.   Effects of an increase in osmolarity of the growth medium on the morphology of gas1 cells. (A and B) WB2d cells (gas1) grown until the stationary phase at 30°C in YNB-glucose or YNB-glucose supplemented with 0.5 M KCl. (C and D) CF staining of exponentially growing WB2d cells under the same conditions.

In order to gain further insight into the effects of the increase of the external osmolarity on gas1 cells, we tested the cell viability and the sensitivity of growth to Calcofluor white (CF) (Sigma, St. Louis, Mo.), a fluorescent stain that binds to nascent chitin molecules and prevents their assembly in microfibrils (9). gas1 mutant is hypersensitive to CF (16). Different dilutions of cells from cultures grown in YNB-glucose medium or in the same medium supplemented with 0.5 M KCl were spotted on YPD (1% peptone, 2% yeast extract, 2% dextrose) plates in the presence or absence of 25 µg of CF per ml. gas1 cells, grown in the presence of 0.5 M KCl, showed a slight increase in cell viability and were less sensitive to the perturbing action of CF, whereas no differences were observed in the parental strain (Fig. 2).

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Cell viability and sensitivity to Calcofluor of gas1 cells. Exponentially growing W303-1B and WB2d cells were grown in YNB-glucose in the absence () or presence (+) of 0.5 M KCl. At a cell density of 5 × 106/ml, 5 µl of a concentrated suspension (lanes 1) and of 10× (lanes 2), 1,000× (lanes 4), and 10,000× (lanes 5) dilutions was spotted on YPD agar plates with or without CF.

To assess if growth in osmotically supported medium affects chitin accumulation, we measured the chitin level in gas1 and its parental strain. The chitin present in the undigestible material obtained after a prolonged treatment with Zymolyase 100T of the alkali-insoluble fraction was measured as described previously (16). The amount of chitin present in gas1 cells grown in YNB-glucose medium was approximately 10-fold higher than in the parental strain, in agreement with the previously reported data (16), whereas in cells grown in 0.5 M KCl, the amount of chitin showed a 50% decrease (Fig. 3). This result is also consistent with that obtained by staining of chitin with CF, which showed a less intense fluorescence on the cell surface of WB2d cells grown in the presence of the osmotic support (Fig. 1C and D).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Chitin levels in gas1 cells grown in an osmotically supported medium. The assays were performed in duplicate, and the values are the means of two different experiments. The standard deviation was no more than 10%.

We have shown in a previous work that GAS1-null mutation increases the sensitivity of cell growth to sodium dodecyl sulfate (SDS), a strong destabilizing agent for the plasma membrane that easily penetrates the mutant due to the increased porosity of the cell wall (16, 18, 23). Cells exponentially growing in YNB-glucose to a density of 5 × 10⁶ cells per ml were plated respectively on YNB-glucose plates containing SDS or SDS plus 1 M sorbitol or, as a control, on plates without SDS (Fig. 4). Cells of the parental strain were unaffected by the presence of 0.005% SDS in both the presence of 1 M sorbitol (Fig. 4A and E) and in the absence of sorbitol (data not shown). On the other hand, gas1 cells were unable to grow with 0.005% SDS (Fig. 4B and F), but they could form microcolonies if sorbitol was present in the plate (Fig. 4G). Thus, sorbitol partially suppresses the lethal effect of SDS on gas1 cells and appears to have a rapid protective effect on the cell wall of the mutant. The opposite shift gave results in agreement with this rapid effect of sorbitol. gas1 cells pregrown in YNB-glucose medium containing 1 M sorbitol (+ sorb in Fig. 4) were not able to grow on plates containing only 0.005% SDS (Fig. 4H). As a control, the cells were also plated in the absence of SDS, and the lack of any effect on growth indicates that the hypo-osmotic shock is not responsible for this observed lethal effect (Fig. 4D).

View larger version (69K): [in this window] [in a new window]   FIG. 4.   Effect of the osmotically stabilized medium on the sensitivity to SDS of a gas1-null mutant. (A, B, C, E, F, and G) W303-1B and WB2d cells exponentially growing in YNB-glucose were diluted, and about 500 cells were plated on solid medium in the presence or absence of SDS and 1 M sorbitol as indicated. (D and H) WB2d cells exponentially growing in YNB-glucose plus 1 M sorbitol (+ sorb) were plated on solid medium in the presence or absence of SDS, but without sorbitol.

Genetic interactions between GAS1 and components of the PKC1-MAP kinase cascade. It has been proposed that the cell wall weakened by the loss of Gas1p could less efficiently counteract the turgor pressure, thus generating the swollen phenotype of gas1 cells (20). This condition would create stress on the plasma membrane that, similarly to the condition of hypotonic shock, could open stretch-activated ion channels, whose presence has been shown in yeast (7, 10), and activate a cellular response. It is known that a decrease in the osmolarity of the growth medium activates the PKC1-mitogen-activated protein (MAP) kinase pathway in S. cerevisiae (8), inducing an increase in tyrosine phosphorylation and protein kinase activity of Slt2p (Mpk1p) (2, 10). Deletion of PKC1 determines an osmotically remedial cell lysis phenotype at all temperatures, while deletion of elements of the downstream MAP kinase cascade, BCK1-MKK1/MKK2-MPK1, determines a milder phenotype for cell lysis that requires a temperature of at least 37°C. It has been proposed that PKC1 could govern a bifurcated pathway (16). We have previously shown that the combination of deletion of GAS1 and PKC1 determines synthetic lethality (16). Here we analyzed the effects of GAS1 deletion and of single elements of the MAP kinase cascade downstream to PKC1. The double heterozygous diploid strains DL247G (relevant genotype, bck1::URA3/BCK1 gas1::LEU2/GAS1) and DL453G (mpk1::TRP1/MPK1 gas1::LEU2/GAS1), both derived from the parental strain 1788 (a/ leu2-3/leu2-3 trp1-1/trp1-1 ura3- 52/ura3-52 his4/his4 can1^r/can1^r), were sporulated and dissected on YPD plates (1% peptone, 2% yeast extract, 2% dextrose) supplemented with 0.5 M KCl, and sporulation was carried out at 24°C to prevent cell lysis. Analysis of the spore progeny of 18 tetrads for each diploid indicated that the bck1 gas1 (Ura⁺ Leu⁺) or mpk gas1 (Trp⁺ Leu⁺) double mutant spores were inviable. Thus, the deletion of GAS1 determines synthetic lethality as well in combination with the inactivation of BCK1 and the last element of the pathway, MPK1. This result suggests that the colethality found between the GAS1 and PKC1 deletions is not simply the sum of severe cell wall defects brought about by the single inactivations. The triggering of the cellular response to cell wall defects of the gas1-null mutant could require the BCK1-MKK1/MKK2-MPK1 module and not other pathways governed by PKC1. Moreover, the lethality of the double-null mutant gas1 pkc1, gas1 bck1, and gas1 mpk1 spores, even in osmotically supported medium, suggests that the increased osmolarity of the growth medium does not completely abolish the dependence from the cell integrity pathway, at least during gas1 spore germination.

112 ura3-52 his3-200 trpl-90 suc29 ade2-101