INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Yeast cells are surrounded by a matrix composed of (1,3)/(1,6)-glucans and mannoproteins as major components and chitin as a minor one (21). Chitin constitutes only 1 to 2% of the cell wall dry weight, but it plays a key role in yeast morphogenesis and is essential for the viability of yeast and fungal cells. During vegetative growth chitin is deposited at the site of bud emergence, forms a ring that surrounds the neck between the mother and daughter cells, and constitutes the primary septum. On the surface of mother cells a chitin ring is still recognizable after cell division, the so-called bud scar, and in the corresponding site on the daughter surface a birth scar is present. A tiny amount of chitin is also layered over the whole of the lateral cell wall, and this occurs in the mother cell.

Three chitin synthase (CS) activities, CSI, CSII, and CSIII, are responsible for the deposition of cell wall chitin. The three isoenzymes differ in certain properties, such as the optimum pH, metal specificity, and susceptibility to inhibitors (6). CSI and CSII activities are determined only by the product of CHS1 and CHS2 genes, respectively, which encode the polypeptides containing the catalytic domain of each chitin synthases. Chs1p is responsible for the synthesis of chitin after cell separation. It plays a repair function, since it counterbalances the acid-induced increase in the chitinase activity that hydrolyzes the chitin present in the primary septum at the end of cytokinesis (3-5, 17, 18). CSI represents about 90% of the in vitro measurable chitin synthase activity, but its contribution to the production of chitin in vivo is negligible. Chs2p is responsible for deposition of the primary septum and is thus necessary for cell division (33, 34). CSIII activity is responsible for the deposition of chitin in the ring and lateral cell walls and contributes to the synthesis of most cell wall chitin during vegetative growth (33). During cell cycle progression the Chs3p level remains constant (10, 38), but its localization changes (10, 31). A complex regulation of synthesis and transport determines the spatial and temporal control of chitin deposition by Chs3p, the catalytic component. The CHS4 to CHS7 genes are involved in this regulation (19, 32, 35, 36, 39). Additionally, CS activities exhibit in vitro zymogenic properties, suggesting that they are regulated at a posttranslational level (6, 8, 9).

In the present study we investigated the increase in chitin accumulation which appears to be part of the responses that a yeast cell activates to counteract cell wall damage. The fks1 mutant, which lost a subunit of the (1,3)-glucan synthase, has a reduced level of (1,3)-glucan and exhibits an induction of chitin accumulation (15, 28). A similar response is present in gas1 cells which lack a (1,3)-glucosyltransferase activity (20) that is important for the correct incorporation of glucan and mannoproteins (see references 15, 24, 28, and 27 for a review). Moreover, the loss of Fks1p or Gas1p induces also the expression of Fks2p, the alternative subunit of the (1,3)-glucan synthase, a 20-fold increase in the cross-links between cell wall mannoproteins and chitin, and a 3-fold increase in CWP1 expression (15, 24, 28). The increase in chitin deposition could be included in a compensation mechanism that mutants defective in cell wall synthesis or assembly activate to maintain cell integrity. In our study we focused on the role of CS activities in the possible mechanism of increased chitin synthesis in the gas1 mutant.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Strains, growth conditions, and genetic methods. The yeast strains used here are listed in Table 1. Standard techniques were used for diploid construction, sporulation, and tetrad dissection. Cells were grown in batches at 30°C in YNB-glucose (Difco yeast nitrogen base without amino acids at 6.7 g/liter, 2% glucose, and the required supplements) or in YEPD (1% yeast extract, 2% Bacto-Peptone, 2% dextrose). For solid media, 2% agar was added. Diploids were sporulated in New Sporulation Medium (8.2 g of sodium acetate, 1.9 g of KCl, 0.35 g of MgSO₄, 1.2 g of NaCl, and 15 g of agar per liter) at 24°C. Spore germination was carried out at 24°C on YEPDAT plates (YEPD, 2% agar, and 100 mg of adenine and 50 mg of tryptophan per liter) containing 0.5 M KCl.

r_km_k

DNA manipulations. Recombinant DNA manipulations were performed using standard techniques (30). Transformation of yeast cells was carried out using the lithium acetate procedure (14) or the Saccharomyces cerevisiae EasyComp Transformation Kit (Invitrogen).

Plasmid and strain construction. The S. cerevisiae haploid strain WB2d was generated from the wild-type strain W303-1B by gene replacement (37). In order to obtain the gas1::HIS3 mutation we used PCR to synthesize a linear fragment which lacked almost the whole of the GAS1 open reading frame (ORF). The upstream primer (5'-TGC GGA CGA TGT TCC AGC GAT TGA AGT TGT TGG TAA TAA GGT CCT GTT CCC TAG CAT GTA-) was designed to join 40 bp corresponding to the region from nucleotides 63 to 102 of the GAS1 ORF with the 5' end from nucleotides 66 to 83 of HIS3. The downstream primer (5'-AGA CTT GGA AGA AGA CCC CGA AGC GTT AGA AGA GGC AGT ACT TGC CAC CTA TCA CCA CCA-) was designed to join 40 bp corresponding to the region from nucleotides 1470 to 1509 of the GAS1 ORF with the 3' end from nucleotides 1446 to 1426 of HIS3. These primers were used to amplify a ~1.2-kbp fragment from YEp6. This PCR product was transformed into W303-1A and Y1306. His⁺ transformants were selected, and correct substitution was tested by PCR analysis. Immunoblot analysis further confirmed the absence of the GAS1 gene product.

RNA extraction and Northern analysis. Total RNA was prepared according to the method of selective precipitation with LiCl (12). Northern analysis was performed as previously described, using nonradioactive or ³²P-radiolabeled single-stranded RNA probes generated by in vitro transcription (25). After hybridization at 50°C, blots were twice washed at 50°C in 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 15 min, once in 1× SSC-0.1% sodium dodecyl sulfate (SDS) for 30 min, and twice in 0.1× SSC-0.1% SDS for 30 min. Two final washes were carried out at 68°C in 0.2× SSC-0.1% SDS for 15 min and 0.2× SSC for 2 min. Densitometric quantification of mRNA was performed by using a computer program. mRNA loading was normalized using the hybridization signal of ACT1.

Preparation of total extracts and membrane fractionation. For total extracts, 2 × 10⁸ cells were collected by filtration, washed, and resuspended in ice-cold deionized water supplemented with a Protease Inhibitor Cocktail (Boehringer Mannheim), one capsule in 25 ml, and 1 mM phenylmethylsulfonyl fluoride. After a 2-min centrifugation at 4°C, the pellets were frozen in dry ice-acetone and stored at 80°C. After thawing, 400 µl of SB-minus buffer (0.0625 M Tris-HCl pH 6.8; 5% SDS), supplemented with the protease inhibitors, was added to each pellet. After the addition of glass beads, cells were broken by shaking on a vortex for four cycles of 1 min alternated with 1 min in ice. After 5 min of centrifugation, the clarified lysate was withdrawn, quickly frozen, and stored at 80°C until use. For the determination of protein concentration, 15-µl aliquots of lysates in duplicate and the DC Protein Assay (Bio-Rad) were used. For SDS-polyacrylamide gel electrophoresis (PAGE) analysis, appropriate amounts of a concentrated solution were added to the lysate in order to bring the samples to a final concentration of 10% glycerol, 5% -mercaptoethanol, and 0.002% bromophenol blue (BFB). Before the loading, samples were denatured at 100°C for 2 min.

Electrophoresis and immunoblotting. Proteins were resolved by SDS-PAGE on 7 or 8% polyacrylamide slab gels. Immunodecoration was carried out as previously described (26). Mouse anti-hemagglutinin (HA) monoclonal HA.11 antibodies (Babco) were used at a 1:1,000 dilution in TBS (0.01 M Tris-0.9% NaCl, pH 7.4) containing 5% bovine serum albumin (BSA) and 0.5% Tween 20 and anti-Gas1p rabbit polyclonal antibodies at a 1:3,000 dilution in TBS, 5% BSA, and 0.1% Tween 20. Horseradish peroxidase-conjugated anti-mouse antibodies (Amersham) or anti-rabbit antibodies (Zymed) were diluted to 1:5,000 and 1:10,000, respectively, in TBS-5% BSA-0.2% Tween 20. Binding was visualized with the ECL Western Blotting Detection Reagent (Amersham) according to the manufacturer's instructions.

Measurement of chitin levels. Pellets corresponding to 5 × 10⁹ cells were collected, resuspended in 4.5 ml of H₂O, and divided into three equal aliquots (one was used for the determination of dry weight, and the other two were centrifuged), and the pellets stored at 20°C until use. After three extractions with 3% NaOH at 75°C, the alkali-insoluble pellet was neutralized and treated for 16 h with 4 mg of Zymolyase 100T per ml at 37°C. The chitin present in the indigestible material of the alkali-insoluble fraction was measured as described previously (24). The micrograms of glucosamine were normalized to the milligrams of dry weight.

Measurement of CS and chitinase activities. For CS activity measurements, cell extracts were obtained according to the protocol described previously (2). CS assays were performed in Tris (pH 7.5) in the presence of Mg²⁺, Co²⁺, or Co²⁺ plus Ni²⁺ in order to discriminate among the three different activities, as described earlier (7). For CSI activity, MES buffer at pH 6.3 was also used (7). The determination of CS activity in cells permeabilized with digitonin was accomplished as described previously (13), except that CSIII activity was measured in the presence of 50 mM Tris (pH 7.5), Co²⁺, and Ni²⁺. Chitinase activity assays were performed as described previously (17).

Microscopic techniques. Chitin was visualized by fluorescence microscopy after being stained with 2 mg of Calcofluor White (CF) per ml (24).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

CSI activity and CHS1 mRNA increases are not responsible for the hyperaccumulation of chitin in gas1 cells. CS activities were measured in the wild-type (W303-1B) and gas1 (WB2d) strains in the presence or absence of trypsin. The results (Table 2) reveal that in the absence of trypsin, the CSI activity doubles in the gas1 mutant with respect to the isogenic strain and that trypsin treatment elicits a six- to sevenfold increase in CSI activity in both strains, a finding in agreement with the zymogenic properties of this enzyme. On the contrary, the CSII and CSIII activities were similar in both strains.

View larger version (74K): [in this window] [in a new window]   FIG. 1.   (A) Northern blot analysis of CHS1 expression in gas1 cells. Total RNA was extracted from the gas1 mutant (WB2d) and its isogenic strain (W303-1B) when cells in YNBglucose at 30°C reached a density of about 8 × 106 to 107 cells/ml. Ca. 8 µg of RNA was loaded into each lane. Hybridization was performed with 32P-labeled CHS1 or ACT1 antisense RNA probes. The autoradiograms were quantitated by densitometry. (B) Suppression of the lysis bud phenotype on the chs1 gas1 mutant. Cells were examined by phase-contrast microscopy. The arrows indicate the refractile cells.

The gas1 null mutation suppresses the lysed-bud phenotype of chs1 null mutants. When chs1 cells are grown in unbuffered minimal medium, numerous small refractile buds can be observed by phase-contrast microscopy (3, 4). The refractile cells have been shown to be lysed cells because of the lack of the Chs1p repair function, which does not counterbalance the acid-induced chitinase activity after cell separation (4, 5). Surprisingly, in the chs1 gas1 double mutant this phenotype could not be observed (Fig. 1B).

Chs3p is responsible for the increase in cell wall chitin levels in the gas1 null mutant. We checked whether deletion of the CHS3 gene might have any effect on the chitin synthesis process in the gas1 mutant. A modification of the CHS3 locus by plasmid targeting had provided the first indication that the phenotype of gas1 cells is severely affected in the double mutant (24). In order to avoid any residual activity of Chs3p, we used a construct in which a large portion of the CHS3 gene had been replaced by the LEU2 gene. A heterozygous chs3 gas1 diploid (LF3) was sporulated, dissection of 20 asci was carried out, and 76 spores were examined. Growth was scored at different times during incubation of the spores at 24°C. Most of the spores gave rise to visible colonies after 48 to 72 h, whereas microcolonies appeared after a further 72 h of incubation (Fig. 2A). The scoring of the phenotype revealed that all of the microcolonies belonged to the His⁺ Leu⁺ class and were therefore chs3 gas1 double mutants. Inoculation of chs3 gas1 spores in liquid YNB-glucose medium had detrimental consequences. The double-mutant cells appeared to be greatly damaged, exhibited an aberrant morphology with many irregularly shaped cells, and were unable to grow. After a prolonged incubation of 4 to 5 days, the double-mutant spores started to grow weakly and in stationary phase rapidly lost viability compared to the wild-type or chs3 spores (Fig. 2B). After CF staining of chs3 gas1 cells only a faint fluorescence was detectable where the primary septum is produced (Fig. 2C). This finding is consistent with the specific loss of Chs3p function. Moreover, many cells were dead and became permeable to the dye (data not shown). The double-mutant cells progressively adapted to growth in liquid medium, and the growth rate defect was gradually suppressed, although not completely. This adaptation did not occur through restoration of the cell wall chitin and is probably due to the selection of second-site suppressors.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Semilethal effects of the double inactivation of CHS3 and GAS1 genes. (A) Representative tetratype tetrads from LF-3 (chs3 gas1 heterozygous diploid) 8 days after dissection. (B) Cell viability assay. Stationary-phase cells from the first inoculum of spores in YNBglucose at 30°C were concentrated to a value of 8 A450. Then, 5 µl of this suspension and four subsequent 10-fold serial dilutions were spotted onto YEPDAT plates. (C) CF staining of four representative spores. Magnifications: upper panels and lower left panel, ×3,200; lower right panel, ×2,000. (D) Effects of CHS3 deletion on chitin levels.

Analysis of Chs3p level in gas1 cells. We studied whether Chs3p might be differentially expressed in gas1 cells with respect to controls. We introduced the gas1::HIS3 mutation into the Y1306 strain, which expresses Chs3p fused at the C-terminal with three HA epitopes (31). By Western blot analysis using anti-HA monoclonal antibodies, we detected the HA-tagged Chs3 protein of 150 kDa in total extracts. Its level slightly decreased in gas1 cells compared to those of the isogenic strain (Fig. 3A). Upon overexposure of the blots additional smeared fragments of about 66 and 60 kDa were detected, and one of about 47 kDa was present only in the mutant (Fig. 3B). The extent of recovery of these fragments was not always the same across a series of experiments, but the pattern was qualitatively reproducible. These results suggest that Ha-tagged Chs3p is more susceptible to proteolytic degradation in the gas1 mutant than in the wild type.

View larger version (74K): [in this window] [in a new window]   FIG. 3.   Western blot analysis of Chs3p in gas1 cells. (A) Total extracts (E; 80 µg) or membrane fractions (P; 40 µg) from Y604, Y1306, or Y1306G (gas1) were subjected to SDS-PAGE. Equal loadings were verified by Ponceau-S staining of the blotted proteins. Blots were immunodecorated with anti-HA monoclonal antibody HA.11. (B) Prolonged exposure of the total extracts shown in panel A. (C) Immunoblot with anti-Gas1p polyclonal antibodies of total extracts shown in panel B. The numbers indicate the molecular masses (in kilodaltons) of the relevant polypeptides.

CSIII activity increases in gas1 mutant cells permeabilized with digitonin. In order to measure CSIII activity under more physiological conditions, whole cells permeabilized with digitonin were used (13). To avoid the high CSI activity level detected under these conditions, we constructed a set of strains that were deleted of the CHS1 gene and were either wild type or mutant for the CHS3 and/or GAS1 genes. A heterozygous diploid (LF5) was constructed by crossing chs1 cells with the suppressed LF3-13D spore (chs3 gas1) and then sporulated. Using this method we were able to detect an increase in CSIII activity in gas1 cells (30.4 ± 5.2 mU/10⁷ cells in the chs1 gas1 mutant spore compared to 9.6 ± 2.3 mU/10⁷ cells in the chs1 spore). No activity was detected in the chs1 chs3 or the chs1 chs3 gas1 control strains.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The aim of this study was to establish the role of the different CSs present in S. cerevisiae in the increase in chitin accumulation in the gas1 mutant. Of the three CS activities, only CSI activity was affected by the presence of the gas1 mutation. The basal activity (measured without adding trypsin) doubled in the gas1 mutant. In contrast to this, the phenotype of the chs1 gas1 double mutant was indistinguishable from the single gas1 mutant in growth properties and morphology. Furthermore, the chitin level was only slightly decreased. We can therefore exclude the possibility that the increase in CSI would be implicated in the chitin response of the mutant. The induction of CSI could be explained in terms of the need to counterbalance the increase in chitinase activity that was unexpectedly found in the mutant. CSI is involved in the repair of damage to the cell wall caused by excessive chitinase activity an acidic pH. The gas1 null mutant could require a higher chitinase activity at the time of cytokinesis due to the higher chitin content in the cell wall. The increase in CSI could subsequently compensate for this increase but would not be essential since the chs1 gas1 double mutant does not show any worsening of the phenotype of gas1 cells. In addition, it is relevant to note that in -factor-treated cells, a well-known condition in which an increase in chitin occurs, CHS1 mRNA was also found to be induced (1), and the expression of myc-Chs1p is approximately threefold higher than in untreated cells (38). Nevertheless, no direct participation of Chs1p in shmoo formation or mating was found, suggesting secondary roles for these changes in the conjugation process (32).

In addition to the lack of any compromise of the growth rate in the chs1 gas1 mutant, we observed a clear suppression of the small-bud-lysis phenotype typical of chs1 cells. Since this phenotypic trait has been ascribed to the loss of the repair function of Chs1p at the birth scar, we interpret this result as being a consequence of the presence of an increased chitin deposition also found in the daughter cells, which could reduce the detrimental effects of the lack of CSI activity after cell division.

Biochemical analysis revealed that CSII and CSIII activities do not change in the mutant. However, we analyzed in detail the phenotypes of yeast cells carrying deletions in the GAS1 and CHS3 genes. The double-mutant cells were severely affected in germination, and the double mutation led to detrimental effects in liquid medium. The progressive adaptation of the cells suggested that suppressors were selected, as was also described for other double null mutants in cell-wall-related genes (for example, kre6 skn1 [29]). Analysis of the chitin content of double-mutant cells clearly demonstrated that the bulk of chitin induced by the presence of the gas1 mutation is produced by CSIII. Since chitinase activity is increased, it can be ruled out that inhibition of degradation of this polysaccharide would contribute to the increase in its accumulation.

We attempted to determine at which level chitin synthesis is regulated in the gas1 mutant. The CHS3 mRNA level did not change in the gas1 mutant, excluding the idea that a transcriptional regulation would be involved. The protein level in total membrane did not change significantly between the mutant and the control. The presence of Chs3p-derived polypeptides of lower mobility in total protein extracts from gas1 cells indicates an increased turnover of the Chs3p full-length protein. For the time being, it is not possible to say whether this effect is associated with a possible activation of the protein, with increased mobilization of the protein through the endocytotic pathway, or whether it might simply be an indirect consequence of pleiotropic effects of the mutation. Preliminary experiments have indicated that the same proteolytic fragments are detectable when the HA-tagged Chs3p is overproduced by the GAL1-GAL10 promoter, indicating that they probably represent endogenous products of proteolysis, which for some reason are slightly stimulated in the mutant.

It is well known that also under other conditions of increase in chitin levels, such as treatment with CF or sporulation, no increase in the Chs3p level is found. By contrast, with -factor treatment of the Chs3p level is sixfold higher, although this does not change the in vitro activity (9, 11). Thus, there is no correlation between the levels of protein and chitin synthesis, probably because other factors, such as posttranslational modifications, mobilization of the enzyme, or interaction with proteins limit the enzymatic activity. It is relevant to note here that a recent study has proposed that the stress-related chitin synthesis probably has a unique targeting and activation mechanism (23). Interestingly, the deposition of chitin by Chs3p in a fks1 mutant was found to be independent from Chs6p (23). Moreover, a putative cell wall sensor protein, Mid2, which functions upstream of the cell integrity pathway, appears to specifically modulate the accumulation of chitin in response to cell wall stress (16).

We measured CSIII activity in cells permeabilized with digitonin. This method was described to measure CSI activity (13); however, the facts that no activity was detected in the chs3 strains and that we obtained reproducible results confirm that it can also be used to detect CSIII activity. Although it is possible that the twofold increase in CSIII activity, detected under these conditions, could be due to different effects of digitonin in the control and the gas1 strains because of their difference in cell wall structure, the results obtained are in agreement with genetic data demonstrating a requirement for CSIII activity in chitin accumulation in the gas1 mutant. The ability to detect such an increase is lost in membrane fractions. This suggests that in the case of the gas1 null mutant a posttranslational modification, an interaction with a regulatory molecule, or a specific ion requirement is not preserved in the membrane preparations used for testing CSIII activity according to the method currently available. In order to understand this posttranslational regulation, experiments to determine the role of other genes involved in the regulation of cell wall biosynthesis in the increase in chitin levels in the gas1 mutant are in progress.