The protein kinase Kic1 affects 1,6-ß-glucan levels in the cell wall of Saccharomyces cerevisiae

Edwin Vinke,a,1, Jack H. Vossene,b,1, Arthur F. J. Ramc,1, Herman van den Ende1, Stephan Brekelmans1, Hans de Nobeld,1 and Frans M. Klis1

Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV, Amsterdam, The Netherlands1

Author for correspondence: Frans M. Klis. Tel: +31 20 525 7834. Fax: +31 20 525 7056. e-mail: klis{at}science.uva.nl


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity. Both over- and underexpressing conditions of KIC1 affected cell wall composition. Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-ß-glucan. When Kic1-deficient cells were crossed with various kre mutants, which also have less 1,6-ß-glucan in their wall, the double mutants displayed synthetic growth defects. However, when crossed with the 1,3-ß-glucan-deficient strain fks1{Delta}, no synthetic growth defect was observed, supporting a specific role for KIC1 in regulating 1,6-ß-glucan levels. Kic1-deficient cells also became highly resistant to the cell wall-degrading enzyme mixture Zymolyase, and exhibited higher transcript levels of the cell wall protein-encoding genes CWP2 and SED1. Conversely, overexpression of KIC1 resulted in increased sensitivity to Zymolyase and in a higher level of 1,6-ß-glucan. Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3. Consistent with this, expression levels of RHO3 correlated with 1,6-ß-glucan levels in the cell wall. Interestingly, expression levels of KIC1 and the MAP kinase kinase PBS2 had opposite effects on Zymolyase sensitivity of the cells and on cell wall 1,6-ß-glucan levels in the wall. It is proposed that Kic1 affects cell wall construction in multiple ways and in particular in regulating 1,6-ß-glucan levels in the wall.

Keywords: KIC1/CWH30, PBS2, RHO3, cell wall synthesis

Abbreviations: HOG, high osmolarity glycerol response; MAPK, mitogen-activated protein kinase; SPB, spindle pole body

e These authors contributed equally to this work.

a Present address: AMC Liver Center, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands.

b Present address: Section of Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318, 1098 SM, Amsterdam, The Netherlands.

c Present address: Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.

d Present address: Genencor International B. V., PO Box 218, 2300 AE, Leiden, The Netherlands.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Saccharomyces cerevisiae is protected from extracellular challenges by its cell wall. These challenges can vary from hypo-osmotic stress to mechanical damage and toxic compounds from other organisms. The yeast cell wall consists of 1,3-ß-glucan, 1,6-ß-glucan, chitin and mannoproteins, which are interconnected in an ordered manner (Klis et al., 2002 ). Cell wall construction and composition are highly dynamic: the composition and structure of the newly formed cell wall are continuously adjusted in response to extracellular conditions, and even to progress in the cell cycle. This indicates that cell wall construction is highly regulated.

The cell wall perturbing agent calcofluor white has been a valuable tool in identifying mutants with a defective cell wall (Roncero et al. 1988 ; Ram et al. 1994 ; Lussier et al. 1997 ; De Groot et al., 2001 ). Analysis of mutants hypersensitive to calcofluor white has resulted in the identification of numerous genes involved in different aspects of cell wall biogenesis (Ram et al., 1995 ; Jiang et al., 1995 , 1996 ; Vossen et al., 1995 ; Van Berkel et al., 1999 ). In addition, screening for mutants resistant to this compound has led to the identification of several genes involved in chitin biosynthesis (Roncero et al., 1988 ).

The KIC1 gene encodes an essential protein kinase, which is involved in cell integrity and morphogenesis. This kinase was identified in a two-hybrid screen with the yeast centrin CDC31. The in vitro kinase activity of Kic1 was found to be dependent on CDC31. However, KIC1 did not share the CDC31 functions in spindle pole body (SPB) duplication, but rather revealed a novel function for CDC31 (Sullivan et al., 1998 ). This was further supported by a mutational analysis of CDC31, which resulted in dissection of the SPB-related functions and KIC1-related functions (Ivanovska & Rose, 2001 ).

The PKC mitogen-activated protein kinase (MAPK) pathway is commonly known as the cell (wall) integrity pathway, although this is certainly not the only MAPK pathway that has an effect on cell wall biosynthesis and composition (Klis et al., 2002 ). For instance, activation of the pheromone response pathway results in the formation of a mating projection and considerable alterations in the cell wall. In this case, at least part of this may be coordinated through the PKC MAPK pathway (Buehrer & Errede, 1997 ). Evidence is also accumulating that the HOG (high osmolarity glycerol response) MAPK pathway plays a role in cell wall assembly. Overexpression of PBS2 – the MAP kinase kinase of this pathway – results in resistance to the K1 killer toxin (Jiang et al., 1995 ) and to laminarinase (Lai et al., 1997 ), an enzyme preparation that contains 1,3-ß-glucanase as its main activity. In addition, mutations in either PBS2 or HOG1 results in calcofluor white resistance (García-Rodriquez et al., 2000 ).

K1 killer toxin is a powerful tool for identifying mutants with defects in 1,6-ß-glucan synthesis and/or assembly (Al-Aidroos & Bussey, 1978 ; Boone et al., 1990 ; Brown et al., 1993 ). This pore-forming protein needs to bind its acceptors (1,6-ß-glucan and Kre1) to perform its lethal action (Bussey, 1991 ; Breinig et al., 2002 ). Mutants defective in the biogenesis of 1,6-ß-glucan hold less of this acceptor, and therefore are resistant to this killer toxin. Several genes have been identified whose gene products mainly localize throughout the secretory pathway (reviewed by Shahinian & Bussey, 2000 ). How the synthesis of 1,6-ß-glucan is regulated is largely unknown.

Here we describe the identification and characterization of CWH30, which is allelic to the previously described KIC1 gene. A Kic1-deficient strain is not only hypersensitive to calcofluor white, but is also resistant to Zymolyase, a cell wall degrading enzyme mixture, indicating that its cell wall is affected. We show that mutation of KIC1 results in K1 killer toxin resistance and decreased levels of 1,6-ß-glucan. Furthermore, KIC1 expression levels were found to correlate with 1,6-ß-glucan levels in the cell wall. Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3 which itself was found to strongly affect 1,6-ß-glucan levels. We propose that KIC1 is involved in regulating the 1,6-ß-glucan levels in the cell.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and media.
The yeast strains used in this study are listed in Table 1. The strains were grown in YPD [1% (w/v) yeast extract, 1% (w/v) Bacto Peptone, 3% (w/v) glucose], YPGal [1% (w/v) yeast extract, 1% (w/v) Bacto Peptone, 3% (w/v) galactose] or in SD [0·17% (w/v) yeast nitrogen base without amino acids and ammonium sulfate, 2% (w/v) glucose, 0·5% (w/v) ammonium sulfate, buffered at pH 6·0 with 1% (w/v) MES] supported with the necessary amino acids, at 28 °C or 37 °C. For solid media, 2% (w/v) Bacto Agar was added. The K1 killer assays were performed in either YPD or SD media, which for this purpose were buffered at pH 4·7 using 3% (w/v) sodium citrate and supplemented with 0·003% methylene blue. Yeast genetics, sporulation and transformation followed established procedures (Sherman & Hicks, 1991 ). Escherichia coli strain DH5{alpha} was used for propagation of all plasmids and was grown in LB medium [1% (w/v) Bacto Tryptone, 1% (w/v) NaCl, 0·5% (w/v) yeast extract]. Yeast extract, Bacto Peptone, Bacto Tryptone, yeast nitrogen base and Bacto Agar were all from Difco Laboratories.


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Table 1. Yeast strains used in this study

 
Strain construction.
Strains JV67 and JV68 were constructed by transforming strains HAB251-15B and AR835 with KIC1 disruption constructs, using the HIS3 and TRP1 markers, respectively. The disruption constructs were created according to Berben et al. (1991) . Primers used for this purpose are listed in Table 2. Correct integration of the disruption constructs was confirmed by Southern analysis, using the 4 kb HindIII fragment from plasmid p14 as a probe (see below). JV80 and JV83 were haploid offspring of JV68 and JV67, respectively. JV142 and JV143 were haploid offspring of JV67 transformed with the PGAL1:KIC1 plasmid (see below). JV144 and JV145 were offspring of JV68 transformed with the PGAL1:KIC1 plasmid. These four haploid strains propagated the PGAL1:KIC1 plasmid even without selective pressure.


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Table 2. Oligonucleotide primers used in this study

 
JV202 was the progeny of the JV143xHAB813 diploid. JV215 was a haploid offspring of the JV143xTR95 diploid. JV220 was a haploid descendant from the JV143xHAB637-1A diploid. The JV168 strain was a haploid descendant from the JV145xAR100 diploid. JV264 was constructed in the FY833 background, using a PBS2 disruption construct with a HIS3 marker. This construct was created as described by Berben et al. (1991) , and correct integration was confirmed by PCR. Primers are all listed in Table 2. JV268 resulted from the JV144xJV264 diploid.

Strains EV116 and EV077 were constructed in the FY834 background, using PCR-generated disruption constructs with the HIS3 marker (Berben et al., 1991 ). Primers are listed in Table 2. Correct integration was confirmed by PCR.

Plasmids, oligonucleotides and recombinant DNA techniques.
For the cloning of the KIC1/CWH30 gene, a YCp50-based genomic library was used (Rose et al. 1987 ). The plasmid that could complement the cwh30-1 mutant was named p14. The 4 kb HindIII fragment containing the complete KIC1 ORF, was subcloned into YEplac195 (Gietz & Sugino, 1988 ) and named p61. Plasmid p62 was isolated from a YEp13-based genomic library based on its ability to complement the cwh30-1 mutant.

DNA handling and manipulation were carried out according to Sambrook et al. (1989) . DNA sequencing was performed as described by Sanger et al. (1977) , using T7 DNA polymerase (Pharmacia). Restriction enzymes, nucleotides, Klenow fragment and alkaline phosphatase were all from Pharmacia. DNA ligase was purchased from Gibco-BRL, SuperTaq polymerase was from HT Biotechnology, Expand high fidelity polymerase was from Boehringer Mannheim and oligonucleotides were from Eurogentec.

Cloning of KIC1 behind the GAL1 promoter.
The Expand high fidelity polymerase was used to create a KIC1 fragment with a 5' XbaI restriction site, followed by three bases of the 5' untranslated region (UTR) of KIC1 and then the KIC1 ORF. The first 500 bases of the 3' UTR were included in this fragment, which was followed by an XhoI restriction site. The primers used for this are listed in Table 2. This PCR generated fragment was digested with XbaI and XhoI, and subsequently cloned into the corresponding sites of the pYEUra3 plasmid (Clontech), resulting in the PGAL1:KIC1 plasmid.

Multicopy suppressor screen.
A high-copy pMA3a-based genomic library [kindly provided by M. Crouzet (University of Bordeaux II, France) & M. F. Tuite (University of Kent, UK)] with a mean insert size of 510 kb was transformed to strain JV141, which contains kic1::TRP1 and carries the PGAL1:KIC1 plasmid. A total of 3500 transformants were replica-plated on selective SD medium containing 100 µg calcofluor white ml-1. Viable colonies were isolated, library plasmids were recovered and retransformed into the JV141 strain. Serial dilutions of transformants of these strains were spotted on selective SD medium containing 50 µg calcofluor white ml-1. The seven plasmids that showed the best suppression were selected for further analysis, and were found to contain five unique inserts (Table 3). Clone 2 contained amongst others the RHO3 gene, which proved to be the gene responsible for the suppression. A 2·2 kb SalI–XhoI fragment containing the RHO3 ORF was removed from clone 2 and cloned into a YEplac181 vector (Gietz & Sugino, 1988 ) resulting in the pEV021 plasmid. The remaining part of clone 2 was religated, and the resulting plasmid lost the ability to suppress the kic1 mutant, whereas plasmid pEV021 retained the ability to suppress the kic1 mutant. Clones 11 and 13 contained an identical insert, and clone 11 was subjected to further analysis. A 2·4 kb XhoI–SalI fragment (the SalI restriction site was located in the pMA3a plasmid, 0·3 kb from the BamHI site which was used for the insertion of the genomic fragments) was cloned into YEplac181 resulting in plasmid pEV017, which contained the MSG5 ORF and could suppress the kic1 mutant. Clone 23 and 44 also showed an identical insert, in which the STB3 gene was the only complete ORF. In addition, 2·3 kb of the 3' end of the SEC7 coding sequence was present in this insert. This 2·3 kb fragment was removed by an XhoI/SalI digest of clone 44, using the XhoI site in the insert and the SalI site in the pMA3a vector, 0·3 kb from the insert. The resulting plasmid (pEV020) retained the ability to suppress the kic1 mutant.


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Table 3. High copy suppressors of KIC1

 
Phenotypic screens.
Calcofluor white sensitivity was analysed as described previously (Ram et al. 1998 ). Precultures were concentrated or diluted to OD530 10. Subsequently, ten-fold dilution series were made of which 4 µl of each dilution was spotted onto YPD or SD plates containing 0, 10 and 50 µg calcofluor white ml-1. Plates were incubated for 3 days at 28 °C.

K1 killer toxin sensitivity was measured using the halo assay (Brown et al. 1994 ), with some modifications. Precultures were concentrated or diluted to OD530 10 and 45 µl cells were seeded in 13 ml killer agar medium. On the surface, 5 µl of a dilution series of 100, 5x10-1, 10-1, 5x10-2 and 10-2 of isolated killer toxin was spotted. Killer toxin was isolated according to Brown et al. (1994) . In short, the medium of the K1 killer toxin-producing strain was concentrated 1000-fold by ultrafiltration using a 10 kDa Amicon filter and was used as such in the halo assay. In all experiments, samples and controls were treated with toxin from the same isolation. Plates were incubated for 4–6 days at 20 °C. The diameters of the haloes were measured for each toxin dilution. Relative apparent sensitivities were calculated according to Reneke et al. (1988) . In short, the diameter of the growth inhibition zone is proportional to the logarithm of the K1 killer toxin dose applied in the centre of the zone. Plotting these parameters against each other allows estimation of the dose required to produce an inhibition zone of a given diameter. The ratio of this dose estimated for wild-type cells divided by the dose found for mutant cells is termed the ’relative apparent sensitivity’.

Zymolyase sensitivities were measured essentially as described by De Nobel et al. (1990) . Yeast strains were grown to equivalent optical densities, and 1 OD530 unit was taken for analysis. Cells were washed once and resuspended in 900 µl 10 mM Tris/HCl pH 7·5. The OD530 was followed for 1 h after the addition of 100 µl Zymolyase 20T (10 mg ml-1 in 10 mM Tris/HCl pH 7·5; Zymolyase 20T was from Kirin Brewery).

Isolation of cell walls.
Cell walls from cells grown to early exponential phase were isolated according to Van Rinsum et al. (1991) . Walls were extracted twice in 50 mM Tris/HCl pH 7·4, 150 mM NaCl, 5 mM EDTA, 2% (w/v) SDS, 0·3% (v/v) ß-mercaptoethanol for 5 min at 100 °C. Walls were extensively washed in distilled water and subsequently freeze-dried.

Determination of 1,6-ß-glucan levels.
The levels of 1,6-ß-glucan in the alkali-insoluble cell wall fraction were basically determined in accordance with Brown et al. (1994) . Essentially, cells were grown for 24 h at 28  °C to stationary phase, harvested and washed twice in distilled water. Samples were split up into four aliquots and three fractions were each three times extracted in 3% (w/v) NaOH at 75 °C for 1 h. The remaining fraction was freeze-dried and used for the determination of the cell dry weight. The alkali-insoluble material was washed twice in 100 mM Tris/HCl, pH 7·5 and then washed in distilled water. The pellet was resuspended in 10 mM Tris/HCl, pH 7·5, 10% (v/v) glycerol, 1 mg Zymolyase 100T ml-1 (Kirin Brewery) and incubated at 37 °C overnight. Following incubation, samples were centrifuged at 14000 g for 5 min, and the supernatant was dialysed against distilled water using a Spectra/POR 3 (6000–8000 molecular mass cut-off) dialysis membrane. The glucose content of the residue – 1,6-ß-glucan and chitin – was determined by the phenol/sulfuric acid method (Dubois et al., 1956 ). In later experiments, the procedure was as follows. Cells were grown for 24 h in YPD to early stationary phase, washed twice in 30 mM Tris/HCl, 1 mM EDTA, pH 7·4, and collected in five aliquots. Two were freeze-dried to quantify the cell dry weight, whereas the three other samples were resuspended in 600 µl 50 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA, pH 7·4 and broken with glass beads. Walls were collected and extracted twice in 50 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA, 2% SDS, 40 mM ß-mercaptoethanol, pH 7·4, at 100  °C for 5 min. Walls were extensively washed in distilled water and suspended in 10 mM Tris/HCl, pH 7·5, with 1 mg Zymolyase 100T ml-1 (w/v) (Kirin Brewery) and incubated at 37 °C for 16 h. Following incubation, solutions were centrifuged for 5 min at 15000 g and the supernatant was dialysed against distilled water using a Spectra/POR 3 (3500 molecular mass cut-off) dialysis membrane. The residue was hydrolysed in 2 M trifluoroacetic acid at 100 °C for 4 h, freeze-dried, and subsequently glucose levels were determined using the D-glucose oxidase assay.

Northern analysis.
RNA was isolated from early exponential phase cells with hot acidic phenol (Ausubel et al., 1998 ). Fifteen microgrammes of RNA was loaded on a 1% agarose gel containing 2·4% formaldehyde. Following electrophoresis, the RNA was blotted onto Hybond-N+ (Amersham) through capillary paper transfer and UV cross-linked to the membrane.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CWH30 is allelic to KIC1
The cwh30-1 mutant was isolated in a general screen for cell wall mutants (Ram et al., 1994 ), based on their hypersensitivity to the cell wall perturbing agent calcofluor white. The gene mutated in cwh30-1 was identified by functional complementation of the hypersensitivity to calcofluor white, using a YCp50-based genomic library. Out of 40000 transformants, only 15 were able to grow in the presence of 50 µg calcofluor white ml-1. The plasmids from these transformants were recovered and upon retransformation only five were able to complement the calcofluor white hypersensitivity of the cwh30-1 mutant. All five complementing plasmids contained the same genomic insert (data not shown). The complementing activity of this insert resided in a 4·4 kb fragment that remained after HindIII truncation of the fragment, but was lost after further truncation to 4·2 kb with XbaI. The 4·4 kb fragment contained the YHR102w ORF, which previously has been named KIC1 for kinase interacting with Cdc31 (Sullivan et al., 1998 ). The XbaI truncation removed part of the 5' upstream region of the KIC1 gene, which seemed essential for complementation of the mutant. Additional evidence that CWH30 was allelic to KIC1 came from the resulting diploid of the cwh30-1 mutant and the kic1 disruption strain JV143. The JV143 strain has been disrupted for kic1 but is supported by a plasmid-borne PGAL1:KIC1 fusion. When the diploid was cultured on glucose-containing medium, the calcofluor white hypersensitivity caused by the recessive cwh30-1 mutation was not complemented (not shown), indicating that CWH30 is allelic to KIC1.

Disruption of KIC1
To generate a KIC1 knock-out mutant, the KIC1 gene was replaced by the TRP1 marker in the diploid AR835 wild-type strain, and by the HIS3 marker in the diploid HAB251-15B wild-type strain. For both heterozygous diploids (JV68 and JV67), tetrad analysis resulted in two wild-type colonies and two very poorly growing mutant colonies (data not shown). The poor growth of the kic1{Delta} strain could not be suppressed by osmotic support of the medium (data not shown). In addition our results show that mutant spores failed to germinate on medium with galactose as the sole carbon source (data not shown). Microscopic analysis showed that kic1{Delta} cells were enlarged and round, forming large clumps, indicating a cell separation defect which is in accordance with previously described phenotypes of kic1 mutants (data not shown). In addition, the cells were very sensitive to pipetting and centrifugation. Fig. 1 shows that the kic1{Delta} mutant displayed calcofluor white hypersensitivity. This was similar to that of the cwh30-1 mutant (Ram et al., 1994 ; data not shown). The morphological defects, the hypersensitivity to calcofluor white and the fragility of the cells are all in agreement with a role in cell wall integrity for KIC1.



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Fig. 1. Both kic1{Delta} and PGAL1:KIC1 strains are calcofluor white hypersensitive. Tenfold serial dilutions of precultures of wild-type (FY834), kic1{Delta} (JV80) and PGAL1:KIC1 (JV144) were spotted onto YEPD and YEPD containing 50 µg calcofluor white ml-1. Cells were grown for 2 days at 28  °C. JV80 grows faster than the original kic1{Delta} strain, probably because it has acquired a second-site suppressor mutation.

 
Construction of PGAL1:KIC1
The kic1{Delta} cells were not only very fragile, but they also had low transformation efficiencies, and even heterozygous diploids sporulated very poorly. In addition, kic1{Delta} cells occasionally developed second-site suppressor mutations. To circumvent the technical problems of working with a kic1{Delta} strain, a repressible KIC1 allele was constructed by placing it under the control of the GAL1 promoter in the pYEUra3 plasmid. The activity of the PGAL1:KIC1 construct was confirmed by its ability to complement the kic1{Delta} growth defect (data not shown). This plasmid was transformed into a diploid heterozygous for kic1 and this strain was sporulated. When germinated on YPGal medium, spores lacking the endogenous KIC1 gene grew indistinguishably from spores with the endogenous KIC1 gene (data not shown). However, when germinated on YEPD medium, the spores lacking the endogenous KIC1 gene had a noticeable growth defect, albeit not as severe as a kic1{Delta} strain without the PGAL1:KIC1 plasmid (data not shown). This suggests that under glucose repression conditions there still is some expression of the KIC1 gene. Under these conditions however, cells still display calcofluor white hypersensitivity and other defects in cell wall integrity (see Figs 1, 2 and 4). The kic1{Delta} mutant carrying the PGAL1:KIC1 plasmid will be referred to as the PGAL1:KIC1 mutant. Interestingly, when grown on nonselective media (e.g. YEPD), the kic1{Delta} strain was not cured of the PGAL1:KIC1 plasmid. In addition, no viable colonies were found when PGAL1:KIC1 cells were put on media containing 5'-FOA (data not shown).



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Fig. 2. KIC1 gene dosage affects cell wall composition. (a) Cells from wild-type ({square}), kic1{Delta} ({diamondsuit}), PGAL1:KIC1 ({bullet}), and wild-type with 2µ KIC1 ({triangleup}) were precultured, washed and incubated in the presence of Zymolyase 20T. (b) Isolated cell walls of wild-type ({square}), kic1{Delta} ({diamondsuit}), PGAL1:KIC1 ({bullet}), and wild-type with 2µ KIC1 ({triangleup}) were incubated in the presence of Zymolyase 20T. (c) KIC1 and PBS2 have opposing effects on Zymolyase sensitivity. Wild-type ({square}), PGAL1:KIC1 ({diamondsuit}), pbs2{Delta} ({bullet}) and PGAL1:KIC1 pbs2{Delta} ({triangleup}) cells were precultured, washed and incubated in the presence of Zymolyase 20T. (d) mRNA levels of cell wall protein encoding genes SED1, CWP2 and CWP1 in wild-type and the PGAL1:KIC1 mutant. Actin mRNA levels are shown as a loading reference.

 


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Fig. 4. KIC1 displays synthetic growth phenotypes with several kre mutants. The PGAL1:KIC1 mutant strain was crossed with kre6{Delta}, kre9{Delta}, kre1{Delta} and fks1{Delta}. Tetrads were dissected on YPGal, and the double and single mutants were tested for growth on YEPD in tenfold serial dilutions.

 
PGAL1:KIC1 mutant cells are resistant to Zymolyase
Zymolyase is a commercial enzyme preparation with both 1,3-ß-glucanase and protease activities, which can be used to assay differences in cell wall structure and composition (De Nobel et al., 1990 ; Ram et al., 1994 ; De Groot et al., 2001 ). Wild-type cells were sensitive to treatment with Zymolyase 20T, whereas Kic1-deficient cells were resistant (Fig. 2a). Conversely, KIC1 expressed from a high-copy plasmid resulted in hypersensitivity to Zymolyase (Fig. 2a). The major substrate for Zymolyase, i.e. 1,3-ß-glucan, forms the inner layer of the cell wall. The outer layer consists of mannoproteins, which in intact cells limits the permeability to macromolecules (Zlotnik et al., 1984 ; De Nobel et al., 1990 ). It is conceivable that (at least part) of the Zymolyase sensitivity of intact cells can be attributed to changes in the protein outer layer and thus in cell wall permeability. One way to investigate this is to compare the Zymolyase sensitivity of intact cells and isolated walls, in which the inner layer is now exposed to Zymolyase. The increase in Zymolyase resistance of cell walls from the PGAL1:KIC1 mutant was much less dramatic compared to intact cells (Fig. 2b), but was still significant. Cell walls derived from a strain with high copy numbers of KIC1 now were more resistant to Zymolyase than wild-type (Fig. 2b). Taken together, these data suggest that KIC1 affects cell wall permeability. This might be caused by altered mannoprotein levels (see below) and possibly to some extent altered glucan levels.

Expression of some cell wall proteins is altered in the kic1 mutant
The mRNA expression levels of some known cell wall proteins in the PGAL1:KIC1 mutant showed increased levels of both SED1 and CWP2 in comparison to wild-type levels (Fig. 2d). CWP1 mRNA levels, however, remained unaffected (Fig. 2d). Interestingly, both sed1{Delta} and cwp2{Delta} mutants are more sensitive to Zymolyase than wild-type (Van der Vaart et al., 1995 ; Shimoi et al., 1998 ). Overexpression of SED1 resulted in Zymolyase resistance (Shimoi et al., 1998 ). This strongly suggests that at least part of the Zymolyase resistance of the PGAL1:KIC1 mutant is caused by increased levels of SED1 and CWP2.

The PGAL1:KIC1 mutant is more resistant to K1 killer toxin
K1 killer toxin has been a powerful tool for identifying genes involved in 1,6-ß-glucan synthesis. Lower sensitivity to this toxin is generally associated with decreased levels of 1,6-ß-glucan in the cell wall, which is a receptor for the toxin (Boone et al., 1990 ; Brown et al., 1993 ). K1 killer toxin sensitivities were compared using the halo assay. Fig. 3(a) shows the halo assays of some strains tested, to exemplify the difference in sensitivity. The K1 killer toxin sensitivity of various strains compared to their corresponding wild-type, is depicted graphically in Fig. 3(b). Note that the overexpression studies were performed on supplemented SD-based media, as opposed to the other strains which were tested on YEPD media. The halo assays performed on SD-based media commonly showed larger haloes than on YEPD media, but the sensitivities compared to wild-type remained consistent.



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Fig. 3. KIC1 expression levels affect K1 killer toxin sensitivity. Relative apparent sensitivity to the K1 killer toxin was determined as described in Methods. (a) Some examples of the halo plate assay. Tenfold dilution series of isolated K1 killer toxin were spotted onto seeded plates. After 4 days at 20 °C, halo diameters were measured and relative apparent sensitivities were calculated. (b) Relative apparent sensitivities of several strains. Strains displayed in the upper panel were grown on SD-based medium with selective amino acid mix. Strains displayed in the lower panel were grown in YEPD-based medium.

 
The sensitivity of the kic1{Delta} mutant (JV83) to the K1 killer toxin was very low (not shown). The PGAL1:KIC1 mutant (JV142) retained a low sensitivity to the toxin, although not to the same extent as the deletion mutant. Conversely, overexpression of KIC1 in wild-type (JV39 with plasmid p62) resulted in an increased sensitivity to the K1 killer toxin (see Fig. 3b). The KIC1 gene dosage effect on K1 killer toxin sensitivity might reflect altered 1,6-ß-glucan levels.

KIC1 affects cell wall 1,6-ß-glucan levels
To determine if the changes in K1 killer toxin sensitivity can be attributed to changes in 1,6-ß-glucan levels, the alkali-insoluble cell wall fraction was analysed. As expected, the kic1{Delta} strain showed a marked decrease in 1,6-ß-glucan levels (Table 4). Consistently, overexpression of KIC1 resulted in a slight increase in 1,6-ß-glucan levels (Table 4). These data are in agreement with the data from the K1 killer toxin assay. The gene dosage effect of KIC1 on 1,6-ß-glucan levels suggests a role for KIC1 in 1,6-ß-glucan deposition.


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Table 4. KIC1 gene dosage affects 1,6-ß-glucan levels

 
The PGAL1:KIC1 mutant displays synthetic growth defects with kre mutants
The proposed role of KIC1 in 1,6-ß-glucan deposition in the cell wall is further supported by the strong genetic interaction occurring between a PGAL1:KIC1 mutant and kre mutants, which have lowered 1,6-ß-glucan levels in the cell wall. The PGAL1:KIC1 mutant was crossed with kre6{Delta}, kre9{Delta} and kre1{Delta} mutants, and the resulting diploids were sporulated. Double mutants were selected on galactose-containing media and the growth phenotypes were analysed on glucose-containing media. kic1 showed a strongly enhanced growth defect with both kre6{Delta} and kre9{Delta}. Interestingly, when the PGAL1:KIC1 mutant was crossed with kre1{Delta}, enhancement of the growth defect was minor (Fig. 4). In contrast, KIC1 did not show an enhanced growth defect with FKS1, a mutant impaired in 1,3-ß-glucan synthesis (Fig. 4). This suggests that the growth defects of Kic1-deficient cells are to a large extent related to the biogenesis of 1,6-ß-glucan, and offers further support for the notion that KIC1 is involved in the regulation of 1,6-ß-glucan biogenesis.

Multicopy suppressor screen of the calcofluor white sensitivity of PGAL1:KIC1 mutant
To further elucidate the regulatory function of KIC1 in cell wall biosynthesis, we introduced a high-copy pMA3a-based genomic library into the PGAL:KIC1 mutant strain JV141. Three thousand five hundred transformants were replica-plated on selective SD medium containing 100 µg calcofluor white ml-1. Plasmids isolated from the surviving colonies were retransformed into JV141 and the original cwh30-1 point mutant to ensure plasmid-dependent suppression of the calcofluor white phenotype. Serial dilutions of cells were spotted onto selective SD containing 50 µg calcofluor white ml-1. Partial sequence analysis of the inserts of the seven strongest suppressors revealed five separate genomic regions (Table 3). Several clones were subjected to subcloning and deletion experiments, identifying the genes responsible for the (partial) suppression of the calcofluor white hypersensitivity of the PGAL:KIC1 mutant (Fig. 5a). These consisted of (1) STB3, a gene encoding a two-hybrid interactor with the Sin3p protein (Kasten & Stillman, 1997 ), (2) MSG5, encoding a dual-specificity protein phosphatase involved in the pheromone adaptation response (Doi et al., 1994 ; Zhan et al., 1997 ) and, in addition, capable of influencing the phosphorylation state of Slt2p (Watanabe et al., 1995 ; Martín et al., 2000 ) and (3) RHO3, encoding a small G-protein, which is known to be involved in bud formation and growth, and organization of the actin cytoskeleton and exocytosis (Matsui & Toh-e, 1992b ; Imai et al., 1996 ; Robinson et al., 1999 ; Adamo et al., 1999 ). JV141 cells were transformed with either of the before-mentioned plasmids and grown on selective medium containing 5'-FOA to determine if the presence of these multicopy suppressor plasmids allowed the loss of the PGAL:KIC1 plasmid. No viable colonies were found, indicating that none of these plasmids could restore (all of) the essential function(s) of KIC1 (data not shown). RHO3 was chosen for further analysis.



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Fig. 5. Multicopy suppressors of the calcofluor white hypersensitivity of the PGAL1:KIC1 mutant. (a) Tenfold serial dilutions of wild-type (FY69)+ YEplac181, PGAL:KIC1 (JV141)+YEplac181, PGAL1:KIC1 (JV141)+2µ RHO3 (pEV021), PGAL1:KIC1 (JV141)+2µ MSG5 (pEV017), PGAL1:KIC1 (JV141)+pMA3a and PGAL1:KIC1 (JV141)+pMA3a-STB3 (pEV020) were spotted onto selective SD media with or without 50 µg calcofluor white ml-1. (b) Tenfold dilution series of wild-type (FY834), rho3{Delta} (EV116), rho4{Delta} (EV077), kic1{Delta}+PGAL1:KIC1 (JV144) and kic1{Delta} (JV80) were spotted onto YPD with or without 50 µg calcofluor white ml-1. Cells were grown for 2–3 days at 30 °C.

 
The rho3 deletant is impaired in cell wall biogenesis
RHO3 was disrupted and the cells were tested for their sensitivity to calcofluor white. Although not to the same extent as Kic1-deficient cells, rho3{Delta} cells showed increased sensitivity to calcofluor white, indicating a defect in cell wall biogenesis (Fig. 5b). In the absence of RHO3, the functionally related RHO4 can suppress the growth defect when overexpressed (Matsui & Toh-e, 1992a ), whereas the rho3 rho4 double mutant is inviable. The double mutant also proved inviable in our genetic background (data not shown). Cells disrupted for RHO4 did not show an increased sensitivity to calcofluor white (Fig. 5b). In addition, overexpression of RHO4 did not restore the calcofluor white hypersensitivity of the PGAL1:KIC mutant (data not shown). This suggests that the function that is suppressed by overexpression of RHO3 in the PGAL1:KIC1 mutant, is not shared by RHO4.

Overexpression of the RHO3 gene in the PGAL1:KIC1 mutant produced an increase in sensitivity to the K1 killer toxin, in contrast to overexpression of the RHO4 gene (Fig. 6a). Deletion of RHO3 resulted in a decrease in K1 killer toxin sensitivity (Fig. 6b), and a decrease of about 40% in cell wall 1,6-ß-glucan (3·3% of cell dry weight in wild-type cells compared to 1·9%). In contrast, deletion of RHO4 had no effect on K1 killer toxin sensitivity (Fig. 6b). Overexpression of the RHO3 gene in wild-type also resulted in an increase in K1 killer toxin sensitivity (Fig. 6c). However, when RHO4 was overexpressed in wild-type, cells displayed a decrease in killer sensitivity (Fig. 6c). A possible explanation for this is that high levels of Rho4 might compete with Rho3. Taken together, the observations suggest that the levels of RHO3 influence the level of cell wall 1,6-ß-glucan and thus the sensitivity of the cells to the K1 killer toxin. This further implicates KIC1 in 1,6-ß-glucan biogenesis, evidently for a part through RHO3.



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Fig. 6. RHO3 gene dosage affects K1 killer toxin sensitivity. Relative apparent sensitivity to the K1 killer toxin was determined as described in Methods. (a) kic1{Delta}+PGAL1:KIC1 (JV144)+YEplac181, kic1{Delta}+PGAL1:KIC1 (JV144)+2µ RHO3 and kic1{Delta}+PGAL1:KIC1 (JV144)+RHO4 are compared. (b) Wild-type (FY834), rho3{Delta} (EV116), rho4{Delta} (EV077) and kic1{Delta}+PGAL1:KIC1 (JV144) was compared. (c) Wild-type (FY834)+YEplac181, wild-type (FY834)+2µ RHO3 and wild-type (FY834)+2µ RHO4 were compared.

 
KIC1 antagonizes the PBS2–HOG1 pathway in cell wall biogenesis
Cell wall phenotypes caused by overexpression of the MAP kinase kinase PBS2 from the HOG pathway show a remarkable resemblance to some of the phenotypes shown by PGAL1:KIC1 mutant cells. Similar to cells that overexpress PBS2 (Jiang et al., 1995 ; Lai et al., 1997 ), PGAL1:KIC1 mutant cells were less sensitive to K1 killer toxin, and showed a modest decrease in 1,6-ß-glucan levels (Fig. 3b; Table 4). The reverse phenotypes were found in the pbs2{Delta} mutant and cells overexpressing KIC1, i.e., hypersensitivity to the K1 killer toxin (Jiang et al., 1995 ; Lai et al., 1997 ; Fig. 3b) and an increase in 1,6-ß-glucan levels (Jiang et al., 1995 ; Table 4). A PGAL1:KIC1 pbs2{Delta} double mutant was generated and this strain displayed an intermediate sensitivity to K1 killer toxin (Fig. 3b). KIC1 deficiency and deletion of PBS2 also reversely affected the sensitivity of the cells to cell wall degrading enzymes (Fig. 2c), whereas the double mutant displayed an intermediate phenotype. In summary, our observations in combination with data from the literature indicate that KIC1 and PBS2 have opposing roles in cell wall biogenesis.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The KIC1 gene was originally identified in a two-hybrid screen interacting with CDC31, which encodes yeast centrin. KIC1, member of the PAK1/Ste20 kinase family, encodes a 116 kDa protein which interacts in vivo with CDC31 and has in vitro kinase activity dependent on CDC31. However, it was shown that KIC1 did not play a role in the SPB duplication function of CDC31. KIC1 rather contributes to its function in cell integrity and morphogenesis, since several kic1 mutants showed aberrant cell wall morphology, wide bud necks, failure in cell separation and cell lysis (Sullivan et al., 1998 ). Previously, cells deleted for KIC1 were found to be inviable (Sullivan et al., 1998 ). However, differences in genetic backgrounds might explain the (albeit very poor) viability of the kic1{Delta} strain in our backgrounds.

The cwh30/kic1 mutant was originally discovered because it was hypersensitive to the cell wall perturbing agent calcofluor white (Ram et al., 1994 ). Interestingly, mutant cells were also resistant to the K1 killer toxin, indicating that their walls contained less 1,6-ß-glucan. As KIC1 encodes a protein kinase, this marked it as a potential regulator of 1,6-ß-glucan biogenesis. The following evidence supports this. KIC1 deficiency resulted in decreased sensitivity to the K1 killer toxin and lower levels of 1,6-ß-glucan in its walls. Conversely, overexpression of KIC1 resulted in increased sensitivity to K1 killer toxin and elevated levels of 1,6-ß-glucan. In addition, the PGAL1:KIC1 mutant crossed with various kre mutants resulted in double mutants with a synthetic growth defect, whereas a combination of the PGAL1:KIC1 mutant with the 1,3-ß-glucan impaired mutant fks1{Delta} did not result in a synthetic growth defect. Taken together, these results imply a role for KIC1 in the regulation of 1,6-ß-glucan biogenesis. Finally, the expression levels of RHO3 correlated with the sensitivity to K1 killer toxin and thus probably with 1,6-ß-glucan levels. In addition, the mutant phenotypes of Kic1-deficient cells were partially suppressed by overexpression of RHO3. This is consistent with the postulated role for KIC1 in regulating 1,6-ß-glucan biogenesis.

Besides the defects in 1,6-ß-glucan deposition, the Kic1-deficient cells also displayed resistance to the cell wall-degrading enzyme mixture Zymolyase, whereas overexpression of KIC1 resulted in hypersensitivity to Zymolyase. This effect may partly be caused by changes in the cell wall mannoprotein composition, since the external layer of mannoproteins in the cell wall determines the porosity and needs to be removed for efficient cell wall degradation (Zlotnik et al., 1984 ; De Nobel et al., 1990 ). There are two lines of evidence that confirm this notion. First, when cell walls were isolated prior to Zymolyase treatment, walls from Kic1-deficient cells showed a much less pronounced resistance to Zymolyase compared to wild-type cell walls. Second, two known cell wall protein encoding genes, SED1 and CWP2, were found to be upregulated in the PGAL1:KIC1 mutant. Interestingly, overexpression of SED1 leads to Zymolyase resistance (Shimoi et al., 1998 ), and, reversely, deletion of both SED1 and CWP2 results in increased sensitivity to Zymolyase (Van der Vaart et al., 1995 ; Shimoi et al., 1998 ). Increased expression of these genes in the Kic1-deficient mutants might thus at least in part explain the resistance to Zymolyase. By which mechanism the expression of these cell wall proteins is induced is unknown. It might reflect the induction of a cell wall repair mechanism as the result of the decrease in 1,6-ß-glucan. (Popolo & Vai, 1999 ; Kapteyn et al., 1999 ; Klis et al., 2002 ), but this normally includes induction of CWP1 expression (Terashima et al., 2000 ; Kapteyn et al., 2001 ). However, CWP1 expression was not induced in the PGAL1:KIC1 mutant. Alternatively, Kic1 might have a regulatory role in multiple cell wall biosynthetic steps and not only in 1,6-ß-glucan biogenesis.

The identification of RHO3 as a multicopy suppressor of the PGAL1:KIC1 mutant suggests that RHO3 might be a downstream target of KIC1 in cell wall biogenesis. This is supported by the RHO3 gene dosage relationship with K1 killer toxin sensitivity and the reduction of 1,6-ß-glucan levels in the rho3{Delta} mutant. Whereas RHO4 contributes to some of the known functions of RHO3 (Matsui & Toh-e, 1992a ; Imai et al., 1996 ), the effects on cell wall biogenesis are not shared by RHO4.

Evidence is accumulating that suggests a role for the PBS2HOG1 pathway in cell wall construction. Overexpression of PBS2 causes resistance to laminarinase, a cell wall degrading enzyme mixture (Lai et al., 1997 ), and deletion results in hypersensitivity (Fig. 2b; Alonso-Monge et al., 2001 ). In addition, PBS2 overexpression results in resistance to the K1 killer toxin and a decrease in cell wall 1,6-ß-glucan levels (Jiang et al., 1995 ). Also, under noninducing conditions the HOG pathway contributes to the maintenance of cell wall architecture (García-Rodriquez et al., 2000 ). Furthermore, overexpression of some cell wall related genes suppress the hyperosmosensitive phenotype of a ste11 ssk2 ssk22 mutant. These include LRE1 and HLR1, which can also suppress the osmosensitivity and the glucanase sensitivity of both pbs2{Delta} and hog1{Delta} mutants (Alonso-Monge et al., 2001 ). Our report further supports a role for PBS2 in cell wall biogenesis. The PGAL1:KIC1 mutant and the pbs2{Delta} single mutant cells had reverse phenotypes in both K1 killer toxin and Zymolyase sensitivities. In the PGAL1:KIC1 pbs2{Delta} double mutant an intermediate phenotype was observed (Fig. 3b). These results suggest that KIC1 and PBS2 play opposing roles in cell wall biogenesis. The mechanism by which KIC1 and PBS2 counteract each other remains obscure. In summary, the protein kinase Kic1 is involved in regulating cell wall construction in multiple ways and seems to have a specific role in controlling 1,6-ß-glucan levels.


   ACKNOWLEDGEMENTS
 
The authors thank all members of the Klis lab for helpful comments and suggestions. Dr Marco Siderius is thanked for many stimulating discussions. We are indebted to Dr Howard Bussey for sharing strains and plasmids, and to Drs Crouzet and Tuite for sharing their pMA3a genomic library.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adamo, J. E., Rossi, G. & Brennwald, P. (1999). The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity. Mol Biol Cell 10, 4121-4133.[Abstract/Free Full Text]

Al-Aidroos, K. & Bussey, H. (1978). Chromosomal mutants of Saccharomyces cerevisiae affecting the cell wall binding site for killer factor. Can J Microbiol 24, 228-237.[Medline]

Alonso-Monge, R., Real, E., Wojda, I., Bebelman, J. P., Mager, W. H. & Siderius, M. (2001). Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects. Mol Microbiol 41, 717-730.[Medline]

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidmen, J. G., Smith, J. A. & Struhl, K. (editors) (1998). Current Protocols in Molecular Biology. New York: Greene Publishing & Wiley Interscience.

Berben, G., Dumont, J., Gilliquet, V., Bolle, P. A. & Hilger, F. (1991). The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7, 475-477.[Medline]

Boone, C., Sommer, S. S., Hensel, A. & Bussey, H. (1990). Yeast KRE genes provide evidence for a pathway of cell wall beta-glucan assembly. J Cell Biol 110, 1833-1843.[Abstract]

Breinig, F., Tipper, D. J. & Schmitt, M. J. (2002). Kre1p, the plasma membrane receptor for the yeast K1 viral toxin. Cell 108, 395-405.[Medline]

Brown, J. L. & Bussey, H. (1993). The yeast KRE9 gene encodes an O glycoprotein involved in cell surface beta-glucan assembly. Mol Cell Biol 13, 6346-6356.[Abstract]

Brown, J. L., Kossaczka, Z., Jiang, B. & Bussey, H. (1993). A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-- 6)-beta-glucan synthesis. Genetics 133, 837-849.[Abstract/Free Full Text]

Brown, J. L., Roemer, T., Lussier, M., Sdicu, A. M. & Bussey, H. (1994). The K1 killer toxin: molecular and genetic applications to secretion and cell surface assembly. In Molecular Genetics of Yeast: a Practical Approach , pp. 217-232. Edited by J. R. Johnston. Oxford:IRL Press at Oxford University Press.

Buehrer, B. M. & Errede, B. (1997). Coordination of the mating and cell integrity mitogen-activated protein kinase pathways in Saccharomyces cerevisiae. Mol Cell Biol 17, 6517-6525.[Abstract]

Bussey, H. (1991). K1 killer toxin, a pore forming protein from yeast. Mol Microbiol 5, 2339-2343.[Medline]

Bussey, H., Saville, D., Hutchins, K. & Palfree, R. G. (1979). Binding of yeast killer toxin to a cell wall receptor on sensitive Saccharomyces cerevisiae. J Bacteriol 140, 888-892.[Medline]

De Nobel, J. G., Klis, F. M., Priem, J., Munnik, T. & Van Den Ende, H. (1990). The glucanase-soluble mannoproteins limit cell wall porosity in Saccharomyces cerevisiae. Yeast 6, 491-499.[Medline]

De Groot, P. W. J., Ruiz, C., Vazquez de Aldana, C. R. & 14 other authors (2001). A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp Funct Genomics 2, 124–142.

Doi, K., Gartner, A., Ammerer, G., Errede, B., Shinkawa, H., Sugimoto, K. & Matsumoto, K. (1994). MSG5, a novel protein phosphatase promotes adaptation to pheromone response in S. cerevisiae. EMBO J 13, 61-70.[Abstract]

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956). Colometric method for determination of sugars and related substances. Anal Chem 28, 350-356.

García-Rodriguez, L. J., Durán, A. & Roncero, C. (2000). Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions. J Bacteriol 182, 2428-2437.[Abstract/Free Full Text]

Gietz, R. D. & Sugino, A. (1988). New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527-534.[Medline]

Imai, J., Toh-e, A. & Matsui, Y. (1996). Genetic analysis of the Saccharomyces cerevisiae RHO3 gene, encoding a rho-type small GTPase, provides evidence for a role in bud formation. Genetics 142, 359-369.[Abstract/Free Full Text]

Ivanovska, I. & Rose, M. D. (2001). Fine structure analysis of the yeast centrin, Cdc31p, identifies residues specific for cell morphology and spindle pole body duplication. Genetics 157, 503-518.[Abstract/Free Full Text]

Jiang, B., Ram, A. F. J., Sheraton, J., Klis, F. M. & Bussey, H. (1995). Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2 action in a pathway that includes modulation of EXG1 transcription. Mol Gen Genet 248, 260-269.[Medline]

Jiang, B., Sheraton, J., Ram, A. F. J., Dijkgraaf, G. J., Klis, F. M. & Bussey, H. (1996). CWH41 encodes a novel endoplasmic reticulum membrane N-glycoprotein involved in beta 1,6-glucan assembly. J Bacteriol 178, 1162-1171.[Abstract]

Kapteyn, J. C., Van Egmond, P., Sievi, E., Van Den Ende, H., Makarow, M. & Klis, F. M. (1999). The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta-1,6-glucan-deficient mutants. Mol Microbiol 31, 1835-1844.[Medline]

Kapteyn, J. C., ter Riet, B., Vink, E., Blad, S., De Nobel, H., Van Den Ende, H. & Klis, F. M. (2001). Low external pH induces HOG1-dependent changes in the organization of the Saccharomyces cerevisiae cell wall. Mol Microbiol 39, 469-479.[Medline]

Kasten, M. M. & Stillman, D. J. (1997). Identification of the Saccharomyces cerevisiae genes STB1-STB5 encoding Sin3p binding proteins. Mol Gen Genet 256, 376-386.[Medline]

Klis, F. M., Mol, P. C., Hellingwerf, K. & Brul, S. (2002). Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol Rev 26, 239-256.[Medline]

Lai, M. H., Silverman, S. J., Gaughran, J. P. & Kirsch, D. R. (1997). Multiple copies of PBS2, MHP1 or LRE1 produce glucanase resistance and other cell wall effects in Saccharomyces cerevisiae. Yeast 13, 199-213.[Medline]

Lussier, M., White, A. M., Sheraton, J. & 17 other authors (1997). Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics 147, 435–450.[Abstract/Free Full Text]

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

Matsui, Y. & Toh-e, A. (1992a). Isolation and characterization of two novel ras superfamily genes in Saccharomyces cerevisiae. Gene 114, 43-49.[Medline]

Matsui, Y. & Toh-e, A. (1992b). Yeast RHO3 and RHO4 ras superfamily genes are necessary for bud growth, and their defect is suppressed by a high dose of bud formation genes CDC42 and BEM1. Mol Cell Biol 12, 5690-5699.[Abstract]

Popolo, L. & Vai, M. (1999). The Gas1 glycoprotein, a putative wall polymer cross-linker. Biochim Biophys Acta 1426, 385-400.[Medline]

Ram, A. F. J., Wolters, A., Ten Hoopen, R. & Klis, F. M. (1994). A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10, 1019-1030.[Medline]

Ram, A. F. J., Brekelmans, S. S. C., Oehlen, L. J. W. M. & Klis, F. M. (1995). Identification of two cell cycle regulated genes affecting the beta-1,3-glucan content of cell walls in Saccharomyces cerevisiae. FEBS Lett 358, 165-170.[Medline]

Ram, A. F. J., Kapteyn, J. C., Montijn, R. C., Caro, L. H. P., Douwes, J. E., Baginsky, W., Mazur, P., Van Den Ende, H. & Klis, F. M. (1998). Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta-1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity. J Bacteriol 180, 1418-1424.[Abstract/Free Full Text]

Reneke, J. E., Blumer, K. J., Courchesne, W. E. & Thorner, J. (1988). The carboxy-terminal segment of the yeast alpha-factor receptor is a regulatory domain. Cell 55, 221-234.[Medline]

Robinson, N. G., Guo, L., Imai, J., Toh-e, A., Matsui, Y. & Tamanoi, F. (1999). Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70. Mol Cell Biol 19, 3580-3587.[Abstract/Free Full Text]

Roemer, T. & Bussey, H. (1991). Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc Natl Acad Sci USA 88, 11295-11299.[Abstract]

Roncero, C., Valdivieso, M. H., Ribas, J. C. & Duran, A. (1988). Isolation and characterization of Saccharomyces cerevisiae mutants resistant to calcofluor white. J Bacteriol 170, 1950-1954.[Medline]

Rose, M. D., Novick, P., Thomas, J. H., Bothstein, D. & Fink, G. R. (1987). A Saccharomyces cerevisiae genomic plasmid bank based on a centromeric-containing shuttle vector. Gene 60, 237-243.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequence with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]

Shahinian, S. & Bussey, H. (2000). ß-1,6-Glucan synthesis in Saccharomyces cerevisiae. Mol Microbiol 35, 477-489.[Medline]

Sherman, F. & Hicks, J. (1991). Micromanipulation and dissection of asci. Methods Enzymol 194, 21-37.[Medline]

Shimoi, H., Kitagaki, H., Ohmori, H., Iimura, Y. & Ito, K. (1998). Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme resistance. J Bacteriol 180, 3381-3387.[Abstract/Free Full Text]

Sullivan, D. S., Biggins, S. & Rose, M. D. (1998). The yeast centrin, Cdc31p, and the interacting protein kinase, Kic1p, are required for cell integrity. J Cell Biol 143, 751-765.[Abstract/Free Full Text]

Terashima, H., Yabuki, N., Arisawa, M., Hamada, K. & Kitada, K. (2000). Up-regulation of genes encoding glycosylphosphatidylinositol (GPI)-attached proteins in response to cell wall damage caused by disruption of FKS1 in Saccharomyces cerevisiae. Mol Gen Genet 264, 64-74.[Medline]

Van Berkel, M. A., Rieger, M., te Heesen, S., Ram, A. F. J., Van Den Ende, H., Aebi, M. & Klis, F. M. (1999). The Saccharomyces cerevisiae CWH8 gene is required for full levels of dolichol-linked oligosaccharides in the endoplasmic reticulum and for efficient N-glycosylation. Glycobiology 9, 243-253.[Abstract/Free Full Text]

Van Der Vaart, J. M., Caro, L. H. P., Chapman, J. W., Klis, F. M. & Verrips, C. T. (1995). Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol 177, 3104-3110.[Abstract]

Van Rinsum, J., Klis, F. M. & Van Den Ende, H. (1991). Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9. Yeast 7, 717-726.[Medline]

Vossen, J. H., Ram, A. F. J. & Klis, F. M. (1995). Identification of SPT14/CHW6 as the yeast homologue of hPIG-A, a gene involved in the biosynthesis of GPI anchors. Biochim Biophys Acta 1243, 549-551l.[Medline]

Watanabe, Y., Irie, K. & Matsumoto, K. (1995). Yeast RLM1 encodes a serum response factor-like protein that may function downstream of the Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol Cell Biol 15, 5740-5749.[Abstract]

Winston, F., Dollard, C. & Ricupero-Hovasse, S. L. (1995). Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11, 53-55.[Medline]

Zhan, X. L., Deschenes, R. J. & Guan, K. L. (1997). Differential regulation of FUS3 MAP kinase by tyrosine-specific phosphatases PTP2/PTP3 and dual-specificity phosphatase MSG5 in Saccharomyces cerevisiae. Genes Dev 11, 1690-1702.[Abstract]

Zlotnik, H., Fernandez, M. P., Bowers, B. & Cabib, E. (1984). Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J Bacteriol 159, 1018-1026.[Medline]

Received 5 August 2002; revised 30 August 2002; accepted 9 September 2002.