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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 SalIXhoI 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 XhoISalI 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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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 46 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 (60008000 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.
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RESULTS |
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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 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
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
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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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
and cwp2
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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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 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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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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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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DISCUSSION |
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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
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 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
and hog1
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
single mutant cells had reverse phenotypes in both K1 killer toxin and Zymolyase sensitivities. In the PGAL1:KIC1 pbs2
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.
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ACKNOWLEDGEMENTS |
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Received 5 August 2002;
revised 30 August 2002;
accepted 9 September 2002.