Center for Fungal Cell Wall Research, Swammerdam Institute for Life Sciences, BioCentrum, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands1
Departamento de Microbiología II, Facultad Farmacia, UCM, Avda. Ramón y Cajal s/n 28040-Madrid, Spain2
Department of Microbiology and Preservation, Unilever Research Laboratory, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands3
Author for correspondence: Hans de Nobel. Tel: +31 20 5257850. Fax: +31 20 5257934. e-mail: nobel{at}bio.uva.nl
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ABSTRACT |
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Keywords: wall damage, wall integrity, Slt2, Mid2, thermotolerance
Abbreviations: CFW, Calcofluor white
a Present address: Fungal & Bacterial Plant Pathology Department, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.
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INTRODUCTION |
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Protein kinase C activates a linear MAP kinase cascade that consists of a MAPKK kinase, Bck1/Slk1; a pair of redundant MAPK kinases, Mkk1 and Mkk2; and a MAP kinase, Slt2/Mpk1 (Gustin et al., 1998 ; Mellor & Parker, 1998
; Heinisch et al., 1999
). Signalling through MAP kinase cascades results in activation of MAP kinases by dual phosphorylation of the conserved threonine and tyrosine residues in subdomain VIII (Cobb & Goldsmith, 1995
). Activation of MAP kinases typically leads to the activation of downstream transcription factors, and consequently to changes in gene expression patterns. Known outputs of Slt2 activation are increased expression of FKS2 and several other genes encoding cell-wall-related proteins (Zhao et al., 1998
; Jung & Levin, 1999
), signalling to the actin cytoskeleton (Helliwell et al., 1998
) and increased thermotolerance after preceding mild heat treatment (Kamada et al., 1995
). Loss of any of the components of the PKC1-MAPK cascade results in osmotic-remedial cell lysis and downregulation of a subset of cell wall genes, demonstrating a regulatory role for this pathway in cell wall construction (Igual et al., 1996
; Gustin et al., 1998
; Mellor & Parker, 1998
).
Activation of the PKC1 pathway seems to be mediated through a family of plasma membrane-localized sensors, Wsc1-4, Mid2 and its homologue Mtl1 (Verna et al., 1997 ; Gray et al., 1997
; Jacoby et al., 1998
; Rajavel et al., 1999
; Ketela et al., 1999
). Cell wall damage and growth at elevated temperatures generate a signal that is transduced from Hcs77/Wsc1 via the GTPase Rho1 to Pkc1 and results in depolarization of the actin cytoskeleton independent of the MAP kinase cascade (Delley & Hall, 1999
). Several cell wall mutants show enhanced GEF activity towards Rho1 (Bickle et al., 1998
) and require Mid2 (Ketela et al., 1999
) or Pkc1 (Roemer et al., 1994
; Garrett-Engele et al., 1995
; Popolo et al., 1997
) for survival, suggesting that cell wall stress in these mutants is perceived and signalled through this pathway.
Here, we analyse Slt2 activation in several cell wall mutants and in cells with an altered cell wall due to treatment with Calcofluor white (CFW), a fluorescent dye that hinders normal cell wall assembly, and with Zymolyase, a cell-wall-degrading enzyme preparation. We found dual phosphorylation of Slt2 and induction of FKS2lacZ expression in an Slt2-dependent manner in response to these wall-weakening conditions. Additionally, we show that Slt2 is required for increased resistance to glucanase digestion and heat shock in response to cell wall stress.
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METHODS |
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Plasmids FKS2lacZ [FKS2(-928 to -1)lacZ] and FKS2706lacZ [FKS2(-706 to -1)lacZ] contain, respectively, 928 bp and 706 bp of the FKS2 upstream non-coding region in front of the bacterial lacZ gene (Zhao et al., 1998 ) and were kindly provided by Dr D. Levin. Plasmid TIR1lacZ (pLGDSRP5'; Marguet & Lauquin, 1986
) contains 2·2 kb of the TIR1 upstream region and the first 58 codons of TIR1 in-frame with the lacZ gene and was a kind gift from Dr G. Lauquin. All three reporter construct plasmids are based on the same yeast episomal plasmid containing a 2µ origin of replication and URA3 as selectable marker (Marguet & Lauquin, 1986
; Zhao et al., 1998
).
Deletions.
Gene deletions were essentially performed using the method described by Baudin et al. (1993) . In short, disruption cassettes consisting of HIS3 flanked by 50 nucleotides identical to the targeted genes, allowing homologous recombination, were constructed in a PCR. The YDp-H plasmid (Berben et al., 1991
) was used as template in the PCR for the disruption cassettes. Oligonucleotides 5'-GTCATGAGTT TTTCACAATT GGAGCAGAAC ATTAAAAAAA AGATAGCCGT GAATTCCCGG GGATCCG (EV21) and 5'-TCTTCATAAA TCCAAATCAT CTGGCATAAA GGAAAATCCT CTAAACTCTT AAGCTAGCTT GGCTGCAG (EV22) were used for the PCR of pkc1::HIS3. Oligonucleotides 5'-GAGATGGCTG ATAAGATAGA GAGGCATACT TTCAAGGTCT TCAATCAAGA GAATTCCCGG GGATCCG (EV25) and 5'-GTCCTAAAAA TATTTTCTAT CTAATCCAAA CTCCAGCTCT TTTTCAAGGT AAGCTAGCTT GGCTGCAG (EV26) were used for the PCR of slt2::HIS3. Deletion of the targeted genes in strain FY834 was confirmed by PCR using primers 5'-GGGGTACCTG TATTTATGAG GCATTGCTAT CTT (EV23) and 5'-GGGGTACCGA GGTTTTTCAT ATGCATGCTC C (EV24) for pkc1::HIS3, and 5'-GGGGTACCGT TCGAATACTT GTGAGCCTA (EV27) and 5'-GGGGTACCAG AGGCGATAAC AAACTTCCG (EV28) for slt2::HIS3.
Growth assays.
The sensitivity of yeast strains to CFW or Zymolyase-100T was assayed as growth inhibition. Cells were precultured in YEPD for 24 h at 28 °C prior to dilution to a concentration of 75000 cells (200 µl YEPD)-1. Growth was continued in the presence of the antifungals in flat-bottom 96-well Greiner PS-microplates at 28 °C without shaking, and growth was determined as OD595 of each culture after resuspension.
ß-Galactosidase assay.
Cells containing the FKS2lacZ or TIR1lacZ plasmids were precultured in selective medium for 24 h at 28 °C. Fresh precultures were used to inoculate liquid YEPD cultures without any addition or with CFW or Zymolyase-100T and the transformants were grown for an additional 47 h at 28 °C. Growth was determined as OD595 and the cells were permeabilized immediately using chloroform/SDS as described previously (Guarente, 1983 ). ß-Galactosidase activity was determined at 30 °C (Guarente, 1983
) and was expressed in Miller units.
Detection of dually phosphorylated Slt2.
Yeast cells were grown overnight to mid-exponential phase in YEPD. The cultures were then diluted in YEPD to OD595 0·3 and growth was continued for 4 h at 24 °C prior to collecting the cells or to treatment with CFW, Congo red or Zymolyase 100-T. Where indicated, sorbitol was added to the media to a final concentration of 1 M. Cells were collected on ice by adding 20 ml of the culture to an equal volume of ice in a Falcon centrifuge tube and pelleted in a refrigerated centrifuge. Cells were then resuspended in 1 ml ice-cold water and transferred to an Eppendorf tube, pelleted and immediately broken or frozen on dry ice. Cells were lysed in 120 µl cold lysis buffer (50 mM Tris/HCl pH 7·5, 10% glycerol, 1% Triton X-100, 0·1% SDS, 150 mM NaCl, 50 mM NaF, 1 mM sodium orthovanadate, 50 mM ß-glycerol phosphate, 5 mM Na pyrophosphate, 5 mM EDTA, 1 mM PMSF and the protease inhibitors tosylphenylalanine chloromethyl ketone, tosyllysine chloromethyl ketone, leupeptin, pepstatin A, antipain and aprotinin, each at 25 µg ml-1) by vigorous shaking with 0·45 mm glass beads in a fast-prep cell breaker (Bio101; level 5·5 for 25 s). Cell extracts were separated from glass beads and cell debris and collected in an Eppendorf tube by centrifugation and further clarified by a 13000 g spin for 15 min at 4 °C. The protein concentration of the supernatants was measured at 280 nm and normalized with lysis buffer. Then, 2xSDS-PAGE sample loading buffer was added and samples were boiled for 5 min. Protein samples (50 µg) were fractionated by SDS-PAGE using 8% polyacrylamide gels and transferred to nitrocellulose membranes (Hybond; Amersham). Membranes were probed with anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (New England Biolabs) to detect active Slt2 at 1/2000 dilution in the presence of 5% non-fat milk for 2 h at room temperature. The primary antibody was detected using a horseradish peroxidase-conjugated anti-rabbit antibody with the ECL detection system. To monitor the amount of Slt2, blots were stripped and reprobed with polyclonal anti-Slt2 antibodies (Martin et al., 1993 ) at 1/1000 dilution, followed by detection as described above.
Thermotolerance.
Cells pregrown in YEPD for 24 h at 28 °C were diluted in YEPD to a density of 0·5x107 cells ml-1 (OD595 0·3) and growth was continued for at least 4 h at 28 °C in the presence or absence of cell-wall-destabilizing compounds. Cultures were washed and resuspended in YEPD, yielding a suspension of OD595 1. Following a heat shock of 020 min at 50 °C, cells were plated on YEPD and incubated for 23 d at 28 °C. Colony forming units were counted and percentage survival was determined relative to cultures that were not shocked at 50 °C.
1,3-ß-Glucanase sensitivity.
Cells were washed and resuspended in 50 mM Tris/HCl pH 7·4 to a concentration of 1·5x107 cells ml-1. ß-Mercaptoethanol was added to a final concentration of 40 mM and the cells were incubated at room temperature for 30 min prior to the addition of 100 U 1,3-ß-glucanase per ml (Quantazyme ylg; Quantum Biotechnologies). The decrease in OD595 was followed in time as a measure of cell lysis and was expressed as a percentage of the OD595 prior to enzyme addition.
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RESULTS |
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When the gas1, fks1
, kre1
and kre9
mutants and their respective wild-types were transformed with a TIR1lacZ reporter construct, no significant ß-galactosidase activity could be detected. TIR1 encodes a cell wall protein that is induced by growth at low temperatures and under anaerobiosis (Donzeau et al., 1996
). The TIR1lacZ reporter plasmid was functional because significant ß-galactosidase activity could be obtained with FY834 and SEY6210 transformants under inductive growth conditions (data not shown). Taken together, these results show that the FKS2lacZ construct but not the TIR1lacZ construct can be used as reporter for cell wall compensation reactions occurring in cell wall mutants.
Enhanced FKS2lacZ expression after cell wall weakening by CFW or Zymolyase
CFW is a negatively charged, fluorescent dye that is unable to pass through the plasma membrane but preferentially binds to chitin in the yeast cell wall and interferes with normal wall assembly (Pringle et al., 1989 ; Ram et al., 1994
). We tested the effect of CFW-induced cell wall perturbation on FKS2lacZ expression. FY834 [FKS2lacZ] and SEY6210 [FKS2lacZ] transformants were transferred from selective medium to YEPD without or with CFW. Induction of ß-galactosidase activity during a 6·5 h incubation period depended on the concentration of CFW but levelled off at concentrations above 50 µg ml-1 (Fig. 2a
). After prolonged incubation, the cultures entered into stationary phase and FKS2lacZ expression increased independently of the presence of CFW (data not shown).
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Addition of 1 M sorbitol to the growth medium partially relieved growth inhibition by CFW or Zymolyase (Fig. 2 and 3
and data not shown) and was associated with reduced FKS2lacZ expression (Fig. 2
). Similar suppression of FKS2lacZ expression was obtained when transformants were cultured in the presence of 0·8 M KCl or 1 M sucrose. The observed suppression of FKS2lacZ expression in an osmotically supported medium suggests that loss of membrane support due to a loss in cell wall strength is a crucial step in sensing CFW- and Zymolyase-induced stress. Unlike the FKS2lacZ transformants, cells transformed with the TIR1lacZ reporter construct did not give rise to detectable ß-galactosidase activity when grown in the presence of identical amounts of CFW or Zymolyase (data not shown).
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The FKS2lacZ reporter construct has been shown to respond to a variety of signals transduced by several different pathways, amongst others the PKC1 pathway, through spatially separated elements within the FKS2 promoter. The FKS2706lacZ reporter, for example, contains a shorter fragment of the FKS2 promoter region and is therefore no longer responsive to calcineurin-transduced signals or stationary phase but remains responsive to high temperature, a known Slt2-transduced signal (Zhao et al., 1998 ). Exponentially growing SEY6210 cells transformed with the FKS2706lacZ reporter construct showed a much reduced ß-galactosidase activity under non-inducing conditions compared to SEY6210 [FKS2lacZ] transformants (0·5 and 20 Miller units, respectively; Figs 1
and 4a
). However, a more than sixfold induction of ß-galactosidase activity was observed when SEY6210 [FKS2706lacZ] cells were grown in the presence of CFW (Fig. 4a
), similar to that observed in SEY6210 [FKS2lacZ] transformants under the same conditions (data not shown). These results are consistent with the proposed role for the Slt2-mediated pathway in upregulating FKS2lacZ expression in response to cell wall perturbation. Next, we examined FKS2lacZ expression in an slt2
strain. Stationary-phase induction of FKS2lacZ occurred to the same extent in both slt2
and FY834 transformants (data not shown). However, unlike FY834 [FKS2lacZ] transformants, the isogenic slt2
[FKS2lacZ] showed no increased ß-galactosidase activity when grown in the presence of sublethal concentrations of CFW or Zymolyase that allowed growth of both strains (Fig. 4b
). These data demonstrate that induction of FKS2lacZ in response to cell wall perturbation depends completely on Slt2, which is not the case for induction by stationary phase.
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Consistent with the above-described enhanced FKS2 expression in cell wall mutants, gas1, fks1
and kre9
mutants displayed a much higher level of dually phosphorylated Slt2 than their wild-type strains (Fig. 5a
). However, whereas the strong Slt2 phosphorylation shown by gas1
mutants was prevented when cells were grown in medium with 1 M sorbitol, osmotic stabilization of the medium greatly reduced but did not totally eliminate the increase in phospho-Slt2 signal shown by fks1
and kre9
mutants (Fig. 5a
). In contrast to these mutants, lack of Kre1p did not result in a detectable increase of Slt2 phosphorylation, possibly because cell wall damage in kre1
cells was relatively mild (Boone et al., 1990
; Brown & Bussey, 1993
; see also Fig. 1
).
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Two putative cell surface sensors, Hcs77(Wsc1/Slg1) and Mid2, have been reported to mediate the activation of the cell integrity pathway (Gray et al., 1997 ; Verna et al., 1997
; Jacoby et al., 1998
; Ketela et al., 1999
; Rajavel et al., 1999
). As shown in Fig. 5(e)
, deletion of MID2 strongly reduced dual phosphorylation of Slt2 in response to CFW-induced stress. This is consistent with recent data showing the requirement of Mid2 for CFW-induced tyrosine phosphorylation of overexpressed Slt2-HA (Ketela et al., 1999
). In contrast, Hcs77(Wsc1/Slg1) seems not to be required for signalling cell wall stress to the Slt2 pathway since loss of this protein did not affect CFW-induced Slt2 activation (Fig. 5e
).
Cell wall perturbation induces thermotolerance in an Slt2-dependent manner
One reported output of Slt2 activation is the acquisition of thermotolerance. Growth at slightly elevated temperatures (37 °C) results in a cellular response that depends in part on Slt2 activation and allows the cells to survive an otherwise lethal heat shock at 50 °C (Kamada et al., 1995 ). We determined whether Slt2 activation in response to cell wall perturbation also resulted in the acquisition of thermotolerance. Loss of FKS1 resulted in a 7-fold, and loss of GAS1 in a 200-fold higher survival after a 15 min heat shock at 50 °C compared to the wild-type strain FY834 (Fig. 6a
). Similarly, enhanced thermotolerance was observed for the kre1
(2-fold) and kre9
(200-fold) mutants compared to their wild-type SEY6210 (data not shown). Growth of FY834 and SEY6210 in the presence of the cell wall perturbants CFW or Zymolyase also resulted in cells that were significantly more resistant to heat than mock-treated cells (Fig. 6b
and data not shown). This response to cell wall perturbants depended on Slt2 because an slt2
mutant in the FY834 background did not acquire thermotolerance (Fig. 6b
). Although upregulation of Fks2 is one of the outputs of Slt2 activation, it is not essential for the concomitant induction of heat resistance because cells lacking Fks2 still became thermotolerant in response to cell wall perturbation (Fig. 6b
).
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DISCUSSION |
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The PKC1 pathway is believed to regulate cell integrity because loss of components in this pathway results in cell lysis that can be prevented by providing osmotic support (Gustin et al., 1998 ; Mellor & Parker, 1998
). This notion is further supported by the observation that the expression of several genes involved in cell wall assembly seems to be controlled by this pathway (Igual et al., 1996
; Jung & Levin, 1999
). Specifically, the temperature-induced expression of Fks2 has been shown to be one of the outputs of Slt2 activation (Zhao et al., 1998
). Activation of the cell integrity pathway has been reported in response to heat and hypotonic stress and during polarized growth (Levin et al., 1994
; Gustin et al., 1998
; Mellor & Parker, 1998
). These conditions might activate the PKC1 pathway by challenging cell wall integrity indirectly. Furthermore, enhanced GDP/GTP exchange activity towards Rho1, an activator of Pkc1 (Nonaka et al., 1995
; Kamada et al., 1996
) has been found in cell wall mutants (Bickle et al., 1998
). In agreement with this, Hcs77, Rho1 and Pkc1 are required for depolarization of the actin cytoskeleton in response to heat stress and cell wall degradation (Delley & Hall, 1999
). These observations suggest that a decrease in cell wall integrity activates Pkc1. Since activation of Slt2 depends on Pkc1 and loss of Slt2 results in fragile cells (Torres et al., 1991
; Lee et al., 1993
; Martin et al., 1993
), a role for Slt2 in transducing the signal elicited by cell wall stress could also be envisioned. However, at present it is unclear whether all the reported outputs generated by cell wall perturbation depend on Slt2. In fact, although stress-induced actin depolarization depends on Pkc1, it does not require the downstream MAP kinase cascade (Delley & Hall, 1999
). In addition, activation of Slt2 as a consequence of cell wall weakening has not been demonstrated unequivocally. For example, Ketela et al. (1999)
recently found increased tyrosine phosphorylation of an overexpressed HA-tagged form of Slt2 in cells grown in the presence of CFW. However, no readily apparent changes in the amount of phosphotyrosine of native Slt2 were detected under those conditions. Furthermore, MAP kinase activation requires dual phosphorylation of threonine and tyrosine residues and the occurrence in yeast of tyrosine-phosphorylated but nevertheless inactive Slt2 has been reported (Buehrer & Errede, 1997
).
Here, we used two readouts for Slt2-mediated signalling: antibodies that detect the dually (threonine and tyrosine) phosphorylated form of Slt2 and FKS2lacZ reporter constructs. Increased dual phosphorylation of Slt2 and Slt2-dependent FKS2lacZ expression were found in gas1, fks1 and kre9 which are cell wall mutants, and in cells treated with cell-wall-destabilizing agents. This response was partially or totally prevented when 1 M sorbitol was added to the medium.
Taken together, these results clearly show the following. (i) Slt2 is activated in response to defects in the cell wall; this activation is likely to mediate the compensation reactions occurring in damaged cells to ensure their integrity. (ii) Osmotic stabilization of the plasma membrane in the presence of a defective cell wall can prevent stimulation of the pathway. This effect depends on the extent of the cell wall damage and indicates that at least a mild cell wall stress is not sensed under such conditions. These conclusions are consistent with gas1 and fks1 mutants requiring a functional Pkc1 pathway for survival (Garrett-Engele et al., 1995 ; Popolo et al., 1997
; Turchini et al., 2000
) and with the hypersensitivity of slt2 mutants to cell wall perturbing agents (Fig. 3
).
Previously, Ketela et al. (1999) reported that tyrosine phosphorylation of overexpressed Slt2-HA in response to CFW-induced cell wall perturbation depends on the putative cell surface sensor Mid2. Our results confirm the role of Mid2 in sensing cell wall stress since we found no activation of Slt2 in mid2
cells stressed with CFW. In contrast, the putative cell integrity sensor, Hcs77, seems not to have a role in sensing cell wall defects and activating the MAP kinase cascade (Fig. 5e
). Interestingly, Hcs77 but not Mid2 is required for Slt2-independent actin depolarization in response to heat or cell wall damage (Delley & Hall, 1999
). These results suggest that cell wall damage can be sensed by at least two different cell surface proteins each triggering a different response. The Hcs77 homologues, Wsc2 and Wsc3 (Verna et al., 1997
), might also be involved in sensing cell wall damage.
The Slt2-mediated increases in thermotolerance and glucanase resistance displayed by cells with an altered cell wall reflect how cells rely on a Slt2-mediated compensation mechanism to face wall-related stress. This also indicates the significance of the modifications in the cell wall architecture of cells in which this mechanism has been triggered. The exact changes in the wall responsible for altered sensitivity to 1,3-ß-glucanase digestion are at present unknown. Induction of Fks2 is not essential because an fks2 strain gave a similar response after cell wall perturbation. Possibly, the strongly increased deposition of chitin in the lateral walls that has been reported for many cell wall mutants, including gas1
and fks1
cells (Dallies et al., 1998
; Kapteyn et al., 1997
, 1999b
) and for cells stressed with CFW (Roncero & Duran, 1985
; Ketela et al., 1999
) is responsible for the increased glucanase resistance. In addition, growth in the presence of CFW reduces the permeability of the yeast wall (De Nobel et al., 1990
), suggesting enhanced incorporation of cell wall mannoproteins that could also account for this resistance. Further study is also required to determine whether increased thermotolerance in these cells depends on concurrent cell wall alterations. Unlike induction by cell wall perturbation, enhanced resistance to degradation by 1,3-ß-glucanase at stationary phase is independent of Slt2. This indicates that alternative signal transduction pathways might be responsible for cell wall alterations induced at this stage.
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ACKNOWLEDGEMENTS |
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Received 15 March 2000;
revised 5 June 2000;
accepted 9 June 2000.