Department of Plant Molecular Biology, Instituto de Biotecnología de la UNAM, 62250 Cuernavaca, Mor., Mexico
Correspondence
Jorge Nieto-Sotelo
jorge{at}ibt.unam.mx
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ABSTRACT |
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INTRODUCTION |
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A large number of studies have implicated the cAMP/protein kinase A (PKA) pathway in the control of these physiological processes (reviewed by Thevelein & de Winde, 1999; Thevelein et al., 2000
). During the exponential phase, the cAMP/PKA pathway downregulates glycogen and trehalose content, stress tolerance, cell-wall resistance to lyticase digestion and expression of genes that are controlled by STRE-boxes in their promoters (Plesset et al., 1987
; Van Dijck et al., 1995
). The cAMP/PKA pathway is also involved in the control of cell-cycle progression at G1 or G0 (Drebot et al., 1990
; Thevelein, 1992
), although it is not clear how this is achieved.
cAMP levels control PKA activity by interacting with its regulatory subunit (Bcy1p), which dissociates from the catalytic subunits (Tpk1p, Tpk2p and Tpk3p), resulting in their activation (Toda et al., 1987a, b
). cAMP levels are controlled by synthesis (through adenylate cyclase) and by degradation (through Pde1p and Pde2p phosphodiesterases) (Ma et al., 1999
). Adenylate cyclase is regulated by the monomeric G-proteins Ras1p and Ras2p, as well as by Svr2p (Fedor-Chaiken et al., 1990
). Activation of Ras proteins requires Cdc25p, a guanine nucleotide-exchange factor that interchanges GDP for GTP (Broek et al., 1987
; Lai et al., 1993
). The Ira1p and Ira2p proteins downregulate Ras by enhancing its intrinsic GTPase activity. Cells containing mutations that cause a decrease in the activity of the cAMP/PKA pathway, such as in adenylate cyclase (cyr1) or Ras (ras2), show slow growth rates, accumulate storage carbohydrates and are heat shock resistant (Iida & Yahara, 1984
; Tatchell, 1986
; Iida, 1988
). In contrast, mutants with an overactive PKA (e.g. bcy1, RAS2val19) fail to arrest in G1 upon nutrient limitation, quickly lose viability in the stationary phase and are very sensitive to different stresses (reviewed by Thevelein & de Winde, 1999
).
A cAMP-independent pathway for controlling PKA targets, referred to as the fermentable growth medium (FGM) pathway, has been proposed. The FGM pathway involves the Sch9p kinase, although its relation to PKA is still not well understood (Thevelein et al., 2000).
In this work, we describe two novel alleles of CDC25 (cdc25-21 and cdc25-22) that show conspicuous phenotypes at 25 °C: elevated tolerance to heat shock and other stresses, a slow growth rate, thick cell walls, glycogen hyperaccumulation, constitutive expression of stress genes and lack of rapid cAMP accumulation upon addition of glucose. These mutants are unable to grow in galactose, in glucose at 40 °C or in non-fermentable media at optimal temperatures. The cdc25-21 mutant lacks 612 aa from the C-terminus, where the catalytic domain resides, but remains viable in glucose. Furthermore, a W303 derivative (cdc25), in which the CDC25 gene has been deleted, is still viable in glucose. We demonstrate that these mutations do not drive cells into the G0 or stationary phase of the cell cycle, but rather that they spend a longer time in G1. Our results indicate that the catalytic C-terminus of Cdc25p plays important roles during fermentative growth in glucose, during growth in non-fermentable carbon sources and in derepression from a glucose-repressed state.
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METHODS |
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Thermotolerance assays.
To evaluate the basal or intrinsic level of thermotolerance, we used the method described by Nicolet & Craig (1991). Heat shocks were given by incubating the cells in a 50 °C water bath for 20 min at 250 r.p.m. To measure cell viability, plates were incubated for 34 days at 25 °C for colony counting. Colony counting was performed by using COVASIAM (Corkidi et al., 1998
). Thermotolerance levels are expressed as the percentage of the number of colonies after a heat shock divided by the number of colonies in the untreated sample. Duplication times were calculated by using the exponential curve-fitting equation (Draper & Smith, 1981
). Growth curves with correlation coefficients >0·99 were used for the calculation.
Other stress assays.
Strains were grown at 25 °C in liquid YPDA medium to mid-exponential phase (an OD600 of 0·4) and treated as follows: for ionic stress treatment, LiCl was added from a sterile stock solution to a final concentration of 0·4 M and the culture was incubated for 24 h. Oxidative stress was tested by adding H2O2 to a final concentration of 20 mM and cultures were incubated for 1 h. Sorbitol was added from a sterile stock solution to a final concentration of 3·5 M and cultures were incubated for 2 h. In all cases, incubations during stress treatment were performed at 25 °C and 250 r.p.m. After stress treatment, cultures were diluted to an OD600 of 0·2 and serial dilutions up to 103 were made. To assess survival, 3 µl of each dilution was dropped onto YPDA plates and incubated at 25 °C for 2 days (for the wild-type strain) or 3 days (for mutant strains).
Mutagenesis of the wild-type yeast strain.
Wild-type strain W303a-LEU+ was mutagenized with EMS according to the method of Lawrence (1991). This strain shows a survival rate of 0·01 % after a heat shock of 20 min at 50 °C. Given the relatively high number of false-positive, heat-shock-resistant cells in a wild-type culture, several rounds of selection were applied to the mutagenized population to enrich for true heat-shock-resistant mutants. Immediately after mutagenesis, cells were resuspended in YPDA medium and divided into 40 samples (1 ml each). Each sample was incubated for 24 h at 25 °C for recovery. Glycerol was added to a final concentration of 15 % (v/v) and samples were frozen at 70 °C until further use. For screening, 0·4 ml aliquots of some samples were used to inoculate 5 ml YPDA medium, grown to mid-exponential phase (an OD600 of 0·40·6) at 25 °C and heat-shocked at 50 °C for 20 min. To start a new round of selection, 0·5 ml of each heat-shocked culture was used to inoculate a new 5 ml culture in YPDA medium and the cycle was repeated successively four more times. After each round of heat shock, aliquots were taken to assess cell viability of the culture. After five heat-shock rounds, the wild-type population displayed a survival rate of 0·1 %, whereas the survival rate in different aliquots of the mutagenized population ranged from 0·41 to 4·1 %. Colonies arising from each aliquot were taken to ensure the isolation of mutants resulting from independent events.
Genetic techniques and nucleic acid manipulations.
Genetic techniques and DNA manipulations were performed according to Guthrie & Fink (1991) and Sambrook et al. (1989)
, respectively. DNA sequencing was performed by using an automated sequencer (model ABI Prism 377-18; Applied Biosystems) with an ABI Prism BigDye Terminator cycle sequencing ready reaction kit. For Northern blot assays, total RNA was prepared from exponentially grown liquid cultures in SD medium at 25 °C or after a heat shock for 15 min at 39 °C by the method of Collart & Oliviero (1993)
. Aliquots (15 µg) of total RNA were separated by electrophoresis on 1·2 % agarose gels that contained formaldehyde, transferred to Hybond-N+ membranes (Amersham Biosciences) and hybridized as described by the manufacturer. HSP104, TPS1, CTT1, GPD1 and IPP1 transcripts were detected by using 32P-radiolabelled DNA probes.
Allele rescue and sequencing of cdc25-21 and cdc25-22 alleles.
A derivative of pJF08 (see below) was used to rescue the mutant alleles in cdc25-21 and cdc25-22 mutants according to the method of Rothstein (1991). pJF08 is in plasmid YCp50 and contains a 9·609 kbp insert of wild-type yeast genomic DNA that includes the whole CDC25 locus (see ScIBT2, ScIBT3 and derivative strains are allelic to CDC25 section in Results). PvuII digestion of pJF08 yields a 9·898 kbp fragment (pJF08
PvuII) that consists of the whole vector plus 1090 bp of the 5' end and 858 bp of the 3' end of the insert. In pJF08
PvuII, the entire CDC25 locus and its flanking regions are deleted. Both mutants were transformed with linear pJF08
PvuII DNA. Plasmids from the ura+ transformants were recovered for DNA sequencing.
Yeast transformation.
Yeast transformations were performed according to the method of Gietz & Woods (2002). For cdc25 mutant strains, 50 ml cultures of YPDA medium were inoculated with small colonies isolated from fresh plates. When they reached mid-exponential phase (an OD600 of 0·40·6), cultures were heat-shocked at 50 °C for 20 min to counterselect thermosensitive revertants that form large colonies. After heat shock, cultures were cooled to 25 °C and 10 µg DNA of a wild-type genomic library in YCp50 (Rose et al., 1987
) was used to transform strain JF099. Cells were plated out in SD medium without uracil and screened after 45 days growth at 25 °C. Wild-type colony size was selected. The wild-type genomic library was a kind gift of Dr Alicia González.
Construction of yeast strains with a full deletion of the CDC25 ORF.
The integrative disruption cassette loxPkanMXloxP was used to generate strains in which the CDC25 gene was deleted (Güldener et al., 1996). The disruption cassette served as template in a PCR that included, as forward oligonucleotide, a hybrid that contained 45 bp of sequence upstream to the ATG codon of CDC25 and 19 bp of sequence at the 5' end of the cassette. As reverse primer, a hybrid containing a 45 bp sequence that is complementary to the region at positions 9651 downstream of the TAA stop codon of the CDC25 ORF, followed by 22 bp of the complementary sequence at the 3' end of the cassette, was used. The PCR product was transformed into the wild-type diploid strains RY008, BY4743 and LRA. Transformants were selected in YPDA medium plus 300 µg geneticin ml1 (Gibco) and replica-plated in the same medium to counterselect false-positives. For growth in liquid media, 200 µg geneticin ml1 was used. Correct insertion of the cassette in the W303 tetrads was verified by PCR and sequence analysis (data not shown). CDC25-deleted mutants are referred to as
cdc25.
cAMP determination.
Cells were grown in SD medium with the appropriate supplements for 48 h. Cultures were centrifuged, resuspended in 0·67 % YNB plus supplements (without glucose) and incubated for 4 h to achieve carbon deprivation. Cellular extracts were obtained by breaking the cells in 8 % TCA. After several ether extractions, extracts were lyophilized and resuspended in 2 ml 50 mM Tris/HCl, 4 mM EDTA (pH 7·5). When necessary, the pH was adjusted to 7·5. Finally, 50 µl aliquots were taken for cAMP determination with the use of a [3H]cAMP assay system (Amersham Biosciences), according to the manufacturer's instructions.
Electron microscopy.
Cells were grown to mid-exponential phase (an OD600 of 0·4) and fixed at 4 °C for 24 h in a solution of 6 % glutaraldehyde and 4 % paraformaldehyde [in 40 mM K2HPO4/KH2PO4 (pH 6·5), 0·5 mM MgCl2]. Cells were post-fixed with 2 % potassium permanganate for 2 h to highlight cell-wall and membrane structures. Spurr resin was used to include the cells. Samples were cut by using a Leica Ultracut R ultramicrotome. Observations were performed with a Zeiss EM900 transmission electron microscope at 80 kV.
Flow-cytometric analysis of DNA content.
Cultures (4 ml) were grown to mid-exponential phase (an OD600 of 0·4) in SD medium with the appropriate supplements. Aliquots of 0·5 ml were fixed in 1·166 ml 100 % ethanol (70 % final concentration). Samples were stored at 4 °C until further use. Cells were washed three times with 0·5 M sodium citrate, pH 7·0; 25 µl RNase (10 mg ml1) was added and samples were incubated for 1 h at 37 °C. Cells were centrifuged for 2 min at 6000 r.p.m. in an Eppendorf microfuge and resuspended in staining buffer [0·05 M sodium citrate (pH 7), 0·01 M NaCl, 0·5 % Nonidet-P40 and 16 µg propidium iodide ml1]. Samples were incubated for at least 6 h at 4 °C. Finally, cells were pelleted as above, resuspended in 1·5 ml EDTA (10 mM, pH 5), sonicated for 15 s and analysed by flow cytometry using a Becton Dickinson flow cytometer. Data acquisition and analysis were done by using CellQuest software.
Lyticase cell-wall digestion assays.
We modified the cell-wall sensitivity assay described by Martegani et al. (1984) by using lyticase instead of glusulase. Lyticase (70 U ml1; Sigma) was added to cells that had been resuspended in 10 mM Tris/HCl, 1 mM DTT to an OD600 of 0·70·8 and the OD600 was measured every 10 min.
Glycogen determination.
Cultures grown on YPDA plates for 3 days at 25 °C were stained with 5 ml of a solution that contained 0·2 % iodine and 0·4 % potassium iodide. Colour pictures were taken 1 min after addition of the iodine/iodide solution to assess the degree of staining (Toda et al., 1985).
Glucose, ethanol and oxygen determination.
To estimate glucose and ethanol levels, cells were cultured in a 10 l reactor in YPDA medium at 25 °C and 250 r.p.m. Sterile air was pumped in at a rate of 10 l min1. The culture was inoculated at an OD600 of 0·05 and samples (5 ml) were taken at the indicated times. Cells were removed by centrifugation and the supernatant was used to quantify glucose and ethanol with a Yellow Spring Instruments biochemistry analyser (model 2700). Membranes YSI2365 and YSI2786 were used for glucose and ethanol determination, respectively. To measure oxygen consumption, cells were grown in batch cultures in YPDA medium at 25 °C and 250 r.p.m. When cells reached the mid-exponential phase (an OD600 of 0·4), an aliquot was taken and placed in a sealed chamber with an oxygen polarographic probe (Mettler Toledo) to monitor dissolved oxygen tension (DOT). Controls were done by injecting a stock solution of potassium cyanide to a final concentration of 1 mM immediately after sealing the chamber. The signal of the DOT probe was amplified and acquired by a Macintosh LC computer via a MacADIOS 411 interface (GW Instruments) and a data-acquisition program written in QuickBasic (Microsoft) (Palomares & Ramírez, 1996).
-Galactosidase assay.
Cells were transformed with plasmid pLA41 to express the lacZ reporter under the GAL1 promoter. Cells were streaked out on plates of SD or SRaf to avoid the accumulation of revertants in liquid cultures (only cells that formed small colonies were analysed from the cdc25 strain). Plates were incubated at 30 °C for 3 days in SD or 5 days on SRaf. To induce the expression of
-galactosidase, collected cells were resuspended in 4 ml SGal and incubated at 25 °C for 75 min. Cultures were centrifuged and pellets were resuspended in 10 µl distilled water and dropped onto nitrocellulose membranes.
-Galactosidase activity was detected by using a filter assay as described by Breeden & Nasmyth (1985)
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RESULTS |
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Genetic analysis of mutant strains ScIBT2 and ScIBT3 indicated that both high thermotolerance and slow growth were recessive traits and that the two phenotypes were the result of a single, nuclear mutation (data not shown). A cross between JF314 and JF411 (ScIBT2 and ScIBT3 derivatives, respectively) showed that neither colony size nor thermotolerance levels were complemented, suggesting that their mutations were allelic (data not shown).
After three consecutive backcrosses to their progenitor wild-type strain, JF099 (ScIBT2 derivative) and JF411 mutant strains retained the high-thermotolerance and slow-growth phenotypes of the original ScIBT2 and ScIBT3 strains. Their basal thermotolerance was several thousand times the level of the wild-type strain (Table 2). Duplication times of the mutants in batch cultures were twice as long as that of the parental strain (Table 2
). Both mutant strains and their progenitors failed to grow in media that contained acetate, galactose or glycerol as the carbon source (data not shown). Both ScIBT2 and ScIBT3 (and their derivatives) grew at 37 °C, but not at 38 °C or above. In contrast, their isogenic wild-type strain (W303a-LEU+) was able to grow at 40 °C (data not shown).
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We cloned the cdc25-21 and cdc25-22 alleles by the allele-rescue procedure (see Methods). The cdc25-21 allele showed a single-base deletion within the CDC25 ORF (adenine 2926). This deletion caused a change in the ORF that resulted in a stop codon 36 nt downstream, plus several other stop codons further downstream. Thus, mutant cdc25-21 is predicted to encode a fusion protein that contains the N-terminal 977 aa of Cdc25p fused to 12 non-native amino acids at the C-terminus (Fig. 1b). In cdc25-22, we found an A
C change at position 4088 of the CDC25 ORF, which changed a histidine to a proline at position 1363 of Cdc25p (Fig. 1b
). To determine whether we had characterized the correct mutant gene, we recreated a deletion strain resembling cdc25-21, where the C-terminal 600 aa of Cdc25 was deleted by insertion of a loxPkanMXloxP gene-disruption cassette (strain JF3210). As for cdc25-21 cells, strain JF3210 was highly thermotolerant and had a growth rate twice as long as that of its isogenic wild-type strain (data not shown).
According to the literature, CDC25 is an essential gene and the catalytic C-terminus of Cdc25p is sufficient for viability (Munder et al., 1988; Lai et al., 1993
). To exclude the possibility of a mistake in the molecular characterization of the cdc25-21 mutant, we disrupted the CDC25 gene in the W303, BY4743 and LRA wild-type diploid backgrounds by integration of a loxPkanMXloxP gene-disruption cassette. Selected diploids were sporulated and asci were dissected in YPDA medium. For RY008 (W303 background), the segregation pattern was 2 : 2 (large vs small colonies) and geneticin resistance always co-segregated with small colony size (Fig. 2a
). In the BY4743 and LRA backgrounds, segregation was 2 : 0 (Fig. 2b, c
) and no geneticin resistance was found in the viable spores. The correct insertion of the cassette in the W303 tetrads was verified by PCR and sequence analysis (data not shown). Basal thermotolerance and growth rate of two small colonies of a tetrad from the W303 background were identical to those of mutant cdc25-21, whilst the two large colonies were wild-type for both phenotypes (data not shown). These data indicate that, in the W303 background, CDC25 is dispensable for growth in glucose, and confirm earlier reports regarding the lethality of CDC25 deletion in other yeast strains. Strain JF3005 (
cdc25) was fully complemented by plasmid pRY002 (Table 2
).
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cdc25-21 does not increase cAMP content upon glucose addition
As Cdc25p is postulated to be an upstream activator of cAMP synthesis (Broek et al., 1987) and
cdc25 cells are capable of growth in glucose media at 25 °C, we asked whether they were able to induce the levels of cAMP in response to glucose addition in glucose-deprived cells. The JF099 strain (cdc25-21) transformed with YCp50 or with pJF08 (carrying the wild-type CDC25 locus) showed no response, whereas the isogenic wild-type strain W303-6B transformed with YCp50 : : LEU2 showed a rapid increase in cAMP levels in response to glucose addition (Fig. 3
). This result suggests that Cdc25p is necessary for the glucose-induced response and reveals that, when expressed in a low-copy plasmid, cdc25-21 behaves as a dominant-negative mutant allele.
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Stationary-phase cells also show high resistance to stresses other than heat shock. We tested for resistance to ionic, oxidative and osmotic stress in cdc25-21 (JF099), cdc25-22 (JF411), cdc25 (JF3005) and cdc25-1 (LRA25) mutants at 25 °C during exponential growth. All four mutants were more tolerant to these stresses when compared with their isogenic wild-type strains (Fig. 4
and data not shown). High resistance to 0·4 M LiCl and 3·5 M sorbitol was also a recessive trait. In contrast, tolerance to oxidative stress by H2O2 treatment was semidominant in all four mutants (Fig. 4
and data not shown). In order to further understand the elevated stress resistance of the cdc25 mutants, we assessed the levels of HSP104, TPS1 (trehalose phosphate synthase), CTT1 (cytosolic catalase T) and GPD1 (glycerol-3-phosphate dehydrogenase) RNAs in the four mutant and isogenic wild-type strains during exponential growth at 25 °C. Expression of these genes in wild-type strains is induced by heat, oxidative and osmotic stress (Wieser et al., 1991
; Albertyn et al., 1994
; Hazell et al., 1995
; Lindquist & Kim, 1996
). Levels of IPP1 (inorganic pyrophosphatase) were used as a control, as it is not induced by stress (Gasch et al., 2000
). In wild-type cells, stress-responsive genes were induced by heat shock and were absent or present in very low amounts at the control temperature of 25 °C (Fig. 5
and data not shown). However, all of these genes, with the exception of GPD1 and IPP1, were overexpressed in the four mutant strains at the control temperature and induced further by heat shock (Fig. 5
and data not shown). These results suggest that the high stress resistance of strains cdc25-21, cdc25-22,
cdc25 and cdc25-1 is due in part to the overexpression of stress-regulated genes. The constitutive expression of stress-inducible genes was a recessive trait (Fig. 5
and data not shown).
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Flow-cytometric analyses of DNA content showed that approximately 61·5±2 % of cells of the cdc25-21 (JF099) strain were in G0+G1 phase during exponential growth at 25 °C (Fig. 8a). In contrast, isogenic wild-type cells that were grown under the same conditions showed 46±0·6 % of the cells in G0+G1 (Fig. 8b
). Microscopic observations of exponentially growing cultures of cdc25-21 (JF337) cells showed that 77±5 % of the cells were non-budding (G0+G1), whereas in isogenic wild-type cultures, 57±5 % were non-budding, correlating with the flow-cytometric analyses.
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CDC25 is not required for fermentation or respiration
The fact that cdc25 mutants lose the ability to grow in non-fermentable carbon sources suggested that they were impaired in their respiratory capacity. To test this idea, we analysed their fermentative and respiratory capacity by measuring growth, glucose utilization, oxygen consumption and ethanol production. Glucose was exhausted completely from mutant cdc25-21 (JF099) cultures by about 38 h, whereas isogenic wild-type cultures (W303-6B) took only 27 h to totally consume glucose (Fig. 10a, b). Ethanol production by the wild-type strain reached a maximum of approximately 6·5 g l1 after about 27 h, corresponding to the glucose exhaustion. Mutant cdc25-21 also produced ethanol concomitantly with glucose depletion, although it could only produce 4·8 g l1 after 38 h (Fig. 10c
). Once glucose was exhausted, cells of both W303-6B and cdc25-21 entered the post-diauxic phase of growth and ethanol concentration in the media started to diminish (Fig. 10a and c
). During exponential growth in YPDA medium, the cdc25-21 mutant consumed oxygen at a lower rate, in agreement with its slower growth rate (Fig. 10d
). Addition of potassium cyanide abolished oxygen consumption in both strains (Fig. 10d
). These data indicate that loss of Cdc25p function does not impair either fermentation or respiration in glucose media.
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DISCUSSION |
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The cdc25-21 allele was predicted to encode a Cdc25p lacking the catalytic, C-terminal domain. The cdc25-22 allele has a histidineproline substitution at position 1363 within the catalytic domain of Cdc25p. We also demonstrated that
cdc25 cells derived from the W303 strain were viable and identical phenotypically to cdc25-21 and cdc25-22 cells. Therefore, the Cdc25p encoded by cdc25-21 and cdc25-22 mutants most probably lacks the capacity to activate Ras. Earlier reports indicated that the C-terminal moiety of Cdc25p is essential for viability (Munder et al., 1988
). Disruptions at a central position of Cdc25p (d3) allow viability, but not gluconeogenic growth or sporulation (Munder et al., 1988
). Our data indicate that, in the W303 background, Cdc25p is dispensable for growth in glucose. We also conclude that the C-terminus of Cdc25p is essential for growth in gluconeogenic carbon sources, such as acetate and glycerol. This finding complements previous reports indicating that the N-terminus is necessary for growth in acetate or glycerol media (Munder et al., 1988
). As the cdc25-21 mutant contains an intact N-terminus and is not able to grow in acetate or glycerol as sole carbon sources, we propose that the full-length Cdc25p is essential for growth in these media. As mutant cdc25-21 grows during the post-diauxic phase by consuming ethanol and oxygen, we suggest that this growth defect is not due to a respiratory deficiency, but possibly to a gluconeogenic defect. Thus, Cdc25p is dispensable for fermentation or respiration in glucose media. The fact that these cdc25 mutants do not grow in galactose suggests that Cdc25p is required to change from a glucose-repressed state to a derepressed state. cdc25-21 (JF099), cdc25-22 (JF411) or
cdc25 (JF3005) cells transformed with pLA41 (which contains a lacZ-UASGAL fusion) showed no detectable
-galactosidase activity during the exponential phase in glucose, implying that, in these cells, the galactose-regulatory system is repressed completely (data not shown). Moreover, when shifted to galactose as the only carbon source, wild-type cells showed the expected enhancement of
-galactosidase activity, whereas mutant cells showed no signs of induction, implying that Cdc25p activity is required for glucose derepression (data not shown). Our data are consistent with previous findings that indicate that overexpression of CDC25 induces derepression of the galactose-regulatory system in the presence of glucose (Van Aelst et al., 1991
; Rudoni et al., 2000
). We suggest that the unique role of Cdc25p in galactose derepression is non-redundant, as inactivation of its function completely abolished the response.
SDC25, which encodes a protein with homology to the catalytic domain of Cdc25p, was originally described as a multicopy suppressor of temperature-sensitive cdc25 mutants (Damak et al., 1991). This homologue could account for the non-lethal phenotype that is observed in
cdc25 mutants in the W303 background. In other backgrounds, such as S288C, SDC25 is a pseudogene (Saccharomyces Genome Database). In the W303 strain, SDC25 encodes a complete ORF that is suggested to be expressed only in non-fermentable carbon sources and upon glucose exhaustion (Boy-Marcotte et al., 1996
). Both cdc25-21 and
cdc25 were able to grow by fermentation in glucose-containing media, suggesting that SDC25 could play an active role during glucose fermentation in the wild-type strain W303. Alternatively, SDC25 expression might be negatively controlled by CDC25. Inactivation of CDC25 could have simply unveiled this relationship.
The transient increase in cAMP in response to glucose was not complemented by the wild-type gene, present in a low-copy plasmid, in the cdc25-21 background. Likewise, resistance to H2O2 by cdc25-21, cdc25-22 and cdc25-1 was semidominant. These dominant and semidominant effects can be explained based on the oligomerization state of Cdc25p. Cdc25p forms dimers (Camus et al., 1997) and its N-terminal domain is capable of dimerizing with the full-length protein, with itself or with the C-terminus (Chen et al., 2000
). Cdc25p can also dimerize with Sdc25p (Camus et al., 1997
). In a heterozygous strain, wild-type homodimers, mutant homodimers and heterodimers must form. If we assume that only wild-type homodimers are fully functional, then it is reasonable to expect that mutant Cdc25p monomers sequester a fraction of wild-type monomers, thus decreasing the levels of wild-type Cdc25p homodimers in the CDC25/cdc25-21 or CDC25/cdc25-22 strain. Low levels of Cdc25p seem to be sufficient to restore wild-type growth rate and basal thermotolerance at 25 °C in glucose, but not to restore the transient elevation of cAMP in response to glucose or sensitivity to H2O2, implying that the full control of these phenomena requires higher Cdc25p activities. The essential role of Cdc25p for growth in non-fermentable carbon sources is similar to that observed for adenylate cyclase and Ras2p (Petitjean et al., 1990
). It is conceivable that when the G protein-coupled receptor (GPCR) system is not functional, as in the absence of glucose, there is a need to maintain a minimum concentration of cAMP to support viability.
cAMP transient response to glucose addition is impaired in cdc25 mutants
Mutant cdc25-21 is able to grow in glucose media, although it does not show the rapid response to glucose addition after carbon deprivation. We suggest that the GPCR system requires Cdc25p to function properly. Our data confirm earlier results that were obtained by studying the cAMP response of temperature-sensitive cdc25-5 mutants after addition of glucose at 37 °C (Van Aelst et al., 1990). By using cdc25-21 cells, we were able to rule out the possible secondary effects of high temperatures in previously described cdc25-5 temperature-sensitive mutants. Interestingly, the transient response to glucose was not complemented by wild-type CDC25 in the cdc25-21 background, possibly as a result of the formation of defective heterodimers, as discussed above. This finding contrasts with earlier reports that indicated full complementation of the cAMP response in cdc25-5 mutants by CDC25 (Van Aelst et al., 1991
).
cdc25 mutants show stationary-phase properties during the exponential phase
Our results show that mutants cdc25-21 and cdc25-22 display some stationary-phase properties. Glycogen hyperaccumulation seen in these mutants is in agreement with previous observations by Petitjean et al. (1990). Martegani et al. (1984)
observed increased cell-wall resistance to glusulase digestion in a cdc25-1 mutant at the restrictive temperature (36 °C). Our data rule out the possibility that cell-wall resistance to digestion by lyticase is a secondary effect of heat shock or cell-cycle arrest in G1. Electron micrographs show that cdc25 mutants do indeed have thicker cell walls at the optimal temperature. The fact that dividing cdc25 cells show thicker cell walls suggests that their stationary phase-like properties are not due to entrance to the stationary phase (see below) and that this property is compatible with cell division.
In exponentially growing cultures, most cdc25-21 and cdc25-22 cells are non-budding. This suggests that cdc25-21 and cdc25-22 cells have an extended G1 phase and/or enter the stationary phase, explaining their slow duplication time. Experiments performed with double cdc25-21 gcs1 and cdc25-22 gcs1 mutants suggest that the slow growth of the single cdc25-21 and cdc25-22 mutants during the exponential phase is not due to a transitory, stationary-phase arrest that is imposed in the whole population. It appears that Cdc25p regulation of basal thermotolerance and other stationary phase-like features (cell-wall changes, stress-gene expression, glycogen accumulation etc.) during the exponential phase do not require the function of Gcs1p. These findings are in disagreement with experiments performed by Drebot et al. (1990); these authors used the temperature-sensitive cdc25-1 mutant and showed that after 9 h incubation at the restrictive temperature of 37 °C, cells entered the stationary phase. In contrast, we observed that during the exponential phase, double cdc25-21 gcs1, cdc25-22 gcs1 or single gcs1 mutant cells, arrested by heat shock at 39 °C, resumed growth immediately after incubation at 14 °C (data not shown). Our results indicate that a conditioning heat shock is neither necessary nor sufficient to impose the stationary-phase arrest. It may be possible that this effect is allele-specific and therefore the cdc25-1 mutation is revealing a new activity for Cdc25p. Alternatively, the conditional phenotype in the double mutant gcs1 sed1 that was studied by Drebot et al. (1990)
may have caused the mentioned effect. If that is the case, we suggest that Sed1p acts synergistically with Cdc25p in controlling entry into the stationary phase. Microscopic observation of individual, non-budded cells from exponentially growing cultures showed that they all divide at the same rate, supporting the idea that cdc25-21 and cdc25-22 cells do not have subpopulations of non-dividing cells in the stationary phase.
The above results suggest that all exponentially growing cdc25-21 and cdc25-22 cells spend a longer time in the G1 phase. This extended period in the G1 phase allows them to follow a distinct developmental programme (i.e. elevated stress resistance, glycogen hyperaccumulation, changes in cell-wall composition and upregulation of stress genes). Alternatively, these stationary phase-like characteristics may be defining a Gsc1p-independent and transitory arrest in the cell cycle that is different from G0, from stationary phase and from G1.
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ACKNOWLEDGEMENTS |
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Received 8 March 2004;
revised 4 June 2004;
accepted 14 June 2004.
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