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Originally published In Press as doi:10.1074/jbc.M404594200 on July 6, 2004 Originally published In Press as doi:10.1074/jbc.M404594200 on June 30, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37973-37981, September 3, 2004
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Sok2p Transcription Factor Is Involved in Adaptive Program Relevant for Long Term Survival of Saccharomyces cerevisiae Colonies*

Libuse Váchová{ddagger}, Frederic Devaux§, Helena Kucerová{ddagger}, Markéta Ricicová¶, Claude Jacq§, and Zdena Palková¶||

From the {ddagger}Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic, the §Laboratoire de Genetique Moleculaire, CNRS 8541, Ecole Normale Superieure, 75005 Paris, France, and the Department of Genetics and Microbiology, Charles University, Vinicná 5, 12844 Prague 2, Czech Republic

Received for publication, April 26, 2004 , and in revised form, June 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Volatile ammonia functions as a long range alarm signal important for the transition of yeast colonies to their adaptive alkali developmental phase and for their consequent long term survival. Cells of aged Saccharomyces cerevisiae sok2 colonies deleted in the gene for Sok2p transcription factor are not able to release a sufficient amount of ammonia out of the cells, they are more fragile than cells of wild type colonies, and they exhibit a survival defect. Genome-wide analysis on gene expression differences between sok2 and WT colonies revealed that sok2 colonies are not able to switch on the genes of adaptive metabolisms effectively and display unbalanced expression and activity of various enzymes involved in cell protection against oxidative damage. Impaired amino acid metabolism and insufficient activation of genes for putative ammonium exporters Ato and of those for some other membrane transporters may be responsible for observed defects in ammonia production. Thus, Sok2p appears to be an important regulator of S. cerevisiae colony development. Gene expression differences caused by its absence in colonies differ from those described previously in liquid cultures, which suggests a pleiotropic effect of Sok2p under different conditions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Yeast colonies growing on complex solid medium produce ammonia, which functions as a volatile signal influencing long term colony development. The pulse of ammonia production and transition of stressed aged colony (e.g. Saccharomyces cerevisiae colony 12 days old) to the alkali developmental phase are accompanied by transient growth arrest followed by the renascent colony growth during the second phase of acidification (1). In neighboring colonies, ammonia induces their ammonia production regardless of their current developmental phase, thus causing synchronization of development of all colonies in the respective territory. Ammonia appears to function as an alarm signal released by the colony, which first feels the stress induced by shortage in nutrients. Then, via the ammonia induction mechanism, this signal is spread through the whole colony population, causing its switch to the alkali phase (2). Our previous findings revealed that the acid-to-alkali colony transition is connected with significant changes in gene expression, indicating a parallel decrease in stress factors and mitochondrial respiratory functions, a switch from the mitochondrial citrate cycle to the methylglyoxylate cycle, enabling more economical exploitation of nutrients, and mobilization of cell reserves by means of {beta}-oxidation of fatty acids in peroxisomes. Additionally, we observed significant changes in the expression of genes coding for various membrane proteins/transporters including those that may be important for ammonia production (Ato transporters) (3). Monitoring the behavior of colonies of strains defective in several particular genes allowed us to identify the gene encoding transcription factor Sok2p as a regulator possibly affecting long term colony development.

Sok2p is a transcriptional repressor of the basic helix-loop-helix family, which was initially characterized as a multicopy suppressor of mutations in the cAMP-dependent protein kinase pathway (4). It has been proposed to counteract the function of Msn2p/Msn4p in the activation of some stationary phase-induced genes (SSA3, GAC1) in the presence of glucose, thus being an actor in glucose repression triggered by the cAMP-dependent protein kinase pathway (5). It was shown that Sok2p can physically interact with the transcription factor Msn2p and is involved in the starvation-induced metabolic changes that control the decision of diploid yeast cells to switch from the mitotic growth to the meiosis and sporulation (6). Additionally, Sok2p was shown to be a negative regulator of sporulation (6) and switch to pseudohyphal growth in diploid cells under conditions of nitrogen limitation (7). In the sok2/sok2 diploid mutant, both sporulation and switch to pseudohyphal growth occur even when a rich nitrogen source is present in the medium. The role of Sok2p in pseudohyphal growth appears to be independent of the cAMP-dependent protein kinase pathway. Microarray and Northern blot experiments indicate that Sok2p negatively regulates the expression of the PHD1, ASH1, and SWI5 genes, which encode positive regulators of the genes required for pseudohyphal growth (e.g. FLO11) (7). As regards these data, Sok2p appears to be a key regulator involved in many processes connected with yeast growth, development, and adaptation to environmental changes, particularly changes in nutrient availability.

Previously, we have shown that colonies of SOK2-deleted strains are not able to produce sufficient amount of ammonia and that they exhibit growth problems at later phases of colony development (3). Here, using cytological, biochemical, and microarray approaches, we observed premature dying of sok2 colonies and significant anomalies in the expression of several groups of genes previously proposed to be involved in ammonia-induced adaptation of S. cerevisiae colonies. We also found that SOK2-deleted colonies have an unbalanced activity of some of the reactive oxygen species detoxification enzymes. Our data show that the loss of SOK2 causes significant pleiotropic disorders already in young colonies. These finally result in faulty transcriptional and physiological reprogramming of aged colonies in their alkali phase of development, leading to failure in colony long term survival and adaptation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Media—Strain S. cerevisiae BY4742 (MAT{alpha}, his3{Delta}1, leu2{Delta}0, lys2{Delta}0, ura3{Delta}0) and the isogenic mutant sok2 are from the EUROSCARF collection. Colonies were grown on GM agar (1% yeast extract, 3% glycerol, 2% agar, 30 mM CaCl2) or GM-BKP1 agar (GM, 0.01% bromcresol purple).

Ammonia Production Measurement—Ammonia released by growing colonies was absorbed into acidic traps as described (1), and the amount of ammonia in various liquid samples was determined by the use of the Nessler reagent.

Determination of the Amount of Ammonium/Ammonia in Cells and in the Agar below Colonies—Colonies were removed at particular time points (see Fig. 1B). The agar surface was thoroughly cleaned of the remaining cells, and discs (diameter of 7 mm) of the entire agar layer below the colonies were cut out. For each parallel measurement, three discs were collected into one Eppendorf tube, and 50 µl of 10% citric acid was immediately added to lower pH and to prevent premature volatilization of ammonia present in the agar. At this step, samples can be stored frozen at -80 °C. To separate ammonia/ammonium from other nitrogen forms present in the agar, was converted to NH3 by the addition of NaOH, causing rapid increase in pH and immediate NH3 volatilization from the agar discs. The precise arrangement was as follows. The agar discs in Eppendorf tube were overlaid by 400 µl of 5 M NaOH, and the tube was immediately closed by the stopper containing a small piece of cotton wool soaked with 200 µl of 10% citric acid. A small piece of perforated nylon cloth was placed between the tube and the stopper to fix the wool in the stopper. Released ammonia was captured into the citric acid solution in the wool for the interval of 10 min. Then, the stopper with the soaked wool was transferred on an empty Eppendorf tube, the liquid was spun down by centrifugation at 1000 rpm for 3 min, and its nitrogen content was determined by using Nessler reagent.



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  		FIG. 1.
Ammonia and ammonium release and ATO1 gene expression are diminished in sok2 colonies. A, the ammonia produced by colonies growing on GM agar was absorbed and measured as described. Each point indicates ammonia produced during the interval of 24 h. B, concentrations of ammonium in agar below the colonies. C, concentration of ammonium in cells of the colonies. D, the differences in the expression of ATO1 in WT and sok2 colonies. Values were obtained by quantification of ATO1 Northern blot (Fig. 4A).

 
The colonies harvested from agar plates were washed and treated in the same way as the agar discs. Concentrated 5 M NaOH was used both for cell disruption and for ammonia volatilization.

Detection of Cells with Permeabilized Membrane and of Disrupted Cells—The whole colony growing on GM-BKP agar was picked up and resuspended in 10 mM MES buffer, pH 6, with 1 M sorbitol, and the percentage of cells stained by BKP was counted under a fluorescence microscope (Leica; filter N2.1). Disrupted cells were photographed either with fluorescence filter N2.1 or under the Nomarski contrast by using a Hitachi camera.

Detection of Proteins Released from Cells of Colonies during Phosphate-buffered Saline Washing—Whole colonies growing on GM agar were picked up at different time points (see Fig. 2D), and wet biomass was weighted and gently suspended in phosphate-buffered saline (8). After centrifugation, proteins of the supernatant were precipitated by methanol/chloroform treatment (9). Sedimented proteins were dissolved in Laemmli sample buffer and run on SDS-PAGE (12% gel). Proteins extracted from 5 mg of wet biomass were loaded on each slot. Silver staining of proteins in gels was performed as described (10).



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  		FIG. 2.
Cells of aged sok2 colonies exhibit defects in survival and in cell integrity. A, morphologies of WT and sok2 colonies after the transition to alkali/abortive alkali phase. Enlarged papillae appear in 14-day-old sok2 colonies. B, changes in the number of cells with permeabilized membrane stained with BKP dye within sok2 colonies (red full line) and WT colonies (blue full line). Values represent an average of two independent experiments; more than 1000 cells were counted at each time point. Dotted lines represent ammonia production (as described in the legend for Fig. 1A) to correlate cell dying with developmental phases. C, disrupted cells in samples picked up from 20-day-old sok2 colonies growing on GM-BKP. A photograph with Nomarski contrast (a) or with fluorescence filter N2.1 (b) is shown. Cells of WT colonies of the same age are shown in parallel. D, SDS-PAGE of proteins from wash extracts of WT and sok2 colonies at different developmental stages.

 
RNA Isolation and Northern Analysis—For total RNA isolation, colonies (approximately 1010 cells) were directly suspended in TES buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 0.5% SDS). The exact isolation procedure is at www.biologie.ens.fr/fr/genetiqu/puces/protocoles_puces.html. The RNA samples were quantified on an Agilent Bioanalyzer 2100 using RNA Nano assay (Agilent Technologies, Palo Alto, CA) (11). mRNA was prepared from total RNA using the Micro Fast track mRNA purification kit (Invitrogen). For Northern blot, 10 µg of total RNA was loaded. The rRNA content was visualized by EtBr staining. The radioactive probes for ATO1, JEN1, POX1, ICL2, TPO2, MSN4, DIA1, CMK2, ARG1, and ACT1 were obtained by random priming on the complete open reading frame (amplified by PCR from the Research Genetics bank of yeast open reading frames) using the Nonaprimer kit. The individual Northern blots were quantified using the TINA (Ray-test) software with ACT1 as the reference.

Microarray Analyses and Biocomputational Analyses of Microarray Data—We used homemade microarray slides containing probes for most of the yeast open reading frames (about 6000 oligonucleotides). The oligonucleotides were supplied by MWG Biotech (Yeast Oligo set). The slides used are Ultragaps from Corning. 4 µg of mRNA was used for each reverse transcription reaction. Detailed protocols are at www.biologie.ens.fr/fr/genetiqu/puces/protocoles_puces.html. The arrays were read by a Genepix 4000 scanner (Axon) and were analyzed with the Genepix 4.0 software. We excluded artifactual spots, saturated spots, and low signal spots. Assuming that most of the genes have unchanged expression, the Cy3/Cy5 ratios were normalized block per block using the LOWESS normalization tool of the Varan software, and genes with significant changes in expression were selected using the confidence interval of 0.999; other parameters were according to the default settings (www.bionet.espci.fr/varan/) (12). The cluster shown on Fig. 3 was generated by Treeview (13). Each microarray result presented here is an average of 5-6 independent biological measurements. The data were deposited in the hybridization array data repository GEO (www.ncbi.nlm.nih.gov/geo/) under the GEO accession number GSE1454 [NCBI GEO] .



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  		FIG. 3.
The expression differences between WT and sok2 colonies in different developmental phases. The difference in expression is indicated by a colored square (colored bar on the bottom right). The corresponding values above the threshold (0.50 for the repression, 2.0 for the induction) are in the supplementary Table 1S.

 
Determination of Catalase and Superoxide Dismutase Activity—Cells of 6-18 giant colonies were harvested and stored at -80 °C until use. Cell lysates were prepared in 10 mM MES buffer, pH 6, supplemented with protease inhibitor mixture Complete, EDTA-free (Roche Applied Science) and 250 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma) by vortexing with glass beads under cooling. Cell debris was removed by centrifugation for 5 min at 13,000 rpm. Protein concentration in supernatant (lysate) was determined using a protein detection kit (Bio-Rad). Supernatant (5-15 µg of proteins/slot) was run on PAGE (9% gel) under non-denaturing conditions (8); SDS was omitted in all steps). The activity of superoxide dismutase in the gel was visualized as described (14). For detection of catalase activity, the method of Clare et al. (15) was used. Enzyme activities were determined by the image analyses using the Un-Scan-It gel program (Silk Scientific Corp.). The commercial catalase from beef liver (Roche Applied Science) and superoxide dismutase from horseradish (Sigma) were used as the standards. Calibration curve using 4-5 different dilutions of the commercial enzyme of the known activity was prepared for each gel.

Determination of Concentrations of Low Molecular Weight Compounds Containing Sulfhydryl Groups—The assay was performed as described (16, 17) with several modifications as follows. Low molecular weight compounds were extracted from colonies by 7.5% trichloroacetic acid. The wet biomass concentration in each sample was 77.5 mg/ml. Extraction was completed by three freeze-thaw cycles; cell debris was removed by centrifugation. 900 µl of 0.4 M Tris-HCl, pH 8.9, was mixed with 100 µl of the extract to adjust the pH in the assay to 8.2-8.3. 10 µl of 10 mM 5,5'-dithiobis-2-nitrobenzoate was added. The absorbance at 412 nm was measured before and 10 min after the addition of 5,5'-dithiobis-2-nitrobenzoate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Physiological Alterations of sok2 Colonies—The first perceptible difference between colonies formed by the S. cerevisiae strain BY4742 (WT colonies) and colonies formed by the isogenic strain deleted in the SOK2 gene (sok2 colonies) appears even in the first acidic phase, when the coloring of the pH dye indicator BKP present in the GM agar indicates more efficient acidification of sok2 colonies in comparison with their WT counterparts (data not shown). As indicated before (3), sok2 colonies are not able to reach the fully developed alkali phase and to produce a significant level of volatile ammonia. The defect in NH3 production can be caused either by an inefficient alkalization of sok2 colony surroundings, leading to an inefficient conversion of protonated to unprotonated NH3 (pKa = 9.25, where Ka is the dissociation constant) (18) and therefore leading to poor NH3 volatilization, or by a defect in the formation/export of out of the sok2 colonies or by a combination of both. As the first possibility would lead to the accumulation of ammonium outside of the cells, we measured the amounts of ammonium in agar below WT and sok2 colonies in different developmental phases (Fig. 1B). The profile of ammonium concentration in the agar below the WT colonies indicates an efficient initiation of ammonium secretion from WT cells in the interval between the 9th and 13th day. Later, the ammonium concentration in the agar transiently decreases, apparently due to the sharp increase in pH, leading to the efficient NH3 volatilization (Fig. 1A). The timing of changes in ammonium concentration in the agar below sok2 colonies is similar to that of WT colonies; however, the amount of ammonium present in agar between the 12th and 13th day (0.04 mg nitrogen/colony) is about half of that in agar below WT colonies (0.1 mg of nitrogen/colony) (Fig. 1B). The sum of ammonia volatilized from WT colonies between the 8th and 13th day is much higher (2.1 mg of nitrogen/colony) than its residual concentration in agar, and it is 11 times lowered in sok2 colonies (0.2 mg of nitrogen/colony). In summary, during the interval of transition of colonies from the acidic to the alkali/abortive alkali phase, the total amount of ammonia released in the sok2 colony (0.24 mg of nitrogen/colony) is about 10% of those released in the WT colony (2.2 mg of nitrogen/colony). There is no dramatic difference in the content of ammonium in WT and sok2 cells (Fig. 1C). At the time of the increased production of extracellular ammonia/ammonium by WT colonies, the ammonium concentration within sok2 cells (0.06 mg of nitrogen/colony) is about 75% of that within WT cells. Later, the intracellular ammonium in sok2 colonies slightly increases, reaching a higher level than that of WT colonies, from which the ammonia/ammonium is efficiently excreted. However, there is no substantial accumulation of ammonium in sok2 cells in concentrations corresponding to the amount of released ammonia in WT colonies (approximately 2 mg of nitrogen/colony). In other words, the inability of sok2 colonies to produce extracellular ammonium/ammonia is not connected with any significant increase of intracellular ammonium, which also means that the production of free ammonium within cells is lowered in sok2 colonies (see below). All these data suggest that besides an inefficient alkalization, sok2 colonies exhibit additional defects in formation and export of ammonium out of the cells. The observed defect in the expression of putative ammonium exporter Ato1p (Fig. 1D) is in agreement with this (for details, see below).

The appearance of an increasing number of papillae, which initiates at the time of the transition to the abortive alkali phase (Fig. 2A), indicates that the coordinated growth of cells within the sok2 colony is impaired and results in irregular colony morphology. This can be the result of the progressive dying of the majority of cells in colonies alongside with the effort of some cells to escape the fate of the majority and to exploit nutrients released from dying cells for their own growth. To prove this hypothesis, we estimated the number of dying cells within sok2 and WT colonies in different phases of the colony development using the BKP dye, which enters only the cells with a permeabilized plasma membrane. Results summarized on Fig. 2B show that there is a significant increase in the number of sok2 cells stained by BKP dye after the switch to the abortive alkali phase (starting approximately at the 13th day). This is accompanied by an increased amount of stained cell debris within samples from sok2 colonies as compared with WT ones (Fig. 2C). Gentle washing of cells picked up from colonies of different ages by phosphate-buffered saline buffer and subsequent analysis of protein content in the washing extract (Fig. 2D) revealed an increased amount of released proteins in extracts from aged sok2 colonies. All these observations indicate that aged sok2 cells became more fragile than WT cells and a large number of them is ultimately disrupted.

Gene Expression Differences between WT and SOK2-deleted Colonies—For microarray comparisons, we isolated transcriptomes from giant colonies (1) of WT strain BY4742 and the isogenic strain deleted in the SOK2 gene. We compared WT and sok2 colonies occurring in five different developmental phases: 1) the early first acidic phase (4th day), 2) the middle first acidic phase (7th day), 3) the neutral phase (11th day), 4) the fully developed alkali phase (WT) and the abortive alkali phase (sok2) (13th day), and 5) the second acidic phase (21st day). We observed differences in the expression of ~500 genes in sok2 colonies at least in one of the five developmental phases (supplementary Table 1S and Fig. 3). Expression changes of some genes representing the main functional categories were confirmed by Northern blot (Fig. 4). The finding that even in the early first acidic phase there are groups of genes that are differently expressed in sok2 colonies when compared with WT colonies is in agreement with the observation that the sok2 versus WT diversity starts even in the first acidic phase. However, the existence of distinct groups of genes differently expressed in any of the particular developmental phases indicates divergences between sok2 and WT during the whole examined interval of colony growth.



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  		FIG. 4.
Time course expression changes of selected genes in WT and sok2 colonies. Northern hybridization of RNAs isolated from WT and sok2 colonies occurring in six developmental time points is shown. The genes were classified into four groups according to the similarities in their expression. The expression curves (graphs on the right) characteristic of the group were created from average values of all genes in the group, using the ACT1 as the reference.

 
The SOK2-deleted Colonies Do Not Switch on Effectively the Genes of Plasma Membrane Transporters Important for Volatile Ammonia Production—Several groups of genes, which were found to be induced during the transition of WT colonies from the acidic phase to the ammonia-producing period of life (3), are down-regulated in sok2 colonies as compared with WT colonies (Fig. 3 and see also Fig. 6). They namely include genes for some transporters and metabolic genes. The genes for phosphate (Pho84p, Pho89p, Pho90p), sulfate (Sul1p), and carboxylic acid (Jen1p) transporters possibly involved in the import of protons into WT cells and initial increase of extracellular pH are down-regulated in sok2 colonies as compared with WT colonies (Fig. 3). Also, the expression of all three genes encoding putative ammonium transporters Ato1p, Ato2p, Ato3p (3) is significantly lowered in sok2 colonies. Northern blot analysis of time course ATO1 gene expression (Fig. 4) revealed that even sok2 colonies start to induce ATO1 gene at approximately the same developmental time as WT colonies. However, in contrast to WT colonies, the expression is much lower and is not sufficiently kept up at later developmental phases. The moderate ATO1 gene induction (Fig. 1D) correlates with the initial appearance of ammonium in the agar below sok2 colonies (Fig. 1B).



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  		FIG. 6.
The scheme of the most prominent differences occurring in colonies of WT and sok2 mutant. A detailed description of the individual processes is given under "Results" and in Figs. 3, 4, 5. The proposed importance of the processes regarding long term survival of yeast colonies is described under "Discussion." Red indicates the activated expression of a particular functional gene group or activation of a specific process, and green indicates the repression. MT, mitochondria; PX, peroxisome; OXPH, oxidative phosphorylation system; mGLC, methylglyoxylate cycle; SUL, transporter of the sulfate; PHO, transporters of the phosphate; DCA, transporter of the carboxylic acids; ATO, putative ammonium exporters; AA, amino acids; TPO, MDR transporters of polyamines.

 
The SOK2-deleted Colonies Do Not Switch on Effectively the Adaptation Program—Groups of metabolic genes possibly involved in the adaptation of WT colonies, which are switched on during their acid-to-alkali transition (3), are not activated during sok2 colony development (Fig. 3). They include genes for peroxisome biogenesis and fatty acid {beta}-oxidation (e.g. FAA1, POX1, IDP3, PXA1, SPS19, CAT2, PEX11, PEX21, FOX2), genes important for non-degradative steps of the citrate cycle and methylglyoxylate cycle in mitochondria (CIT1, CIT2, CIT3, ICL2), and genes encoding various dehydrogenases (ADH2, ADH1, FDH1) of cytosol and mitochondria, which might participate in NADH regeneration during the escape of cells from oxidative stress (18). Differences in the expression of POX1 (encoding acyl-CoA oxidase, an enzyme involved in fatty acid {beta}-oxidation in peroxisomes) and ICL2 (encoding isocitrate lyase, a key enzyme of the methylglyoxylate cycle) detected on Northern blots of RNAs isolated from sok2 and WT colonies in different developmental phases are even more expressive (Fig. 4). These findings indicate that sok2 colonies are not able to adapt efficiently to a shortage of nutrients. They seem not to be able to switch on the economical mitochondrial methylglyoxylate pathway as well as to activate genes of metabolisms providing essential substrates, acetyl-CoA, and oxaloacetate. The later is demonstrated by the absence of activation of genes essential for utilization of fatty acids (important for acetyl-CoA production) and by the absence of activation of the gene JEN1 for the plasma membrane transporter of carboxylic acids and genes of the cytosolic glyoxylate cycle MLS1, ICL1, and MDH3 (important for the import of carboxylic acids and their conversion to oxaloacetate).

The SOK2-deleted Colonies Remain Stressed—The gene encoding Msn4p, one of the major activators (in concert with Msn2p) of various stress-response genes via their stress-response element sequence (19), is strongly activated in sok2 colonies as compared with WT colonies, starting even in the `middle' first acidic phase (Figs. 3 and 4). Various stress genes, which are induced in the sok2 mutant (Fig. 3), contain a stress-response element in their promoter and seem to be under the Msn4p control (e.g. CTT1, HOR2). The gene YER130c encoding the Msn4p homologue, a transcription factor of unknown function, is also strongly activated. The YER130c gene was shown to be activated by Haa1p, a transcription factor that also activates the TPO2 gene encoding the polyamine transport protein (see below) (20).

Expression changes indicate that sok2 colonies are not able to switch on the adaptation program and become more stressed by reactive oxygen species than WT colonies. This prediction was checked by the direct analysis of the activity of important oxidative stress defense enzymes, namely cytosolic catalase Ctt1p and superoxide dismutase Sod1p (Fig. 5A). The enzymatic assays revealed the higher activity of Ctt1p in cell lysates of sok2 colonies in comparison with WT colonies in all estimated phases. On the other hand, the activity of Sod1p in sok2 colonies is almost identical with that of WT colonies at later developmental phases (phases 3-5), whereas it is significantly lower in sok2 colonies occurring at the early and middle first acidic phases. The prediction of cumulative oxidative stress in sok2 colonies is also supported by the observation that the amount of low molecular weight compounds containing sulfhydryl groups (e.g. glutathione) is lowered in sok2 colonies during the first acidic phase (Fig. 5B)



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  		FIG. 5.
Stress-related differences in WT and sok2 colonies. A, enzymatic activities of cytosolic catalase Ctt1p and cytosolic superoxide dismutase Sod1p in different times of development of WT and sok2 colonies. The upper part shows activity in gels, the lower part shows quantified activity as described under "Experimental Procedures." B, time course of relative concentration of low molecular thiols in WT (full line) and in sok2 (dotted line) colonies. The concentration in WT colonies in each time point was considered as 100%. Values represent an average of three independent experiments and four measurements of each.

 
Alterations in Expression of Amino Acid Metabolic Genes and Possible Role of TPO Genes—Amino acids and their metabolism appear to be important for the proper transition of colonies from acidic to alkali phase as well as for ammonia production (1, 21). Microarray analyses revealed that there are differences in the expression of a large number of genes connected with amino acid metabolism between sok2 and WT colonies (e.g. GDH3, ARG3, ARG1, ARG4, LEU4, HIS4, ILV6, LYS4, and several others are repressed in neutral phase sok2 colonies, Fig. 3), which indicates that amino acid metabolic pathways are unbalanced by the absence of Sok2p. This may be another reason for the observed defect in ammonia production.

Additionally, improper coordination of individual metabolic reactions can lead to the accumulation of various intermediates of amino acid metabolism, some of which are toxic. This view is supported by finding that the genes TPO1, TPO2, TPO3, and TPO4, which belong to the major facilitator superfamily of the multidrug resistance genes, are induced in sok2 colonies as compared with WT colonies (Figs. 3 and 6). TPO genes encode plasma membrane-bound exporters involved in the detoxification of the excess of spermidine in yeast cells (22). Northern blot detection of time course TPO2 gene expression in WT and sok2 colonies (Fig. 4) suggests quantitative differences in the level of its expression. In WT colonies, TPO2 is expressed predominantly in the early first acidic phase, and then its expression gradually decreases. Conversely, TPO2 expression in sok2 appears to peak in neutral phase colonies. Polyamines are synthesized from ornithine, one of the intermediates of arginine metabolism. Interestingly, the expression of genes for Arg3p (ornithine carbamoyltransferase), Arg1p (argininosuccinate synthase), and Arg4p (argininosuccinate lyase), which are important for the resynthesis of arginine from ornithine, the precursor of synthesis of the polyamines putrescine, spermidine, and spermine, is down-regulated in sok2 colonies. The expression of the ARG1 gene strongly peaks at neutral phase WT colonies, whereas it is only moderately increased in sok2 colonies (Fig. 4). Another reason for the increased rate of polyamine formation may be the necessity to handle ammonium produced by amino acid catabolism, which is not, in contrast to WT colonies, exported into surroundings.

Regulation Differences between sok2 and WT Colonies—In contrast to relatively weak changes in the expression of cell regulators (transcription factors and kinases) observed during the WT colony development (3), the expression of a large group of genes involved in various regulations quite strongly differs in sok2 colonies as compared with the WT colonies (Fig. 3). The group includes genes encoding transcription regulators, kinases, and proteins modulating kinase activity. Differences in the expression of particular regulators between WT and sok2 colonies are detectable in each of the five estimated developmental phases (Fig. 3) and appear even at the early acidic phase. This supports the prediction that Sok2p absence starts to influence colony development relatively early and indicates that Sok2p primarily regulates the expression of other transcription factors and kinases. Besides genes for transcription factors involved in stress response (Msn4p, Yer130p), in addition, the genes encoding Ime1p (inductor of meiosis), Tye7p (a transcription factor that can suppress the requirement of the Gcr1p activator for the expression of glycolytic genes), and several others change their expression in sok2 colonies in comparison with WT colonies. The group of genes involved in signaling cascades that alter their expression in sok2 colonies includes the genes for Bag7p (the Rho GTPase activator involved in the control of cell wall synthesis), for Rck1p kinase, Tos3p kinase, Cmk2p (calcium/calmoduline-dependent kinase), Rgs2p (GTPase activator), and several others (Fig. 3). The induction of the gene for Rgs2p correlates with the observed activation of some of the stress-related genes. Rgs2p negatively regulates glucose-induced activation of the cAMP signaling pathway, probably by binding to Gpa2p (an {alpha}-subunit of G-protein), thus stimulating its GTPase activity. Overexpression of RGS2 generates phenotypes consistent with low activity of cAMP-dependent protein kinase and elevated expression of stress-response element-controlled genes (23). The induction coefficient of transcription factors extend up to 4.9 (MSN4), whereas the induction of some of the genes encoding factors involved in signaling is even stronger (up to 7.7 in the case of RCK1) (supplementary Table 1S).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
sok2 colonies exhibit wild type-like fitness and growth until they reach the time period when WT colonies perform the acidic-to-alkali transition, which has been proposed to be the prerequisite of colony longevity. The marked morphological and physiological changes of sok2 cells and colonies appear only after they pass their abortive alkali phase transition. Since then, the proportion of fragile and dead cells in sok2 colonies significantly increases, and some of the remaining viable cells appear to be able to grow and divide on behalf of nutrients from dying cells, thus forming small microcolonies (`papillae') (Fig. 2). At least some of these changes of aged sok2 colonies appear to be a reflection of gene expression alterations that result in the inability of sok2 colonies to reprogram efficiently their metabolism (see below). The observed activation of some of the genes of the cell wall integrity pathway (e.g. WSC4, MID2, PST1, YLR194c) (Fig. 3 and supplementary Table 1) in sok2 colonies could be an unsuccessful attempt to cope with defects in the cell wall leading to cell destabilization and fragility. Despite of the fact that phenotypic changes emerge at later developmental phases, the expression alterations in sok2 colonies initiate much earlier, even in the early first acidic phase. Some of these changes escalate later, in the abortive alkali phase when additional changes affiliate. Also, the second acidic developmental phase of sok2 colonies is characterized by distinct gene expression differences as compared with WT colonies of the same age. This suggests that early consequences of SOK2 gene deletion are not rescued and persist throughout whole investigated period of the sok2 colony life. The finding that in each particular phase there are marked alterations in the expression of large groups of genes with different regulatory functions imply that the absence of Sok2p in young colonies disrupts regulation cascades that control the later phases of colony development. This leads to pleiotropic changes in the expression of several gene groups and finally negatively affects colony survival.

Expression experiments suggest that sok2 colonies are not able to switch on metabolic events, which have been proposed to be important for long term colony survival (Fig. 6). In particular, sok2 colonies seem to switch neither the mitochondrial methylglyoxylate cycle nor pathways presumably supplying its main substrates acetyl-CoA (fatty-acid {beta}-oxidation in peroxisomes) and oxaloacetate (carboxylic acids import, amino acid metabolism, and cytoplasmic glyoxylate cycle) nor amino acid metabolism (Figs. 3 and 4). These defects can be related to the fact that sok2 colonies do not produce a significant amount of volatile ammonia, the proposed signal of nutrient exhaustion, of transient growth arrest, and of changes allowing cells to economize their metabolism (1, 3). The process of ammonia production can be influenced at least at three steps: (i) formation of free intracellular ammonium, (ii) export of ammonium out of the cells, and (iii) conversion of to NH3 and its volatilization. It seems that sok2 cells exhibit defects at all these steps (Fig. 1). As it was proposed that amino acids are important for the ammonia production in WT colonies (1, 21), the impaired amino acid metabolism in sok2 colonies may not provide the sufficient amount of ammonium designated for export. This can explain the absence of high level accumulation of ammonium in sok2 cells (Fig. 1C) due to a defect in its export. On the other hand, one cannot exclude a possibility that a slight increase of intracellular ammonium (which cannot be efficiently exported) switches off the additional intracellular production of free ammonium. The low expression of putative ammonium exporters Ato1p, Ato2p, and Ato3p in sok2 colonies, leading to a low amount of respective proteins in plasma membrane and thus to the low ammonium export capability of the cells, can be the second reason for the very low production of ammonia. Additionally, an inefficient increase in the pH of sok2 colonies, probably caused by the lowered expression of other transporters (e.g. Pho, Sul), does not allow efficient conversion and NH3 volatilization. Our data do not allow us to distinguish whether the Sok2p directly activates genes of important transporters and metabolic genes or (more probably) acts indirectly via controlling the regulators of these genes. The finding that ATO3 appears to be under the control of Gcn4p (24), the general regulator of various amino acid metabolic genes, suggests an interesting relation between the regulation of ATO genes and regulation of amino acid metabolic genes under conditions of limited nutrients.

A side effect of unbalanced amino acid metabolism can be the high level accumulation of some metabolic intermediates in sok2 colonies, e.g. of polyamines (Fig. 6). The finding that genes encoding MDR transporters involved in polyamine resistance (TPO genes) are activated in sok2 colonies (Figs. 3 and 4) is in agreement with this. Polyamines are essential compounds in all prokaryotic and eukaryotic cells; however, they are toxic in higher concentrations. Thus, their intracellular level is tightly regulated both by controlling their metabolism and by controlling their transport into intracellular compartments or out of the cells. In S. cerevisiae, the polyamines putrescine, spermidine, and spermine are transported into the vacuole (25). Additionally, it was found that S. cerevisiae cells excrete putrescine during their fermentative growth (22). Mutations in TPO genes rendered the cells more sensitive to an increased level of polyamines (26). The induction of genes of Tpo transporters might therefore reflect the effort of sok2 cells to eliminate the escalating amount of toxic polyamine metabolites (which cannot be reutilized because of unbalanced amino acid metabolic enzymes) by extruding them out of the cells.

Another trouble of sok2 colonies possibly connected with their inability to switch on adaptive metabolic events seems to be an increase of overall stress (Fig. 6). The stress-related problems of sok2 colonies start probably quite early at the first acidic phase. The expression of some of the stress-related genes (e.g. MSN4, Figs. 3 and 4) is increased in sok2 colonies as well as the activity of cytosolic catalase Ctt1p, one of the stress defense enzymes (Fig. 5A). On the contrary, the cytosolic superoxide dismutase Sod1p (involved in the conversion of the superoxide radical to hydrogen peroxide) exhibits lower activity in sok2 colonies during the first acidic phase than in WT colonies. This may result in the early increase of the superoxide radical (), which can cause the primal cell damages in sok2 colonies. This problem cannot be rescued by catalase Ctt1p converting hydrogen peroxide to H2O and O2. The observed decrease in the level of low molecular weight compounds containing sulfhydryl groups (e.g. glutathione) in first acidic phase sok2 colonies is another indication of increased oxidative stress in sok2 colonies (Fig. 5B). It suggests that a larger portion of glutathione is oxidized during the reparation of an enhanced amount of oxidized proteins arising by the action of reactive oxygen species in sok2 colonies than in WT colonies. Additionally, also several genes encoding various dehydrogenases (and other oxidoreductases), which might participate in NADH regeneration in mitochondria, are repressed in sok2 colonies. Some of these enzymes may be important for protecting mitochondria and for the recovery of WT colonies from oxidative stress (18).

In summary, we observed extensive transcriptional differences between sok2 and WT colonies during the whole investigated period of colony development. However, we found few overlaps between the effect of SOK2 deletion on gene expression in colonies of haploid cells growing on complex solid medium with glycerol (Fig. 3 and supplementary Table 1S) and those that were described previously (7). We did not notice any change in the expression of the genes of pseudohyphal growth regulators Phd1p, Ash1p, and Swi5p, of the stationary phase-inducible genes SSA3 and GAC1, or of the sporulation-specific gene SPO13. On the other hand, the main effects that we observed (e.g. overexpression of MSN4 and TPO) were not encountered in previous microarray experiments performed on yeast diploid cells growing in nitrogen-limited liquid medium (7). This absence of overlap arising from different culture conditions points more probably toward the pleiotropic role of Sok2p in the regulation of various aspects of yeast growth and development. In colonies, as in the case of pseudohyphal regulation, Sok2p appears to act independently of the cAMP-dependent protein kinase pathway as neither the inactivation (gene deletion) nor the permanent activation (the presence of the constitutively activated gene allele) of Ras2p or Gpa2p leads to any observable defect in ammonia signaling and colony growth (1, 21).

It appears that a cumulation of individual defects in the adaptation process dooms the sok2 colonies to death (Fig. 6). Aged colonies of strains deleted individually in genes CTT1, SOD1, or MSN4 exhibit partial grow defects at the later second acidic phase, so these individual defects are significantly less prominent than defects caused by the absence of Sok2p.2 Sok2p probably does not control the timing of the initiation of acidic-to-alkali transition since sok2 colonies try to switch on the alternative metabolic pathways at the same time as wild type colonies; however, this attempt appears to be abortive (Fig. 4). The SOK2 deletion thus affects the competence of aged cells to achieve the adaptive program as well as the fully developed alkali phase of ammonia production.


   FOOTNOTES
 
* This work was supported by the Czech Grant Agency Grant 204/02/0650, CAS Grant Agency Grant S5020202, Research Concept Grant AV0 Z5020903, Ministry of Education of the Czech Republic Grants LA141 and J13/98:113100003, and a grant from European Molecular Biology Organization Young Investigator Programme (to Z. P.). The microarray facilities used in this work are from the Service de Genomique du Departement de Biologie of the Ecole Normale Superieure, which is part of the Genopole Ile de France. Exchange stays by Z. P. and F. D. were supported by BARRANDE project 2002-039-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains a supplementary table. Back

|| To whom correspondence should be addressed. Tel.: 420-2-21951721; Fax: 420-2-21951724; E-mail: zdenap{at}natur.cuni.cz.

1 The abbreviations used are: BKP, bromcresol purple; MDR, multidrug resistance; WT, wild type; MES, 4-morpholineethanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. Back

2 L. Váchová and Z. Palková, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Vojtech Závada for critical reading of the manuscript, and Vladimíra Haislová and Alexandra Pokorná for the technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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