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New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1
FEMS Yeast Research 3 (2003) 77^84
www.fems-microbiology.org
New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1 Cristina Rodr|¤guez a
a;1
, Pascual Sanz b , Carlos Gancedo
a;
Instituto de Investigaciones Biome¤dicas ‘Alberto Sols’ CSIC-UAM, Unidad de Bioqu|¤mica y Gene¤tica de Levaduras, C/Arturo Duperier 4, 28029 Madrid, Spain b Instituto de Biomedicina de Valencia, CSIC, 46010 Valencia, Spain Received 22 May 2002 ; received in revised form 24 July 2002; accepted 26 July 2002 First published online 28 August 2002
Abstract We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose^ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation. 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Glucose signalling ; Catabolite repression ; Snf1; Protein kinase; Pyruvate carboxylase; MTH1
1. Introduction Saccharomyces cerevisiae is found in wine cellars and breweries. When fermentations start the yeast ¢nds media rich in sugars that in time become depleted of them and enriched in ethanol. The presence of glucose and related hexoses has an important in£uence on the expression of many yeast genes and consequently on the metabolism of the organism. In the presence of these sugars, the expression of a large number of genes, mainly those encoding enzymes required to use alternative carbon sources, is repressed. This phenomenon, known as catabolite repression, has been studied in deep and a broad picture of it is emerging [1^3]. Central actors in the repression process
* Corresponding author. Tel. : +34 (91) 585 46 20; Fax : +34 (91) 585 45 87. E-mail address : [email protected] (C. Gancedo). 1
Present address: Bioferma Technological Center, Avda. Europa s/n, 30840 Alhama de Murcia, Murcia, Spain.
are the Mig1 repressor and the Snf1 kinase complex that counteracts it and whose activity is necessary for derepression of glucose-repressed genes [2,4]. Glucose availability in the medium is assessed by the sensor proteins Snf3 and Rgt2 [5^7] that transmit information about the external situation to the internal cellular machinery. These sensors interact with the proteins Std1 and Mth1, relieving their repressive e¡ect on the expression of the genes encoding glucose transporters [8,9]. In the search for new elements involved in glucose signalling in yeast, one strategy used has been the isolation of suppressors of the toxic e¡ects of glucose on di¡erent glycolytic mutants [10^12]. A common result of these approaches has been the isolation of mutated forms of MTH1 less sensitive to the presence of glucose in the medium [9,13]. A possible way to avoid the isolation of mutants a¡ected in MTH1 is to start the search of new mutants in a strain lacking MTH1. This is in principle feasible since deletion of MTH1 does not produce an appreciable e¡ect on growth or gene expression in di¡erent media [14]. We have constructed a triple mutant strain with disruptions in the genes PYC1 PYC2 that en-
1567-1356 / 02 / $22.00 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 5 6 - 3
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code isoenzymes of pyruvate carboxylase [15,16] and in the gene MTH1, and used it to isolate new genes implicated in catabolite repression or glucose signalling. The rationale of our approach is based on the ¢nding that pyc1 pyc2 mutants do not grow in a minimal medium with glucose as carbon source and ammonium as nitrogen source [15]. This behavior is due to the fact that in these conditions pyruvate carboxylase is the only enzyme able to produce oxaloacetate to replenish the tricarboxylic acid cycle, since glucose represses the alternative anaplerotic enzymes isocitrate lyase and malate synthase [17] (Fig. 1). Mutations that abolish catabolite repression would allow growth of pyc1 pyc2 mutants in glucose^ammonium medium because the genes encoding the enzymes of the glyoxylate cycle would be expressed. We report here the isolation and analysis of mutants that partially alleviate the repressive e¡ect not only of glucose but also of galactose.
2. Materials and methods 2.1. Yeast strains, culture conditions and genetic methods The S. cerevisiae strains used in this work are shown in Table 1. Gene disruptions were done according to Rothstein [18]. To construct strain YCR006 an EcoRI^EcoRI fragment from plasmid pMTH1: :TRP1 [9] was introduced into strain YCR005, and to construct strains YCR012 and YCR013 a BamHI^BamHI fragment from pUCsnf1D : :KanMX4 (see below) was introduced into strains W303-1A and YCR011, respectively. Yeasts were grown
at 30‡C either in a rich medium (YP, 1% yeast extract, 2% peptone) or in a minimal medium (YNB, Difco) with 40 mM ammonium sulfate or aspartate and the adequate requirements. The indicated carbon sources were used unless indicated otherwise at 2%. Mutagenesis of strain YCR003 was done by ethyl methane sulfonate as in Lawrence [19]. After mutagenesis the cells were spread directly on minimal glucose^ammonium plates. Colonies appearing after 3^4 days were picked and puri¢ed on plates of the same composition. Crosses between strains, pre-sporulation and sporulation media and micromanipulation of spores were done by standard procedures. Transformation was done by the lithium acetate method [20]. 2.2. Plasmids and library Plasmid pUC-snf1D : :KanMX4 contains an inner fragment of 537 bp of the SNF1 gene replaced by the KanMX4 selection marker [21]. Other plasmids used in this study were pSH18-18 (6lexAoplacZ) and pLR1D1 (0lexAoplacZ) [22], LexA-Snf1, LexA-Snf1K84R and Snf4-GAD [23], pACTII (GAD) [24], pEG202 (LexA) [25] and FBP1-lacZ [26]. The yeast genomic library GRF88, based in YCp50, was obtained from the ATCC collection (Rockville, MD, USA). PCR products were cloned in plasmid pGEM-T (Promega, Madison, WI, USA). 2.3. RNA extraction and Northern analysis Total RNA was extracted from 50 mg cells (wet weight) with the Gibco BRL Trizol reagent (Invitrogen, Carlsbad,
Table 1 Strains used in this work Strain
Name in text
Genotype
Origin or reference
W303-1A 22BHL
Wild-type pyc1 pyc2
Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 MatK ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 can1-100 mth1: :URA3 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 mth1: :URA3 MatK ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 MatK ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 mth1: :TRP1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 mth1: :TRP1 esc1-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 esc1-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 mth1: :TRP1 ESC3-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 ESC3-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 snf1: :KanMX4 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 ESC3-1 snf1: :KanMX4
[42] [12]
MJ002 YCR003
pyc1 pyc2 mth1
YCR005 YCR006 YCR008 YCR009
esc1-1
YCR010 YCR011
ESC3-1
YCR012
snf1
YCR013
ESC3-1 snf1
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Opposite sex to MJL5 [9] This work. From a cross 22BHLUMJ002 This work. From a cross 22BHLUMJ002 This work. Disruption of MTH1 with pMTH1: :TRP1 in YCR005 This work This work This work This work This work. Disruption of SNF1 in W303-1A with KanMX4 This work. Disruption of SNF1 in YCR011 with KanMX4
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CA, USA). The RNAs were separated in 1.5% formaldehyde agarose gels and transferred to Hybond Nþ ¢lters (Amersham, Little Chalfont, UK). Probes were labelled with 32 P using conventional techniques [27]. The probes used were those described by Diderich et al. [28], kindly supplied by A.L. Kruckeberg (Amsterdam, The Netherlands). 2.4. Preparation of extracts and enzymatic assays Cell extracts were prepared by vortexing at 4‡C four times for 1 min, with 1-min intervals in ice about 100 mg wet weight yeast cells with 1 g glass beads (0.4^0.5 mm diameter) and 0.5 ml of 20 mM imidazole, pH 7. After centrifugation in the cold at 8000Ug for 15 min, the supernatants were used for enzyme assays and determination of protein concentration. Fructose-1,6-bisphosphatase was assayed as in Gancedo and Gancedo [29], glutamate dehydrogenase as in Witt et al. [17] and isocitrate lyase as in Dixon and Kornberg [30]. Invertase activity was assayed in whole cells as described by Jiang and Carlson [23]. L-Galactosidase activity was assayed in permeabilized cells and expressed in Miller units [31]. Protein was assayed using the commercial Pierce (Rockford, IL, USA) assay. 2.5. Measurement of glucose consumption, fermentation and respiration Glucose consumption was determined by following glucose disappearance in yeast suspensions (20 mg ml31 ) in 50 mM phosphate bu¡er, pH 6, at 30‡C. Fermentation and respiration were measured at 30‡C in a conventional Warburg respirometer in the same conditions.
3. Results 3.1. Isolation and genetic characterization of mutations that suppress the pyc1 pyc2 phenotype To isolate new genes of S. cerevisiae involved in catabolite repression or glucose signalling and to avoid the isolation of mutations in the gene MTH1 (see Section 1) we started our search with a triple pyc1 pyc2 mth1 mutant strain (YCR003, see Table 1). S. cerevisiae pyc1 pyc2 mth1 mutants do not grow in glucose^ammonium but do so in glucose^aspartate (Fig. 1). After conventional mutagenesis we isolated several independent mutants able to grow in glucose^ammonium. Each mutant was backcrossed to a pyc1 pyc2 mth1 isogenic strain (YCR006, see Table 1) of opposite mating type; the diploids were isolated and tested for growth in glucose^ammonium. Ten diploids derived from mutants with a clear phenotype were sporulated and 12^15 complete tetrads of each cross were scored for growth in glucose^ammonium medium. All mu-
Fig. 1. Situation and role of pyruvate carboxylase in yeast metabolism. During growth in media with ammonium as nitrogen source, inorganic nitrogen enters metabolism via NADPþ -glutamate dehydrogenase (Gdh) that drains away K-ketoglutarate from the tricarboxylic acid cycle to form glutamate. If glucose or other repressing sugars are used as carbon sources, pyruvate carboxylase (Pyc) is the only enzyme able to replenish the cycle due to the repression of isocitrate lyase (Icl) and malate synthase (Mls). Therefore pyc1 pyc2 mutans do not grow in minimal glucose^ammonium medium; they may grow, however, on glucose^aspartate. During growth on non-repressing carbon sources the joint action of isocitrate lyase and malate synthase replenishes the tricarboxylic acid cycle.
tants that produced a progeny indicative of a monogenic mutation were rechecked. Finally we retained one mutant carrying a recessive mutation and another one carrying a semidominant one ; we named the observed phenotype esc (escape of sugars control), the genes responsible for it ESC1 and ESC3, respectively, and the corresponding alleles esc1-1 and ESC3-1. During the analysis of tetrads derived from diploids heterozygous for the ESC3-1 mutation we observed a 2þ :23 segregation of small-size colonies; all the segregants forming small colonies carried the ESC3-1 allele. Minimal galactose^ammonium medium is also a nonpermissive medium for pyc1 pyc2 mth1 mutants due to the repressive e¡ect of galactose on the genes encoding the enzymes of the glyoxylate cycle [32]. However, the strains containing either the esc1-1 or the ESC3-1 mutations grew also in this medium, indicating that the mutations a¡ect some enzymatic or regulatory step common to the two sugars. The esc1-1 and ESC3-1 alleles were placed in a PYC1 PYC2 MTH1 background by adequate genetic crosses and all the studies reported here were carried out with these strains (YCR009 and YCR011, see Table 1). These strains
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Table 2 Some physiological characteristics of the esc1-1 and ESC3-1 mutants Characteristic
Strain
Generation time (min) YP glucose YP galactose YNB glucose Glucose consumption (Wmol g31 min31 ) Fermentation (Wmol CO2 g31 min31 ) Respiration (Wmol O2 g31 min31 )
Wild-type
esc1-1
ESC3-1
90 120 120 26 26 3
90 130 120 16 5 8
150 200 210 14 5 8
The yeasts were grown in the media indicated and the di¡erent parameters determined as described in Section 2. Values are the mean of three independent experiments. Variation between individual values was 6 15%.
are referred to in the text as esc1-1 and ESC3-1, respectively. 3.2. Physiological characteristics of the mutants While the esc1-1 mutant grew as the wild-type in rich or minimal glucose or galactose media, the ESC3-1 mutant grew always slower in those media with an increase of about 1.7-fold in generation time (Table 2). The rate of glucose utilization was lower than that of the wild-type in both mutants and the glucose fermentation rate was strongly decreased (Table 2). Although the rate of glucose respiration increased (Table 2) the mutants were able to grow in the presence of antimycin A, at di¡erence with other strains carrying suppressors that relieve the e¡ect of glucose [11] or galactose [32]. The mutants were not thermosensitive and their microscopic morphology appeared normal in the conditions used. 3.3. The mutations esc1-1 and ESC3-1 decrease repression by glucose and galactose Since pyruvate carboxylase is the only known anaplerotic enzyme that replenishes the tricarboxylic acid cycle during growth in repressing sugars, the fact that the suppressor mutations allowed growth in glucose or galactose^ ammonium media implies that the enzymes of the glyoxy-
late cycle, the alternative anaplerotic pathway (Fig. 1), should be at least partially active. We tested the activity of isocitrate lyase as well as that of other enzymes encoded by catabolite-repressed genes during growth in glucose or galactose and found that all of them were partially derepressed. The degree of derepression was di¡erent for the di¡erent enzymes but the derepression was always higher in the ESC3-1 mutant (Table 3). It is noteworthy that in all cases the level of enzymes found in derepressing conditions was higher in the esc1-1 mutant than in the wild-type. Curiously, although no activity of fructose1,6-bisphosphatase was found in the assay in the esc1-1 mutant grown in glucose, we found that in these conditions a FBP1-lacZ fusion was expressed at low level in the mutant but not in the wild-type (result not shown). This di¡erence between the two situations may indicate that although some derepression of FBP1 occurs in the mutant, fructose-1,6-bisphosphatase activity is not detected due to the e¡ect of catabolite inactivation that affects the yeast enzyme but not the L-galactosidase reporter enzyme [33]. High levels of glucose repress invertase but low levels of the sugar have an inducing e¡ect [34]. As shown in Table 3, low levels of glucose were unable to produce induction of invertase in the mutants. Also in the ESC3-1 mutant at high glucose the level of activity remained similar to that found at 0.05% glucose, indicating a di⁄culty in receiving or processing adequately the glucose signal.
Table 3 Speci¢c activities in the esc mutants of some enzymes repressed by glucose and galactose Relevant genotype
Wild-type esc1-1 ESC3-1
FbPase (mU (mg protein)31 ) GDH (mU (mg protein)31 )
ICL (mU (mg protein)31 )
Invertase (nmol glucose (mg yeast)31 min31 )
Glu
Gal
Gly
Glu
Gal
Gly
Glu
Gal
Gly
2% Gluc
0.5% Gluc
61 61 8
1 10 10
66 90 40
9 30 68
40 60 80
246 340 205
61 4 9
1.5 10 10
38 80 76
3 3 12
230 17 20
For the assay of fructose-1,6-bisphosphatase (FbPase), NAD-glutamate dehydrogenase (GDH) and isocitrate lyase (ICL), yeasts were grown in rich medium with 2% glycerol (Gly), glucose (Glu) or galactose (Gal) as carbon source as described in Section 2, and harvested during the exponential phase of growth. For the invertase assay yeasts were grown on YPD, harvested during the exponential phase of growth and resuspended in YP 2% glucose and YP 0.05% glucose, respectively, for 5 h. Assays for the activities reported were as described in Section 2. Values are the mean of three independent cultures done on di¡erent days. Variation between individual values was 6 15%.
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Fig. 2. Northern blot analysis of the transcripts of the HXT1 and HXT2 genes in strains bearing the mutations esc1-1 and ESC3-1. Total RNA was prepared from cells grown in YP glucose (2% or 0.2%) or YP glucose and treated as indicated in Section 2. Each lane contained about 50 Wg of total RNA. Hybridization was carried out with the speci¢c probes described in Section 2. As an internal control of mRNA charge the amount of ribosomal RNA is shown.
3.4. Expression of the genes encoding sugar transporters in the esc1-1 and ESC3-1 mutants S. cerevisiae has several glucose transporters that are di¡erentially expressed as a function of the glucose concentration in the medium [35]. We studied in the esc mutants the expression of HXT1 and HXT2 as representatives of a low- and a high-a⁄nity transporter. As seen in Fig. 2, expression of HXT1 that is induced at high glucose had a pattern of expression similar in the wild-type and in the mutants; however, expression of HXT2 that is induced by low glucose and repressed by high glucose [36] was no longer repressible in the mutants. Neither HXT1 nor HXT2 was expressed during growth on galactose (Fig. 2), giving evidence that in the mutants the induction pathway controlled by glucose was intact.
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not grow in these conditions since the GAL genes are not repressed and metabolism of the toxic analogue will be possible. Therefore overexpression of genes that complement the ESC3-1 mutation would allow growth of a transformed-complemented mutant in fructose+2-deoxygalactose because the GAL genes will be repressed and 2-deoxygalactose will not be metabolized. We transformed an ESC3-1 mutant with a yeast genomic library selecting for the URA3 marker of the library plasmid on galactose plates and we then replica-plated about 15 000 transformants to plates with 25 mM fructose+1 mM 2-deoxygalactose. On these plates 10 colonies grew; plasmids were isolated from them and analyzed. Two di¡erent restriction patterns with an overlapping region were found; this region contained the sequence of REG1, a gene encoding a regulatory subunit of the phosphatase Glc7 [37]. Overexpression of REG1 restored a normal colony size to the ESC3-1 mutant during growth on glucose and allowed growth of the mutant on fructose in the presence of 2-deoxygalactose. It was important to verify if ESC3-1 was an allelic form of REG1 or if REG1 was an extragenic suppressor. To discriminate between these possibilities we used two approaches. In one of them we made use of the fact that REG1 is linked to TRP1, therefore if ESC3-1 is an allelic form of REG1, in a cross ESC3-1 trp1UESC3 TRP1 most of the tetrads should be of parental di-type and the majority of the ESC3-1 segregants sensitive to 2-deoxygalactose should require tryptophan due to the trp1 mutation. We analyzed 13 complete tetrads and found a segregation of the markers indicative of non-linked genes, demonstrating that although overexpression of REG1 suppressed the phenotype studied, REG1 was not the gene a¡ected in the mutant. In the other approach we studied the e¡ect of the expression of the REG1 gene isolated from the ESC3-1 mu-
3.5. Cloning of REG1 as a suppressor of the phenotype of the ESC3-1 mutant We attempted to clone the genes responsible for the suppressor phenotype found. The high rate of spontaneous reversion of the esc1-1 phenotype precluded a direct approach to the cloning of the ESC1 gene; an indirect approach using the derepression of a FBP1-lacZ fusion in the mutant also failed. In the case of the ESC3 gene we tried to take advantage of our ¢nding that glucose did not repress enzymes needed for galactose utilization in the ESC3-1 mutant (e.g. speci¢c activity of galactokinase in a glucose+galactose culture was 740 mU (mg protein)31 in the mutant while it was below detection level in the wild-type). Our rationale was the following: a wild-type yeast pregrown in a medium with galactose will grow when transferred to a medium with fructose and the toxic analogue 2-deoxygalactose because the genes of the galactose pathway are repressed by fructose; however, a strain with the ESC3-1 mutation will
Table 4 Two-hybrid interactions between Snf1 and Snf4 and transcriptional activation by LexA-Snf1 in an ESC3-1 mutant Strain and LexA fusions Wild-type (6lexAoplacZ) LexA-Snf1 LexA-Snf1 ESC3-1 (6lexAoplacZ) LexA-Snf1 LexA-Snf1 LexA-Snf1K84R LexA ESC3-1 (0lexAoplacZ) LexA-Snf1
GAD fusions
L-Galactosidase (U)
Snf4-GAD GAD
61 61
Snf4-GAD GAD GAD GAD
68 1050 4 2
GAD
61
A wild-type and an ESC3-1 mutant were transformed with plasmids expressing the indicated fusions and grown on 4% glucose. They were harvested during the exponential phase of growth and L-galactosidase was assayed as described in Section 2 ; values are average activities of four to six transformants, with deviation among values lower than 15% in all cases.
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tant by PCR on the behavior of a pyc1 pyc2 mutant. When a pyc1 pyc2 strain was transformed with a plasmid bearing the REG1 gene, none of the transformants grew in glucose^ammonium medium and all of them were resistant to 2-deoxygalactose, indicating that REG1 is di¡erent from ESC3. This result makes clear that REG1 was not mutated in the ESC3-1 mutant, and supports the conclusion reached with the classical genetic approach. 3.6. The ESC3-1 mutation acts upstream of the Snf1 complex Derepression of glucose-repressed genes requires an active Snf1 complex [2]. Since in the esc mutants glucose repression was decreased, we studied in the ESC3-1 mutant, that presents the strongest phenotype, the interaction of Snf1 with Snf4, the activating subunit of the Snf1 kinase complex [23]. As shown in Table 4, expression of a LexA-dependent reporter gene during growth on high glucose was negligible in a wild-type strain expressing LexASnf1 and Snf4-GAD fusion proteins, and high in the ESC3-1 mutant expressing the same proteins. We observed also that in the ESC3-1 background a LexA-Snf1 fusion was by itself a potent transcriptional activator of the reporter gene (Table 4). This activation was dependent on Snf1 activity, as it was not observed when a fusion with a mutated, inactive SNF1 gene (SNF1K84R) was used (Table 4). Kuchin et al. [22] have reported that a hyperactive Snf1 bound as a LexA fusion to a promoter activates transcription in a glucose-regulated way. Our results suggest that the mutation ESC3-1 results in a permanently active Snf1. As proposed by Kuchin et al. [22], this activated Snf1 may interact directly with the RNA polymerase holoenzyme and thereby activate transcription. Although Snf4 has been shown to be required for the direct activation by a LexA-Snf1 fusion [22], our results indicate that in the ESC3-1 mutant, the simultaneous expression of Snf4-GAD interferes with the activation by LexA-Snf1. This is a surprising result for which we do not have a straightforward explanation. We have checked that the characteristics of the ESC3-1 mutant are not due to mutations in the SNF1 or the SNF4 genes. For this we obtained by PCR the SNF1 and SNF4 genes from the ESC31 mutant and transformed either a snf1 or a snf4 mutant with independent PCR products. Expression of invertase and of FBP1-lacZ in the transformants was regulated by glucose as in the wild-type, indicating that the behavior observed in the ESC3-1 mutant was not due to mutations in SNF1 or SNF4 (results not shown). We also veri¢ed that expression of glucose-repressed genes did not occur in a double ESC3-1 snf1 mutant (Table 5). Therefore we conclude that the ESC3-1 mutation does not bypass the requirement for Snf1. The mutation likely acts upstream of the Snf1 complex, either facilitating the activation of Snf1 or interfering with its glucose-dependent inactivation. As described in the Section 3.5, an
Table 5 E¡ect of an snf1 mutation on the derepression of di¡erent genes in the ESC3-1 mutant Relevant genotype
Speci¢c activity (mU (mg protein)31 ) of: Invertase
Wild-type snf1 ESC3-1 ESC3-1 snf1
61 61 5 61
FBP1-lacZ 61 61 255 61
The yeasts were grown in minimal medium with 4% glucose and harvested in the exponential phase of growth. Enzymatic assays were as described in Section 2. For the L-galactosidase assays, values are the mean of four to six independent transformants, with di¡erences among values lower than 10% in all cases.
excess of the regulatory protein Reg1 counteracts this action of the ESC3-1 mutation.
4. Discussion We have isolated mutants from S. cerevisiae that relieve, partially and simultaneously, the repression produced by glucose and by galactose. Although this is not generally realized, galactose is able to produce in yeast many of the e¡ects caused by glucose, although with less intensity [32,38]. The isolation of the mutants reported here suggests that both sugars share some common step(s) in their respective repression pathways. Mutants with the characteristics described here have not been reported previously, in spite of various searches for mutants that resist the glucose or the galactose e¡ects [10,11,32]. In the case of glucose, mutant alleles of MTH1 have appeared in di¡erent screens, while in those used to search for galactose resistance, mutations a¡ecting either GAL2 or GAL4 have been found [32]. These results indicate that an interference with the glucose signal, which involves the glucose sensors Snf3 and Rgt2, as well as Mth1 [8,9], produces the strongest mutants, and that only a deletion of the MTH1 gene allows recovery of mutations in other genes. It is noteworthy, however, that the mutations described in this work acted even in a MTH1 wild-type background. A marked decrease in glucose transport can release glucose repression in yeast [11]. However, this mechanism is unlikely to account for the phenotypes observed here, since an important although reduced glucose consumption was measured in the mutants. Also the release of galactose repression argues against such a mechanism. Moreover, HXT1 expression was similar in the mutants and in the wild-type, and HXT2 had lost its repression by glucose. The characteristics of the mutants make it also unlikely that they are alleles of recently isolated mutations conferring resistance to catabolite repression such as gsf2 or GSF4 [39,40]. The recessive gsf2 mutation causes a syn-
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thetic lethal phenotype with snf1, which was not the case with the ESC3-1 mutant as shown in Table 5. The GSF4 mutants exhibit a 20-fold higher invertase than the wildtype when grown at high glucose levels [40], making them also di¡erent from the mutants reported here. According to present ideas, two conditions shall be met for derepression of genes repressed by both glucose and galactose. First, the protein Snf1 shall be in its active, phosphorylated state and second, Snf1 shall be localized in the nucleus [41]. This localization occurs only when the protein Gal83, a component of the Snf1 complex, is in a certain conformation. The mutant ESC3-1 behaved as having a permanently active Snf1 as shown by the derepression of galactokinase in the presence of glucose. Moreover, the signi¢cant derepression of genes encoding gluconeogenic enzymes observed in the mutants indicates that at least part of the Snf1 protein is localized in the nucleus both in glucose and in galactose cultures. A possible explanation of the results is that the mutation in£uences some signal that a¡ects the phosphorylation of Snf1 and the conversion of Gal83 to its active conformation. It is worth remarking that the phenotype of the ESC3-1 mutant is completely dependent on the presence of the protein kinase Snf1: double mutants ESC3-1 snf1 are permanently repressed. Moreover, theESC3-1 mutation is not localized in SNF1 or in SNF4; it appears therefore that the mutation acts upstream of Snf1 and that ESC3-1 could encode a protein implicated in the function of the Snf1 complex. The isolation of REG1 as a suppressor of the ESC3-1 phenotype is also consistent with an upregulated activity of Snf1 due to a mutated Esc3 protein. Reg1 is a regulatory subunit of the Glc7 phosphatase complex that targets it to speci¢c substrates. When REG1 is overexpressed, the complex Glc7/Reg1 is able to convert the active form of Snf1 to the inactive one, thus causing the reappearance of a normal phenotype in the mutant. An additional, unexpected feature found in the mutant ESC3-1 was that in this background, a LexA-Snf1 fusion was by itself a potent transcriptional activator of a LexA fusion gene reporter in glucose-growing cells. Although it has been described that a hyperactive Snf1 kinase, Snf1G53R, bound to a LexA fusion was also a potent transcriptional activator, its activity was otherwise regulated as in a wild-type [22], in contrast with the behavior observed in the ESC3-1 mutant. Curiously, expression of Snf4-GAD interfered with the activation by LexA-Snf1, a result for which we presently do not have an adequate explanation. The results presented indicate that there are still unknown elements in the signal transduction pathways for sugars and give evidence that there is a common link in the pathways that transduce the glucose and galactose signals.
83
Acknowledgements We thank Juana M. Gancedo for discussions and active help in the preparation of the manuscript, Carmen-Lisset Flores and O. Zaragoza for discussions along the work, and A.L. Kruckeberg (Amsterdam, The Netherlands) for sending the probes to assay expression of genes encoding hexose transporters. This work was partially supported by Grants PB97-1213-CO2-01 and PB98-0846 from the Spanish Government to C.G. and to P.S., respectively. C.R. bene¢tted from a predoctoral Fellowship from the Fundacio¤n Ramo¤n Areces (Madrid, Spain).
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[30] Dixon, G.H. and Kornberg, H.L. (1959) Assay methods for enzymes of the glyoxylate cycle. Biochem. J. 72, 3. [31] Ludin, K., Jiang, R. and Carlson, M. (1998) Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 95, 6245^6250. [32] Rodr|¤guez, C. and Flores, C.L. (2000) Mutations in GAL2 or GAL4 alleviate catabolite repression produced by galactose in Saccharomyces cerevisiae. Enz. Microb. Technol. 26, 748^755. [33] Gamo, F.J., Navas, M.A., Bla¤zquez, M.A., Gancedo, C. and Gancedo, J.M. (1994) Catabolite inactivation of heterologous fructose-1,6bisphosphatases and fructose-1,6-bisphosphatase-L-galactosidase fusion proteins in Saccharomyces cerevisiae. Eur. J. Biochem. 222, 879^884. º zcan, S., Vallier, L.G., Flick, J.S., Carlson, M. and Johnston, M. [34] O (1997) Expression of the SUC2 gene of Saccharomyces cerevisiae is induced by low levels of glucose. Yeast 13, 127^137. [35] Boles, E. and Hollenberg, C.P. (1997) The molecular genetics of hexose transport in yeasts. FEMS Microbiol. Rev. 21, 85^111. º zcan, S. and Johnston, M. (1995) Three di¡erent regulatory mech[36] O anisms enable yeast hexose transporter (HXT) genes to be induced by di¡erent levels of glucose. Mol. Cell. Biol. 15, 1564^1572. [37] Tu, J. and Carlson, M. (1995) REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae. EMBO J. 14, 5939^5946. [38] GoncXalves, P.M., Gri⁄oen, G., Bebelman, J.P. and Planta, R.J. (1997) Signalling pathways leading to transcriptional regulation of genes involved in the activation of glycolysis in yeast. Mol. Microbiol. 25, 483^493. [39] Sherwood, P.W. and Carlson, M. (1999) E⁄cient export of the glucose transporter Hxt1p from the endoplasmic reticulum requires Gsf2p. Proc. Natl. Acad. Sci. USA 96, 7415^7420. [40] Sherwood, P.W., Katic, I., Sanz, P. and Carlson, M. (2000) A glucose transporter chimera confers a dominant negative glucose starvation phenotype in Saccharomyces cerevisiae. Genetics 155, 989^992. [41] Vincent, O., Townley, R., Kuchin, S. and Carlson, M. (2001) Subcellular localization of the Snf1 kinase is regulated by speci¢c beta subunits and a novel glucose signaling mechanism. Genes Dev. 15, 1104^1114. [42] Thomas, B.J. and Rothstein, R. (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56, 619^630.
FEMSYR 1512 13-2-03
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New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1 Cristina Rodr|¤guez a
a;1
, Pascual Sanz b , Carlos Gancedo
a;
Instituto de Investigaciones Biome¤dicas ‘Alberto Sols’ CSIC-UAM, Unidad de Bioqu|¤mica y Gene¤tica de Levaduras, C/Arturo Duperier 4, 28029 Madrid, Spain b Instituto de Biomedicina de Valencia, CSIC, 46010 Valencia, Spain Received 22 May 2002 ; received in revised form 24 July 2002; accepted 26 July 2002 First published online 28 August 2002
Abstract We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose^ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation. 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Glucose signalling ; Catabolite repression ; Snf1; Protein kinase; Pyruvate carboxylase; MTH1
1. Introduction Saccharomyces cerevisiae is found in wine cellars and breweries. When fermentations start the yeast ¢nds media rich in sugars that in time become depleted of them and enriched in ethanol. The presence of glucose and related hexoses has an important in£uence on the expression of many yeast genes and consequently on the metabolism of the organism. In the presence of these sugars, the expression of a large number of genes, mainly those encoding enzymes required to use alternative carbon sources, is repressed. This phenomenon, known as catabolite repression, has been studied in deep and a broad picture of it is emerging [1^3]. Central actors in the repression process
* Corresponding author. Tel. : +34 (91) 585 46 20; Fax : +34 (91) 585 45 87. E-mail address : [email protected] (C. Gancedo). 1
Present address: Bioferma Technological Center, Avda. Europa s/n, 30840 Alhama de Murcia, Murcia, Spain.
are the Mig1 repressor and the Snf1 kinase complex that counteracts it and whose activity is necessary for derepression of glucose-repressed genes [2,4]. Glucose availability in the medium is assessed by the sensor proteins Snf3 and Rgt2 [5^7] that transmit information about the external situation to the internal cellular machinery. These sensors interact with the proteins Std1 and Mth1, relieving their repressive e¡ect on the expression of the genes encoding glucose transporters [8,9]. In the search for new elements involved in glucose signalling in yeast, one strategy used has been the isolation of suppressors of the toxic e¡ects of glucose on di¡erent glycolytic mutants [10^12]. A common result of these approaches has been the isolation of mutated forms of MTH1 less sensitive to the presence of glucose in the medium [9,13]. A possible way to avoid the isolation of mutants a¡ected in MTH1 is to start the search of new mutants in a strain lacking MTH1. This is in principle feasible since deletion of MTH1 does not produce an appreciable e¡ect on growth or gene expression in di¡erent media [14]. We have constructed a triple mutant strain with disruptions in the genes PYC1 PYC2 that en-
1567-1356 / 02 / $22.00 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 5 6 - 3
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code isoenzymes of pyruvate carboxylase [15,16] and in the gene MTH1, and used it to isolate new genes implicated in catabolite repression or glucose signalling. The rationale of our approach is based on the ¢nding that pyc1 pyc2 mutants do not grow in a minimal medium with glucose as carbon source and ammonium as nitrogen source [15]. This behavior is due to the fact that in these conditions pyruvate carboxylase is the only enzyme able to produce oxaloacetate to replenish the tricarboxylic acid cycle, since glucose represses the alternative anaplerotic enzymes isocitrate lyase and malate synthase [17] (Fig. 1). Mutations that abolish catabolite repression would allow growth of pyc1 pyc2 mutants in glucose^ammonium medium because the genes encoding the enzymes of the glyoxylate cycle would be expressed. We report here the isolation and analysis of mutants that partially alleviate the repressive e¡ect not only of glucose but also of galactose.
2. Materials and methods 2.1. Yeast strains, culture conditions and genetic methods The S. cerevisiae strains used in this work are shown in Table 1. Gene disruptions were done according to Rothstein [18]. To construct strain YCR006 an EcoRI^EcoRI fragment from plasmid pMTH1: :TRP1 [9] was introduced into strain YCR005, and to construct strains YCR012 and YCR013 a BamHI^BamHI fragment from pUCsnf1D : :KanMX4 (see below) was introduced into strains W303-1A and YCR011, respectively. Yeasts were grown
at 30‡C either in a rich medium (YP, 1% yeast extract, 2% peptone) or in a minimal medium (YNB, Difco) with 40 mM ammonium sulfate or aspartate and the adequate requirements. The indicated carbon sources were used unless indicated otherwise at 2%. Mutagenesis of strain YCR003 was done by ethyl methane sulfonate as in Lawrence [19]. After mutagenesis the cells were spread directly on minimal glucose^ammonium plates. Colonies appearing after 3^4 days were picked and puri¢ed on plates of the same composition. Crosses between strains, pre-sporulation and sporulation media and micromanipulation of spores were done by standard procedures. Transformation was done by the lithium acetate method [20]. 2.2. Plasmids and library Plasmid pUC-snf1D : :KanMX4 contains an inner fragment of 537 bp of the SNF1 gene replaced by the KanMX4 selection marker [21]. Other plasmids used in this study were pSH18-18 (6lexAoplacZ) and pLR1D1 (0lexAoplacZ) [22], LexA-Snf1, LexA-Snf1K84R and Snf4-GAD [23], pACTII (GAD) [24], pEG202 (LexA) [25] and FBP1-lacZ [26]. The yeast genomic library GRF88, based in YCp50, was obtained from the ATCC collection (Rockville, MD, USA). PCR products were cloned in plasmid pGEM-T (Promega, Madison, WI, USA). 2.3. RNA extraction and Northern analysis Total RNA was extracted from 50 mg cells (wet weight) with the Gibco BRL Trizol reagent (Invitrogen, Carlsbad,
Table 1 Strains used in this work Strain
Name in text
Genotype
Origin or reference
W303-1A 22BHL
Wild-type pyc1 pyc2
Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 MatK ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 can1-100 mth1: :URA3 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 mth1: :URA3 MatK ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 MatK ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 mth1: :TRP1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 mth1: :TRP1 esc1-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 esc1-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 pyc1: :LEU2 pyc2: :HIS3 mth1: :TRP1 ESC3-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 ESC3-1 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 snf1: :KanMX4 Mata ade2-1 his3-11,15 leu2-3,112 ura3-52 trp1-1 ESC3-1 snf1: :KanMX4
[42] [12]
MJ002 YCR003
pyc1 pyc2 mth1
YCR005 YCR006 YCR008 YCR009
esc1-1
YCR010 YCR011
ESC3-1
YCR012
snf1
YCR013
ESC3-1 snf1
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Opposite sex to MJL5 [9] This work. From a cross 22BHLUMJ002 This work. From a cross 22BHLUMJ002 This work. Disruption of MTH1 with pMTH1: :TRP1 in YCR005 This work This work This work This work This work. Disruption of SNF1 in W303-1A with KanMX4 This work. Disruption of SNF1 in YCR011 with KanMX4
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CA, USA). The RNAs were separated in 1.5% formaldehyde agarose gels and transferred to Hybond Nþ ¢lters (Amersham, Little Chalfont, UK). Probes were labelled with 32 P using conventional techniques [27]. The probes used were those described by Diderich et al. [28], kindly supplied by A.L. Kruckeberg (Amsterdam, The Netherlands). 2.4. Preparation of extracts and enzymatic assays Cell extracts were prepared by vortexing at 4‡C four times for 1 min, with 1-min intervals in ice about 100 mg wet weight yeast cells with 1 g glass beads (0.4^0.5 mm diameter) and 0.5 ml of 20 mM imidazole, pH 7. After centrifugation in the cold at 8000Ug for 15 min, the supernatants were used for enzyme assays and determination of protein concentration. Fructose-1,6-bisphosphatase was assayed as in Gancedo and Gancedo [29], glutamate dehydrogenase as in Witt et al. [17] and isocitrate lyase as in Dixon and Kornberg [30]. Invertase activity was assayed in whole cells as described by Jiang and Carlson [23]. L-Galactosidase activity was assayed in permeabilized cells and expressed in Miller units [31]. Protein was assayed using the commercial Pierce (Rockford, IL, USA) assay. 2.5. Measurement of glucose consumption, fermentation and respiration Glucose consumption was determined by following glucose disappearance in yeast suspensions (20 mg ml31 ) in 50 mM phosphate bu¡er, pH 6, at 30‡C. Fermentation and respiration were measured at 30‡C in a conventional Warburg respirometer in the same conditions.
3. Results 3.1. Isolation and genetic characterization of mutations that suppress the pyc1 pyc2 phenotype To isolate new genes of S. cerevisiae involved in catabolite repression or glucose signalling and to avoid the isolation of mutations in the gene MTH1 (see Section 1) we started our search with a triple pyc1 pyc2 mth1 mutant strain (YCR003, see Table 1). S. cerevisiae pyc1 pyc2 mth1 mutants do not grow in glucose^ammonium but do so in glucose^aspartate (Fig. 1). After conventional mutagenesis we isolated several independent mutants able to grow in glucose^ammonium. Each mutant was backcrossed to a pyc1 pyc2 mth1 isogenic strain (YCR006, see Table 1) of opposite mating type; the diploids were isolated and tested for growth in glucose^ammonium. Ten diploids derived from mutants with a clear phenotype were sporulated and 12^15 complete tetrads of each cross were scored for growth in glucose^ammonium medium. All mu-
Fig. 1. Situation and role of pyruvate carboxylase in yeast metabolism. During growth in media with ammonium as nitrogen source, inorganic nitrogen enters metabolism via NADPþ -glutamate dehydrogenase (Gdh) that drains away K-ketoglutarate from the tricarboxylic acid cycle to form glutamate. If glucose or other repressing sugars are used as carbon sources, pyruvate carboxylase (Pyc) is the only enzyme able to replenish the cycle due to the repression of isocitrate lyase (Icl) and malate synthase (Mls). Therefore pyc1 pyc2 mutans do not grow in minimal glucose^ammonium medium; they may grow, however, on glucose^aspartate. During growth on non-repressing carbon sources the joint action of isocitrate lyase and malate synthase replenishes the tricarboxylic acid cycle.
tants that produced a progeny indicative of a monogenic mutation were rechecked. Finally we retained one mutant carrying a recessive mutation and another one carrying a semidominant one ; we named the observed phenotype esc (escape of sugars control), the genes responsible for it ESC1 and ESC3, respectively, and the corresponding alleles esc1-1 and ESC3-1. During the analysis of tetrads derived from diploids heterozygous for the ESC3-1 mutation we observed a 2þ :23 segregation of small-size colonies; all the segregants forming small colonies carried the ESC3-1 allele. Minimal galactose^ammonium medium is also a nonpermissive medium for pyc1 pyc2 mth1 mutants due to the repressive e¡ect of galactose on the genes encoding the enzymes of the glyoxylate cycle [32]. However, the strains containing either the esc1-1 or the ESC3-1 mutations grew also in this medium, indicating that the mutations a¡ect some enzymatic or regulatory step common to the two sugars. The esc1-1 and ESC3-1 alleles were placed in a PYC1 PYC2 MTH1 background by adequate genetic crosses and all the studies reported here were carried out with these strains (YCR009 and YCR011, see Table 1). These strains
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Table 2 Some physiological characteristics of the esc1-1 and ESC3-1 mutants Characteristic
Strain
Generation time (min) YP glucose YP galactose YNB glucose Glucose consumption (Wmol g31 min31 ) Fermentation (Wmol CO2 g31 min31 ) Respiration (Wmol O2 g31 min31 )
Wild-type
esc1-1
ESC3-1
90 120 120 26 26 3
90 130 120 16 5 8
150 200 210 14 5 8
The yeasts were grown in the media indicated and the di¡erent parameters determined as described in Section 2. Values are the mean of three independent experiments. Variation between individual values was 6 15%.
are referred to in the text as esc1-1 and ESC3-1, respectively. 3.2. Physiological characteristics of the mutants While the esc1-1 mutant grew as the wild-type in rich or minimal glucose or galactose media, the ESC3-1 mutant grew always slower in those media with an increase of about 1.7-fold in generation time (Table 2). The rate of glucose utilization was lower than that of the wild-type in both mutants and the glucose fermentation rate was strongly decreased (Table 2). Although the rate of glucose respiration increased (Table 2) the mutants were able to grow in the presence of antimycin A, at di¡erence with other strains carrying suppressors that relieve the e¡ect of glucose [11] or galactose [32]. The mutants were not thermosensitive and their microscopic morphology appeared normal in the conditions used. 3.3. The mutations esc1-1 and ESC3-1 decrease repression by glucose and galactose Since pyruvate carboxylase is the only known anaplerotic enzyme that replenishes the tricarboxylic acid cycle during growth in repressing sugars, the fact that the suppressor mutations allowed growth in glucose or galactose^ ammonium media implies that the enzymes of the glyoxy-
late cycle, the alternative anaplerotic pathway (Fig. 1), should be at least partially active. We tested the activity of isocitrate lyase as well as that of other enzymes encoded by catabolite-repressed genes during growth in glucose or galactose and found that all of them were partially derepressed. The degree of derepression was di¡erent for the di¡erent enzymes but the derepression was always higher in the ESC3-1 mutant (Table 3). It is noteworthy that in all cases the level of enzymes found in derepressing conditions was higher in the esc1-1 mutant than in the wild-type. Curiously, although no activity of fructose1,6-bisphosphatase was found in the assay in the esc1-1 mutant grown in glucose, we found that in these conditions a FBP1-lacZ fusion was expressed at low level in the mutant but not in the wild-type (result not shown). This di¡erence between the two situations may indicate that although some derepression of FBP1 occurs in the mutant, fructose-1,6-bisphosphatase activity is not detected due to the e¡ect of catabolite inactivation that affects the yeast enzyme but not the L-galactosidase reporter enzyme [33]. High levels of glucose repress invertase but low levels of the sugar have an inducing e¡ect [34]. As shown in Table 3, low levels of glucose were unable to produce induction of invertase in the mutants. Also in the ESC3-1 mutant at high glucose the level of activity remained similar to that found at 0.05% glucose, indicating a di⁄culty in receiving or processing adequately the glucose signal.
Table 3 Speci¢c activities in the esc mutants of some enzymes repressed by glucose and galactose Relevant genotype
Wild-type esc1-1 ESC3-1
FbPase (mU (mg protein)31 ) GDH (mU (mg protein)31 )
ICL (mU (mg protein)31 )
Invertase (nmol glucose (mg yeast)31 min31 )
Glu
Gal
Gly
Glu
Gal
Gly
Glu
Gal
Gly
2% Gluc
0.5% Gluc
61 61 8
1 10 10
66 90 40
9 30 68
40 60 80
246 340 205
61 4 9
1.5 10 10
38 80 76
3 3 12
230 17 20
For the assay of fructose-1,6-bisphosphatase (FbPase), NAD-glutamate dehydrogenase (GDH) and isocitrate lyase (ICL), yeasts were grown in rich medium with 2% glycerol (Gly), glucose (Glu) or galactose (Gal) as carbon source as described in Section 2, and harvested during the exponential phase of growth. For the invertase assay yeasts were grown on YPD, harvested during the exponential phase of growth and resuspended in YP 2% glucose and YP 0.05% glucose, respectively, for 5 h. Assays for the activities reported were as described in Section 2. Values are the mean of three independent cultures done on di¡erent days. Variation between individual values was 6 15%.
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Fig. 2. Northern blot analysis of the transcripts of the HXT1 and HXT2 genes in strains bearing the mutations esc1-1 and ESC3-1. Total RNA was prepared from cells grown in YP glucose (2% or 0.2%) or YP glucose and treated as indicated in Section 2. Each lane contained about 50 Wg of total RNA. Hybridization was carried out with the speci¢c probes described in Section 2. As an internal control of mRNA charge the amount of ribosomal RNA is shown.
3.4. Expression of the genes encoding sugar transporters in the esc1-1 and ESC3-1 mutants S. cerevisiae has several glucose transporters that are di¡erentially expressed as a function of the glucose concentration in the medium [35]. We studied in the esc mutants the expression of HXT1 and HXT2 as representatives of a low- and a high-a⁄nity transporter. As seen in Fig. 2, expression of HXT1 that is induced at high glucose had a pattern of expression similar in the wild-type and in the mutants; however, expression of HXT2 that is induced by low glucose and repressed by high glucose [36] was no longer repressible in the mutants. Neither HXT1 nor HXT2 was expressed during growth on galactose (Fig. 2), giving evidence that in the mutants the induction pathway controlled by glucose was intact.
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not grow in these conditions since the GAL genes are not repressed and metabolism of the toxic analogue will be possible. Therefore overexpression of genes that complement the ESC3-1 mutation would allow growth of a transformed-complemented mutant in fructose+2-deoxygalactose because the GAL genes will be repressed and 2-deoxygalactose will not be metabolized. We transformed an ESC3-1 mutant with a yeast genomic library selecting for the URA3 marker of the library plasmid on galactose plates and we then replica-plated about 15 000 transformants to plates with 25 mM fructose+1 mM 2-deoxygalactose. On these plates 10 colonies grew; plasmids were isolated from them and analyzed. Two di¡erent restriction patterns with an overlapping region were found; this region contained the sequence of REG1, a gene encoding a regulatory subunit of the phosphatase Glc7 [37]. Overexpression of REG1 restored a normal colony size to the ESC3-1 mutant during growth on glucose and allowed growth of the mutant on fructose in the presence of 2-deoxygalactose. It was important to verify if ESC3-1 was an allelic form of REG1 or if REG1 was an extragenic suppressor. To discriminate between these possibilities we used two approaches. In one of them we made use of the fact that REG1 is linked to TRP1, therefore if ESC3-1 is an allelic form of REG1, in a cross ESC3-1 trp1UESC3 TRP1 most of the tetrads should be of parental di-type and the majority of the ESC3-1 segregants sensitive to 2-deoxygalactose should require tryptophan due to the trp1 mutation. We analyzed 13 complete tetrads and found a segregation of the markers indicative of non-linked genes, demonstrating that although overexpression of REG1 suppressed the phenotype studied, REG1 was not the gene a¡ected in the mutant. In the other approach we studied the e¡ect of the expression of the REG1 gene isolated from the ESC3-1 mu-
3.5. Cloning of REG1 as a suppressor of the phenotype of the ESC3-1 mutant We attempted to clone the genes responsible for the suppressor phenotype found. The high rate of spontaneous reversion of the esc1-1 phenotype precluded a direct approach to the cloning of the ESC1 gene; an indirect approach using the derepression of a FBP1-lacZ fusion in the mutant also failed. In the case of the ESC3 gene we tried to take advantage of our ¢nding that glucose did not repress enzymes needed for galactose utilization in the ESC3-1 mutant (e.g. speci¢c activity of galactokinase in a glucose+galactose culture was 740 mU (mg protein)31 in the mutant while it was below detection level in the wild-type). Our rationale was the following: a wild-type yeast pregrown in a medium with galactose will grow when transferred to a medium with fructose and the toxic analogue 2-deoxygalactose because the genes of the galactose pathway are repressed by fructose; however, a strain with the ESC3-1 mutation will
Table 4 Two-hybrid interactions between Snf1 and Snf4 and transcriptional activation by LexA-Snf1 in an ESC3-1 mutant Strain and LexA fusions Wild-type (6lexAoplacZ) LexA-Snf1 LexA-Snf1 ESC3-1 (6lexAoplacZ) LexA-Snf1 LexA-Snf1 LexA-Snf1K84R LexA ESC3-1 (0lexAoplacZ) LexA-Snf1
GAD fusions
L-Galactosidase (U)
Snf4-GAD GAD
61 61
Snf4-GAD GAD GAD GAD
68 1050 4 2
GAD
61
A wild-type and an ESC3-1 mutant were transformed with plasmids expressing the indicated fusions and grown on 4% glucose. They were harvested during the exponential phase of growth and L-galactosidase was assayed as described in Section 2 ; values are average activities of four to six transformants, with deviation among values lower than 15% in all cases.
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tant by PCR on the behavior of a pyc1 pyc2 mutant. When a pyc1 pyc2 strain was transformed with a plasmid bearing the REG1 gene, none of the transformants grew in glucose^ammonium medium and all of them were resistant to 2-deoxygalactose, indicating that REG1 is di¡erent from ESC3. This result makes clear that REG1 was not mutated in the ESC3-1 mutant, and supports the conclusion reached with the classical genetic approach. 3.6. The ESC3-1 mutation acts upstream of the Snf1 complex Derepression of glucose-repressed genes requires an active Snf1 complex [2]. Since in the esc mutants glucose repression was decreased, we studied in the ESC3-1 mutant, that presents the strongest phenotype, the interaction of Snf1 with Snf4, the activating subunit of the Snf1 kinase complex [23]. As shown in Table 4, expression of a LexA-dependent reporter gene during growth on high glucose was negligible in a wild-type strain expressing LexASnf1 and Snf4-GAD fusion proteins, and high in the ESC3-1 mutant expressing the same proteins. We observed also that in the ESC3-1 background a LexA-Snf1 fusion was by itself a potent transcriptional activator of the reporter gene (Table 4). This activation was dependent on Snf1 activity, as it was not observed when a fusion with a mutated, inactive SNF1 gene (SNF1K84R) was used (Table 4). Kuchin et al. [22] have reported that a hyperactive Snf1 bound as a LexA fusion to a promoter activates transcription in a glucose-regulated way. Our results suggest that the mutation ESC3-1 results in a permanently active Snf1. As proposed by Kuchin et al. [22], this activated Snf1 may interact directly with the RNA polymerase holoenzyme and thereby activate transcription. Although Snf4 has been shown to be required for the direct activation by a LexA-Snf1 fusion [22], our results indicate that in the ESC3-1 mutant, the simultaneous expression of Snf4-GAD interferes with the activation by LexA-Snf1. This is a surprising result for which we do not have a straightforward explanation. We have checked that the characteristics of the ESC3-1 mutant are not due to mutations in the SNF1 or the SNF4 genes. For this we obtained by PCR the SNF1 and SNF4 genes from the ESC31 mutant and transformed either a snf1 or a snf4 mutant with independent PCR products. Expression of invertase and of FBP1-lacZ in the transformants was regulated by glucose as in the wild-type, indicating that the behavior observed in the ESC3-1 mutant was not due to mutations in SNF1 or SNF4 (results not shown). We also veri¢ed that expression of glucose-repressed genes did not occur in a double ESC3-1 snf1 mutant (Table 5). Therefore we conclude that the ESC3-1 mutation does not bypass the requirement for Snf1. The mutation likely acts upstream of the Snf1 complex, either facilitating the activation of Snf1 or interfering with its glucose-dependent inactivation. As described in the Section 3.5, an
Table 5 E¡ect of an snf1 mutation on the derepression of di¡erent genes in the ESC3-1 mutant Relevant genotype
Speci¢c activity (mU (mg protein)31 ) of: Invertase
Wild-type snf1 ESC3-1 ESC3-1 snf1
61 61 5 61
FBP1-lacZ 61 61 255 61
The yeasts were grown in minimal medium with 4% glucose and harvested in the exponential phase of growth. Enzymatic assays were as described in Section 2. For the L-galactosidase assays, values are the mean of four to six independent transformants, with di¡erences among values lower than 10% in all cases.
excess of the regulatory protein Reg1 counteracts this action of the ESC3-1 mutation.
4. Discussion We have isolated mutants from S. cerevisiae that relieve, partially and simultaneously, the repression produced by glucose and by galactose. Although this is not generally realized, galactose is able to produce in yeast many of the e¡ects caused by glucose, although with less intensity [32,38]. The isolation of the mutants reported here suggests that both sugars share some common step(s) in their respective repression pathways. Mutants with the characteristics described here have not been reported previously, in spite of various searches for mutants that resist the glucose or the galactose e¡ects [10,11,32]. In the case of glucose, mutant alleles of MTH1 have appeared in di¡erent screens, while in those used to search for galactose resistance, mutations a¡ecting either GAL2 or GAL4 have been found [32]. These results indicate that an interference with the glucose signal, which involves the glucose sensors Snf3 and Rgt2, as well as Mth1 [8,9], produces the strongest mutants, and that only a deletion of the MTH1 gene allows recovery of mutations in other genes. It is noteworthy, however, that the mutations described in this work acted even in a MTH1 wild-type background. A marked decrease in glucose transport can release glucose repression in yeast [11]. However, this mechanism is unlikely to account for the phenotypes observed here, since an important although reduced glucose consumption was measured in the mutants. Also the release of galactose repression argues against such a mechanism. Moreover, HXT1 expression was similar in the mutants and in the wild-type, and HXT2 had lost its repression by glucose. The characteristics of the mutants make it also unlikely that they are alleles of recently isolated mutations conferring resistance to catabolite repression such as gsf2 or GSF4 [39,40]. The recessive gsf2 mutation causes a syn-
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thetic lethal phenotype with snf1, which was not the case with the ESC3-1 mutant as shown in Table 5. The GSF4 mutants exhibit a 20-fold higher invertase than the wildtype when grown at high glucose levels [40], making them also di¡erent from the mutants reported here. According to present ideas, two conditions shall be met for derepression of genes repressed by both glucose and galactose. First, the protein Snf1 shall be in its active, phosphorylated state and second, Snf1 shall be localized in the nucleus [41]. This localization occurs only when the protein Gal83, a component of the Snf1 complex, is in a certain conformation. The mutant ESC3-1 behaved as having a permanently active Snf1 as shown by the derepression of galactokinase in the presence of glucose. Moreover, the signi¢cant derepression of genes encoding gluconeogenic enzymes observed in the mutants indicates that at least part of the Snf1 protein is localized in the nucleus both in glucose and in galactose cultures. A possible explanation of the results is that the mutation in£uences some signal that a¡ects the phosphorylation of Snf1 and the conversion of Gal83 to its active conformation. It is worth remarking that the phenotype of the ESC3-1 mutant is completely dependent on the presence of the protein kinase Snf1: double mutants ESC3-1 snf1 are permanently repressed. Moreover, theESC3-1 mutation is not localized in SNF1 or in SNF4; it appears therefore that the mutation acts upstream of Snf1 and that ESC3-1 could encode a protein implicated in the function of the Snf1 complex. The isolation of REG1 as a suppressor of the ESC3-1 phenotype is also consistent with an upregulated activity of Snf1 due to a mutated Esc3 protein. Reg1 is a regulatory subunit of the Glc7 phosphatase complex that targets it to speci¢c substrates. When REG1 is overexpressed, the complex Glc7/Reg1 is able to convert the active form of Snf1 to the inactive one, thus causing the reappearance of a normal phenotype in the mutant. An additional, unexpected feature found in the mutant ESC3-1 was that in this background, a LexA-Snf1 fusion was by itself a potent transcriptional activator of a LexA fusion gene reporter in glucose-growing cells. Although it has been described that a hyperactive Snf1 kinase, Snf1G53R, bound to a LexA fusion was also a potent transcriptional activator, its activity was otherwise regulated as in a wild-type [22], in contrast with the behavior observed in the ESC3-1 mutant. Curiously, expression of Snf4-GAD interfered with the activation by LexA-Snf1, a result for which we presently do not have an adequate explanation. The results presented indicate that there are still unknown elements in the signal transduction pathways for sugars and give evidence that there is a common link in the pathways that transduce the glucose and galactose signals.
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Acknowledgements We thank Juana M. Gancedo for discussions and active help in the preparation of the manuscript, Carmen-Lisset Flores and O. Zaragoza for discussions along the work, and A.L. Kruckeberg (Amsterdam, The Netherlands) for sending the probes to assay expression of genes encoding hexose transporters. This work was partially supported by Grants PB97-1213-CO2-01 and PB98-0846 from the Spanish Government to C.G. and to P.S., respectively. C.R. bene¢tted from a predoctoral Fellowship from the Fundacio¤n Ramo¤n Areces (Madrid, Spain).
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