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Department of Molecular and Cellular Biology, College of Biological Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada
Correspondence
George van der Merwe
gvanderm{at}uoguelph.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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mutant in response to high concentrations of NaCl, implying that NSF1 is also needed for the yeast response to sodium stress. The carbon- and NaCl-mediated transcriptional activation of ENA1 is dependent on Nsf1. This finding implies that the yeast response to non-fermentable carbon and salt stress is at least partially dependent on NSF1.
These authors contributed equally to this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gancedo, 1998; Schuller, 2003).
The genome-wide transcriptional profile of yeast during fermentative growth differs greatly from that during respiratory growth. The specific signalling pathways that control different sets of transcription factors required to elicit specific transcriptional responses to utilize the available carbon sources have been reviewed extensively (Carlson, 1999; Gancedo, 1998; Schuller, 2003). Briefly, in the presence of abundant glucose the transcriptional repressor Mig1 is dephosphorylated and located in the nucleus, where it represses the transcription of glucose-repressible genes, including TCA cycle, glyoxylate cycle and gluconeogenic genes, needed for growth in the presence of non-fermentable carbon sources (De Vit et al., 1997). Under these conditions the Snf1 kinase complex is found in an autoinhibited, inactive conformation (Jiang & Carlson, 1996). This is made possible through the activation of protein phosphatase type 1 (PP1; Reg1/Glc7), which dephosphorylates Snf1, resulting in the formation of the inactivated conformation of the complex (Sanz et al., 2000a, b). In contrast, when glucose is limiting or only non-fermentable carbon sources are present, Snf1 is phosphorylated and found in its activated conformation (Jiang & Carlson, 1996). Active Snf1 phosphorylates Mig1, which exits the nucleus, thereby enabling the derepression of glucose-repressible genes such as members of the GAL and MAL gene families and the transcriptional activator CAT8 (DeVit & Johnston, 1999; Hedges et al., 1995; Klein et al., 1998; Mercado et al., 1991; Scholer & Schuller, 1993; Schuller, 2003; Treitel et al., 1998). In addition, active Snf1 enables the binding of the transcriptional activator Adr1 to DNA (Young et al., 2002). The actions of Cat8 and Adr1, either independently or in combination, are involved in the derepression of many glucose-repressed genes (Carlson, 1999; Young et al., 2003).
The transcriptional repressor Nrg1 inhibits the transcription of many functionally diverse genes. Its function has been linked to glucose repression (Kuchin et al., 2002; Zhou & Winston, 2001), adaptation to stress via the regulation of STRE (STress REsponse) element-containing genes (Vyas et al., 2005), and adaptation to alkaline pH and salt stress (Lamb & Mitchell, 2003). Nrg1 and its homologue Nrg2 have both been shown to interact with Snf1 (Vyas et al., 2001). Snf1 inactivates Nrg1 (Kuchin et al., 2002; Vyas et al., 2001), and the transcription of NRG1 is inhibited by Rim101 under alkaline pH and salt stress (Lamb & Mitchell, 2003). However, the levels of NRG1 transcription and subsequent protein synthesis increase in the presence of non-fermentable carbon sources, while the level of Nrg2 decreases (Berkey et al., 2004). These findings imply a function for Nrg1 in derepressive carbon conditions.
The phosphorylation and activation of Snf1 is not limited to glucose-depletion conditions. For example, treatment of yeast cells with 0.8 M NaCl results in the phosphorylation of Snf1 on Thr210, thereby activating the kinase (McCartney & Schmidt, 2001). When yeast cells are exposed to increasing concentrations of sodium ions, the cells respond by inducing the transcription of ENA1, which encodes a Na+/Li+ efflux pump needed for sodium and lithium tolerance and growth in alkaline media (Cyert, 2003; Haro et al., 1991; Ruiz & Arino, 2007). Many signalling pathways affect the transcription of ENA1. Amongst these are Snf1, which activates ENA1 transcription by negatively regulating Mig1 and Nrg1 (Alepuz et al., 1997; Lamb & Mitchell, 2003), and Rim101, which represses NRG1 transcription, resulting in the derepression of ENA1 transcription (Lamb & Mitchell, 2003; Vyas et al., 2005). In addition, the snf1 mutant is hypersensitive to NaCl (1.2 M) and LiCl (300 mM), and supports only partial transcriptional activation of ENA1 in response to sodium stress (Alepuz et al., 1997). The transcription of several genes needed to maintain ion homeostasis in the cell is also controlled by carbon conditions. For example, the transcription of ENA1 is glucose-repressed but activated in a manner dependent on Snf1 when galactose is the sole carbon source (Alepuz et al., 1997). The mechanism of ENA1 transcriptional activation by galactose is not fully understood.
S. cerevisiae experiences many challenges during the course of wine fermentation, including osmotic stress due to the high sugar and solute content of the grape juice, and continuously changing nutrient conditions as fermentable sugars are converted to ethanol and nitrogen becomes limiting (reviewed by Boulton et al., 1998). Not surprisingly, the transcriptome of S. cerevisiae undergoes significant changes during the course of fermentation. DNA microarray experiments analysing the transcriptional response of S. cerevisiae during fermentations have identified a variety of uncharacterized genes that have altered transcription profiles as fermentation proceeds (Rossignol et al., 2003; Marks et al., 2008). These genes could play important roles in the adaptation of the yeast to changing conditions during fermentation. One of these poorly characterized genes, YPL230w, is the focus of this study. We renamed YPL230w to NSF1 (nutrient and stress response factor 1) as we provide evidence that Nsf1 functions in response to changing nutrient and sodium stress conditions. A previous search that screened the S. cerevisiae genome for Cys2-His2 (C2H2)-type zinc fingers, structures that are known DNA-binding motifs, found such a motif in Ypl230w/Nsf1 (Bohm et al., 1997). The increased transcription of YPL230w/NSF1 during the course of fermentation led us to investigate the role of Nsf1 in the context of yeast carbon metabolism when fermentable carbon sources are depleted.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. AVY4 was constructed using a PCR-based integrative transformation procedure described elsewhere (Longtine et al., 1998). GFP for fusion to NSF1 was amplified from pFA6a-GFP(S65T)-His3MX6 according to Longtine et al. (1998). Transformants were selected on SC minus histidine solid media [0.17 % (w/v) yeast nitrogen base (YNB) without amino acids and ammonium sulphate (Difco), 0.5 % (w/v) ammonium sulphate, 2 % (w/v) glucose, 0.79 g l–1 complete supplement mixture (CSM)–HIS dropout mix (Q-Biogene), 2 % agar (Difco)]. All integration events were confirmed by PCR.
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Yeasts were transformed by electroporation as described elsewhere (Ausubel et al., 2002). Escherichia coli strain DH5
was used for cloning and plasmid amplification, as described by Ausubel et al. (2002).
Plasmid construction.
Primers NSF1-F1 (5'-TCTCGGTACCACGAAATGGAAAATACCACG-3') and NSF1-R1 (5'-TCTCGGATCCAGACGCGGGAGTTTAAAAT-3') were used to amplify the NSF1 coding sequence from BY4742 genomic DNA using the PfuTurbo DNA polymerase (Stratagene) according to the manufacturer's specifications. The PCR product was cloned as a KpnI/BamHI fragment (restriction sites underlined) behind the GAL1 promoter in pYES2 (Invitrogen) to generate pRPS112. The integrity of the plasmid was confirmed by sequencing.
The 985 nt promoter region of ADY2 was amplified from BY4742 genomic DNA using primers ADY2-lacZ5 (5'-TATAGGATCCTGGAAATCCTAACCATCGG-3') and ADY2-lacZ3 (5'-TATAGGATCCGTTTCCGCTCGTTTGTT-3') and the Pwo DNA polymerase (Roche) according to the manufacturer's recommendations. The PCR product was cloned as a BamHI fragment and fused in-frame with the lacZ gene in pVAN2 (van der Merwe et al., 2001b) to yield pADY2-lacZ (CEN, URA3). The integrity of the plasmid was confirmed by sequencing.
RNA extraction and Northern analysis.
Yeast strains were cultured to early exponential phase (OD600 
0.35–0.55) in the indicated media prior to RNA extraction. The phenol-based RNA extraction procedure, electrophoresis and RNA transfer to nylon membrane have been described previously (van der Merwe et al., 2001a). DIG-labelled DNA probes were generated using a PCR DIG Probe Synthesis kit (Roche) according to the manufacturer's recommendations. The sequences of primers used for probe synthesis are available upon request. Pre-hybridization was performed at 50 °C using 10 ml DIG Easy Hyb solution (Roche) for 30 min. Denatured probes were added to 5 ml DIG Easy Hyb solution and hybridized for 18 h. Membranes were washed twice for 5 min in 2x SSC : 0.1x SDS at room temperature and twice for 15 min in 0.1x SSC : 0.1x SDS at 50 °C. Membranes were blocked for 30 min, followed by a 30 min incubation with anti-DIG antibodies (1 : 10 000 in blocking solution; Roche) and detected with CDP-Star (Roche) for 5 min. Membranes were exposed to autoradiography film for visualization.
Fluorescence microscopy.
Transformants of AVY4 with pNOP1-dsRED (Strub et al., 2007) were precultured to exponential phase (OD600 0.5–0.6) in SD minus leucine media. Cells were harvested, washed and transferred to the indicated media and further incubated. Samples were collected and analysed using a Nikon Eclipse E600 fluorescence microscope at x1000 magnification. Images were collected with a Photometrics Coolsnapfx monochrome charge-coupled device (CCD) digital camera (Roper Scientific) and processed with Metamorph (Universal Imaging, Version 5.0).
β-Galactosidase assays.
Fresh pADY2-lacZ transformants of the wild-type and nsf1 strains were inoculated into minimal synthetic media containing either 2 % (w/v) glucose or 1.5 % (w/v) potassium acetate as carbon sources and cultured at 30 °C before samples were collected for β-galactosidase assays.
For analysis of ENA1 expression, pKC201 ( µ, URA3) (Cunningham & Fink, 1996) transformants of the wild-type and nsf1 strains were pre-cultured in selective synthetic glucose media to early exponential phase (OD600 0.35–0.55). Cells were harvested, washed and distributed to yeast extract and peptone with glucose (YPD) or yeast extract and peptone containing 2 % galactose (YPGal) media containing the indicated concentrations of NaCl. All media used in these assays were adjusted to pH 5.5. Cultures were incubated at 30 °C for 90 min, at which point cells were harvested. β-Galactosidase assays were performed as described previously (van der Merwe et al., 2001b). The units of activity were normalized to the OD600 of the culture and calculated as described elsewhere (Miller, 1972). Averages and SDs were determined from replicates of at least three independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Marks et al., 2008). We analysed the transcription of NSF1 in response to different carbon conditions by growing the wild-type strain to early exponential phase in rich media with glucose (YPD), acetate (YPA), ethanol (YPE) or glycerol (YPGlyc) as carbon source. Total RNAs were extracted and probed for NSF1 and ACT1. These analyses showed that the NSF1 transcript was present at a low basal level in glucose, but was more abundant when acetate, ethanol or glycerol was supplied as a sole carbon source (Fig. 1a). These findings suggested that the expression of NSF1 in exponentially growing cells is regulated by the quality of the available carbon source.
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). Similar results were obtained when NSF1 was overexpressed in the 
1278b genetic background (data not shown). Overexpression of NSF1 therefore results in slower growth of the yeast.
Nsf1 localizes to the nucleus and activates gene expression in non-fermentable carbon sources
Based on the observations of Bohm et al. (1997), Ypl230w is proposed to be a putative transcription factor. We investigated a potential nuclear role for Nsf1 by analysing its subcellular localization. GFP was fused to NSF1 in the wild-type strain (AVY4; NSF1–GFP) and used to determine whether Nsf1 localized to the nucleus in a manner dependent on the available carbon sources. Consistent with the transcriptional profile of NSF1, Nsf1–GFP was actively expressed and detected when AVY4 was grown with acetate as a sole carbon source, but was not detected in the cells when glucose was the carbon source (Fig. 2a). Nsf1–GFP clearly co-localized with Nop1–dsRED, a nucleolar protein, when AVY4 was grown in the presence of non-fermentable carbon sources, thereby indicating that Nsf1 localizes to the nucleus (Fig. 2a). Nsf1–GFP was also present in the nucleus when either ethanol or glycerol was the sole carbon source (data not shown).
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). ACS1, IDH1 and CIT2 are genes needed to assimilate acetate into the intermediates of the TCA cycle as part of respiratory metabolism, while FBP1 is needed in gluconeogenesis to generate glucose. Total RNAs were extracted from the wild-type and nsf1 mutant strains cultured with acetate as the carbon source and analysed for the transcription of ACS1, CIT2, IDH1 and FBP1. The levels of CIT2, IDH1 and ACS1 mRNA were clearly lower in the nsf1 mutant than in the wild-type strain (Fig. 2b). In contrast, the transcription of FBP1 was not affected by the absence of NSF1 (Fig. 2b). We further analysed the effect of NSF1 deletion on the transcription of ADY2, which encodes the acetate permease of S. cerevisiae and is transcriptionally repressed by glucose, but activated by non-fermentable carbon sources, specifically acetate (Paiva et al., 2004). Transformants of the wild-type and nsf1 mutant with pADY2-lacZ were grown to early exponential phase in selective media with either glucose or acetate as the sole carbon source. Samples were collected and used for β-galactosidase assays. While the ADY2 promoter only supported transcription as a low basal level in the presence of glucose, it was highly activated with acetate as the carbon source in the wild-type strain. The latter level of activation was decreased by 32 % in the absence of NSF1 (Fig. 2c).
The nuclear localization of Nsf1–GFP in cells grown in glycerol medium suggested a similar transcriptional activation role for this protein during growth with glycerol. Total RNAs extracted from the wild-type and nsf1 mutant strains grown in glycerol were probed for CIT2, IDH1, ACS1 and FBP1. The levels of CIT2, IDH1 and FBP1 mRNA decreased slightly in the nsf1 mutant in comparison to the wild-type strain (Fig. 2b), suggesting that Nsf1 plays a role in the transcriptional activation of these genes when glycerol is the carbon source. Interestingly, the transcription of ACS1 was not affected by NSF1 deletion (Fig. 2b). In combination, these observations suggest that Nsf1 is involved in the transcriptional activation of some glucose-repressed genes when non-fermentable carbon sources support growth. The lack of transcription observed in the nsf1 mutant seemed more pronounced in acetate than in glycerol, implying that Nsf1 might have a more important role when acetate is the carbon source.
Adr1 and Snf1 are needed to activate NSF1 transcription
The carbon-regulated transcription of NSF1 in early exponential phase implies that several nutrient signalling pathways are involved in the transcriptional regulation of this gene. We used The Promoter Database of Saccharomyces cerevisiae (SCPD; http://rulai.cshl.edu/SCPD/) to interrogate the promoter region of NSF1 for potential transcription factor-binding sites (TFBSs) to provide insights into the transcriptional regulation of this gene. We focussed on the major regulators of carbon-related transcriptional regulation, and identified TFBSs for Adr1 and Nrg1 (Table 2).
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, adr1, nrg1 and snf1 mutant strains with that of the wild-type strain when these cells were incubated with glycerol as the sole carbon source. The transcription of NSF1 decreased in both the adr1 and nrg1 mutants compared with the wild-type strain (Fig. 3). In contrast, the cat8 mutant strain supported almost wild-type levels of NSF1 transcription. The Snf1 kinase regulates several downstream transcriptional regulators, including Adr1 and Nrg1. Deletion of SNF1 resulted in the greatest loss of NSF1 transcription, as the snf1 mutant supported 40 % of the wild-type level of NSF1 transcriptional activation (Fig. 3b). Consistent with these results, Nsf1–GFP was nuclear in the snf1 mutant, but the level of fluorescence intensity was low (data not shown). In combination, these observations indicate that the Snf1 signalling pathway is needed for the transcriptional activation of NSF1 under non-fermentable carbon conditions.
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; Lamb et al., 2001; Platara et al., 2006), was suggestive of saline-related transcriptional regulation of NSF1. Interestingly, the transcription of NSF1 has been reported to increase slightly when cells are treated with 0.8 M NaCl (Yoshimoto et al., 2002). Upon closer analysis of NSF1 transcription in response to higher concentrations of NaCl, we observed low levels of NSF1 mRNA in YPD, while NSF1 mRNA was present at above basal levels in 0.8 and 1.0 M NaCl, with the highest abundance being observed with 1.2 M NaCl (Fig. 4a). Consistent with these findings, Nsf1–GFP localized to the nucleus in response to treatment with 1.0 M NaCl (Fig. 4b). These findings suggested a nuclear function for Nsf1 under salt-stress conditions also.
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; Cyert, 2003; Lamb et al., 2001; Platara et al., 2006). To analyse the potential involvement of Nsf1 in the transcriptional activation of ENA1, we cultured pKC201 (ENA1–lacZ) transformants of the wild-type and nsf1 mutant strains in selective synthetic glucose media to exponential phase before shifting the cells to salt-stress conditions (see Methods). The wild-type cells expressed ENA1–lacZ at low levels in YPD media devoid of salt, but induced its transcription in response to treatment with either 1.0 or 1.2 M NaCl. However, in the nsf1 mutant the salt-induced β-galactosidase activities were 45 and 70 % lower than those of the wild-type when cells were treated with 1.0 and 1.2 M NaCl, respectively (Fig. 5a).
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, the transcription of ENA1–lacZ was activated with galactose as the carbon source. However, this level of activation was decreased by 27 % in the nsf1 mutant (Fig. 5b). When analysing the response to salt stress in galactose-containing medium, NSF1 was clearly needed for the transcriptional activation of ENA1–lacZ, as the levels of activation decreased by more than 60 % in the nsf1 mutant when the cells were treated with either 1.0 or 1.2 M NaCl (Fig. 5b). Surprisingly, in our analysis the wild-type strain did not show an increase in transcriptional induction of ENA1–lacZ in YPGal containing NaCl in comparison with YPGal, although this effect was observed by Alepuz et al. (1997). Consistent with our observations of Nsf1 having a nuclear localization and transcriptional activation function, Nsf1–GFP also localized to the nucleus in galactose media in the presence or absence of NaCl (data not shown). Collectively these observations suggest that NSF1 is involved in the response of yeast to salt stress. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and during the alcoholic fermentation process (Rossignol et al., 2003; Marks et al., 2008). However, this gene was poorly characterized, despite the presence of a clear C2H2 zinc finger DNA-binding motif. Here we show that Nsf1 localizes to the nucleus in response to either gluconeogenic carbon conditions or the presence of salt stress, and affects the transcriptional activation of several carbon- or salt-regulated genes.
Our analysis of NSF1 transcription showed the classic profile of a glucose-repressed gene. During the exponential growth phase, NSF1 is weakly expressed in medium containing glucose, but is activated in the presence of non-fermentable carbon sources. The latter transcriptional activation of NSF1 is partially dependent on the Snf1 signalling pathway, as the deletion of SNF1 results in a loss of 60 % of NSF1 transcription. Snf1 controls the activity of several downstream transcriptional regulators, including Adr1, Cat8 and Nrg1. We identified two copies of the consensus Adr1-binding site in the promoter of NSF1, and the transcription of NSF1 decreased in the adr1 mutant in glycerol. In contrast, the cat8 mutant showed no effect on the transcriptional activation of NSF1. Consistent with this, no copies of the carbon source response element (CSRE; Haurie et al., 2001), which serves as the binding site for Cat8, were identified in the NSF1 promoter.
Snf1 also positively regulates NRG1 at the RNA and protein levels (Berkey et al., 2004). Nrg1 has been described as mainly a transcriptional repressor. It is present at increased levels in cells grown on non-fermentable carbon sources, and has been suggested to bind to the sequences CCCCT and CCCTC (Lamb & Mitchell, 2003; Park et al., 1999). Five potential Nrg1-binding sites are present in the 650 nt promoter region of NSF1. Our results indicate that Nrg1 participates in the transcriptional activation of NSF1. This is consistent with the findings of Vyas et al. (2005), who reported a potential positive role for Nrg1 in the transcription of carbon-regulated genes, as 187 genes showed decreased expression in the nrg1 mutant. The exact mechanism by which Nrg1 results in the positive regulation of gene transcription is not known. Other signalling pathway(s) could function in parallel to the Snf1 pathway to regulate NSF1 transcription under non-fermentable carbon conditions. It is currently not known which other pathways participate in the transcriptional regulation of NSF1.
We observed that the transcriptional activation of NSF1 and the localization of Nsf1–GFP to the nucleus occurred in a condition-specific manner. Glucose as a carbon source represses the transcription of NSF1 and we could not detect Nsf1–GFP under these conditions. In contrast, Nsf1–GFP is found in the nucleus in conditions under which NSF1 is actively transcribed, such as growth with non-fermentable carbon sources or salt stress. It is under these conditions that Nsf1 has an effect on condition-specific transcriptional regulation. More specifically, Nsf1 participates in the transcriptional activation of some known glucose-repressed genes during exponential growth in non-fermentable carbon conditions. The transcriptional activation of three of the 40 most highly glucose-repressed genes, FBP1, ADY2 and ACS1 (Young et al., 2003), as well as IDH1 and CIT2, was tested in this study. Adr1 and Cat8 contribute to varying degrees to the transcriptional regulation of these genes under non-fermentable carbon conditions. Our analysis showed that Nsf1 also participates in the transcriptional activation of these genes. More specifically, the transcription of ADY2 is known to be Adr1-dependent (Young et al., 2003), while that of ACS1 is co-activated by both Adr1 and Cat8 (Tachibana et al., 2005). The transcriptional activation of both ACS1 and ADY2 decreased in the nsf1 mutant under non-fermentable carbon conditions, despite the presence of both ADR1 and CAT8. In contrast, the transcription of CIT2 was upregulated in an adr1 mutant (Young et al., 2003), but decreased when NSF1 was deleted (Fig. 2b). Since Adr1 is a known transcriptional activator, its effect on CIT2 transcription could be indirect, while that of Nsf1 is direct. Interestingly, the transcription of other Adr1-dependent genes, such as GUT1 and GUT2 (Young et al., 2003), is not affected by NSF1 deletion (data not shown). In combination these observations provide further evidence that Nsf1 is needed for the transcriptional activation of some, but not all, glucose-repressed genes.
Despite the clear involvement of Nsf1 in the derepression of glucose-repressed genes, the wild-type and nsf1 mutant strains showed similar growth rates with all the carbon sources used in this study (data not shown). This observation emphasizes the redundant function of Nsf1 under non-fermentable carbon conditions. The relationship of Nsf1 to known transcription regulators, like Adr1, Cat8 and Nrg1, in the activation of glucose-repressed genes is currently not known. Nsf1 contains a putative C2H2 zinc finger DNA-binding motif (Bohm et al., 1997). Specific amino acid residues contained in the finger–loop regions of these motifs are known to make base-specific contacts with the binding site to enable high-affinity binding with the target site (Bohm et al., 1997). The amino acid residues of Msn2, Msn4 and Nrg1 needed for base-specific binding are conserved in the Nsf1 C2H2 zinc finger pair, indicating that Nsf1 could be a DNA-binding protein with binding sites similar to those of these known transcription factors (Bohm et al., 1997) (Fig. 6). Consistent with this, several specific characteristics of yeast transcription factors, such as stretches of glutamine residues and serine-rich regions, have been shown recently to be shared by the transcription factors analysed (Titz et al., 2006). Although those authors did not identify Nsf1, this protein contains a Ser-rich region (amino acids 111–130) and a Gln-rich region (amino acids 185–197) shared by many of the activators identified in their screen. In combination, the amino acid sequence characteristics of Nsf1 therefore support our findings that it functions as a potential transcriptional activator. Although Nsf1 has all the characteristics of a transcription factor, evidence of its direct binding to DNA is still lacking. Identifying the precise DNA-binding sequence for Nsf1 could help clarify the relationship between Nsf1 and known transcriptional regulators.
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). In contrast, the deletion of NSF1 had no significant effect on the transcriptional activation of ENA1–lacZ when cells were exposed to 0.75 M NaCl (data not shown). Genome-wide transcriptional analysis has shown that YPL230w transcription is slightly increased by 0.8 M NaCl treatment (Yoshimoto et al., 2002). In addition, those authors showed that the treatment of a crz1 mutant with 0.8 M NaCl results in reduced activation of NSF1 transcription, suggesting that the transcriptional activator Crz1 is needed for the transcriptional activation of NSF1. However, our analysis of the NSF1 promoter failed to identify a Crz1 DNA-binding element, thereby suggesting that this transcriptional activation might be indirect.
The promoter of ENA1 is the target of multiple signalling pathways. The TOR (via Gln3/Ure2) (Crespo et al., 2001), Snf1 (via Mig1/Mig2 and Nrg1/Nrg2) (Platara et al., 2006), Rim101 (via Nrg1/Nrg2) (Lamb et al., 2001; Lamb & Mitchell, 2003; Platara et al., 2006) and the calcineurin pathways all converge on the ENA1 promoter (Cyert, 2003; Haro et al., 1991; Ruiz & Arino, 2007). The TOR and Snf1 pathways respond to nutrient availability, and the calcineurin pathway responds to intracellular ion concentrations, while Rim101 and Snf1 also regulate the yeast response to alkaline stress. The regulation of ENA1 is therefore complex, as it responds to multiple stimuli. The transcriptional activators Crz1 and Gln3, controlled by the calcineurin and TOR pathways, respectively, have been shown to activate ENA1 transcription, while repressors such as Mig1/Mig2 and Nrg1 have been linked to its repression. Our data indicate that Nsf1 is a novel regulator involved in the transcriptional activation of ENA1 in response to non-fermentable carbon sources and sodium stress. Our analysis of ENA1–lacZ transcription was performed at pH 5.5, thereby limiting the role of alkaline stress and the Rim101 pathway in the activation. Identifying the signalling pathway(s) other than Snf1 that control the function(s) of NSF1 would greatly enhance our understanding of the role of Nsf1 in the salt-stress response of S. cerevisiae.
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ACKNOWLEDGEMENTS |
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Edited by: M. Schweizer
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Received 28 April 2008;
accepted 7 May 2008.
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