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Department of Cell and Molecular Biology/Microbiology, University of Gothenburg, Box 462, S-40530 Göteborg, Sweden
Correspondence
Stefan Hohmann
stefan.hohmann{at}gu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1999; Serrano & Rodriguez-Navarro, 2001).
The most important system for the detoxification of sodium and lithium ions in yeast is the Ena1p Na+-ATPase, which mediates export of these ions from the cell. Mutants lacking ENA1 exhibit hypersensitivity to sodium and lithium (Haro et al., 1991). Transcription of the ENA1 gene is controlled by multiple signalling transduction pathways, such as the HOG pathway (Proft & Serrano, 1999; Rep et al., 2001), the calcineurin pathway (Hirata et al., 1995; Mendizabal et al., 2001; Mendoza et al., 1994, 1996), the TOR pathway (Crespo et al., 2001; Withee et al., 1998), the Rim101p pathway (Lamb & Mitchell, 2003; Platara et al., 2006), the protein kinase A (PKA) pathway (Hirata et al., 1995; Marquez & Serrano, 1996) and the Snf1p glucose repression pathway (Alepuz et al., 1997; Ye et al., 2006). In many instances, transcription factors involved in regulation of ENA1 expression have been identified. For instance, the osmosensing HOG pathway controls ENA1 expression via Sko1p (Proft & Serrano, 1999; Proft et al., 2001; Rep et al., 2001), the calcineurin protein phosphatase controls expression of ENA1 via Crz1p (Hirata et al., 1995; Mendizabal et al., 2001; Mendoza et al., 1994; Serrano et al., 2002) and glucose repression is mediated via Snf1p and Mig1p (Alepuz et al., 1997; Proft & Serrano, 1999; reviewed by Ruiz & Arino, 2007). A possible candidate for a transcription factor linking Snf1p to ENA1 expression under salt stress is Nrg1p. Nrg1p has been reported to mediate Snf1p-dependent responses (Berkey et al., 2004). NRG1 expression is upregulated by high pH stress and salt stress, and deletion of NRG1 leads to increased ENA1 expression under these conditions (Causton et al., 2001; Lamb & Mitchell, 2003; Platara et al., 2006; Vyas et al., 2005). Nrg1p is phosphorylated under salt stress and this phosphorylation is mediated by protein kinase CK2 (Berkey & Carlson, 2006; Vyas et al., 2001). The role of Nrg1p in salt stress responses has not yet been fully elucidated. Nrg2p, closely related to Nrg1p, plays a role in the response to a variety of environmental conditions and its deletion results in an increased NaCl tolerance (Vyas et al., 2005).
Snf1p is a highly conserved protein kinase, homologous to mammalian AMP-activated protein kinases (reviewed by Carling, 2004; Carlson, 1999; Hedbacker & Carlson, 2008). Snf1p plays a key role in glucose repression by deactivation of the transcriptional repressor Mig1p (Nehlin & Ronne, 1990), the best-characterized downstream target of Snf1p. Mig1p is deactivated through phosphorylation by Snf1p, leading to export of Mig1p from the nucleus to the cytosol (De Vit et al., 1997; Ostling & Ronne, 1998). The Snf1p protein kinase is a heterotrimeric complex, comprising the catalytic 
-subunit Snf1p, the regulatory 
-subunit Snf4p, and any of the three distinct β-subunits Sip1p, Sip2p or Gal83p (Amodeo et al., 2007; Jiang & Carlson, 1997; Vincent & Carlson, 1999; Vincent et al., 2001; Yang et al., 1994). Snf1p is activated in a multi-step process, where the Snf4p regulatory -subunit plays a role in eliminating the auto-inhibitory interaction of the C-terminal regulatory domain with the N-terminal catalytic domain of Snf1p (Celenza & Carlson, 1989; Celenza et al., 1989; Jiang & Carlson, 1996; McCartney & Schmidt, 2001). Phosphorylation on threonine 210 (T210) by any one of the redundant upstream kinases Sak1p, Tos3p or Elm1p is required for activation of Snf1p (Hong et al., 2003; McCartney & Schmidt, 2001; Nath et al., 2003; Sutherland et al., 2003). Under conditions tested so far, Sak1p is the most important Snf1p-activating kinase (Hong & Carlson, 2007; McCartney et al., 2005). Although it has been reported that Snf1p and Mig1p, which control glucose repression/derepression, also affect ENA1 expression (Alepuz et al., 1997; Proft & Serrano, 1999), little is known about the mechanisms by which the Snf1p glucose repression pathway contributes to the detoxification of sodium. We have recently reported that Gis4p is a new component of the regulatory system controlling ENA1 expression and that Gis4p collaborates with Snf1p (Ye et al., 2006), but the underlying mechanism is not yet understood.
Here we report that Snf1p is rapidly phosphorylated following sodium treatment and this activation is needed for acquisition of salt tolerance. Our observations confirm and extend a recent report by the Carlson laboratory (Hong & Carlson, 2007) that also showed salt-stress stimulation of Snf1p activity. We report evidence that salt-stress activation of Snf1p does not lead to inactivation of Mig1p but mediates ENA1 upregulation via different regulators, possibly including the Nrg1p repressor. Moreover it appears that one of the three Snf1p upstream kinases, Elm1p, plays a role in the control of ENA1 expression that is independent of Snf1p.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Strains were generated either by transformation of PCR product or by crossing and tetrad analysis. The deletion of genes was confirmed by the generation of different-sized products in PCRs with different primers (Table 2).
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; Schmidt & McCartney, 2000) and the centromeric plasmid pGFP-MIG1 was tagged to encode green fluorescent protein (GFP) at the N-termini (De Vit et al., 1997); these plasmids were kindly provided by Martin Schmidt (University of Pittsburgh School of Medicine) and Mark Johnston (Washington University, St Louis). The centromeric plasmid pUG35 (Niedenthal et al., 1996) and pUG35-ENA1 were kindly provided by Lennart Adler (Wadskog et al., 2006) (a gift from Martha Cyert and Victoria Heath).
Plasmid pELM1 was constructed by ligation of a BamHI/NotI-digested genomic copy of ELM1 by using PCR amplification and plasmid pCM188 (Gari et al., 1997). The construct was confirmed by complementation of the cell elongation phenotype of the elm1
mutant.
For epitope tagging of the genomic copy of NRG1, a sixfold HA epitope was integrated into the NRG1 gene using a PCR product of a 6HA-HIS3MX6 cassette in the pYM15 vector (Janke et al., 2004). The construction was confirmed by PCR with different primers (Table 2
), generating products of different sizes.
Growth conditions and growth assays.
Yeast cells were routinely grown in medium either containing 2 % peptone and 1 % yeast extract supplemented with 2 % glucose as a carbon source (YPD) or containing YNB (yeast nitrogen base without amino acids) supplemented with necessary amino acid(s) and with 2 % glucose or 2 % raffinose. Selection and growth of transformants carrying a plasmid was performed in YNB medium supplemented with the required amino acid (Sherman et al., 1983). Plate growth assays were performed by pregrowing cells in YPD or YNB medium supplemented with the required amino acid. Cells were resuspended in the same medium to an OD600 of 1.0 or (for the single elm1 mutant or any combination with the elm1 mutant) 2.0. Five-microlitre aliquots of a 10-fold serial dilution of this culture were spotted onto agar plates supplemented with 2 % glucose, 2 % raffinose and 1 M NaCl, as indicated. Growth was monitored after 2–3 days at 30 °C.
Western blot analysis.
Cells (10 ml) were grown to OD600 0.5–0.8 and harvested by centrifugation. Cells were then resuspended in 500 µl 2 M NaOH supplemented with 7 % 2-mercaptoethanol and incubated for 2 min at room temperature. Five hundred microlitres of 50 % trichloroacetic acid was added, and the samples were vortexed and sedimented. Samples were washed in 500 µl 1 M Tris/HCl (pH 7.5) and resuspended in 120 µl 1x SDS sample buffer (62.5 mM Tris/HCl pH 6.8, 3 % SDS, 10 % glycerol, 5 % β-mercaptoethanol, 0.001 % bromophenol blue) and incubated for 5 min at 100 °C. Forty micrograms of the supernatant was separated by SDS-PAGE using 7.5 % and 12.5 % polyacrylamide gels for Mig1p and Nrg1p, respectively, and analysed by immunoblotting using anti-HA antibody (Sigma) and secondary anti-mouse IgG antibody conjugated to horseradish peroxidase. Lumi-Light Western blotting substrate (Roche) and a FUJIFILM LAS-1000 camera were used for visualization.
For phosphorylation of Snf1, cells (25 ml) were grown to OD600 0.5–0.8. NaOH was added to the culture to a final concentration 0.1 M prior to the protein extraction procedure described above. For immunoprecipitation of Snf1-HA, 400 µg of the supernatant was dialysed and immunoprecipitated as described previously (McCartney & Schmidt, 2001). The supernatant was separated by SDS-PAGE using 7.5 % polyacrylamide gels and analysed by immunoblotting using our anti-phospho-Snf1 antiserum (prepared as described by McCartney & Schmidt, 2001; Open Biosystems) and a secondary anti-rabbit IgG antibody (Sigma) conjugated to horseradish peroxidase, and 40 µg of the supernatant was separated and analysed by immunoblotting against anti-HA antibody as a loading control. Lumi-Light Western blotting substrate (Roche) and a FUJIFILM LAS-1000 camera were used for visualization.
Gene expression analysis.
To estimate ENA1 promoter activity, the ENA1-lacZ construct pKC201 (Cunningham & Fink, 1996) was transformed into relevant yeast strains. Cells were grown to an OD600 of 1.0, NaCl was added to a final concentration of 0.8 M, and samples were taken for protein extraction at different time points. The specific activity of β-galactosidase was determined as described previously (Rose et al., 1990), and the protein concentration was determined using a Dc protein assay kit (Bio-Rad). Data represent the means and standard deviations of results from three biological replicates.
Microscopy and staining methods.
Cells were grown to exponential phase in selective media and visualized with a Leica DMRXA microscope using bright-field, GFP or 4',6'-diamidino-2-phenylindole (DAPI) filters. DNA was strained by DAPI (1 µg ml–1) for 10 min, and cells were spun down and visualized. Cells exposed to a final concentration of 0.8 M NaCl or 0.05 % glucose were observed after 5 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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mutant grows more poorly on agar plates containing NaCl or LiCl (Alepuz et al., 1997; Ye et al., 2006). In order to study if this is due to a specific role of Snf1p in signalling salt stress we tested if Snf1p becomes phosphorylated at T210 following NaCl treatment. Indeed Snf1p became phosphorylated within 1 min and remained highly phosphorylated during the following 180 min (Fig. 1a). Phosphorylation of T210 is required for cells to acquire salt tolerance since a mutant in which T210 is replaced by the non-phosphorylatable residue alanine is as salt sensitive as a snf1 mutant (Fig. 1b). Even lower levels of NaCl or 0.1 M LiCl (data not shown) caused T210 phosphorylation. Moreover, osmotic stress and to a lesser extent oxidative stress, but not heat stress, also caused T210 phosphorylation (Fig. 1c), showing that Snf1p phosphorylation is not a general stress response. Since a snf1 mutant is not osmosensitive (data not shown) and since low levels of NaCl and LiCl that do not cause osmotic stress stimulate Snf1p phosphorylation, we believe that the salt effect on Snf1p phosphorylation is not an osmotic response.
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; Hedbacker & Carlson, 2008; Johnston, 1999). Following a shift from high to low glucose Mig1p becomes rapidly phosphorylated on up to four sites (Fig. 2a) in a Snf1p-dependent manner (Ahuatzi et al., 2007; Ostling & Ronne, 1998) and exits the nucleus (Fig. 2b) (De Vit et al., 1997). Adding 0.8 M NaCl to yeast cells grown on 2 % glucose caused only minor changes to the Mig1p phosphorylation pattern (Fig. 2a) and did not cause Mig1p to exit the nucleus (Fig. 2b).
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), and it relocalized to the cytoplasm (Fig. 2b). The observations reported above contradict the well-established correlation between Snf1p- and Mig1p-phosphorylation. Although Snf1p is rapidly phosphorylated and activated under salt stress, this does not lead to Mig1p phosphorylation. This suggests that proteins might exist that prevent Mig1p from becoming phosphorylated under salt stress.
We tested two candidate proteins. It has been reported that hexokinase PII, Hxk2p, controls access of Snf1p to Mig1p (Ahuatzi et al., 2007; Moreno et al., 2005). Although deletion of HXK2 affected the Mig1p phosphorylation pattern under low and high glucose, it did not result in salt-stimulated Mig1p phosphorylation (Fig. 2c). Gis4p is a newly recognized component of the system controlling acquisition of salt tolerance and seems to function together with Snf1p (Ye et al., 2006). Deletion of GIS4 had no apparent effect on Mig1p phosphorylation (Fig. 2d) and nuclear exit (Fig. 2e) following a shift to low glucose, consistent with our previous observation that it does not affect glucose derepression (Ye et al., 2006). Deletion of GIS4 also did not cause a major change in the Mig1p phosphorylation pattern (Fig. 2d) or nuclear residence (Fig. 2e) following a shift to NaCl in the presence or the absence of glucose. Hence, the factor(s) that presumably prevent salt-induced phosphorylation of Mig1p by Snf1p remain(s) to be identified.
Deletion of NRG1 suppresses the growth defect of the snf1 mutant under salt stress
Nrg1p is a C2H2 zinc-finger repressor protein and a downstream regulator of Snf1p regulating several stress-responsive genes (Vyas et al., 2001). Nrg1p affects ENA1 expression under salt and high pH stress (Lamb & Mitchell, 2003; Platara et al., 2006; Vyas et al., 2005). To investigate if Nrg1p might be a downstream effector of Snf1p under salt stress we tested if deletion of NRG1 suppressed the growth defect on NaCl of a snf1 mutant. The single nrg1 mutant showed improved salt tolerance (Fig. 3a) and an increased level of ENA1-lacZ expression (Fig. 3b), which, however, remained salt-regulated. Deletion of NRG2, which has previously been reported to mediate NaCl tolerance both alone and in combination with a NRG1 deletion (Vyas et al., 2005), did not result in any increased salt tolerance. The discrepancy with previous results might be explained by strain differences (W303 in our hands and the S288C background in Vyas et al., 2005). The snf1 mutant showed salt sensitivity and strongly reduced, albeit still regulated, ENA1-lacZ expression. The double snf1 nrg1 mutant showed behaviour similar to the wild-type both for growth on 1 M NaCl and for ENA1-lacZ expression, although the basal expression was increased. While these results indicate that Nrg1p might function as a downstream repressor of Snf1p in controlling ENA1 expression, this result needs to be interpreted with caution because deletion of NRG1 stimulated ENA1 expression even in a SNF1 wild-type background (Fig. 3b).
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mutant was NaCl sensitive and showed very low levels of ENA1 expression. The double mutant gis4 nrg1 showed improved growth on NaCl (Fig. 3a) and an essentially wild-type ENA1-lacZ expression profile (Fig. 3b), consistent with Nrg1p acting downstream of Gis4p.
As with Mig1p, the Nrg1p phosphorylation state can be monitored with the help of an HA-tagged version of the protein and the banding pattern in Western blot analysis. It appears that the upper, phosphorylated band of Nrg1p becomes stronger after salt stress but this pattern looks the same in wild-type and the snf1 mutant (Fig. 3c). We noted, however, that the total level of Nrg1p strongly increased 60 min after salt addition in a Snf1p- and partly Gis4p-dependent fashion (Fig. 3c). Upregulation of NRG1 mRNA levels in response to salt has previously been reported (Causton et al., 2001; Vyas et al., 2005).
Sak1p is the most important kinase for Snf1p phosphorylation under salt stress
It has been demonstrated that one of the three upstream kinases Sak1p, Tos3p or Elm1p is essential for activation of Snf1p under glucose-limited conditions. The phosphorylation of Snf1p is completely abolished in a sak1 tos3 elm1 strain (Hong et al., 2003; McCartney & Schmidt, 2001; Nath et al., 2003; Sutherland et al., 2003). This led us to investigate whether the three Snf1p-activating kinases are also necessary under salt stress and if any one of them is specifically important. Indeed, phosphorylation of Snf1p at T210 was completely abolished in the absence of the three upstream kinases, which therefore are required for salt-induced Snf1p phosphorylation also (Fig. 4a). As is the case for glucose regulation, and as was observed also for salt regulation by the Carlson group (Hong & Carlson, 2007), Sak1p is the most important upstream kinase for Snf1p phosphorylation following salt treatment (Fig. 4b). Elm1p and Tos3p by themselves provide only very low levels of phosphorylation.
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tos3 elm1 mutant was stronger than that of the snf1 mutant (Fig. 5a), suggesting that one or several of the upstream kinases played a specific role in salt tolerance acquisition. Further analysis revealed that Elm1p plays this special role. Deletion of ELM1 by itself and in combination with other kinases caused strong NaCl sensitivity (Fig. 5a) and low ENA1-lacZ expression (Fig. 5b). Testing NaCl sensitivity of snf1, sak1, tos3 and elm1 mutants in the BY strain background resulted in similar although not very severe growth phenotypes (data not shown). In contrast to deletion of SNF1, deletion of ELM1 abolished salt regulation of ENA1-lacZ expression. Consistently, the mRNA level of ENA1, monitored by RT-PCR, initially increased but dramatically dropped after 45 min of salt treatment in the elm1 mutant, whereas in the wild-type the level continuously increased up to 90 min after salt stress (data not shown). The initial induction of ENA1 expression in the mutants might be due to the activation of the calcineurin and HOG pathway. It is noteworthy that the snf1 elm1 double mutant had a very similar phenotype to the elm1 single mutant, suggesting that Elm1p plays a more important role in salt tolerance than does Snf1p. Taken together the data are consistent with a scenario where Elm1p affects salt tolerance to a minor extent via Snf1p but largely in a pathway parallel to Snf1p.
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mutant and slightly improved NaCl tolerance of the wild-type. This plasmid restored to wild-type levels the salt tolerance of the snf1 and elm1 mutants (Fig. 6a), consistent with ENA1 being a target of the Elm1p-Snf1p pathways. Further consistent with ENA1 expression being a downstream target of Snf1p, additional deletion of Snf1 did not increase salt sensitivity of the ena1 strain (Fig. 6b).
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mutant. This was not the case (Fig. 6c). Hence, even though Elm1p seems to perform its function in salt tolerance mainly in parallel with Snf1p its overexpression in the absence of Snf1p does not seem to be sufficient to restore ENA1 expression. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-subunit Snf4p as well as phosphorylation at T210 by one of the three upstream kinases (reviewed by Carling, 2004; Hedbacker & Carlson, 2008). Recent data suggest that the glucose signal is mediated via the dephosphorylation reaction on T210 rather than by the kinases (Rubenstein et al., 2008).
We report here that Snf1p is robustly phosphorylated in response to treatment with NaCl (and LiCl; data not shown) even in the presence of glucose. Similar results have recently been reported by the Carlson group (Hong & Carlson, 2007). Our data confirm and extend their observations.
Phosphorylation of Snf1p seems to be required for the acquisition of normal salt tolerance since a T210A mutation results in inability to grow on raffinose (which requires Snf1p activity) and in NaCl sensitivity. Remarkably, activation of Snf1p by NaCl in the presence of glucose leads neither to phosphorylation and nuclear exit of Mig1p or Snf1p (McCartney & Schmidt, 2001), nor to glucose derepression (data not shown). However, NaCl does not seem to prevent Mig1p phosphorylation or nuclear export since this occurs even in the presence of NaCl at low glucose levels. This observation suggests that for glucose derepression Snf1p activation is required but not sufficient. Cells grown in high glucose with salt do not display nuclear enrichment of Snf1p (Hong & Carlson, 2007), which might provide the simplest explanation why Mig1p is not phosphorylated under those conditions. On the other hand, cells lacking GAL83, which is the β-subunit required for nuclear localization of Snf1p, are able to phosphorylate Mig1p normally under low-glucose conditions (Schmidt & McCartney, 2000). This suggests that some factor either specifically recruits Snf1p to salt-relevant targets and hence away from Mig1p or actively prevents Snf1p from accessing or phosphorylating Mig1p. We have tested two possible candidates, namely Hxk2p and Gis4p. Our data do not support the idea that either of those two proteins performs such a function. It also does not appear that any of the three β-subunits Gal83p, Sip1p or Sip2p performs a salt-specific role in targeting Snf1p since any of the three seems to be sufficient to mediate salt tolerance (data not shown). Interestingly, it has recently been proposed that a presently unknown factor mediates the glucose response to Snf1p via Reg1p-Glc7p (Rubenstein et al., 2008). Hence it appears that the full complement of proteins that take part in Snf1p-mediated signalling has not yet been identified despite the fact that the protein complex has been studied for almost 30 years. It is well possible that such factors can be found among the many different proteins that have been reported to physically and/or genetically interact with Snf1p (GRID database: http://www.thebiogrid.org/).
As is the case for Snf1p phosphorylation at low glucose concentrations, the Sak1p upstream kinase is most important for Snf1p phosphorylation under salt stress while Elm1p and Tos3p seem to contribute to a minor extent. This is in accordance with data from the Carlson laboratory (Hong & Carlson, 2007). Unexpectedly, deletion of ELM1 causes strong salt sensitivity and almost completely abolishes ENA1 induction under salt stress, an effect that cannot be mediated via Snf1p (alone). Hence, Elm1p, which plays a role in cellular morphogenesis, septin behaviour and cytokinesis (Bouquin et al., 2000; Garrett, 1997; Koehler & Myers, 1997), appears to play a specific role in controlling ENA1 expression, understanding of which requires further studies.
Since salt-mediated Snf1p activation does not lead to phosphorylation and nuclear export of Mig1p, some other transcription factor must mediate the Snf1p effect on ENA1 expression. In fact, for unknown reasons deletion of MIG1 causes some salt sensitivity and not salt tolerance as might have been expected for a downstream repressor (Ye et al., 2006). Such an effect is mediated by Nrg1p. Deletion of NRG1 causes improved salt tolerance and enhanced ENA1 expression, suppression of the salt sensitivity of the snf1 mutant and restoration of almost wild-type ENA1 expression in the snf1 nrg1 double mutant. Expression of ENA1 remains salt regulated in that mutant, probably due to the involvement of several other signalling pathways in controlling its expression. However, whether Nrg1p is truly mediating the Snf1p effect remains to be demonstrated. Our data do not support the idea that Snf1p phosphorylates Nrg1p in vivo. Snf1p is, however, needed for a salt-stimulated increase in Nrg1p levels, which might be relevant for downregulation of gene expression during adaptation. In addition, it has been shown that Nrg1p is also regulated by Rim101p in response to salt and alkaline pH stress (Lamb et al., 2001; Lamb & Mitchell, 2003; Platara et al., 2006).
Taking the results together, it appears that Snf1p is involved in controlling ENA1 expression via a pathway that may involve any of the three upstream kinases (where Elm1p, in addition, has a separate role), any of the three β-subunits, Gis4p and perhaps Nrg1p. Further work is needed to elucidate which proteins target activated Snf1p to the salt response and prevent glucose repression under salt stress in the presence of glucose.
An interesting question concerns the reason why Snf1p is involved in the control of ENA1. Maybe the cell needs to coordinate expression of ENA1 with the energy balance. The sodium export function of Ena1p requires ATP. The main role of Snf1p and its orthologues is to coordinate energy-producing and -consuming effects. While it does not appear that salt-induced activation of Snf1p causes glucose derepression it might target other functions that lead to higher energy generation to satisfy the needs for sodium detoxification. Our data also suggest that other, although not all, stress conditions activate Snf1p. This opens the way for most interesting studies towards better understanding of the apparently far more complex functions of the conserved Snf1p kinase in yeast physiology.
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ACKNOWLEDGEMENTS |
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Edited by: K. Kuchler
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Received 30 April 2008;
accepted 5 May 2008.
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1.0, adjusted to exactly OD600 1.0 and 5 µl aliquots were spotted in 1 : 10 dilution steps onto the indicated media. The result was scored after 2 days. The snf1
