Glc7 is the only catalytic subunit of the protein phosphatase type 1 in the yeast S. cerevisiae and, together with its regulatory subunits, is involved in many essential processes. Analysis of the non-essential mutants in the regulatory subunits of Glc7 revealed that the lack of Reg1, and no other subunit, causes hypersensitivity to unfolded protein response (UPR)-inducers, which was concomitant with an augmented UPR element-dependent transcriptional response. The Glc7–Reg1 complex takes part in the regulation of the yeast AMP-activated serine/threonine protein kinase Snf1 in response to glucose. We demonstrate in the present study that the observed phenotypes of reg1 mutant cells are attributable to the inappropriate activation of Snf1. Indeed, growth in the presence of limited concentrations of glucose, where Snf1 is active, or expression of active forms of Snf1 in a wild-type strain increased the sensitivity to the UPR-inducer tunicamycin. Furthermore, reg1 mutant cells showed a sustained HAC1 mRNA splicing and KAR2 mRNA levels during the recovery phase of the UPR, and dysregulation of the Ire1-oligomeric equilibrium. Finally, overexpression of protein phosphatases Ptc2 and Ptc3 alleviated the growth defect of reg1 cells under endoplasmic reticulum (ER) stress conditions. Altogether, our results reveal that Snf1 plays an important role in the attenuation of the UPR, as well as identifying the protein kinase and its effectors as possible pharmacological targets for human diseases that are associated with insufficient UPR activation.

INTRODUCTION

The endoplasmic reticulum (ER) is responsible for the synthesis, folding, modification and quality control of secreted and membrane proteins in eukaryotic cells [1,2]. The ER is also involved in other important biological functions, such as lipid biosynthesis or calcium storage [3]. Accumulation of improperly modified/folded proteins represents a stress that leads, through the unfolded protein response (UPR), to the decrease in the ER-targeted protein synthesis and to an increase in the synthesis of chaperones. The UPR is an stress response evolutionarily conserved from yeast to metazoans [4,5]. The only known system in yeast cells to monitor the quality of protein folding in the ER is Ire1, a transmembrane protein that also contains a serine/threonine protein kinase and endoribonuclease domains in its cytoplasmic tail. Accumulation of misfolded proteins in the ER lumen triggers the oligomerization of Ire1, trans-autophosphorylation and an increase in its RNase activity, which leads to the atypical splicing reaction of the mRNA encoding the Hac1 transcription factor. HAC1i, the spliced and induced form of the HAC1 mRNA, is then translated and the Hac1 protein imported to the nucleus, where it binds to the unfolded protein response element (UPRE) in the responsive gene promoters, such as KAR2, initializing a well-characterized transcriptional response [6,7].

Although the activation of the kinase activity of Ire1 requires the autophosphorylation of the activation loop in the kinase domain and its di- or oligo-merization, there is no agreement about whether autophosphorylation of Ire1 triggers its oligomerization or whether the oligomerization causes an increase in its kinase activity. Likewise, the mechanism of inactivation of Ire1 after restoration of normal protein folding is still unknown [8]. While an Ire1 kinase-activity-driven mechanism has been proposed [9], a number of phosphatases have been reported as candidates for the dephosphorylation of Ire1, which might support a dephosphorylation-dependent model for Ire1 attenuation [10]. For example, mammalian PP2Ce possesses intrinsic substrate specificity towards IRE1 Ser-724 in vitro and in vivo [11]. In yeast cells, Ptc2, another member of the PP2C family, dephosphorylates Ire1 in vitro [12]. Furthermore, the yeast phosphoesterase Dcr2 has also been involved in the down-regulation of the UPR, since it interacts with Ire1 in vivo and dephosphorylates Ire1 in vitro [13]. Nevertheless, the role of Ptc2 and Dcr2 is probably shared with other phosphatases, since the combined loss of both causes a weak growth defect in the presence of tunicamycin [13]. Moreover, the mechanisms and effectors that regulate Ptc2 and Dcr2 activity in response to ER stress and target the phosphatases to Ire1 remain unknown.

In yeast cells, GLC7 is the only catalytic subunit of the protein phosphatase type 1 encoded in the Saccharomyces cerevisiae genome. Glc7 is a conserved and essential protein that is involved in numerous cellular functions [14]. The specific regulation of these functions, involving modification of Glc7 localization and/or substrate recognition, is achieved by its interaction with a diversity of an expanding number of Glc7-interacting proteins or regulatory subunits. The interaction of some of these regulatory proteins with Glc7 requires a conserved binding site, defined as (R/K)(V/I)X(F/W) or RVXF, which is necessary for their interaction with Glc7 [15,16]. The R260A-encoding substitution in GLC7 is responsible for several phenotypes of glc7-109 [17], one of 20 GLC7 alleles isolated by alanine-scanning mutagenesis [18]. The residue Arg-260 is in the vicinity of the Glc7 hydrophobic channel that makes contact with the aromatic residue of the described binding-partner motif [17]. Thus, the Glc7R260A mutant phosphatase cannot interact with its regulatory subunits through their RVXF motifs.

One of the well-characterized functions of Glc7 is the adaptation to a non-preferred carbon source by regulating the Snf1 protein kinase, the core element of the yeast carbon catabolite-repression pathway [19], a conserved pathway in eukaryotes [20]. Snf1 forms part of a complex together with Snf4 and one of the three alternative β subunits Sip1/Sip2/Gal83 [21]. Yeast cells prefer glucose to other carbon sources, and it has been proposed that, in the presence of high concentrations of glucose, Glc7 together with its RVXF-containing regulatory subunit Reg1 dephosphorylate the residue Thr-210 in the activation loop of Snf1, inactivating the protein kinase [22]. Inactive Snf1 leads to the transcriptional repression of the genes involved in the metabolism of alternative carbon sources, in a process that depends on the regulation of the transcriptional repressors Mig1/2 by Snf1 [23]. Protein phosphatases, other than Glc7–Reg1, have been reported as important for the Snf1 regulation in yeast cells [2426].

In S. cerevisiae, Snf1 also plays an important role in the cellular response to various environmental stresses [21]. Snf1 regulates acetyl-CoA carboxylase 1 (ACC1) and the global histone acetylation [27], and is required for cell protection against situations that decrease the ratio of reduced to oxidized glutathione [28], affect the cell wall integrity [29] and the sphingolipid metabolism [30], promote the Swi4–Swi6 cell cycle box-binding factor (SBF) and MluI cell cycle box-binding factor (MBF)-dependent transcription [31], or mediate the short-term transcriptional response of alkali-induced genes [32]. It has been shown that Snf1 plays an important glucose-dependent role in regulating, through the action of the mitogen-activated protein kinase (MAPK) Hog1, the traffic of the lipid phosphatase Sac1 between ER and Golgi membranes [33]. Sac1 regulates PI(4)PPtdIns4P homoeostasis at the interface between plasma membrane and the cortical ER, co-ordinating the secretory capacity of ER and Golgi membranes in response to growth conditions [34]. On the other hand, Ire1 and its mammalian homologue Ire1α have been recognized to have important functions in metabolism, including ER-to-Golgi protein transport, expression of key metabolic transcriptional regulators and enzymes involved in triacylglycerol biosynthesis [35]. Finally, it has been reported that glucose deprivation activates the UPR in various mammalian cell lines [36]. This evidence suggests that regulatory events mediated by Snf1 might be important in the adaptive yeast response to accumulation of unfolded or misfolded proteins in the ER.

In the present study, we uncover an unexpected role for the Snf1 protein kinase in the regulatory mechanisms that control UPR signalling. Our results indicate that activation of Snf1 provokes hypersensitivity to different ER-stress-inducer agents, and defective kinetics of deactivation of the UPR. To our knowledge, this is the first evidence regarding the connection between the Snf1, the master controller of the nutritional status, and the regulation of the UPR.

MATERIALS AND METHODS

Growth of Escherichia coli and yeast strains

Yeast cells were incubated at 28°C in YPD medium (1% yeast extract, 2% peptone and 2% glucose) or in synthetic medium [37] containing 2% (w/v) glucose and lacking the appropriate selection requirements. YPD medium was adjusted to pH 5.5 when indicated. Plates containing other carbon sources were prepared by replacing the glucose by 2% (v/v) of ethanol, glycerol or the specified concentrations of raffinose plus galactose. Yeast strains used in the present study are defined in Table 1. E. coli DH5α cells were used as plasmid DNA host and were grown at 37°C in LB broth supplemented with 50 μg/ml ampicillin, when required. Bacterial and yeast cells were transformed using standard methods. Standard recombinant DNA techniques were performed as described elsewhere [38].

Table 1
Strains used in the present study
Strain Genotype Reference or source 
BY4741 MAT ahis3∆1 leu2met15ura3∆ EUROSCARF 
 BY4741 hac1::kanMX4 EUROSCARF 
 BY4741 ire1::kanMX4 EUROSCARF 
 BY4741 snf1::kanMX4* EUROSCARF 
 BY4741 reg1::kanMX4* EUROSCARF 
YJFD31 BY4741 reg1::kanMX4 snf1::nat1 This work 
YJFD97 BY4741+YCplac111 This work 
YJFD98 BY4741+pJFD11 This work 
YJFD17 BY4741 glc7::nat1 + pJFD11 This work 
DBY746 MATαleu 2-3, 112 his3Δ1 trp1-289 ura 3-52 D. Botstein 
MP030 DBY746 reg1:: KanMX4 J. Ariño 
CCV180 DBY746 snf1::LEU2 J. Ariño 
CCV181 DBY746 reg1::KanMX4 snf1::LEU2 J. Ariño 
Strain Genotype Reference or source 
BY4741 MAT ahis3∆1 leu2met15ura3∆ EUROSCARF 
 BY4741 hac1::kanMX4 EUROSCARF 
 BY4741 ire1::kanMX4 EUROSCARF 
 BY4741 snf1::kanMX4* EUROSCARF 
 BY4741 reg1::kanMX4* EUROSCARF 
YJFD31 BY4741 reg1::kanMX4 snf1::nat1 This work 
YJFD97 BY4741+YCplac111 This work 
YJFD98 BY4741+pJFD11 This work 
YJFD17 BY4741 glc7::nat1 + pJFD11 This work 
DBY746 MATαleu 2-3, 112 his3Δ1 trp1-289 ura 3-52 D. Botstein 
MP030 DBY746 reg1:: KanMX4 J. Ariño 
CCV180 DBY746 snf1::LEU2 J. Ariño 
CCV181 DBY746 reg1::KanMX4 snf1::LEU2 J. Ariño 

*These strains were checked for the appropriate gene deletion.

The sensitivity of yeast cells to tunicamycin and soraphen A in solid media was evaluated by drop test growth on agar plates at 28°C, as previously described [39]. The sensitivity to tunicamycin in liquid media was evaluated in cell cultures in 96-well plates. Fresh cultures in 250 μl of YPD were prepared at D660 values of 0.01 and 0.02 from saturated cultures, and grown for 16–24 at 28°C in the presence of the indicated concentration of tunicamycin (AppliChem) or DMSO, used as a vehicle. Growth was monitored by measuring the D650 in a Multiskan Ascent (ThermoFisher) and data are represented as growth in the presence of tunicamycin relative to growth of the same strain in DMSO-containing media.

Plasmids

Plasmids used in this work are described in Table 2. To make the pJFD11 plasmid (YCplac111-glc-109) the BamHI–PstI DNA fragment containing the glc7-109 gene of the pJFD10 plasmid (Table 2) was cloned in the same restriction sites of the centromeric LEU2-marked YCplac111.

Table 2
Plasmids used in the present study
Plasmid Description Reference or source 
pJFD11 YCp111-glc7-109 This work 
pJFD10 YCplac33-glc-109 [93
pMCZ-Y UPRE-CYC1-lacZ on a multicopy URA3-marked vector [40
YEp-REG1 YEplac195-ADH1p-REG1 P. Sanz 
YEplac195-PTC2 Yep195-PTC2 [94
pRJ65 pRS424-LexA-Reg1 [73
LexA-Reg1F468R pRS424-LexA-Reg1F468R [50
LexA-Reg1F468D pRS424- LexA-Reg1F468D [50
pKK1 Single-copy LEU2-marked vector [95
pMS109 pKK1-pHAC1i [95
pCE108 YCp50-Snf1 [96
pCE108-Y106A YCp50-Snf1Y106A [53
pCE108-G53R YCp50-Snf1G53R [53
pSK119 pWS93-ADH1p-3xHA-SNF1 [97
pSK119A pSK119-Snf1T210A [54
pSK120 pSK119-Snf1K84R [97
pIRE1-HA pRS315-IRE1-HA [98
pIRE1-Flag pRS426-IRE1-Flag [99
YEp357-SUC2 YEp357-SUC2p-lacZ [100
pRS-4-4-20 pRS316-FLAG-4-4-20 [49
pBG18-PIS1 pBG1805-PIS1 [101
Plasmid Description Reference or source 
pJFD11 YCp111-glc7-109 This work 
pJFD10 YCplac33-glc-109 [93
pMCZ-Y UPRE-CYC1-lacZ on a multicopy URA3-marked vector [40
YEp-REG1 YEplac195-ADH1p-REG1 P. Sanz 
YEplac195-PTC2 Yep195-PTC2 [94
pRJ65 pRS424-LexA-Reg1 [73
LexA-Reg1F468R pRS424-LexA-Reg1F468R [50
LexA-Reg1F468D pRS424- LexA-Reg1F468D [50
pKK1 Single-copy LEU2-marked vector [95
pMS109 pKK1-pHAC1i [95
pCE108 YCp50-Snf1 [96
pCE108-Y106A YCp50-Snf1Y106A [53
pCE108-G53R YCp50-Snf1G53R [53
pSK119 pWS93-ADH1p-3xHA-SNF1 [97
pSK119A pSK119-Snf1T210A [54
pSK120 pSK119-Snf1K84R [97
pIRE1-HA pRS315-IRE1-HA [98
pIRE1-Flag pRS426-IRE1-Flag [99
YEp357-SUC2 YEp357-SUC2p-lacZ [100
pRS-4-4-20 pRS316-FLAG-4-4-20 [49
pBG18-PIS1 pBG1805-PIS1 [101

β-Galactosidase reporter assays

The indicated strains were transformed with the reporter plasmid pMCZ-Y (Table 2), which contains the UPRE fused to lacZ gene [40]. Cultures were grown up to saturation in the appropriate drop-out medium to maintain the plasmid, and then cells were inoculated on YPD (at pH 5.5) to give a D660 of 0.15. Growth was resumed until the D660 reached 0.6–0.7, then tunicamycin (or the vehicle) was added at the indicated concentrations and incubated for the specified time. Cells were collected by centrifugation (5 min at 750g), the β-galactosidase activity was measured as described previously [41], and results are expressed as Miller units, referring to the number of cells [42].

RNA preparation, Northern blot analysis and qRT-PCR

The total RNA purification, separation and transfer to nylon membranes was performed as previously described [39]. For the Northern blot experiments the digoxigenin-labelled probes used contained sequences of KAR2 (+151 to +751), HAC1 (+18 to +657) or U1 (-124 to +536). Labelling of the probes, washes and image capture were performed as described [39].

For quantitative reverse transcription–PCR (qRT-PCR) experiments, the RNA was prepared as described in [43]. Changes in the expression of KAR2 were analysed by qRT-PCR using 15 ng of total RNA and the QuantiTect SYBR Green RT-PCR kit (Qiagen). Reactions, in a final volume of 10 μl, were performed in a CFX96 Real-Time PCR apparatus (Bio-Rad Laboratories). The amplification primers used for quantification of KAR2 mRNA were KAR2-F (5′-ACTGTTATCGGTATTGAC-3′) and KAR2-R (5′-ATAGACAATAGAGAGACATC-3′). Primers RT_ACT_up2 (5′-TGCTGTCTTCCCATCTATCG-3′) and RT_ACT_do2 (5′-ATTGAGCTTCATCACCAAC-3′) were used for quantification of ACT1 mRNA. The levels of RNA expression of KAR2 were normalized against the expression levels of actin RNA.

Protein preparation, co-immunoprecipitation and immunodetection

Yeast cells co-transformed with the pRS423-IRE1-HA and the pRS426-IRE1-Flag plasmids (Table 2; gifts from Y. Kimata, NAIST-GSBS, Ikoma, Nara, Japan) grown in YPD medium (pH 5.5) were treated with 1 μg/ml tunicamycin for 1 h. Cells were washed with fresh medium and resuspended in the same volume of YPD (pH 5.5) medium. The equivalent to cells at 10 D660 units was collected for protein extract preparation at each time point. Protein extracts from collected cells and the immunoprecipitation of Ire1–HA (haemagglutinin) were performed as previously described [44] using the anti-HA antibody (Roche Diagnostics). Protein samples were resolved using standard 6% acrylamide SDS/PAGE, blotted on to nitrocellulose membranes, and filters were blocked with 5% (w/v) non-fat dried milk in TBST (10 mM Tris/HCl, pH 8.0, 150 mM NaCl and 0.1% Tween 20). Ire1–Flag and Ire1–HA were detected using anti-Flag (1:2000 dilution) and anti-HA (1:2000 dilution) antibodies, respectively. As secondary antibody, we used horseradish peroxidase-coupled sheep anti-mouse IgG (1:25000 dilution; GE Healthcare). Blots were developed using the ECL Western blotting detection kit from GE Healthcare. Images were captured with the VersaDoc 4000 MP Image System (Bio-Rad Laboratories) and analysed with the Quantity One 1-D software (Bio-Rad Laboratories).

For detection of phospho-Snf1, wild-type and reg1 mutant cells were transformed with either the empty multicopy YEplac195 [45] or the same plasmid containing the PTC2 gene. Ten millilitres of fresh cell cultures in YPD medium at D660 0.8 were split into two aliquots and cells were cultured for 15 min in either YPD (pH 5.5) or YP containing 0.05% glucose (pH 5.5). Whole cell protein extract of each sample were precipitated with trichloroacetic acid (TCA), and the phosphorylation state of Snf1 and total amount of this protein was detected as previously described [46].

RESULTS

Cells expressing a mutated form of Glc7 display tunicamycin sensitivity and impaired UPR-instigated transcription

We investigated the implication of the protein phosphatase Glc7 in the response of yeast cells to ER stress. To do this, we examined growth in the presence of agents that induce unfolded protein stress using a strain containing the glc7-109 allele as the only source of Glc7. The protein Glc7K259A, R260A, encoded by the glc7-109 allele, is mutated in the hydrophobic channel that makes contact with the aromatic residue of the RVXF motif present in many of its regulatory subunits and, consequently, is impaired in the binding to them [17]. As shown in Figure 1(A), glc7-109 cells were more sensitive to tunicamycin, a natural inhibitor of N-linked glycosylation that is widely employed as an inducer of ER stress [47], than those containing the genomic wild-type copy of the GLC7 gene. Cells of the mutant strain were also more sensitive to the presence of other less specific ER-stress inducers, such as β-mercaptoethanol (Figure 1A). We reasoned that the hypersensitivity provoked by the glc7-109 allele could be a consequence of a defective transcriptional response to the ER stress driven by the UPR. To explore this possibility, we measured the activation of an UPRE::lacZ reporter, which contains the UPRE (CAGCGTG) fused to the E. coli lacZ gene [40], in wild-type and mutant cells exposed to 2 μg/ml tunicamycin. As shown in Figure 1(B), mutation of the binding site of Glc7 to its RVXF motif-containing regulatory subunits failed to normally activate the transcription driven by the UPRE-regulated expression in response to tunicamycin. Hence, fully functional yeast Glc7 is required to mediate a proper UPR and to cope with chemical agents that generate accumulation of unfolded or misfolded proteins in the ER.

The interaction of Glc7 with its regulatory subunits is required for the proper adaptation to UPR-inducers

Figure 1
The interaction of Glc7 with its regulatory subunits is required for the proper adaptation to UPR-inducers

(A) Three microlitres of 1:5 serial dilutions of cultures of GLC7+YCp111 (YJFD97), GLC7+YCp-glc7-109 (YJFD98) and glc7+YCp-glc7-109 (YJFD17) cells at a D660 of 0.05 (D660 values of 0.05, 0.01 and 0.002) were spotted on to plates containing YPD, YPD plus DMSO, or the described concentrations of the UPR-inducer tunicamycin or β-mercaptoethanol. Growth at 28°C was monitored after 3 days for tunicamycin plates or 6 days for the β-mercaptoethanol plates. (B) The strains YJFD97 (white bars), YJFD98 (grey bars) and YJFD17 (black bars) were transformed with the pMCZ-Y plasmid (Table 2) containing a UPRE-β-galactosidase reporter gene [40]. Cell cultures were grown until reaching a D660 of 0.8 in YPD (pH 5.5). Cells were transferred to fresh YPD containing 2.0 μg/ml tunicamycin, grown at 28°C and samples were taken at the indicated times. β-Galactosidase activity was measured in permeabilized cells as described in the Materials and methods section. Data are means±S.E.M. for six independent transformants.

Figure 1
The interaction of Glc7 with its regulatory subunits is required for the proper adaptation to UPR-inducers

(A) Three microlitres of 1:5 serial dilutions of cultures of GLC7+YCp111 (YJFD97), GLC7+YCp-glc7-109 (YJFD98) and glc7+YCp-glc7-109 (YJFD17) cells at a D660 of 0.05 (D660 values of 0.05, 0.01 and 0.002) were spotted on to plates containing YPD, YPD plus DMSO, or the described concentrations of the UPR-inducer tunicamycin or β-mercaptoethanol. Growth at 28°C was monitored after 3 days for tunicamycin plates or 6 days for the β-mercaptoethanol plates. (B) The strains YJFD97 (white bars), YJFD98 (grey bars) and YJFD17 (black bars) were transformed with the pMCZ-Y plasmid (Table 2) containing a UPRE-β-galactosidase reporter gene [40]. Cell cultures were grown until reaching a D660 of 0.8 in YPD (pH 5.5). Cells were transferred to fresh YPD containing 2.0 μg/ml tunicamycin, grown at 28°C and samples were taken at the indicated times. β-Galactosidase activity was measured in permeabilized cells as described in the Materials and methods section. Data are means±S.E.M. for six independent transformants.

ER-stress resistance depends on the Glc7-regulatory subunit Reg1

It was then examined whether Glc7 needs the binding to its regulatory subunits to properly respond to UPR-inducers. A set of single deletion mutants on genes coding for non-essential regulatory subunits of Glc7 was assayed for growth under ER-stress conditions. Mutation of most regulatory subunits had no effect on the survival of yeast cells to tunicamycin or β-mercaptoethanol (Supplementary Figure S1). Lack of Bni4, Shp1 and Sla1 generated a certain sensitivity to the reducing agent, and only the absence of Reg1 resulted in notable hypersensitivity to both chemical ER-stress inducers (Figure 2A and Supplementary Figure S1). Quite remarkably, the effect caused by the absence of REG1 was as pronounced as that observed in ire1 and hac1 mutant cells (Figure 2A), which lack the main effectors of the UPR [4]. ER stress can also be promoted by overexpression of either membrane proteins or the single-chain antibody 4-4-20 (scFv) fragment [48,49]. Overexpression of the ER membrane protein Pis1 or heterologous expression of the single-chain antibody 4-4-20 caused stronger growth defects in reg1 mutant cells than in wild-type cells (Figures 2B and 2C, and Supplementary Figure S2), suggesting that a lack of Reg1 triggers hypersensitivity to both, chemical and non-chemical induction of the UPR. The phenotype of the reg1 strain was specifically due to the lack of the REG1 gene, and not to any other factor, since it was observed in other genetic backgrounds (results not shown), and the overexpression of REG1 in reg1 mutant cells completely rescued the hypersensitivity to tunicamycin (Figure 2D). Hence, Reg1 is essential to promote cell survival and adaptation under conditions that disrupt ER function.

reg1 mutant cells are hypersensitive to tunicamycin

Figure 2
reg1 mutant cells are hypersensitive to tunicamycin

(A) Drop test of BY4741 wild-type (WT) and its indicated isogenic derivatives on YPD or YPD plus the indicated concentrations of tunicamycin. Three microlitres of four 1:5 serial dilutions (D660 values of 0.05, 0.01, 0.002 and 0.0004) of cell cultures were spotted on to each plate. Growth at 28°C was recorded after 3 (0.25 μg/ml tunicamycin) or 4 days (1.0 μg/ml tunicamycin). (B) BY4741 wild-type (WT) and reg1 cells were transformed with either the pRS316-FLAG-4-4-20 (pRS-4-4-20) plasmid [49] to express a model single-chain antibody fragment under control of the GAL1-10 promoter or with the empty pRS316 plasmid (Control). Cell cultures were diluted (D660 values of 0.05, 0.01, 0.002 and 0.0004) and 3 μl of each dilution was spotted on to plates containing 2% glucose (SCD) or 2% galactose plus 1% raffinose (SCRaf+Gal). Growth at 28°C was monitored after 2 or 3 days, respectively. (C) Yeast strains described in (B) were transformed with the plasmid pBG18-PIS1 expressing the phosphatidylinositol synthase PIS1 gene under control of the GAL1-10 promoter [101], or with the empty BG1805 plasmid (Control), and spotted on to plates as described in (B). (D) BY4741 wild-type (WT) and reg1 mutant cells were transformed with the empty YEplac195 plasmid (Control) or the same plasmid expressing the wild-type REG1 gene under the control of the ADH1 promoter (Table 2; a gift from P. Sanz). Three microlitres of three 1:5 serial dilutions of cultures at D660 values of 0.05, 0.01 and 0.002 were spotted on to YPD plates containing the indicated concentrations of tunicamycin. Cell growth at 28°C was monitored after 2 (YPD), 3 (0.1 μg/ml tunicamycin) or 4 days (1.0 μg/ml tunicamycin).

Figure 2
reg1 mutant cells are hypersensitive to tunicamycin

(A) Drop test of BY4741 wild-type (WT) and its indicated isogenic derivatives on YPD or YPD plus the indicated concentrations of tunicamycin. Three microlitres of four 1:5 serial dilutions (D660 values of 0.05, 0.01, 0.002 and 0.0004) of cell cultures were spotted on to each plate. Growth at 28°C was recorded after 3 (0.25 μg/ml tunicamycin) or 4 days (1.0 μg/ml tunicamycin). (B) BY4741 wild-type (WT) and reg1 cells were transformed with either the pRS316-FLAG-4-4-20 (pRS-4-4-20) plasmid [49] to express a model single-chain antibody fragment under control of the GAL1-10 promoter or with the empty pRS316 plasmid (Control). Cell cultures were diluted (D660 values of 0.05, 0.01, 0.002 and 0.0004) and 3 μl of each dilution was spotted on to plates containing 2% glucose (SCD) or 2% galactose plus 1% raffinose (SCRaf+Gal). Growth at 28°C was monitored after 2 or 3 days, respectively. (C) Yeast strains described in (B) were transformed with the plasmid pBG18-PIS1 expressing the phosphatidylinositol synthase PIS1 gene under control of the GAL1-10 promoter [101], or with the empty BG1805 plasmid (Control), and spotted on to plates as described in (B). (D) BY4741 wild-type (WT) and reg1 mutant cells were transformed with the empty YEplac195 plasmid (Control) or the same plasmid expressing the wild-type REG1 gene under the control of the ADH1 promoter (Table 2; a gift from P. Sanz). Three microlitres of three 1:5 serial dilutions of cultures at D660 values of 0.05, 0.01 and 0.002 were spotted on to YPD plates containing the indicated concentrations of tunicamycin. Cell growth at 28°C was monitored after 2 (YPD), 3 (0.1 μg/ml tunicamycin) or 4 days (1.0 μg/ml tunicamycin).

The absence of Reg1 misregulates the transcriptional response to ER stress

It was then analysed whether Reg1 could be involved in the UPR signalling. First, we tested whether the overexpression of the spliced mRNA (active form) of the transcription factor HAC1 (HAC1i) could provide protection to cells lacking REG1. As shown in Figure 3(A), the presence of the spliced and active form of HAC1 mRNA did not provide protection to reg1 mutant cells exposed to tunicamycin. Thus, the hypersensitivity of reg1 mutant cells to ER stress does not appear to be associated with a defect in the activation of the HAC1 mRNA splicing.

The response to tunicamycin is altered in reg1 mutant cells

Figure 3
The response to tunicamycin is altered in reg1 mutant cells

(A) Drop test of wild-type BY4741 and reg1 cells transformed with the empty pKK1 plasmid (Table 2) or the same plasmid expressing the mature/spliced and active mRNA of HAC1 (pHAC1i) in plates containing YPD plus the indicated concentrations of tunicamycin. Three microlitres of four 1:5 serial dilutions (D660 values of 0.05, 0.01, 0.002 and 0.0004) were spotted on to plates and growth at 28°C was monitored after the specified time. (B) Cells from the indicated strains transformed with the reporter plasmid pMCZ-Y [40] were treated with 2.5 μg/ml tunicamycin (Tn; empty bars), 100 mM DTT (grey bars) or 50 mM β-mercaptoethanol (β-ME; black bars) for 90 min. β-Galactosidase activity was measured in these and in untreated cells, as described in the Materials and Methods section, and shown as fold change using untreated cells as a reference. Results are means±S.E.M. for nine independent transformants. The data were subjected to a two-tailed Student's t-test and statistical significance was indicated by “**” for P < 0.01 and “*” for P < 0.05. (C) Cells from wild-type (WT; filled circles), as well as reg1 (empty squares), and hac1 (empty triangles) mutant strains were grown until reaching a D660 of 0.8 in YPD (pH 5.5). Cells were transferred to fresh YPD containing 2.0 μg/ml tunicamycin and cell samples were taken at the indicated times. β-Galactosidase activity was measured as described in the Materials and methods section. Data means±S.E.M. for three independent transformants.

Figure 3
The response to tunicamycin is altered in reg1 mutant cells

(A) Drop test of wild-type BY4741 and reg1 cells transformed with the empty pKK1 plasmid (Table 2) or the same plasmid expressing the mature/spliced and active mRNA of HAC1 (pHAC1i) in plates containing YPD plus the indicated concentrations of tunicamycin. Three microlitres of four 1:5 serial dilutions (D660 values of 0.05, 0.01, 0.002 and 0.0004) were spotted on to plates and growth at 28°C was monitored after the specified time. (B) Cells from the indicated strains transformed with the reporter plasmid pMCZ-Y [40] were treated with 2.5 μg/ml tunicamycin (Tn; empty bars), 100 mM DTT (grey bars) or 50 mM β-mercaptoethanol (β-ME; black bars) for 90 min. β-Galactosidase activity was measured in these and in untreated cells, as described in the Materials and Methods section, and shown as fold change using untreated cells as a reference. Results are means±S.E.M. for nine independent transformants. The data were subjected to a two-tailed Student's t-test and statistical significance was indicated by “**” for P < 0.01 and “*” for P < 0.05. (C) Cells from wild-type (WT; filled circles), as well as reg1 (empty squares), and hac1 (empty triangles) mutant strains were grown until reaching a D660 of 0.8 in YPD (pH 5.5). Cells were transferred to fresh YPD containing 2.0 μg/ml tunicamycin and cell samples were taken at the indicated times. β-Galactosidase activity was measured as described in the Materials and methods section. Data means±S.E.M. for three independent transformants.

The expression of the UPRE::lacZ reporter triggered by tunicamycin exposure was then measured. As expected, mutant cells lacking Hac1 displayed a marginal transcriptional response to the UPR-inducer. It was also noted that the basal transcriptional level in reg1 mutant cells was substantially lower than that in wild-type cells (65 and 99 Miller units, respectively). However, deletion of REG1 increased the tunicamycin-induced level of β-galactosidase activity when compared with wild-type cells (Figure 3B). Indeed, whereas tunicamycin induced the transcription of the reporter gene about 11-fold in wild-type cells, the induction in reg1 mutant cells was about 25-fold (Figure 3B). Furthermore, a similar effect was observed in reg1 mutant cells exposed to β-mercaptoethanol or DTT (Figure 3B). This was in some way surprising since, as we showed above, the glc7-109 allele displayed a reduced expression of the UPRE::lacZ reporter (Figure 1A). In this respect, it is worth pointing out that the glc7-109 allele is impaired in the binding of a number of regulatory subunits to Glc7 [17], some of which regulate cellular processes that could, in turn, alter the UPRE-mediated transcriptional response.

Lack of Reg1 also affected the kinetics of the transcriptional response to tunicamycin. As observed in Figure 3(C), the basal levels, and those of 20 min-treated cells, were lower in the reg1 than in the wild-type strain. However, deletion of REG1 increased and misregulated the long-term expression of the UPRE::lacZ reporter. Indeed, after 200 min of treatment with tunicamycin, the level of β-galactosidase activity in reg1 mutant cells was still strongly increasing, whereas wild-type cells appeared to attenuate the expression of the reporter (Figure 3C). Thus, absence of Reg1 decreases the basal transcriptional level driven by the UPRE and impairs the deactivation of the UPR in yeast.

Binding of Reg1 to Glc7 is important for the proper cellular response to tunicamycin

We examined whether the effects of reg1 on the UPRE-mediated transcription were a direct consequence of a misregulated Glc7. For this, wild-type and reg1 mutant cells were transformed with expression plasmids carrying either the native Reg1 (pLexA-Reg1) or mutated versions containing a point mutation of Phe-468, Reg1F468D and Reg1F468R, and growth in the presence of tunicamycin was tested. It has been described that point mutations of Phe-468 made Reg1 unable to target Glc7 to its substrate Hxk2 [50] without an apparent effect on its stability [22]. Consistent with the observations shown in Figure 2(D), expression of REG1 in reg1 mutant cells almost fully restored the tunicamycin resistance observed in wild-type cells carrying the empty pLexA plasmid. In comparison, the expression of either Reg1F468D or Reg1F468R provided some tolerance to the drug, but reg1 mutant cells were still unable to grow, even at tunicamycin doses of 0.4 μg/ml (Figure 4). These results indicate that the disruption of the binding of Glc7 to Reg1 greatly affects the sensitivity to tunicamycin.

Interaction of Reg1 with Glc7 is needed for adaptation to UPR-inducers

Figure 4
Interaction of Reg1 with Glc7 is needed for adaptation to UPR-inducers

Wild-type BY4741 and its isogenic reg1 strains were transformed with the empty plasmid pLexA or with the same plasmid expressing Reg1 (pLexA-Reg1), Reg1F468D or Reg1F468R mutant version (Table 2). Three microlitres of three 1:5 serial dilutions of cultures at D660 values of 0.05, 0.01 and 0.002 were spotted on to YPD plates containing the indicated concentrations of tunicamycin. Growth at 28°C was monitored after 4 days.

Figure 4
Interaction of Reg1 with Glc7 is needed for adaptation to UPR-inducers

Wild-type BY4741 and its isogenic reg1 strains were transformed with the empty plasmid pLexA or with the same plasmid expressing Reg1 (pLexA-Reg1), Reg1F468D or Reg1F468R mutant version (Table 2). Three microlitres of three 1:5 serial dilutions of cultures at D660 values of 0.05, 0.01 and 0.002 were spotted on to YPD plates containing the indicated concentrations of tunicamycin. Growth at 28°C was monitored after 4 days.

The hypersensitivity of reg1 mutant cells to ER stressors depends on the presence of a functional Snf1 protein kinase

We reasoned that the function of Reg1 in the UPR might be related to its well-known role as a regulator of the Snf1 complex [22]. The sensitivity to tunicamycin of snf1 and reg1 snf1 mutant cells was then tested. As expected, a functional Snf1 kinase was required for the growth of yeast cells on ethanol or glycerol as sole carbon sources (Figure 5A). On the contrary, absence of Snf1 in cells of the BY4741 or DBY476 background had no major effects on the yeast growth tested on solid or liquid tunicamycin-containing medium, respectively (Figures 5A and 5B). Moreover, deletion of SNF1 in reg1 mutant cells completely rescued the hypersensitivity to tunicamycin of reg1 mutant cells. In consonance with this, reg1 snf1 double mutant cells of the DBY476 strain displayed a wild-type-like expression of the UPRE::lacZ reporter both under basal and tunicamycin- or DTT-induced ER-stress conditions (Figure 5C). Finally, the hypersensitivity of reg1 mutant cells to ER stress caused by overexpression of the ER membrane protein Pis1 was also significantly reduced when SNF1 was deleted (Supplementary Figure S2). From these results, similar in the two different genetic backgrounds used, it can be concluded that the alterations in the sensitivity to tunicamycin and in the transcriptional response triggered by UPR-inducers in reg1 mutant cells greatly depend on the presence of Snf1.

The hypersensitivity and aberrant transcriptional response of reg1 cells to tunicamycin depends on the presence of Snf1

Figure 5
The hypersensitivity and aberrant transcriptional response of reg1 cells to tunicamycin depends on the presence of Snf1

(A) Drop test of cells (at D660 values of 0.05, 0.01 and 0.002) of the indicated mutant strains in the BY4741 genetic background on YPD, YPD plus DMSO, the specified concentrations of tunicamycin or YP plus 2% (v/v) of ethanol or glycerol as the only carbon source. Growth at 28°C was monitored after 3 days. (B) Growth in liquid cultures of DBY476 wild-type (WT; filled circles), and their isogenic reg1 mutant cells (empty squares), snf1 mutant cells (empty triangles) and reg1 snf1 double mutant cells (empty diamonds) in YPD plus the indicated concentrations of tunicamycin. Relative growth is represented as a percentage with respect to the growth of means strain in YPD plus DMSO. Nine independent transformants were analysed and the means±S.E.M. are represented. (C) The pMCZ-Y plasmid (Table 2) was introduced into wild-type DBY746 and its derivatives. Cells in liquid cultures in YPD (pH 5.5) at a D660 of 0.6 were treated with 2 μg/ml tunicamycin (Tn; grey bars) or 5 mM DTT (black bars) for 90 min. Control cells (white bars) means only the DMSO solvent. β-Galactosidase activity was measured as indicated in the text. Data are means±S.E.M. for six independent clones. The numbers in parentheses indicate the fold induction of the activity caused by the UPR-inducers in each strain.

Figure 5
The hypersensitivity and aberrant transcriptional response of reg1 cells to tunicamycin depends on the presence of Snf1

(A) Drop test of cells (at D660 values of 0.05, 0.01 and 0.002) of the indicated mutant strains in the BY4741 genetic background on YPD, YPD plus DMSO, the specified concentrations of tunicamycin or YP plus 2% (v/v) of ethanol or glycerol as the only carbon source. Growth at 28°C was monitored after 3 days. (B) Growth in liquid cultures of DBY476 wild-type (WT; filled circles), and their isogenic reg1 mutant cells (empty squares), snf1 mutant cells (empty triangles) and reg1 snf1 double mutant cells (empty diamonds) in YPD plus the indicated concentrations of tunicamycin. Relative growth is represented as a percentage with respect to the growth of means strain in YPD plus DMSO. Nine independent transformants were analysed and the means±S.E.M. are represented. (C) The pMCZ-Y plasmid (Table 2) was introduced into wild-type DBY746 and its derivatives. Cells in liquid cultures in YPD (pH 5.5) at a D660 of 0.6 were treated with 2 μg/ml tunicamycin (Tn; grey bars) or 5 mM DTT (black bars) for 90 min. Control cells (white bars) means only the DMSO solvent. β-Galactosidase activity was measured as indicated in the text. Data are means±S.E.M. for six independent clones. The numbers in parentheses indicate the fold induction of the activity caused by the UPR-inducers in each strain.

The inappropriate activation of Snf1 in reg1 mutant cells increases its sensitivity to tunicamycin

The above results suggested that the effects observed in tunicamycin-exposed reg1 mutant cells might reflect the inappropriate activity of the protein kinase Snf1. In agreement with this, the absence of the Glc7-regulatory subunit induced the basal expression of a construct containing the lacZ gene fused to the promoter of the S. cerevisiae SUC2 under repressing conditions (Figure 6A), an effect that was abolished by the combined deletion of REG1 and SNF1 genes. Moreover, the repression process was properly maintained in hac1 and ire1 mutants that did not respond to tunicamycin, which indicates that the UPR pathway is not required for the regulation of the transcriptional response to limited glucose (Figure 6A). In addition, the UPR-inducer tunicamycin did not activate the Snf1 pathway in wild-type cells (Figure 6A).

Snf1 is inappropriately activated in reg1 mutant cells

Figure 6
Snf1 is inappropriately activated in reg1 mutant cells

(A) BY4741 wild-type (WT) and the indicated isogenic mutant strains transformed with a lacZ reporter plasmid containing the SUC2 promoter (Table 2) were grown in YPD (pH 5.5) until reaching a D660 of 0.8 and the cells were then washed with YP medium, transferred to fresh YPD (as a negative control, white bars), YP containing 0.05% (w/v) glucose (grey bars) or YPD plus 2.5 μg/ml tunicamycin (black bars) and grown at 28°C for 90 min. β-Galactosidase activity was measured as described in the Materials and methods section. Data are means±S.E.M. for six independent transformants. (B) Drop test of cells (at D660 values of 0.05, 0.01, 0.002 and 0.0004) of the indicated mutant strains in the BY4741 genetic background on YPD or YPD plus the indicated concentrations of soraphen A. Growth at 28°C was monitored after 3 days.

Figure 6
Snf1 is inappropriately activated in reg1 mutant cells

(A) BY4741 wild-type (WT) and the indicated isogenic mutant strains transformed with a lacZ reporter plasmid containing the SUC2 promoter (Table 2) were grown in YPD (pH 5.5) until reaching a D660 of 0.8 and the cells were then washed with YP medium, transferred to fresh YPD (as a negative control, white bars), YP containing 0.05% (w/v) glucose (grey bars) or YPD plus 2.5 μg/ml tunicamycin (black bars) and grown at 28°C for 90 min. β-Galactosidase activity was measured as described in the Materials and methods section. Data are means±S.E.M. for six independent transformants. (B) Drop test of cells (at D660 values of 0.05, 0.01, 0.002 and 0.0004) of the indicated mutant strains in the BY4741 genetic background on YPD or YPD plus the indicated concentrations of soraphen A. Growth at 28°C was monitored after 3 days.

Then, we tested the growth of yeast cells in the presence of soraphen A, a natural product that inhibits ACC1, the first enzyme in the fatty acid synthesis pathway, which is phosphorylated and inactivated by Snf1 [51,52]. As can be seen in Figure 6(B), absence of SNF1 in either snf1 and reg1 snf1 mutants decreased sensitivity to the ACC1-specific inhibitor, soraphen A. On the contrary, reg1 mutant cells showed increased sensitivity to the drug which indicates the inactivation of ACC1 by an active Snf1 (Figure 6B). Thus, Snf1 is inappropriately active in reg1 mutant cells and this activation of Snf1 regulates the function of different and unrelated targets.

We also analysed whether the activation state of Snf1 does affect the UPR and, consequently, can modulate cellular sensitivity to tunicamycin. Wild-type and reg1 mutant cells were transformed with plasmids carrying hyperactive or inactive alleles of SNF1, and their sensitivity to tunicamycin was analysed. The point mutation Snf1Y106A relieves glucose inhibition of its phosphorylation, resulting in the phosphorylation of Thr-210, even in the presence of high levels of glucose. Snf1R53G is more active in both high and low glucose concentrations [53]. As shown in Figure 7(A), expression of any of the hyperactive alleles slightly increased the sensitivity to tunicamycin, not only of wild-type cells, but also in those lacking Reg1, where Snf1 is already active. On the contrary, expression in these cells of inactive alleles of SNF1, such as Snf1T210A, that prevents the phosphorylation of Thr-210, or Snf1K84R, with a mutation in the ATP-binding site [54], increased the tolerance to tunicamycin (Figure 7B). This effect is more pronounced in reg1 mutant cells, where Snf1 is active. This could indicate that the inactive Snf1T210A and Snf1K84R isoforms titrate the β and γ regulatory subunits of Snf1 away from the wild-type Snf1 kinase complex, thus reducing its activity. However, increased growth in tunicamycin-containing medium was also observed in cells of the snf1 mutant expressing the inactive alleles of SNF1 (Figure 7B). Therefore, it seems that the inactive Snf1 complex is still able to interact with key targets, altering their localization and/or activity and promoting tunicamycin resistance in yeast cells.

Sensitivity to tunicamycin depends on the Snf1 activity

Figure 7
Sensitivity to tunicamycin depends on the Snf1 activity

(A) BY4741 wild-type (WT) and its derivative reg1 mutant strain were transformed with the empty YCp50 plasmid or the same plasmid expressing the wild-type SNF1, or the Y106A or the G53R point mutations (Table 2) that produce active protein kinases. Three microlitres of three 1:5 serial dilutions of cultures at D660 values of 0.05, 0.01 and 0.002 were spotted on to YPD plates containing the indicated concentrations of tunicamycin (or the DMSO vehicle). Cell growth at 28°C was monitored after 4 days. (B) As in (A), but cells were transformed with an empty pWS93 plasmid or the same plasmid expressing the wild-type SNF1or the T210A and K84R point mutations (Table 2) that produce inactive protein kinases. Cell growth at 28°C was monitored after 2 days. (C) Three 1:5 serial dilutions of the BY4741 wild-type were spotted on to plates containing YP plus either 2% (w/v) glucose, 0.05% (w/v) glucose or 2% (v/v) ethanol in the presence of the indicated concentrations of tunicamycin. Cells were incubated at 28°C and the growth was monitored after 3 days.

Figure 7
Sensitivity to tunicamycin depends on the Snf1 activity

(A) BY4741 wild-type (WT) and its derivative reg1 mutant strain were transformed with the empty YCp50 plasmid or the same plasmid expressing the wild-type SNF1, or the Y106A or the G53R point mutations (Table 2) that produce active protein kinases. Three microlitres of three 1:5 serial dilutions of cultures at D660 values of 0.05, 0.01 and 0.002 were spotted on to YPD plates containing the indicated concentrations of tunicamycin (or the DMSO vehicle). Cell growth at 28°C was monitored after 4 days. (B) As in (A), but cells were transformed with an empty pWS93 plasmid or the same plasmid expressing the wild-type SNF1or the T210A and K84R point mutations (Table 2) that produce inactive protein kinases. Cell growth at 28°C was monitored after 2 days. (C) Three 1:5 serial dilutions of the BY4741 wild-type were spotted on to plates containing YP plus either 2% (w/v) glucose, 0.05% (w/v) glucose or 2% (v/v) ethanol in the presence of the indicated concentrations of tunicamycin. Cells were incubated at 28°C and the growth was monitored after 3 days.

Finally, we examined the sensitivity to tunicamycin of yeast cells grown on low glucose or ethanol as sole carbon source, conditions that trigger the activation of Snf1. As shown in Figure 7(C), the sensitivity of wild-type cells to tunicamycin was markedly increased when they were grown under de-repressing conditions, where Snf1 is more active. We can conclude that the activity of the Snf1 protein kinase does affect the cellular sensitivity to ER stress caused by tunicamycin exposure.

Deactivation of the UPR is defective in reg1 mutant cells in a Snf1-dependent manner

The results shown above suggested a defect in the attenuation of the UPR in reg1 mutant cells. Both the intensity of the response and the duration of activation appeared to be affected by the absence of Reg1. In order to confirm this point, we determined the expression levels of KAR2 in cells that were initially exposed to tunicamycin for 1 h and then incubated in the absence of the drug at different times. KAR2 encodes a heat-shock protein of the ER lumen [55], the expression of which upon ER stress depends on the Ire1–Hac1 system [6,56]. As expected, the induction of KAR2 by tunicamycin, measured by qRT-PCR, was higher in reg1 mutant cells (13-fold) than in wild-type cells (5-fold) (Figure 8A). Furthermore, the quantity of this mRNA in wild-type cells returned to basal levels 6 h after the compound was washed out from the medium. However, in reg1 mutant cells, KAR2 mRNA levels remained elevated even after 8 h of growth in the absence of the UPR-inducer. Finally, additional deletion of SNF1 completely restored the quantity of mRNA of KAR2 to levels similar to those in wild-type cells. Comparable results were found when the levels of mRNA of KAR2 were measured by Northern bloting (Figure 8B, top panel). Hence, the lack of REG1 alters the kinetics of disappearance of KAR2 mRNA levels induced by tunicamycin, and this effect is completely dependent on the presence of a functional Snf1.

reg1 mutant cells are defective in the deactivation of the UPR

Figure 8
reg1 mutant cells are defective in the deactivation of the UPR

(A) Quantification of the KAR2 mRNA by qRT-PCR in the indicated strains at different times after removal of tunicamycin from the culture medium. Wild-type (WT) strain in the BY4741 genetic background (white bars) and its isogenic derivatives reg1 (light gray), snf1 (dark gray) and reg1 snf1 (YJFD31, black bars) were grown in YPD (pH 5.5) until a D660 of 0.6 was reached (NI sample). The UPR was induced by incubation in the presence of tunicamycin (1 μg/ml) for 1 h. Cells were washed and transferred to fresh YPD (pH 5.5) (0 h sample). Total RNA from the cell samples, collected at the indicated times (3, 6 and 8 h), was purified and 15 ng of total RNA were used to quantify the amount of mRNA corresponding to KAR2 means and ACT1 genes. Data are means±S.E.M. for three independent reactions for each sample (normalized to ACT1 expression). (B) Wild-type strain (BY4741) and its isogenic derivatives reg1, snf1 and reg1 snf1 (YJFD31) were grown in YPD (pH 5.5) until reaching a D660 of 0.6 and control samples were taken (NI). Cell cultures were treated with 1 μg/ml tunicamycin for 1 h and then transferred to fresh YPD (pH 5.5). Samples were taken at different times after the tunicamycin was washed out from the medium (0, 3 and 6 h). Total RNA was prepared from each sample and Northern blot analyses were performed using the probes for KAR2, HAC1 and U1 detailed in the Materials and methods section.

Figure 8
reg1 mutant cells are defective in the deactivation of the UPR

(A) Quantification of the KAR2 mRNA by qRT-PCR in the indicated strains at different times after removal of tunicamycin from the culture medium. Wild-type (WT) strain in the BY4741 genetic background (white bars) and its isogenic derivatives reg1 (light gray), snf1 (dark gray) and reg1 snf1 (YJFD31, black bars) were grown in YPD (pH 5.5) until a D660 of 0.6 was reached (NI sample). The UPR was induced by incubation in the presence of tunicamycin (1 μg/ml) for 1 h. Cells were washed and transferred to fresh YPD (pH 5.5) (0 h sample). Total RNA from the cell samples, collected at the indicated times (3, 6 and 8 h), was purified and 15 ng of total RNA were used to quantify the amount of mRNA corresponding to KAR2 means and ACT1 genes. Data are means±S.E.M. for three independent reactions for each sample (normalized to ACT1 expression). (B) Wild-type strain (BY4741) and its isogenic derivatives reg1, snf1 and reg1 snf1 (YJFD31) were grown in YPD (pH 5.5) until reaching a D660 of 0.6 and control samples were taken (NI). Cell cultures were treated with 1 μg/ml tunicamycin for 1 h and then transferred to fresh YPD (pH 5.5). Samples were taken at different times after the tunicamycin was washed out from the medium (0, 3 and 6 h). Total RNA was prepared from each sample and Northern blot analyses were performed using the probes for KAR2, HAC1 and U1 detailed in the Materials and methods section.

It was then investigated whether the effects on the induced mRNA level of KAR2 caused by the lack of Reg1 could be the consequence of a lack of attenuation of the UPR. We first monitored the change in the relative abundance of the precursor (HAC1u) and mature (spliced, HAC1i) forms of HAC1 mRNA. It has already been described that the levels of HAC1i decline after tunicamycin wash-out, during the recovery phase [10]. As observed in Figure 8(B) (middle panel), treatment with tunicamycin triggered the splicing of the HAC1 mRNA in all the strains tested. In wild-type cells, the spliced form was completely absent 6 h after removal the drug from the medium. Interestingly, it was still clearly detectable in reg1 mutant cells, indicating a defect in the inactivation of the pathway. In addition, this defect, as well as the abnormal levels of KAR2 mRNA, were Snf1-dependent, since reg1 snf1 double mutant cells displayed similar kinetics of splicing and quantity of KAR2 mRNA as wild-type cells (Figures 8A and 8B). Thus, our results indicate that the active form of Snf1 present in reg1 mutant cells is the cause of the observed abnormal kinetics of both the deactivation of HAC1 splicing and KAR2 transcriptional response.

Reg1 is important for the proper de-oligomerization of the Ire1 receptor

ER stress, caused by the presence of unfolded proteins, induces the oligomerization of the Ire1 receptor, increases its kinase activity and trans-autophosphorylation which, in turn, causes a conformational change that increases its RNase activity and triggers the atypical splicing of the HAC1 mRNA [5759]. Since reg1 mutant cells display abnormally high HAC1i levels during the deactivation phase, we wanted to know whether this effect could be due to dysregulation of the Ire1-oligomeric equilibrium. In order to determine the quantity of Ire1 that is present as a complex (oligomerized), we carried out experiments of co-immunoprecipitation, as previously described [60], with wild-type and reg1 mutant cells transformed with two plasmids that simultaneously express HA- and Flag-tagged versions of Ire1. As shown in Figure 9, we were able to detect a notable increase in the quantity of complexed Ire1 in both wild-type and reg1 mutant cells after the treatment for 1 h with tunicamycin (comparison of ‘NI’ with ‘0’ lines). The quantity of oligomerized Ire1 in wild-type cells then started to slowly decline about 2 h after the drug wash-out, being greatly reduced after 20 h (36% of the complexed Ire1 detected at the wash-out time). In comparison, the quantity of the complexed form of Ire1 in reg1 mutant cells continued increasing even after 20 h in the absence of tunicamycin (228% of the complexed Ire1 detected at the wash-out time).

Deactivation of the Ire1 receptor is impaired in reg1 mutant cells

Figure 9
Deactivation of the Ire1 receptor is impaired in reg1 mutant cells

Wild-type and reg1 mutant cells in the BY4741 genetic background were co-transformed with the pRS423 plasmid expressing HA- and Flag-tagged versions of Ire1 (Table 2). Cell cultures were grown in YPD (pH 5.5) until reaching a D660 of 0.6, when the untreated samples were taken (NI sample). Cell cultures were treated with 2 μg/ml tunicamycin for 1 h and then transferred to fresh YPD (pH 5.5). Cell samples were taken at different times after the tunicamycin was washed out from the medium (0, 2, 4, 6, 8 and 20 h). Total protein from cells at an equivalent of 10 D660 units was used for immunoprecipitation with the anti-HA antibody (Input) and the anti-HA immunoprecipitation products (Anti-HA IP) were analysed by Western bloting using both anti-HA and anti-Flag antibodies.

Figure 9
Deactivation of the Ire1 receptor is impaired in reg1 mutant cells

Wild-type and reg1 mutant cells in the BY4741 genetic background were co-transformed with the pRS423 plasmid expressing HA- and Flag-tagged versions of Ire1 (Table 2). Cell cultures were grown in YPD (pH 5.5) until reaching a D660 of 0.6, when the untreated samples were taken (NI sample). Cell cultures were treated with 2 μg/ml tunicamycin for 1 h and then transferred to fresh YPD (pH 5.5). Cell samples were taken at different times after the tunicamycin was washed out from the medium (0, 2, 4, 6, 8 and 20 h). Total protein from cells at an equivalent of 10 D660 units was used for immunoprecipitation with the anti-HA antibody (Input) and the anti-HA immunoprecipitation products (Anti-HA IP) were analysed by Western bloting using both anti-HA and anti-Flag antibodies.

The growth defect of reg1 mutant cells on tunicamycin is alleviated by overexpression of the protein phosphatases Ptc2 and Ptc3

Since autophosphorylation is a key event in the activation of the Ire1 receptor, we analysed whether its extended activation observed in reg1 cells could be associated with a defect in the inactivation by dephosphorylation of the RNase Ire1. In fact, it has been reported that Ptc2, a member of the PP2C family of protein phosphatases, interacts and is required for the dephosphorylation of Ire1 [12]. According to this, overexpression of Ptc2 slightly alleviated the sensitivity to tunicamycin of reg1 mutant cells. Overexpression of Ptc3, a close Ptc2 paralogue [61], also contributed to relieving, although to lesser extent, the hypersensitivity of reg1 mutant cells to the UPR-inducer (Supplementary Figure S3). Overexpression of Ptc2/Ptc3 could contribute to dephosphorylate the P-loop of Snf1, causing its inactivation. However, previously published data indicate that Ptc1 is the only PP2C able to dephosphorylate Snf1 [25]. We also observed that overexpression of Ptc2 does not affect the Snf1 phosphorylation (Supplementary Figure S4). Thus, the hypersensitivity to tunicamycin observed in reg1 mutant cells could be attributable, at least in part, to the high levels of phosphorylation of Ire1. Rescue of this hypersensitivity by expressing Ptc2 or Ptc3 is far from complete and could be explained by the existence of other protein phosphatases involved in Ire1 dephosphorylation [13].

DISCUSSION

The precise adjustment of signalling pathways is critical to regulate their final output and the diverse biological responses they control. Attenuation of the signal is essential for normal cell functioning, and their deregulation often results in human diseases [62]. Proper attenuation of HAC1 mRNA splicing during UPR signalling determines cell survival after recovery of the ER protein-folding capacity [10], and its dysregulation may compromise human health [63]. Evidence is shown here, for the first time, which supports the hypothesis that activation of the Snf1 pathway causes a dramatic defect in the ability of yeast cells to attenuate the UPR signalling. Lack of Reg1, the regulatory subunit of the Glc7 protein phosphatase, which down-regulates the activity of Snf1, resulted in sustained HAC1 mRNA splicing and high levels of the transcript of KAR2. The importance of this defect is supported by the finding that reg1 mutant cells are as sensitive to UPR-inducer drugs as cells lacking Ire1 or Hac1, the main regulators of the UPR signalling. Hence, our study represents a significant advance in the understanding of the mechanisms and effectors involved in the regulation of the UPR.

The UPR signalling can be triggered by treating cells with different chemical agents, from which tunicamycin, which blocks N-linked glycosylation, is likely to be the most commonly used ER-stress inducer [47]. Nevertheless, it seems evident that different UPR activators induce defined types of stress, as is evidenced by their regulation of common and unique targets, and cause specific physiological effects [64]. In fact, it has been described that tunicamycin activates the Ca2+ influx through a mechanism that is independent of Ire1 and Hac1 [65], resulting in a calcineurin-dependent response element (CDRE)-driven transcription activation [66]. Tunicamycin also affects the cell wall integrity and the high osmolarity pathways, leading to the activation of the corresponding MAPKs, Slt2 and Hog1, in a manner that is independent of a functional canonical UPR pathway [39,67]. Thus, tunicamycin exposure might have other UPR-unrelated cellular effects. On the other hand, side effects of tunicamycin uptake or disposal/excretion could be evoked to explain the results observed in reg1 mutant cells. We show that growth in the presence of DTT or β-mercaptoethanol, which disrupts protein folding in the ER by preventing disulfide bond formation, was compromised by knockout of REG1. Exposure of yeast cells to either of these activators increased the expression of the UPR-lacZ reporter, and the up-regulation (as fold induction compared with untreated cells) was significantly higher in the reg1 than in the wild-type strain. We also show that heterologous expression of a model single-chain antibody fragment (scFv, 4-4-20) on yeast cells caused a growth defect that was more pronounced in cells lacking Reg1. This recombinant protein contains four cysteines and two disulfide bonds, but does not display N-glycosylation sites [49]. Its expression saturates the capacity of cells to properly fold protein, initiating UPR signalling [68], which is required for the degradation of protein substrates from the ER [49,69]. Similarly, lack of Reg1 reduced the capacity of yeast cells to deal with the overproduction of PIS1, the gene encoding phosphatidylinositol synthase [70,71], a membrane-associated protein which is not N-glycosylated [72]. Hence, our results suggest a general effect of Reg1/Snf1 on protein folding in the ER. It is recognized that Reg1 is important in the performance of multiple cellular functions [7377] by directing Glc7 to substrates such as Snf1, Hxk2, Pda1 and Hsp60 [14]. In particular, Glc7-Reg1 plays a major role in maintaining the Snf1 activation loop in the dephosphorylated state during growth on high glucose [21]. Consequently, blockage of the Snf1 dephosphorylation by knockout of REG1 or disruption of the binding between Glc7 and Reg1 leads to improper activation of Snf1. Consistent with this, the reg1 mutant cells were only resistant to ER stress when SNF1 was disrupted, or when SNF1 alleles with reduced catalytic activity were expressed. We also found that absence of Snf1 had no noticeable effect on the growth of yeast cells on tunicamycin-containing medium. Furthermore, exposure of wild-type yeast cells to tunicamycin or other chemicals that induce ER stress did not alter the glucose signalling to the SNF1 pathway. The glucose repression process was also properly maintained in hac1 and ire1 mutants that do not respond to tunicamycin. Hence, our results suggest that neither the UPR pathway contributes to the regulation of the transcriptional response to limited glucose nor Snf1 is required to cope with the accumulation of unfolded or misfolded proteins.

Our results, however, clearly show that the activity of Snf1 is physiologically relevant under ER-stress conditions, and that the protein kinase plays a functional role in the proper attenuation of the Ire1–Hac1 system. Therefore, there must be cell conditions where the convergent activation of Snf1 and the UPR contribute to the regulation of key cellular functions. In mammalian cells, one of the major physiological cell conditions leading to UPR activation is glucose deprivation [35]. Cholesterol- or fatty-acid-induced macrophage or pancreatic β-cells apoptosis or alterations in ER fatty acid and lipid composition activates the UPR [7880]. Perturbation of the yeast cellular lipid composition such as low unsaturated fatty acid levels or depletion of inositol in yeast cells [8183] also activates Ire1. Finally, Ire1 and its mammalian homologue Ire1α have been implicated in various important metabolic functions [34], and there is increasing evidence that the UPR has a broad influence on cellular physiology [84].

The UPR could also contribute to integrate different metabolic signals. Several pathways, including metabolic signalling routes, contribute to regulate the Ire1 pathway, (see [85] for a recent review). Ire1α, which is phosphorylated by the glucagon-stimulated protein kinase PKA (protein kinase A) at Ser-724 in the liver, seems to be coupled to glucose metabolism [86]. In yeast cells, Snf1 is inhibited by the PKA pathway, since Sak1, one of the Snf1-activating kinases, is negatively regulated by PKA [87] and Glc7-Reg1 is activated by PKA [26]. Snf1 regulates the traffic of the PtdIns4P phosphatase Sac1 between ER and Golgi membranes, a function that, in turn, depends on Hog1 [32]. Finally, Snf1 and Ire1 appear to be required for the activation of Hog1 under metabolic respiration conditions [88].

Nevertheless, we were unable to show the activation of the UPRE-mediated transcription in low glucose-grown cells (results not shown). It should be stressed that there are many distinct physiological UPR activation states, and that these represent the first step in generating diverse outputs [84]. For example, activation of the Ire1–Hac1 system by inositol depletion is essentially different from that induced by DTT [83]. Thus, stress stimuli causing accumulation of unfolded proteins in the ER activate Ire1 via its interaction with the aberrant proteins, whereas lack of inositol, a main component of phospholipids which causes membrane- or lipid-related aberrations, activates Ire1 without this interaction with aberrant proteins [83]. Furthermore, the activation of Ire1 upon treatment with DTT or tunicamycin is rapid (within 30 min or 1 h of stimulus onset), whereas the cellular mechanism activating Ire1 upon inositol depletion per se is slow in responding (around 5 h) [83]. It is also possible that different stimuli may result in the activation of different target genes. Expansion of ER membrane requires UPR signalling, is driven by lipid biosynthesis and occurs independently of an increase in ER chaperone levels [89]. Hence, it is reasonable to speculate with the possibility that the UPR during glucose limitation is activated in a specific manner and its output modulated by effectors and signals other than those associated with the ER protein load.

But how does Snf1 modulate the UPR signalling? Our results suggest that the defect in the deactivation of the atypical splicing of the HAC1 mRNA could be attributable to a defective de-oligomerization of the Ire1 receptor. Similar defects on the attenuation of the UPR in yeast have been observed in cells expressing Ire1 carrying phosphomimetic mutations within the kinase-activation loop [10], indicating that the dephosphorylation of these serine residues of Ire1 appeared to be necessary for Ire1 inactivation. A Snf1-regulated dephosphorylation of Ire1 by Ptc2 and Dcr2 may explain, at least in part, its deactivation. As we show in the present study, overexpression of Ptc2 or Ptc3 alleviated the tunicamycin sensitivity of reg1 mutant cells. We thus speculate that Snf1 plays a direct or indirect role in regulating the phosphorylation state of Ire1 leading to changes in its self-association/dissociation state. It could be hypothesized that activation of Snf1 in the reg1 mutant might impair the interaction of Ire1 with Kar2, an event that would lead to the formation of higher-order oligomeric forms of Ire1 [60]. Nevertheless, Snf1 might regulate the attenuation of the UPR signalling at different levels and imply several effectors. However, we cannot rule out an indirect effect of Snf1 on the deactivation mechanism(s). It is well documented that yeast Snf1 regulates lipid, fatty acid and sphingolipid metabolism [30,9092]. Consequently, Snf1-mediated changes in the ER lipid composition could also contribute to the Ire1 attenuation, as has been demonstrated by its activation upon inositol depletion [83]. More work is required to further elucidate the exact mechanism(s) involved in the regulation by Snf1 of UPR attenuation.

AUTHOR CONTRIBUTION

Antonio Casamayor and José Prieto designed the study. Jofre Ferrer-Dalmau, Francisca Randez-Gil, Maribel Marquina and Antonio Casamayor carried out the research, and Antonio Casamayor and José Prieto analysed the data and wrote the paper.

We thank J. Ariño (UAB, Barcelona, Spain), M. Carlson (Columbia University, New York, U.S.A.), Y. Kimata (NAIST-GSBS, Ikoma, Nara, Japan), K. Mori (Kyoto University, Kyoto, Japan), A.S. Robinson (University of Delaware, Newark, DE, U.S.A.), P. Sanz (IBV, València, Spain), M. Valkonen (VTT Biotechnology, Espoo, Finland) and H. Zhu (Johns Hopkins University, Baltimore, MD, U.S.A.) for provision of reagents. The excellent technical assistance of Montserrat Robledo at the Universitat Autònoma de Barcelona is acknowledged. We are also grateful to the Servei de Genòmica Bioinformàtica from the IBB (Universitat Autònoma de Barcelona),

FUNDING

This work was supported by the Ministry of Science and Innovation, Spain, and ERDF [grant numbers BFU2009-11593 and AGL2010-17516 (to A.C. and F.R.-G.)]; and the Generalitat de Catalunya [grant number 2009 SGR-1091 (to A.C.)].

Abbreviations

     
  • ACC1

    acetyl-CoA carboxylase 1

  •  
  • CDRE

    calcineurin-dependent response element

  •  
  • ER

    endoplasmic reticulum

  •  
  • HA

    haemagglutinin

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • PKA

    protein kinase A

  •  
  • qRT-PCR

    quantitative reverse transcription–PCR

  •  
  • UPR

    unfolded protein response

  •  
  • UPRE

    unfolded protein response element

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Supplementary data