Induction of Ptp2 and Cmp2 protein phosphatases is crucial for the adaptive response to ER stress in Saccharomyces cerevisiae

Mizuno, Tomoaki; Nakamura, Meyu; Irie, Kenji

doi:10.1038/s41598-018-31413-6

Download PDF

Article
Open access
Published: 30 August 2018

Induction of Ptp2 and Cmp2 protein phosphatases is crucial for the adaptive response to ER stress in Saccharomyces cerevisiae

Tomoaki Mizuno¹,
Meyu Nakamura¹ &
Kenji Irie¹

Scientific Reports volume 8, Article number: 13078 (2018) Cite this article

1284 Accesses
8 Citations
Metrics details

Subjects

Abstract

Expression control of the protein phosphatase is critically involved in crosstalk and feedback of the cellular signaling. In the budding yeast ER stress response, multiple signaling pathways are activated and play key roles in adaptive reactions. However, it remains unclear how the expression level of the protein phosphatase is modulated during ER stress response. Here, we show that ER stress increases expression of Ptp2 tyrosine phosphatase and Cmp2 calcineurin phosphatase. Upregulation of Ptp2 is due to transcriptional activation mediated by Mpk1 MAP kinase and Rlm1 transcription factor. This induction is important for Ptp2 to effectively downregulate the activity of Hog1 MAP kinase. The budding yeast genome possesses two genes, CMP2 and CNA1, encoding the catalytic subunit of calcineurin phosphatase. CMP2 is more important than CNA1 not only in ER stress response, but also in salt stress response. Higher promoter activity of CMP2 contributes to its relative functional significance in ER stress response, but is less important for salt stress response. Thus, our results suggest that expression control of Ptp2 and Cmp2 protein phosphatases at the promoter level is crucial for adaptive responses to ER stress.

Potassium nutrient status drives posttranslational regulation of a low-K response network in Arabidopsis

Article Open access 23 January 2023

Tolerance to alkaline ambient pH in Aspergillus nidulans depends on the activity of ENA proteins

Article Open access 31 August 2020

The role of transcriptional repressor activity of LexA in salt-stress responses of the cyanobacterium Synechocystis sp. PCC 6803

Article Open access 15 October 2020

Introduction

The endoplasmic reticulum (ER) is responsible for folding and modification of nascent secretory and transmembrane proteins. When ER functions are perturbed by increased influx of newly synthesized polypeptides or exposure to stressors causing defects of glycosylation and disulfide bond formation, aberrant proteins accumulate in the ER lumen and membrane. This condition is referred to as ER stress. To restore ER homeostasis, cells under ER stress conditions activate transcription of a variety of genes, including genes encoding ER-resident chaperones and proteins functioning in the secretory pathway or ER-associated degradation^1,2. In the budding yeast Saccharomyces cerevisiae, the unfolded protein response (UPR) signaling pathway composed of Ire1 and Hac1 plays a principal role in broad transcriptional response to ER stress^1,2.

Protein phosphorylation and dephosphorylation catalyzed by protein kinases and phosphatases, respectively, are fundamental mechanisms by which cells respond to changes of the extracellular environment. Particularly, regulation of phosphorylation/dephosphorylation is essential for cells to initiate adaptive responses to environmental stresses. Previous studies using the budding yeast revealed that several kinases, including the stress responsive MAP kinases (MAPKs), such as Mpk1 and Hog1, and the Snf1 AMP-activated protein kinase (AMPK), are involved in ER stress response^{3,4,5,6,7,8,9,10}. Furthermore, it has been shown that the calcineurin phosphatase participates in protection of yeast cells from ER stress¹¹. It is well-known that many protein kinases can be activated through binding of their activator proteins and phosphorylation mediated by their upstream kinases. Indeed, such mechanisms operate in the budding yeast ER stress response^{3,4,5,6,7,8,9}. In contrast, modulation of the expression levels is frequently observed in the situations where the phosphatase activity is regulated^12,13. Furthermore, expression control of protein phosphatases is involved in feedback regulation of the signal and crosstalk between the signaling pathways¹³. In Saccharomyces cerevisiae, there are approximately thirty genes encoding known or putative protein phosphatases^14,15. Previous genome-wide microarray analyses suggested that the mRNA levels of a subset of yeast protein phosphatases increase upon exposure to ER stress^16,17. However, none of them were categorized as the target of the UPR pathway. Thus, little is known about regulation of the expression level of protein phosphatases during the budding yeast ER stress response and, furthermore, its physiological significance.

In this study, we analyzed mRNA expression patterns of 35 genes encoding yeast protein phosphatases in ER stress response. We found that the mRNA levels of 8 genes were more than two-fold increased by ER stress. Of these, PTP2 and CMP2 were involved in cellular sensitivity to ER stress. PTP2 and CMP2 encode a tyrosine phosphatase of MAPKs and a catalytic subunit of calcineurin phosphatase, respectively^18,19,20,21. Our data presented here suggest that the PTP2 and CMP2 promoters are activated in response to ER stress and that promoter activation is required for the PTP2 and CMP2 genes to effectively fulfill their roles in ER stress response. Thus, regulation of the expression levels of protein phosphatases is critically involved in adaptive responses to ER stress.

Results

The mRNA levels of PTP2 and CMP2 are upregulated by ER stress

In Saccharomyces cerevisiae, 35 genes encode the known or putative catalytic components of protein phosphatases^14,15 (Table 1). We first monitored their mRNA levels during ER stress response. Wild-type yeast cells were treated with tunicamycin, which causes ER stress by inhibiting N-linked glycosylation, and then harvested up to 7.5 hr every 1.5 hr; a quantitative real-time RT-PCR (qRT-PCR) analysis was carried out to quantitate the mRNA levels. We found that 10 mRNAs (CMP2, MIH1, PPZ2, PTC5, PTC6, PTP1, PTP2, SAL6, SDP1 and YCH1) increased more than two-fold within 7.5 hr after tunicamycin treatment (Fig. 1A and Supplementary Figs 1–5). We performed similar experiments using dithiothreitol (DTT), which causes ER stress by inhibition of the disulfide bond formation. Exposure to DTT increased the mRNA levels of 12 genes, including PPZ1 and PTP3 in addition to 10 genes whose expression was increased after exposure to tunicamycin (Fig. 1B and Supplementary Figs 6–10). We next attempted to examine the mRNA levels in cells which were ER-stressed by genetic alterations. Previous reports showed that activation of the UPR pathway, which is interpreted as an indication of ER stress conditions, was caused by the block of transport form the ER to the Golgi^22,23,24. The SEC12 gene is essential for the initiation of coat protein complex II (COPII)-coated vesicle formation in ER-to-Golgi transport²⁵, and its temperature-sensitive allele, sec12-4, causes the UPR activation²³ (data not shown). Therefore, we analyzed expression of protein phosphatases in the sec12-4 mutant: Wild-type and sec12-4 mutant cells were incubated for 4 hr at 37 °C; their total RNAs were prepared and subjected to qRT-PCR analysis. The changes in gene expression patterns caused by sec12-4 mutation closely resembled those induced by treatment with chemical ER stressors, with some exceptions such that MIH1 expression was strongly induced by sec12-4 mutation (Fig. 1C and Supplementary Figs 11–15). Similar results were obtained using the sec13-1 and sec16-2 mutants (Supplementary Figs 11–15), which are also defective in vesicle formation from the ER²³. Remarkably, among 10 genes whose mRNAs were increased by both tunicamycin and DTT, expression of 8 genes (CMP2, MIH1, PPZ2, PTC6, PTP1, PTP2, SAL6, and SDP1) was upregulated more than two-fold in the sec12-4

Table 1 Saccharomyces cerevisiae genes encoding the protein phosphatase.

Full size table

mutant cells compared to wild-type cells. Thus, approximately 25% of mRNAs for protein phosphatases are upregulated under ER stress conditions.

Next, we extracted protein phosphatases that are physiologically important in ER stress response from 10 protein phosphatases that were induced by both tunicamycin and DTT. We examined ER stress sensitivity of cells deleted for each gene. Cells were plated on medium containing tunicamycin as an inducer of ER stress, and their growth was monitored. Among them, the cmp2 mutant displayed hypersensitivity to tunicamycin, but the ptp2 mutant was resistant to tunicamycin (Fig. 1D). The CMP2 gene encodes a catalytic subunit of calcineurin phosphatase^18,19. Previous studies using an inhibitor of the calcineurin phosphatase activity revealed that the budding yeast calcineurin acts to confer ER stress tolerance¹¹. Furthermore, it has been reported that ptp2 deletion results in increased resistance to ER stress⁶. Thus, our observations are consistent with previous findings.

PTP2 expression is induced by the Mpk1-Rlm1 signaling axis during ER stress response

We attempted to identify the regulator of PTP2 expression. In the budding yeast ER stress response, the UPR pathway composed of Ire1 and Hac1 activates a broad transcriptional program^1,2. Therefore, we examined whether the UPR pathway induces expression of the PTP2 gene. However, the mRNA levels of PTP2 were apparently unaffected by hac1 mutation (Supplementary Fig. 16A). Previous studies demonstrated that several signaling pathways, including Mpk1, Hog1 and Snf1, become activated in budding yeast ER stress response^{3,4,5,6,7,8,9,10}. Therefore, we tested their involvement in regulation of PTP2 expression. We found that induction of PTP2 mRNA upon ER stress was impaired in the mpk1 mutant, but not in hog1 or snf1 mutant cells (Fig. 2A, and Supplementary Fig. 16B,C). Previous studies revealed that Mpk1 phosphorylates and activates Rlm1, a transcription factor belonging to the MADS (MCM1, Agamous, Deficiens, and SRF) family²⁶. Furthermore, it has been reported that Rlm1 acts downstream of Mpk1 in ER stress response⁷. We therefore examined whether Rlm1 is involved in PTP2 upregulation during ER stress response. Similar to mpk1 mutation, rlm1 mutation significantly reduced the mRNA levels of PTP2 (Fig. 2A and Supplementary Fig. 16D). We next examined the protein levels of Ptp2 during ER stress response. We used the strain expressing the carboxyl-terminally Myc-tagged Ptp2 for western blot analysis. As shown in our previous report⁹, Ptp2 was increased following exposure to ER stressors (Fig. 2B,C). However, Ptp2 induction was diminished by mpk1 and rlm1 mutations (Fig. 2B,C, and Supplementary Fig. 16E). These results indicate that ER stress-activated Mpk1-Rlm1 pathway upregulates the expression level of the PTP2 gene.

Previous reports showed that PTP2 expression is induced by heat shock in a manner dependent on Mpk1 and Rlm1^27,28. Hence, it has been assumed that Rlm1 transcriptionally activates the PTP2 gene in response to heat shock; however, it remains unclear whether the PTP2 promoter is really activated. Therefore, we investigated whether ER stress increases PTP2 promoter activity. To test this, we constructed the P_PTP2-GFP reporter (Fig. 2D), consisting of the 5′ upstream region of the PTP2 gene to drive GFP expression, and monitored the mRNA levels of GFP by qRT-PCR (Fig. 2F). GFP expression from the P_PTP2-GFP reporter was increased after treatment with ER stressors (Fig. 2F and Supplementary Fig. 16F). However, GFP induction was impaired by rlm1 mutation (Fig. 2F). This suggests that the PTP2 promoter is activated by ER stress in a manner dependent on Rlm1. We further explored the mechanism by which Rlm1 controls PTP2 promoter activity. Previous analysis revealed that Rlm1 binds the consensus sequence, CTA(T/A)(T/A)(T/A)(T/A)TAG²⁹. Our sequence analysis utilizing JASPAR, a database of transcription factor binding profiles (http://jaspar.genereg.net/), showed that two putative binding motifs for Rlm1 exist in the 5′ upstream region of the PTP2 gene (Fig. 2E). We designated them as the Rlm1-binding motifs (RBMs). To examine whether the RBMs are really important for PTP2 induction during ER stress response, we deleted each RBM or both in the P_PTP2-GFP reporter. Deletion of RBM1, the sequence spanning from −282 to −273, clearly suppressed GFP induction (Fig. 2G). On the other hand, deletion of RBM2, the sequence spanning from −42 to −33, alone failed to change GFP expression pattern, but inhibited GFP upregulation in combination to RBM1 deletion (Fig. 2G and Supplementary Fig. 16F). This result supports the model whereby Rlm1 transcriptionally activates the PTP2 gene during ER stress response.

Ptp2 induction is required for effective downregulation of Hog1 activity in ER stress response

To investigate whether ER stress-induced upregulation of Ptp2 is critical for its role in ER stress response, we examined ER stress sensitivity of cells in which PTP2 induction is impaired. The recent study revealed that Mpk1 is involved in the ER stress surveillance (ERSU) pathway, which prevents transmission of stressed ER into daughter cells, and the defect in ERSU causes hypersensitivity to ER stress⁷. Rlm1 has been reported to act downstream of Mpk1 in ER stress response^4,7. Indeed, hypersensitivity to ER stress was observed in the rlm1 mutant⁴ (Supplementary Fig. 16G). Thus, we considered that the mpk1 and rlm1 mutants are not suitable to be used as cells with diminished PTP2 induction. Therefore, we generated a series of P_PTP2-PTP2 constructs, which express PTP2 under the control of wild-type or mutated PTP2 promoter, and compared their ability to complement the ER stress resistant phenotype caused by ptp2 mutation (Fig. 3A). The ptp2 mutant was resistant to ER stress. When harboring wild-type P_PTP2-PTP2 integration, ER stress sensitivity of the ptp2 mutants was comparable to wild-type cells. Deletion of each RBM had little effect on the ability of P_PTP2-PTP2 integration to complement the ptp2 mutant phenotype. However, the P_PTP2-PTP2 integration in which both RBMs were deleted only partly complement ER stress resistance caused by ptp2 mutation. These results suggest that ER stress-induced Ptp2 upregulation is important for its role in ER stress response.

Ptp2 is known to dephosphorylate and inactivate Hog1 MAPK^20,21. A previous report showed that loss of Ptp2 causes enhanced ER stress resistance in a HOG1-dependent manner⁶. This phenomenon was seen in our strain background (data not shown). On the other hand, it has also been proposed that Ptp2 functions in dephosphorylation and inactivation of Mpk1^27,28. Therefore, we examined the effects of ptp2 mutation on the activities of Hog1 and Mpk1 during ER stress response. It is well-known that antibodies against the phosphorylated form of mammalian p38 and p42/p44 MAPKs can be utilized to detect the phosphorylated form of the budding yeast Hog1 and Mpk1, respectively. We therefore performed western blot analysis using anti-phospho-p38 antibodies to monitor Hog1 activity. As observed previously^5,9, tunicamycin treatment increased Hog1 activity in wild-type cells (Fig. 3B). In the ptp2 mutant cells, tunicamycin-induced Hog1 activation was significantly enhanced (Fig. 3B), indicating that Ptp2 dephosphorylates and inactivates Hog1 in ER stress response. We next determined Mpk1 activity by western blot analysis using anti-phospho-p42/p44 antibodies. Consistent with previous observations^4,7, Mpk1 became activated by tunicamycin exposure (Fig. 3B). The activated Mpk1 level in the ptp2 mutant was comparable to that in wild-type cells (Fig. 3B). Western blot analysis also showed that the amount of Mpk1 was increased after ER stress treatment (Fig. 3B). This increase has been reported to result from Rlm1-mediated transcriptional activation of the MPK1 gene⁷. Similar to wild-type cells, Mpk1 upregulation was observed in the ptp2 mutants, suggesting that Ptp2 is not involved in regulation of the Mpk1-Rlm1 pathway in ER stress response. Thus, Ptp2 acts as a negative regulator of Hog1 in ER stress response.

We next examined how Rlm1-mediated transcriptional activation of the PTP2 gene influences Hog1 activity in ER stress response. Hog1 activation upon tunicamycin exposure was upregulated in the rlm1 mutant (Fig. 3C). This result suggests that Rlm1 is involved in downregulation of Hog1 activity during ER stress response. To test whether that ER stress-induced activation of the PTP2 promoter is important for Hog1 downregulation, we compared the Hog1 activity in the ptp2 mutant cells harboring P_PTP2-PTP2 integrations (Fig. 3D). When carrying wild-type P_PTP2-PTP2 integration, the ptp2 mutant displayed Hog1 activity at comparable levels to wild-type cells. However, Hog1 activation was upregulated by deleting both RBMs from the P_PTP2-PTP2 integration. Taken together, these results indicate that Rlm1-mediated transcriptional activation of the PTP2 gene is critically involved in Hog1 downregulation during ER stress response.

The CMP2 promoter is important for ER stress response, but not for salt stress response

The CMP2 gene encodes a catalytic subunit of calcineurin phosphatase^18,19. Calcineurin is a heterodimer composed of the catalytic and regulatory subunits³⁰. The budding yeast contains another gene encoding a catalytic subunit of calcineurin, which is termed CNA1^18,19. The calcineurin regulatory subunit is encoded by the CNB1 gene³¹. As shown above, cells lacking Cmp2 exhibited hypersensitivity to ER stress (Fig. 1D). To investigate whether Cna1 is also involved in ER stress response, we examined tunicamycin sensitivity of the cna1 mutant cells, together with the cnb1 mutant cells (Fig. 4A). The cna1 mutation alone did not lead to ER stress hypersensitivity, but significantly enhanced ER stress sensitive phenotype caused by cmp2 deletion. ER stress hypersensitivity of the cmp2 cna1 double mutant cells was indistinguishable from that of the cnb1 mutant cells. These results indicate that Cmp2 and Cna1 act as the major and minor catalytic subunit of calcineurin in ER stress response, respectively.

We next asked why CMP2 is more important in ER stress response than CNA1. We compared the expression levels of Cmp2 and Cna1 using yeast strains carrying the carboxyl-terminally Myc-tagged genes. Consistent with an increase in CMP2 mRNA levels after ER stress treatment, Cmp2 expression was upregulated by ER stress (Fig. 4B). In contrast, Cna1 expression remained relatively unchanged following exposure to ER stress (Fig. 4B). Under ER stress conditions, Cmp2 was expressed at significantly higher levels than Cna1 (Fig. 4B). Next, we examined whether the difference in the protein levels between Cmp2 and Cna1 is due to the difference in the promoter activity between the CMP2 and CNA1 genes. To address this, we generated two reporter constructs, P_CMP2-GFP and P_CNA1-GFP, which express GFP under the control of the CMP2 and CNA1 promoters, respectively (Fig. 4C). GFP expression from the P_CMP2-GFP reporter, but not the P_CNA1-GFP reporter, was increased after ER stress treatment (Fig. 4D). The CMP2 promoter was more active than the CNA1 promoter under ER stress conditions (Fig. 4D). Thus, the difference in the promoter activity between CMP2 and CNA1 is reflected in their protein levels.

It may be possible that higher functional significance of Cmp2 attributes to higher activity of the CMP2 promoter. To explore this possibility, we generated two integrations: one expresses CMP2 from its own promoter; the other expresses CMP2 from the CNA1 promoter. We compared their ability to rescue ER stress hypersensitivity associated with cmp2 deletion (Fig. 4E). The P_CMP2-CMP2 integration could fully rescue ER stress sensitive phenotype of the cmp2 mutant. However, the P_CNA1-CMP2 integration failed to restore ER stress hypersensitivity caused by cmp2 mutation, suggesting that the CMP2 promoter is critical for the role of the CMP2 gene in ER stress response. We next examined whether CNA1 driven by the CMP2 promoter can compensate for loss of CMP2. However, the cmp2 mutant cells carrying the P_CMP2-CNA1 integration exhibited hypersensitivity to ER stress. This result suggests that not only the promoter but also the sequence including the coding and 3′ untranslated regions are important for the role of the CMP2 gene in ER stress response.

We next compared the functional significance of Cmp2 and Cna1 in other stress response except for ER stress. Previously, it has been reported that the yeast calcineurin functions in adaptation to high salt conditions^32,33. Consistent with the previous report³², hypersensitivity to sodium ions was observed in the cmp2 single mutant, but not in the cna1 single mutant (Fig. 4F). The cmp2 cna1 double mutant cells exhibited hypersensitivity to sodium ions indistinguishable from that of the cnb1 mutant cells (Fig. 4F). These results indicate that similar to ER stress response, Cmp2 is more important than Cna1 in salt stress response. However, unlike ER stress response, not only CNA1 but also CMP2 mRNA levels were largely unchanged in salt stress response (Fig. 4G,H). We next asked whether the CMP2 promoter is critical for the role of the CMP2 gene in salt stress response. The P_CMP2-CMP2 integration completely rescued salt stress sensitive phenotype seen in cmp2 mutant cells (Fig. 4I). We found that even when CMP2 was expressed from the CNA1 promoter, salt stress hypersensitivity associated with cmp2 mutation could be effectively restored (Fig. 4I). Thus, the CMP2 promoter is less important for salt stress response compared with ER stress response.

Discussion

Reversible protein phosphorylation exerted by the antagonistic activity of protein kinases and phosphatases is one of the most well-analyzed post-translational modifications. Numerous studies have demonstrated that proper modulation of phosphorylation is required for eukaryotic cells to adapt to the environmental stress. In controlling the function of protein phosphatases, regulation of their expression levels is frequently utilized. Under ER stress conditions, yeast cells facilitate the expression of a variety of genes functioning to restore ER homeostasis. However, it remains unclear whether the expression levels of protein phosphatases are regulated in yeast ER stress response.

In this study, we examined the mRNA expression patterns of the budding yeast protein phosphatases during ER stress response. Of 35 mRNAs encoding known or putative protein phosphatases, 8 mRNAs (CMP2, MIH1, PPZ2, PTC6, PTP1, PTP2, SAL6, and SDP1) were upregulated by both chemical and genetic ER stressors; 2 mRNAs (PTC5 and YCH1) were increased by chemical ER stressors, but not by the genetic ER stressor. To investigate the physiological role of ER stress-induced protein phosphatases in ER stress response, we examined ER stress sensitivity associated with each deletion mutant. Contrary to our expectation, only two mutations, ptp2 and cmp2, altered cellular sensitivity to ER stress. In the previous study³⁴, double mutants for 30 genes encoding non-essential yeast protein phosphatases were constructed in 435 combinations, and 4 double mutant cells displayed synthetic growth defects under specific conditions. In 10 protein phosphatases that were identified here as those induced by chemical ER stressors, for instance, two mitochondrial protein phosphatases, Ptc5 and Ptc6, are included^35,36,37. Additionally, the budding yeast possesses Ptc7, the third protein phosphatase residing within mitochondria^35,36. Therefore, failure of single mutation to alter ER stress sensitivity may be due to the functional redundancy between more than two protein phosphatases.

Here, we revealed that the Mpk1-Rlm1 axis promotes PTP2 expression in ER stress response. Previous reports showed that PTP2 expression is induced by environmental stresses, such as heat shock and osmotic stresses^21,27,28,38. Upon heat shock, PTP2 is upregulated in a manner dependent on Mpk1 and Rlm1^27,28. Thus, it is likely that a similar mechanism operates in regulation of PTP2 expression in various stress responses. However, the roles of PTP2 in heat shock and ER stress responses seem to differ from each other: Ptp2 acts to downregulate Mpk1 in heat shock response, whereas Ptp2 functions in Hog1 inactivation during ER stress response^6,27,28. To date, the physiological necessity of PTP2 upregulation in heat shock response remains to be elucidated. We showed here that the ER stress resistant phenotype of the ptp2 mutants was only partially rescued by PTP2 expression from mutated PTP2 promoter in which two putative Rlm1-binding motifs were deleted. This result indicates that PTP2 induction is required for its full function in ER stress response. Taken together, our results clearly demonstrated that the mechanism by which Hog1 is downregulated by the Mpk1-Rlm1 axis through Ptp2 induction exists in the budding yeast ER stress response (Supplementary Fig. 16H). Previous studies have revealed that constitutive activation of Hog1 leads to cell lethality^39,40. However, under our laboratory conditions used in this study, Hog1 upregulation caused by ptp2 mutation advantageously functions in adaptation to ER stress, since the ptp2 mutant cells were resistant to ER stress. Furthermore, PTP2 overexpression failed to suppress ER stress hypersensitivity caused by rlm1 mutation, but rather enhanced it (Supplementary Fig. 16G). Nevertheless, why do yeast cells possess the mechanism by which Hog1 is negatively regulated through Ptp2 induction in response to ER stress? Previous studies suggested that ER stress sensitivity of the budding yeast is altered by the extracellular environments, including the composition of the culture medium^8,41. Furthermore, it is well-known that the activity of Hog1 and Mpk1 are dynamically modulated by various environmental conditions^42,43. Accordingly, the mechanism of Hog1 downregulation whereby the Mpk1-Rlm1 axis activates PTP2 expression may be required for yeast cells under certain conditions (for example, the combined stress conditions) to facilitate adaptation to ER stress.

Previously, it has been shown that the budding yeast calcineurin phosphatase confers resistance to ER stress^3,11. However, it remained unclear how two alternative calcineurin catalytic subunits, Cna1 and Cmp2, operate in ER stress response, because the previous results were mainly based on the experiment using an inhibitor of calcineurin. Here, we found that Cmp2 makes a greater contribution to ER stress response than Cna1. Similar relative contribution was seen in salt stress response³² (Fig. 4F). Furthermore, it has been reported that Cmp2 is more important for recovery from the mating factor-induced growth arrest than Cna1⁴⁴. Thus, Cmp2 and Cna1 act as the major and minor catalytic subunits in distinct biological processes, respectively. However, the determinant of the relative functional significance appears to be different between, at least, salt and ER stress responses. Our reporter analysis revealed that the CNA1 promoter is less active than the CMP2 promoter. Nevertheless, Cmp2 expression from the CNA1 promoter could almost completely rescue the salt sensitive phenotype associated with cmp2 mutation. This result suggests that higher promoter activity is not required for the CMP2 gene to confer salt tolerance. In contrast, the promoter activity is likely to be a key determinant of the functional significance in ER stress response, since replacement of the CMP2 promoter with the CNA1 promoter considerably reduced the function of CMP2 in ER stress response. Additionally, the CMP2 gene may possess another element that is functionally important for ER stress response. This idea is derived from our observation that Cna1 expression from the CMP2 promoter hardly rescued cmp2 hypersensitivity to ER stress. Our results presented here suggest that the adaptive response to ER stress requires a higher amount of Cmp2 than salt stress. One plausible explanation for this phenomenon is as follows: the Cmp2 targets are different between ER and salt stress responses; more abundant Cmp2 is required for interacting physically with its target in ER stress response. In the budding yeast, a major target of the calcineurin is Crz1 transcription factor^45,46. Crz1 acts downstream of the calcineurin in salt stress response, whereas Crz1 is unlikely to transduce a signal from the calcineurin in ER stress response^41,45,46,47. The previous study suggests that under ER-stressed conditions, the calcineurin dephosphorylates Cch1, a subunit of the high affinity calcium channel³. However, it remains obscure whether Cch1 dephosphorylation is related to the physiological role of the calcineurin in ER stress response. Thus, identification of the calcineurin target in ER stress response should be needed to elucidate why higher promoter activity is required for the function of CMP2 in ER stress response. Additionally, since CMP2 induction occurred in the hac1, rlm1, hog1 and snf1 mutant cells (Mizuno et al., unpublished data), identification of the components involved in CMP2 expression should be needed for the understanding of the regulatory mechanisms of the calcineurin in ER stress response.

Materials and Methods

Plasmids

The PTP2, CMP2 and CNA1 genes were amplified from the Saccharomyces cerevisiae W303 derivative⁴⁸ by PCR with the following primers: 5′-CTCTAGAGGATCCCCGGGGGACACTCGTTTAATTTAGC-3′ and 5′-TCGAGCTCGGTACCCGGGTATGGGTACTGACATCTCTG-3′ for PTP2; 5′-CTCTAGAGGATCCCCGGGCGTCCCAAAAAGGAAATAGC-3′ and 5′-TCGAGCTCGGTACCCGGGTCTCTGAGTCAGACAGTGTC-3′ for CMP2; 5′-CTCTAGAGGATCCCCGGGGATTTTGAAGATACTAGTGC-3′ and 5′- TCGAGCTCGGTACCCGGGTTGGTCGCACAAGGTGTCTC-3′ for CNA1. The amplified PTP2, CMP2 and CNA1 DNA fragments were inserted into the YCplac33 vector⁴⁹ by In-Fusion cloning kits (Takara), yielding the YCplac33-P_PTP2-PTP2, YCplac33-P_CMP2-CMP2 and YCplac33-P_CNA1-CNA1 plasmids, respectively. To make the P_PTP2-GFP, P_CMP2-GFP and P_CNA1-GFP constructs, 1000-bp, 1000-bp and 997-bp genomic fragments containing the 5′ upstream sequences of the PTP2, CMP2 and CNA1 genes, respectively, were amplified by PCR with the following primers: 5′-CTCTAGAGGATCCCCGGGGGACACTCGTTTAATTTAGC-3′ and 5′-TAACCCGGGGATCCGATCCATCAATAGCAACGTCGATC-3′ for P_PTP2-GFP; 5′-CTCTAGAGGATCCCCGGGCGTCCCAAAAAGGAAATAGC-3′ and 5′-TAACCCGGGGATCCGAGACATTGCGGGTTCAAGAAG-3′ for P_CMP2-GFP; 5′-CTCTAGAGGATCCCCGGGGATTTTGAAGATACTAGTGC-3′ and 5′-TAACCCGGGGATCCGCGACATTGGCGTTGAGAGTG-3′ for P_CNA1-GFP. The DNA fragment encoding GFP followed by the ADH1 terminator (T_ADH1) was amplified from the pFA6a-GFP-HIS3MX6 vector by PCR with the following primers: 5′-CGGATCCCCGGGTTAATTAAC-3′ and 5′-TCGAGCTCGGTACCCGGGAGATCTATATTACCCTGTTATCC-3′. The amplified 5′ upstream sequences of the PTP2, CMP2 and CNA1 genes, together with the GFP-T_ADH1 DNA fragment, were fused to the YCplac33 vector by In-Fusion cloning kits (Takara), yielding the YCplac33-P_PTP2-GFP, YCplac33-P_CMP2-GFP and YCplac33-P_CNA1-GFP plasmids, respectively. Deletions of RBMs in the PTP2 promoter were generated by oligonucleotide-directed PCR using the YCplac33-P_PTP2-GFP plasmid as a template. The primers used to delete RBMs are: 5′-CTACACATAAAGTTCCATAAAGCAG-3′ and 5′-GAACTTTATGTGTAGTACACCTAAC-3′ for RBM1 deletion; 5′-TGTAAACACTGGGGATCGGACCTAG-3′ and 5′-TCCCCAGTGTTTACAATAAAATAGG-3′ for RBM2 deletion. The P_ACT1-PTP2 construct was generated as follows. A 728-bp genomic fragment containing the ACT1 promoter was amplified from the Saccharomyces cerevisiae W303 derivative by PCR with the following primers: 5′- CTCTAGAGGATCCCCGGGAAGGGAACGTCAACCTGAAG-3′ and 5′-TGCTATGCGATCCATTGTTAATTCAGTAAATTTTCG-3′. The coding region of the PTP2 gene together with the 3′ downstream sequence was amplified by PCR with the following primers: 5′- ATGGATCGCATAGCACAGCAATATCG-3′ and 5′-TCGAGCTCGGTACCCGGGTATGGGTACTGACATCTCTG-3′. The amplified P_ACT1 and PTP2 DNA fragments were fused with the YCplac33 vector by In-Fusion cloning kits (Takara), yielding the YCplac33-P_ACT1-PTP2 plasmid. The P_CNA1-CMP2 construct was generated as follows. The CNA1 promoter was amplified by PCR with the following primers: 5′-CTCTAGAGGATCCCCGGGGATTTTGAAGATACTAGTGC-3′ and 5′-AGCGTCTGAAGACATTGGCGTTGAGAGTGTTTTATGG-3′. The coding region of the CMP2 gene together with the 3′ downstream sequence was amplified by PCR with the following primers: 5′-ATGTCTTCAGACGCTATAAGAAATAC-3′ and 5′-TCGAGCTCGGTACCCGGGTCTCTGAGTCAGACAGTGTC-3′. The amplified P_CNA1 and CMP2 DNA fragments were fused with the YCplac33 vector by In-Fusion cloning kits (Takara), yielding the YCplac33-P_CNA1-CMP2 plasmid. Similarly, the YCplac33-P_CMP2-CNA1 plasmid was constructed. The primers used to amplify P_CMP2 and CNA1 are: 5′-CTCTAGAGGATCCCCGGGCGTCCCAAAAAGGAAATAGC-3′ and 5′-CAAGTCTTTCGACATTGCGGGTTCAAGAAGAAG-3′ for P_CMP2; 5′-ATGTCGAAAGACTTGAATTCTTCACG-3′ and 5′-TCGAGCTCGGTACCCGGGTTGGTCGCACAAGGTGTCTC-3′ for CNA1. To generate the integrations, the inserts in the YCplac33 plasmids were subcloned into the pRS306 vector⁵⁰. Plasmids used in this study are described in Supplementary Table 1.

Strains

Standard procedures were followed for yeast manipulations⁵¹. Yeast strains harboring the complete gene deletions (MIH1, SDP1, YCH1, PTC5, PTC6, CMP2, PPZ2, SAL6, PTP1, MPK1, RLM1, CNA1 and CNB1) and carboxyl-terminally Myc-tagged genes (CMP2 and CNA1) were generated by a PCR-based method as described previously^52,53. Primer sets were designed such that 46 bases at the 5′ end of primers were complementary to those at the corresponding region of the target gene, and 20 bases at their 3′ end were complementary to the pFA6a sequence, 5′-TGCAGTACTCTGCGGGTGTATACAG-3′ or 5′- ATTTGACTGTATTACCAATGTCAGC-3′. All strains produced by a PCR-based method were verified by colony PCR amplification to confirm that replacement had occurred at the expected locus. Strains carrying the integrations, P_PTP2-GFP, P_CMP2-GFP, P_CNA1-GFP, P_PTP2-PTP2, P_ACT1-PTP2, P_CMP2-CMP2, P_CNA1-CNA1, P_CNA1-CMP2, and P_CMP2-CNA1, were constructed by integrating the linearized plasmids, pRS306-P_PTP2-GFP, pRS306-P_CMP2-GFP, pRS306-P_CNA1-GFP, pRS306-P_PTP2-PTP2, pRS306-P_ACT1-PTP2, pRS306-P_CMP2-CMP2, pRS306-P_CNA1-CNA1, pRS306-P_CNA1-CMP2, and pRS306-P_CMP2-CNA1, respectively. The sec12-4, sec13-1 and sec16-2 strains of the YPH499 derivative were kind gifts from Dr. Akihiko Nakano (Riken). Strains used in this study are listed in Supplementary Table 2.

RNA isolation and RT–PCR

Preparation of total RNA and generation of cDNA were performed as described previously⁹. The cDNAs were quantitated by a quantitative real-time RT-PCR (qRT-PCR) method using a 7500 fast real-time RT-PCR system (Applied Biosystems) with SYBR Premix Ex Taq (Takara), and levels of gene expression were normalized to ACT1 expression. Primers used to analyze the mRNA level are described in Supplementary Table 3.

Protein extraction, western blot analysis and antibodies

Preparation of protein extracts and Western blot analysis were performed as described previously⁹. Anti-phospho-p38 MAPK monoclonal antibody D3F9 (Cell Signaling), anti-Hog1 polyclonal antibody y-215 (Santa Cruz), anti-phospho-p44/42 MAPK polyclonal antibody (Cell Signaling), anti-Mpk1 polyclonal antibody yN-19 (Santa Cruz), anti-Myc monoclonal antibody 9E10 (Santa Cruz) and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Western (Merck Millipore) or the Odyssey Imaging Systems (LI-COR Biosciences). Signal intensities were quantified by the Odyssey Imaging Systems, and statistical analysis was performed with Excel (Microsoft).

Stress sensitivity

Assays for tunicamycin and sodium chloride toxicity were carried out as follows. Cells were grown to exponential phase, and cultures were adjusted to an optical density of 0.5. Cell cultures were then serially diluted 5-fold, spotted onto normal plates or plates containing the indicated concentrations of tunicamycin, followed by incubation at 25 °C for 3 days (for plates lacking stressors) or more than 5 days (for plates containing stressors).

References

Walter, P. & Ron, D. The unfolded protein response: from stress pathway to homeostatic regulation. Science. 334, 1081–1086 (2011).
Article ADS PubMed CAS Google Scholar
Mori, K. Signalling pathways in the unfolded protein response: development from yeast to mammals. J. Biochem. 146, 743–750 (2009).
Article PubMed CAS Google Scholar
Bonilla, M. & Cunningham, K. W. Mitogen-activated protein kinase stimulation of Ca(2+) signaling is required for survival of endoplasmic reticulum stress in yeast. Mol. Biol. Cell 14, 4296–4305 (2003).
Article PubMed PubMed Central CAS Google Scholar
Chen, Y. et al. Identification of mitogen-activated protein kinase signaling pathways that confer resistance to endoplasmic reticulum stress in Saccharomyces cerevisiae. Mol. Cancer Res. 3, 669–677 (2005).
Article PubMed CAS Google Scholar
Bicknell, A. A., Tourtellotte, J. & Niwa, M. Late phase of the endoplasmic reticulum stress response pathway is regulated by Hog1 MAP kinase. J. Biol. Chem. 285, 17545–17555 (2010).
Article PubMed PubMed Central CAS Google Scholar
Torres-Quiroz, F., García-Marqués, S., Coria, R., Randez-Gil, F. & Prieto, J. A. The activity of yeast Hog1 MAPK is required during endoplasmic reticulum stress induced by tunicamycin exposure. J. Biol. Chem. 285, 20088–20096 (2010).
Article PubMed PubMed Central CAS Google Scholar
Babour, A., Bicknell, A. A., Tourtellotte, J. & Niwa, M. A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance. Cell. 142, 256–269 (2010).
Article PubMed PubMed Central CAS Google Scholar
Ferrer-Dalmau, J., Randez-Gil, F., Marquina, M., Prieto, J. A. & Casamayor, A. Protein kinase Snf1 is involved in the proper regulation of the unfolded protein response in Saccharomyces cerevisiae. Biochem. J. 468, 33–47 (2015).
Article PubMed CAS Google Scholar
Mizuno, T., Masuda, Y. & Irie, K. The Saccharomyces cerevisiae AMPK, Snf1, negatively regulates the Hog1 MAPK Pathway in ER stress response. PLoS Genet. 11, e1005491 (2015).
Article PubMed PubMed Central CAS Google Scholar
Kimura, Y., Irie, K. & Mizuno, T. Expression control of the AMPK regulatory subunit and its functional significance in yeast ER stress response. Sci Rep. 7, 46713 (2017).
Article ADS PubMed PubMed Central Google Scholar
Bonilla, M., Nastase, K. K. & Cunningham, K. W. Essential role of calcineurin in response to endoplasmic reticulum stress. EMBO J. 21, 2343–2353 (2002).
Article PubMed PubMed Central CAS Google Scholar
Fiscella, M. et al. Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. Proc. Natl. Acad. Sci. USA 94, 6048–6053 (1997).
Article ADS PubMed CAS Google Scholar
Caunt, C. J. & Keyse, S. M. Dual-specificity MAP kinase phosphatases (MKPs): shaping the outcome of MAP kinase signalling. FEBS J. 280, 489–504 (2013).
Article PubMed PubMed Central CAS Google Scholar
Sakumoto, N. et al. A series of protein phosphatase gene disruptants in Saccharomyces cerevisiae. Yeast 15, 1669–1679 (1999).
Article PubMed CAS Google Scholar
Breitkreutz, A. et al. A global protein kinase and phosphatase interaction network in yeast. Science. 328, 1043–1046 (2010).
Article ADS PubMed PubMed Central CAS Google Scholar
Travers, K. J. et al. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell. 101, 249–258 (2000).
Article PubMed CAS Google Scholar
Kimata, Y., Ishiwata-Kimata, Y., Yamada, S. & Kohno, K. Yeast unfolded protein response pathway regulates expression of genes for anti-oxidative stress and for cell surface proteins. Genes Cells. 11, 59–69 (2006).
Article PubMed CAS Google Scholar
Liu, Y. et al. The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B. Mol. Gen. Genet. 227, 52–59 (1991).
Article PubMed CAS Google Scholar
Cyert, M. S., Kunisawa, R., Kaim, D. & Thorner, J. Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase. Proc. Natl. Acad. Sci. USA 88, 7376–7380 (1991).
Article ADS PubMed CAS Google Scholar
Wurgler-Murphy, S. M., Maeda, T., Witten, E. A. & Saito, H. Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases. Mol. Cell. Biol. 17, 1289–1297 (1997).
Article PubMed PubMed Central CAS Google Scholar
Jacoby, T. et al. Two protein-tyrosine phosphatases inactivate the osmotic stress response pathway in yeast by targeting the mitogen-activated protein kinase, Hog1. J. Biol. Chem. 272, 17749–17755 (1997).
Article PubMed CAS Google Scholar
Higashio, H., Kimata, Y., Kiriyama, T., Hirata, A. & Kohno, K. Sfb2p, a yeast protein related to Sec24p, can function as a constituent of COPII coats required for vesicle budding from the endoplasmic reticulum. J. Biol. Chem. 275, 17900–17908 (2000).
Article PubMed CAS Google Scholar
Sato, M., Sato, K. & Nakano, A. Evidence for the intimate relationship between vesicle budding from the ER and the unfolded protein response. Biochem. Biophys. Res. Commun. 296, 560–567 (2002).
Article PubMed CAS Google Scholar
Chang, H. J., Jesch, S. A., Gaspar, M. L. & Henry, S. A. Role of the unfolded protein response pathway in secretory stress and regulation of INO1 expression in Saccharomyces cerevisiae. Genetics. 168, 1899–1913 (2004).
Article PubMed PubMed Central CAS Google Scholar
Nakano, A., Brada, D. & Schekman, R. A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J. Cell Biol. 107, 851–863 (1988).
Article PubMed CAS Google Scholar
Watanabe, Y., Irie, K. & Matsumoto, K. Yeast RLM1 encodes a serum response factor-like protein that may function downstream of the Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol. Cell. Biol. 15, 5740–5749 (1995).
Article PubMed PubMed Central CAS Google Scholar
Mattison, C. P., Spencer, S. S., Kresge, K. A., Lee, J. & Ota, I. M. Differential regulation of the cell wall integrity mitogen-activated protein kinase pathway in budding yeast by the protein tyrosine phosphatases Ptp2 and Ptp3. Mol. Cell. Biol. 19, 7651–7660 (1999).
Article PubMed PubMed Central CAS Google Scholar
Hahn, J. S. & Thiele, D. J. Regulation of the Saccharomyces cerevisiae Slt2 kinase pathway by the stress-inducible Sdp1 dual specificity phosphatase. J Biol. Chem. 277, 21278–21284 (2002).
Article PubMed CAS Google Scholar
Dodou, E. & Treisman, R. The Saccharomyces cerevisiae MADS-box transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein kinase pathway. Mol. Cell. Biol. 17, 1848–1859 (1997).
Article PubMed PubMed Central CAS Google Scholar
Cohen, P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 58, 453–508 (1989).
Article PubMed CAS Google Scholar
Cyert, M. S. & Thorner, J. Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone. Mol. Cell. Biol. 12, 3460–3469 (1992).
Article PubMed PubMed Central CAS Google Scholar
Nakamura, T. et al. Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. EMBO J. 12, 4063–4071 (1993).
Article PubMed PubMed Central CAS Google Scholar
Mendoza, I., Rubio, F., Rodriguez-Navarro, A. & Pardo, J. M. The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. J. Biol. Chem. 269, 8792–8796 (1994).
PubMed CAS Google Scholar
Sakumoto, N. et al. A series of double disruptants for protein phosphatase genes in Saccharomyces cerevisiae and their phenotypic analysis. Yeast. 19, 587–599 (2002).
Article PubMed CAS Google Scholar
Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature. 425, 686–691 (2003).
Article ADS PubMed CAS Google Scholar
Sickmann, A. et al. The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. USA 100, 13207–13212 (2003).
Article ADS PubMed CAS Google Scholar
Krause-Buchholz, U., Gey, U., Wünschmann, J., Becker, S. & Rödel, G. YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. FEBS Lett. 580, 2553–2560 (2006).
Article PubMed CAS Google Scholar
Chang, M. et al. Rck1 up-regulates Hog1 activity by down-regulating Slt2 activity in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 440, 119–124 (2013).
Article PubMed CAS Google Scholar
Maeda, T., Tsai, A. Y. & Saito, H. Mutations in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause a synthetic growth defect in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 5408–5417 (1993).
Article PubMed PubMed Central CAS Google Scholar
Maeda, T., Wurgler-Murphy, S. M. & Saito, H. A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature. 369, 242–245 (1994).
Article ADS PubMed CAS Google Scholar
Dudgeon, D. D., Zhang, N., Ositelu, O. O., Kim, H. & Cunningham, K. W. Nonapoptotic death of Saccharomyces cerevisiae cells that is stimulated by Hsp90 and inhibited by calcineurin and Cmk2 in response to endoplasmic reticulum stresses. Eukaryot. Cell. 7, 2037–2051 (2008).
Article PubMed PubMed Central CAS Google Scholar
Levin, D. E. Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway. Genetics. 189, 1145–1175 (2011).
Article PubMed PubMed Central CAS Google Scholar
Saito, H. & Posas, F. Response to hyperosmotic stress. Genetics. 192, 289–318 (2012).
Article PubMed PubMed Central CAS Google Scholar
Foor, F. et al. Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast. Nature. 360, 682–684 (1992).
Article ADS PubMed CAS Google Scholar
Stathopoulos, A. M. & Cyert, M. S. Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes Dev. 11, 3432–3444 (1997).
Article PubMed PubMed Central CAS Google Scholar
Matheos, D. P., Kingsbury, T. J., Ahsan, U. S. & Cunningham, K. W. Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. Genes Dev. 11, 3445–3458 (1997).
Article PubMed PubMed Central CAS Google Scholar
Ruiz, A., Yenush, L. & Ariño, J. Regulation of ENA1 Na(+)-ATPase gene expression by the Ppz1 protein phosphatase is mediated by the calcineurin pathway. Eukaryot. Cell. 2, 937–948 (2003).
Article PubMed PubMed Central CAS Google Scholar
Tadauchi, T., Matsumoto, K., Herskowitz, I. & Irie, K. Post-transcriptional regulation through the HO 3′-UTR by Mpt5, a yeast homolog of Pumilio and FBF. EMBO J. 20, 552–561 (2001).
Article PubMed PubMed Central CAS Google Scholar
Gietz, R. D. & Sugino, A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 74, 527–534 (1988).
Article PubMed CAS Google Scholar
Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for effcient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122, 19–27 (1989).
PubMed PubMed Central CAS Google Scholar
Kaiser, C. A., Adams, A. & Gottschling, D. E. Methods in yeast genetics. Cold Spring Harbor Laboratory Press. (1994).
Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 14, 953–961 (1998).
Article PubMed CAS Google Scholar
Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 21, 947–962 (2004).
Article PubMed CAS Google Scholar

Download references

Acknowledgements

We thank K. Kurokawa and A. Nakano for materials, and the members of our laboratory for their help. This research was supported by JSPS KAKENHI Grant Number JP16K07336 (to T.M.) and the Takeda Science Foundation (to T.M.).

Author information

Authors and Affiliations

Department of Molecular Cell Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
Tomoaki Mizuno, Meyu Nakamura & Kenji Irie

Authors

Tomoaki Mizuno
View author publications
You can also search for this author in PubMed Google Scholar
Meyu Nakamura
View author publications
You can also search for this author in PubMed Google Scholar
Kenji Irie
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

T.M. designed research strategies, performed experiments with M.N., analyzed data and wrote manuscript. K.I. provided solutions.

Corresponding author

Correspondence to Tomoaki Mizuno.

Ethics declarations

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplemental information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Mizuno, T., Nakamura, M. & Irie, K. Induction of Ptp2 and Cmp2 protein phosphatases is crucial for the adaptive response to ER stress in Saccharomyces cerevisiae. Sci Rep 8, 13078 (2018). https://doi.org/10.1038/s41598-018-31413-6

Download citation

Received: 11 June 2018
Accepted: 20 August 2018
Published: 30 August 2018
DOI: https://doi.org/10.1038/s41598-018-31413-6

This article is cited by

Not just the wall: the other ways to turn the yeast CWI pathway on
- Elena Jiménez-Gutiérrez
- Estíbaliz Alegría-Carrasco
- Humberto Martín
International Microbiology (2020)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.