Genetic dissection of the signaling pathway required for the cell wall integrity checkpoint

The cell wall is an indispensable structure for the proliferation of plant and fungal cells, and provides tensile flexibility and physical protection that define the morphology of the cell (Klis et al., 2002; Levin, 2011). During budding and cell cycle progression in the budding yeast Saccharomyces cerevisiae, the cell wall is synthesized and deposited to the growing bud in a cell cycle-dependent manner, and thus cell wall synthesis is tightly coordinated with the cell division cycle (Keaton and Lew, 2006; Negishi and Ohya, 2010). Unless timely remodeling of the cell wall materials and proper bud morphogenesis occur, the cell fails to prepare sufficient space for the daughter nucleus. The regulation of this coordination, named the cell wall integrity checkpoint, is considered a cell cycle checkpoint (Suzuki et al., 2004), involving fundamental processes that underlie the duplication and segregation of genetic material and cellular components.

Deficiency of 1,3-β-glucan, a major cell wall filament, during bud emergence and in the growing bud results in arrest of the budding cycle and the accumulation of unbudded and small-budded cells. This activates the cell wall integrity checkpoint and the cell cycle arrests with duplicated and unseparated spindle pole bodies (SPBs). Cell cycle arrest with unseparated SPBs is achieved by transcriptional downregulation of the mitotic cyclin CLB2 by phosphorylation and degradation of the S-phase transcription factor Hcm1, and transcriptional regulation of the transcription factors Fkh2 and Ndd1 (Negishi et al., 2016). The dynactin complex has also been shown to play an essential role in the cell wall integrity checkpoint (Suzuki et al., 2004). S. cerevisiae dynactin is composed of at least three proteins – Arp1, Nip100 and Jnm1 (Geiser et al., 1997; Kahana et al., 1998; McMillan and Tatchell, 1994) – which all play a role in the cell wall integrity checkpoint (Suzuki et al., 2004). It was previously shown that dynactin acts with dynein for nuclear migration (Hildebrandt and Hoyt, 2000; Kahana et al., 1998; Moore et al., 2008), but the components of dynein – including dynein heavy chain (Dyn1), dynein intermediate chain (Pac11), Num1, Pac1 and Bik1 – are not required for the cell wall integrity checkpoint (Suzuki et al., 2004). Mutational analysis of Arp1 clearly demonstrated a separation of the two functions of nuclear migration and the cell wall integrity checkpoint (Igarashi et al., 2005). These results suggested that, in addition to its role in nuclear migration, the dynactin complex also has specific functions in the cell wall integrity checkpoint, possibly through additional protein–protein interactions.

Visual screening of cells synchronized in the G₁ phase of the cell cycle has been used to isolate cell wall integrity checkpoint mutants. Checkpoint mutants in the temperature-sensitive 1,3-β-glucan synthase mutant background (fks1ts fks2Δ) primarily exhibited SPB separation and bipolar spindle formation at a restrictive temperature. An Arp1 point mutant (arp1-P268L), as well as deletion mutants of the components (Arp1, Jnm1 and Nip100) of the dynactin complex, induced a checkpoint defect (Suzuki et al., 2004), demonstrating that the dynactin complex is essential for the checkpoint. In contrast, gain-of-function HCM1 mutations and overexpression of FKH2 and CLB2 also resulted in checkpoint defects (Negishi et al., 2016; Suzuki et al., 2004), indicating that these genes negatively regulate the checkpoint. However, the checkpoint mutants have not been thoroughly isolated and a comprehensive understanding of the checkpoint pathway remains elusive.

To obtain a better understanding of the cell wall integrity checkpoint pathway, we further isolated and characterized checkpoint mutants. To isolate additional mutants, we have taken two approaches. One to test the possibility that interacting partners of the dynactin complex function in the cell wall integrity checkpoint, and the second to test whether the mitogen-activated protein kinase (MAPK) signaling pathways are involved in the checkpoint. By applying both biochemical and genetic analyses, we have elucidated the hierarchical relationship of signaling proteins essential for checkpoint function.

To evaluate checkpoint activity, we used a temperature-sensitive 1,3-β-glucan synthase mutant, fks1-1154 harboring an fks1-ts allele integrated into the fks1Δ fks2Δ background, because the FKS1 and FKS2 products have overlapping functions (Inoue et al., 1995; Mazur et al., 1995). Because a high temperature shift resulted in cell wall stress conditions (Martin et al., 1993), we compared the fks1-1154 strain with the corresponding wild-type strain (FKS1) after shifting to the restrictive temperature of 37°C. When the FKS1 strain was synchronized in G₁ by centrifugal elutriation followed by release at 37°C, cells with medium or large buds were increased (Fig. 1A) and bipolar spindle formation was observed (Fig. 1B,E). However, when the fks1-1154 strain was synchronized in G₁ by centrifugal elutriation followed by release at 37°C, unbudded or small-budded cells with unseparated SPBs accumulated, preventing the formation of cells with medium or large buds (Fig. 1A) and bipolar spindle formation (Fig. 1B,E) after 200 min or more. In contrast, the checkpoint mutation arp1-P268L resulted in a failure to accumulate unseparated SPBs, representing unbudded or small-budded cells (Fig. 1A) with elongated bipolar spindles (Fig. 1B,E), as described previously (Suzuki et al., 2004).

As indicated in the Saccharomyces Genome Database, two-hybrid, reconstituted complex, protein-fragment complementation assay and affinity capture mass spectrometry experiments have revealed a number of proteins that physically interact with components of the dynactin complex (Table S1). To identify other checkpoint mutants, we generated deletion mutants of the gene products that interacted with the dynactin complex shown in Table S1. We then evaluated bud growth and spindle elongation at the restrictive temperature in the fks1-1154 background. When BZZ1, a member of the Schizosaccharomyces pombe cdc15 homology (PCH) family, was deleted in the fks1-1154 background, the appearance of medium- or large-budded cells was inhibited, similarly to fks1-1154 (Fig. 1A), and cells formed bipolar spindles (Fig. 1B,E), similar to the other checkpoint mutants. Bzz1 was isolated as a protein that physically interacts with Las17, which associates with Vrp1, Myo3 and Myo5 to form the Las17 complex and regulates actin polymerization at the cell cortex (Lechler et al., 2000; Soulard et al., 2002). To determine whether Bzz1 plays a role in the checkpoint through the Las17 complex, we investigated whether Vrp1, a component of the Las17 complex, is involved in the checkpoint. Deletion of VRP1 resulted in a defective checkpoint phenotype (Fig. 1A,B,E), with cells accumulating with elongated bipolar spindles. We were unable to construct MYO3 or MYO5 deletions in the fks1-1154 and FKS1 background in our strain background (YPH499).

As for the other gene products that interacted with dynactin complex, we constructed deletion mutants of SRO77, RHO2 and END3 in the fks1-1154 background, and these showed failure to exhibit a strong checkpoint defect (Fig. 1A,B,E). We failed to construct SMI1 and GON7 deletions in the fks1-1154 background, and therefore used an alternative approach to test the involvement of Smi1 and Gon7 in the checkpoint. Following treatment of FKS1 strains with echinocandin B, an inhibitor of 1,3-β-glucan synthase, G₁-synchronized cells were collected and bud growth and spindle elongation were evaluated. Unlike the arp1-P268L mutant, the smi1 and gon7 deletion mutants did not accumulate unbudded or small-budded cells with elongated spindles (Fig. 1C,D), indicating that Smi1 and Gon7 are not required for the checkpoint.

Like other eukaryotic organisms, budding yeast has several MAPK pathways that transduce extracellular signals into distinct nuclear responses (Molina et al., 2010). Because the cell wall integrity checkpoint probably senses abnormalities in the cell wall, and transduces a signal into the nucleus, we tested the possibility that some of the MAPKs are involved in the cell wall integrity checkpoint. There are at least five major MAPK pathways in budding yeast, namely, the mating-pheromone response pathway, the filamentous growth pathway, the cell wall integrity (CWI) pathway, the sporulation/nutritional deprivation pathway and the HOG pathway (Hohmann, 2002).

We tested the involvement of these MAPK pathways in the cell wall integrity checkpoint. The significant checkpoint defect was not observed when disrupting the FUS3 and SMK1 MAPK genes of the mating-pheromone response pathway and the sporulation/nutritional deprivation pathway, respectively, in the fks1-1154 background (Fig. S1). The deletion mutant of the KSS1 MAPK of the filamentous growth pathway failed to accumulate unbudded or small-budded cells (Fig. S1), indicating that kss1Δ was not suited for the cell wall integrity checkpoint assay. These results suggested that Fus3 and Smk1 MAPK pathways were not major pathways controlling the checkpoint. However, we found that the HOG MAPK pathway was required for the checkpoint (Fig. 2B-E; Fig. S1). When HOG1, the MAPK gene of the HOG pathway, was deleted in the fks1-1154 background, the cells mostly accumulated as unbudded or small-budded cells (Fig. 2C,E; Fig. S1) with elongated bipolar spindles (Fig. 2B,D,E; Fig. S1) after 200 min or more, a typical checkpoint-defective phenotype.

There are two major branches upstream of Hog1 kinase, the SHO1 and SLN1 branches (Brewster et al., 1993; Maeda et al., 1994, 1995) (Fig. 2A). The components of the SHO1 branch are predominantly localized to the cytoplasmic membrane at regions of polarized growth, such as the emerging bud and the bud neck (Raitt et al., 2000; Reiser et al., 2000, 2003), and are absolutely required for the activation of Hog1 MAPK under hyperosmotic conditions (Maeda et al., 1994, 1995; O'Rourke and Herskowitz, 2002; Posas and Saito, 1997; Posas et al., 1998; Raitt et al., 2000; Reiser et al., 2000). In contrast, components of the SLN1 branch are uniformly distributed throughout the plasma membrane and are involved in phosphorylation and activation of Pbs2 MAPK kinase in response to lower osmotic stress via the activation of Ssk1 (Posas and Saito, 1998). When the genes encoding the components of the SHO1 branch – SHO1, STE50, STE11 and PBS2 – were deleted, the cell wall integrity checkpoint was defective, similar to the HOG1 deletion mutant (Fig. 2C,D). We further confirmed that simultaneous deletion of STE20 and CLA4, or MSB2 and HKR1, which are thought to have redundant functions (Cvrckova et al., 1995; Tatebayashi et al., 2006, 2007), also resulted in a defective checkpoint, because unbudded or small-budded cells with bipolar spindles accumulated 4 h after the release from G₁ under checkpoint conditions (Fig. 2E). On the other hand, the SLN1 branch mutations ssk1Δ and ssk2Δ ssk22Δ resulted in an accumulation of cells with unseparated SPBs (Fig. 2E), indicating that the components of the SLN1 branch were not required for the checkpoint. Finally, deletion of each downstream transcription factor of Hog1, smp1Δ, msn2Δ/4Δ, sko1Δ and hot1Δ, as well as deletion of all these transcription factors, resulted in an accumulation of cells with unseparated SPBs (Fig. 2E), suggesting the existence of another downstream target(s) of Hog1 in the cell wall integrity checkpoint.

To test whether the kinase activity of Hog1 MAPK is required for the cell wall integrity checkpoint, we examined the phosphorylation status of Hog1 when the checkpoint was induced. Hog1 MAPK is activated following its phosphorylation (Brewster et al., 1993). We found that Hog1 is weakly phosphorylated in fks1-1154 cells only in the early stages of the cell cycle under checkpoint-inducing conditions (Fig. 3A, 0 and 30 min after release). No phosphorylation of Hog1 was observed in wild-type FKS1 cells (Fig. 3A). To verify the importance of the Hog1 kinase activity suggested by this phosphorylation, we used the hog1-as mutant (analog sensitive; replacement of threonine 100 with an alanine) (Westfall and Thorner, 2006) to inhibit Hog1 kinase activity with the addition of the cell-permeable adenine analog compound, 1NM-PP1, to the culture medium. Cells with the hog1-as mutation accumulated as unbudded or small-budded cells with bipolar spindles only in the presence of 1NM-PP1 (Fig. 3B,C), indicating a checkpoint-defective phenotype. Kinase-dead mutants of Hog1, hog1-K52R and hog1-D144A (Ferrigno et al., 1998; Reiser et al., 1999; Westfall and Thorner, 2006) were also defective in checkpoint function (Fig. 3D,E), further indicating the requirement for Hog1 kinase activity in the checkpoint.

With the hog1-as mutant, we next investigated when Hog1 kinase activity was required for the checkpoint. Strains were grown in YPD medium (containing yeast extract, polypeptone and glucose), or YPD medium supplemented with 1NM-PP1, synchronized in G₁ by elutriation, and released in checkpoint-inducing conditions. 1NM-PP1 was added immediately following synchronization, and the formation of bipolar spindles was observed 240 min after the release (Fig. 3F). Our results revealed that the cell wall checkpoint functioned, provided Hog1 was phosphorylated at the initial stage (0–30 min) of the checkpoint mechanism (Fig. 3F). Hog1 kinase activity was no longer required for the checkpoint at 60 min or more following the release. When cells were pre-grown in YPD medium supplemented with 1NM-PP1, immediate removal of 1NM-PP1 following synchronization resulted in a checkpoint-arrest phenotype (Fig. 3F). Taking these findings together, we conclude that Hog1 kinase activity is required transiently for 30 min after the release from G₁.

We also investigated the involvement of the CWI MAPK pathway using several deletion mutants (Fig. 4A). When MAPKKK, MAPKK or MAPK of the CWI MAPK pathway – namely, BCK1, MKK1/2 or SLT2 – was deleted in the fks1-1154 strain, the cells exhibited a typical checkpoint-defective phenotype, primarily accumulating as unbudded or small-budded cells with elongated bipolar spindles (Fig. 4B; Fig. S1) after 4 h. The sensor proteins of the CWI MAPK pathway – Mid2, Wsc1, Wsc2, Wsc3 and Mtl1 – and the transcription factors Swi4/6 and Rlm1 are not directly involved in the checkpoint (Suzuki et al., 2004; Fig. 4B). Slt2 MAPK is activated by phosphorylation (Lee et al., 1993), and the requirement for Slt2 MAPK in the cell wall integrity checkpoint prompted us to investigate whether the phosphorylation of Slt2 was altered upon activation of the checkpoint. Phosphorylation of Slt2 in fks1-1154 cells was evident in asynchronous cultures, but was markedly higher than that in wild-type cells following release from G₁ (Fig. 4C), suggesting that Slt2 MAPK is also activated upon activation of the checkpoint.

We have demonstrated that several cellular components including the dynactin and Las17 complexes, the HOG MAPK and CWI MAPK pathways, and many transcriptional regulators (Hcm1, Fhk2 and Ndd1) are involved in the cell wall integrity checkpoint. To investigate the hierarchical relationship between these components, we constructed double-deletion mutants and examined their checkpoint phenotypes. It has previously been shown that deletion of the transcription factor of CLB2 (fkh2Δ) rescues the checkpoint defect caused by the absence of the dynactin complex (Suzuki et al., 2004). Similarly, although a checkpoint defect was observed in the absence of HOG1 or SLT2, the concomitant deletion of FKH2 rescued the checkpoint-defective phenotype (Fig. 5A,C). Concomitant deletion of FKH2 also rescued the checkpoint-defective phenotype of the bzz1Δ mutant (Fig. 5A,C), suggesting that Fkh2 is a negative factor of the checkpoint downstream of Hog1, Slt2 and Bzz1.

Next, we examined whether the checkpoint defect caused by deletion of the dynactin or Las17 complexes can be rescued by hyperactivation of the HOG and CWI MAPK pathways. We employed a constitutively active allele of Hog1 MAPK (HOG1-F318S) and Bck1 MAPKKK (BCK1-20) with an independent intrinsic catalytic activity (Bell et al., 2001; Lee and Levin, 1992). We found that the defective checkpoint caused by the absence of Arp1, a component of the dynactin complex, could not be rescued by hyperactivation of Hog1 (Fig. 5B,D). Likewise, the defective checkpoint caused by the absence of Bzz1, a component of the Las17 complex, could not be rescued by hyperactivation of Hog1 (Fig. 5B,D), suggesting that HOG MAPK functions upstream of the dynactin and Las17 complexes. The defective checkpoint caused by the deletion of BZZ1, however, was rescued by hyperactivation of CWI MAPKKK (BCK1-20) (Fig. 5B,D), suggesting that the CWI MAPK pathway acts downstream of the dynactin and Las17 complexes.

To confirm the order of signaling factors from a transcriptional perspective, we examined the expression of CLB2 in hog1Δ, hog1Δ fkh2Δ, slt2Δ and slt2Δ fkh2Δ. We found that CLB2 expression was upregulated in hog1Δ and slt2Δ cells and downregulated in hog1Δ fkh2Δ and slt2Δ fkh2Δ cells (Fig. 5E), implying that the defective cell wall integrity checkpoint is caused by a failure to regulate CLB2 expression. We also examined the phosphorylation of Slt2 in hog1Δ, bzz1Δ and arp1Δ cells, and found that Slt2 phosphorylation was decreased in cells of these mutants just after release from G₁ (Fig. 5F), further supporting the idea that the CWI MAPK pathway acts downstream of the HOG MAPK pathway, and the Las17 and dynactin complexes. We detected induction of Slt2 phosphorylation at 60 min after release from G₁ (Fig. 5F), when Hog1 kinase activity was no longer required.

It is widely believed that genetic interaction networks highlight mechanistic connections between genes and their corresponding pathways (Costanzo et al., 2016). To reveal functional connections between cell wall-constructing enzymes and the cell wall integrity checkpoint, we investigated a coherent set of gene interactions with a global interaction dataset (Costanzo et al., 2016). Genes involved in N-glycosylation (Jungmann and Munro, 1998; Jungmann et al., 1999), 1,3-β-glucan synthesis (Douglas et al., 1994; Inoue et al., 1995; Mouyna et al., 2000) and 1,6-β-glucan synthesis (Brown et al., 1993; Hill et al., 1992; Page et al., 2003) displayed many genetic interactions with genes of the HOG MAPK pathway, the dynactin and Las17 complexes, and the CWI MAPK pathway (Fig. 6A). Overall, more negative (73.92%) than positive (26.08%) interactions were observed (Fig. 6B). Thus, the genetic interaction network traced the cooperative functional connections whereby cell wall synthetic defects were monitored by the cell wall integrity checkpoint.

Overall, our results suggest that perturbation of cell wall synthesis transmits a signal through the SHO1 branch of the HOG MAPK pathway, which is then transduced through the dynactin and Las17 complexes, activating the CWI MAPK pathway, eventually inducing a decrease in the expression of the mitotic cyclin Clb2. It has previously been shown that activation of the CWI MAPK pathway results in phosphorylation and decreased levels of the S-phase transcription factor Hcm1, resulting in decreased levels of Clb2 and the mitotic transcription factor Fkh2 (Negishi et al., 2016). Our working model of the cell wall integrity checkpoint pathway is shown in Fig. 6C.

The dynactin complex, M-phase transcription factor Fkh2, M-phase cyclin Clb2 (Suzuki et al., 2004) and S-phase transcription factor Hcm1 are involved in the cell wall integrity checkpoint. Upon activation of this checkpoint, the Hcm1 protein level is downregulated to prevent the transcription of FKH2, which leads to the downregulation of CLB2 (Negishi et al., 2016). This study demonstrated that a number of other gene products – including components of the Las17 complex, Bzz1 and Vrp1, the HOG MAPK pathway, and the CWI MAPK pathway – function in the cell wall integrity checkpoint. Our analysis showed that this checkpoint utilized the connection of the well-known HOG and CWI MAPK signaling pathways, which are essential for responding to environmental stimuli. Epistatic analysis of checkpoint genes suggested their order of action, revealing the regulatory hierarchy in the checkpoint pathway. Tight cooperative interactions were observed between newly discovered cell wall integrity checkpoint genes and genes important in cell wall construction and assembly.

It will be challenging to determine the sensor protein that monitors the perturbation of cell wall synthesis. We found that components of the SHO1 branch of the HOG MAPK pathway are the most upstream factors in the cell wall integrity pathway. In response to conditions of high osmolarity, the yeast mucin proteins Hkr1 and Msb2 are thought to act as functionally redundant single-path membrane proteins that work upstream of the SHO1 branch (Cvrckova et al., 1995; Tatebayashi et al., 2006, 2007). Both proteins exhibit a highly O-glycosylated Ser/Thr-rich extracellular domain that is embedded in the cell wall (Tatebayashi et al., 2007). Recent analyses have revealed that the Opy2–Msb2 complex (Yamamoto et al., 2016) and a four-transmembrane-domain protein, Sho1 (Tatebayashi et al., 2015), act as an osmosensor upon hyperosmotic stress. Because the deletion of SHO1 and the simultaneous deletion of HKR1 and MSB2 both resulted in a defective checkpoint, our results suggest that Sho1, Hkr1 and Msb2 also act as sensors during the cell wall integrity checkpoint. Sensors in the SHO1 branch are predominantly localized to the plasma membrane at regions of polarized growth (Raitt et al., 2000; Reiser et al., 2003, 2000), and thereby might sense defective synthesis of cell wall materials, such as mannoproteins and β-glucan, in the bud.

We observed weak phosphorylation of HOG1 MAPK during checkpoint activation. Defects in cell wall synthesis induced phosphorylation of HOG1 MAPK that was significantly weaker than that under hyperosmotic conditions. However, this weak phosphorylation turned out to be important for activation of the checkpoint based on the analysis of kinase-dead mutants. Active Hog1 might function differently, owing to the weak and temporary phosphorylation observed during checkpoint activation compared with the conventional response of Hog1. Similar weak Hog1 phosphorylation was observed following treatment with the cell wall lytic enzyme zymolyase (Bermejo et al., 2008). These results suggested that not only the synthetic deficiency, but also the digestion of 1,3-β-glucan, induces a similar cell cycle response. Use of the analog-sensitive HOG1 mutant strongly suggested the importance of its weak phosphorylation early in the cell cycle. The phosphorylation of Hog1 early in the cell cycle, more precisely 0–30 min after the release from G₁, was important for the cell wall integrity checkpoint. In other words, activation of Hog1 was not required for the checkpoint after the first 30 min, probably due to irreversible activation of downstream signals. It should be noted that none of the Hog1 downstream transcriptional factors is involved in the checkpoint. Therefore, it is possible that weak Hog1 phosphorylation resulted in irreversible activation of different cellular signaling processes in the cytoplasm. Recently, cytosolic localization of Hog1 was also reported following heat stress (Shiraishi et al., 2017). Among the interacting partners of the dynactin complex, Bzz1, but none of the other interacting proteins tested, played a role in the cell wall integrity checkpoint. The Bzz1-interacting protein Vrp1, however, was involved in the checkpoint. Although both Bzz1 and Vrp1 interact with Las17 (Soulard et al., 2002), a factor that promotes the nucleation of actin by activating the Arp2/3 protein complex (Lechler et al., 2000; Tyler et al., 2016), it is not clear whether Arp2/3 and actin organization are required for the checkpoint. However, given that Bzz1 has many interacting partners, presumably through its SH3 domain (Soulard et al., 2002), it is possible that Bzz1 is involved in the scaffolding or localization of active Hog1 in the checkpoint.

Although analysis of double-mutant phenotypes suggested that the dynactin and Las17 complexes function downstream of the HOG MAPK pathway, there is no direct evidence of their direct and physical interaction. Future genetic and biochemical analyses will be required to address these issues.

In addition to the HOG MAPK pathway, the CWI MAPK pathway was also shown to be involved in the cell wall integrity checkpoint. Genetic analysis revealed that MAPKKK, MAPKK and MAPK of the CWI MAPK pathway are all required for the checkpoint. Epistatic analysis suggested that perturbation of the cell wall transduces a signal in the following order: the HOG MAPK pathway, the dynactin and Las17 complexes, and finally the CWI MAPK pathway. Phosphorylation of CWI MAPK was decreased in the absence of Hog1, Bzz1 and Arp1 just after release from G₁ synchronization, further supporting this order. It has been shown that the upstream sensors of the CWI pathway do not function in the checkpoint (Suzuki et al., 2004; Fig. 4B). Taking these findings together, the sequential connection of the HOG and CWI pathways is important for the checkpoint, representing another example of sequential regulation of MAP kinases in budding yeast (Leng and Song, 2015; Sanz et al., 2017).

To accomplish cell division properly under internal and external conditions of stress, eukaryotic cells use several conventional cell cycle checkpoint mechanisms, including the DNA damage checkpoint (Foiani et al., 2000), the DNA replication checkpoint (Longhese et al., 2003), the spindle assembly checkpoint (Musacchio and Salmon, 2007) and the cytokinesis checkpoint (McCollum and Gould, 2001). Accumulating evidence has indicated that other checkpoint systems exist in budding yeast to ensure that progression of the cell cycle is coordinated with duplication, inheritance and organelle integrity. Functional vacuoles are crucial for cell cycle progression in G₁ (Jin and Weisman, 2015). Cell cycle arrest in G₁ occurs following damage and repair of the plasma membrane (Kono et al., 2016). Duplication of extracellular materials is required for progression of the cell cycle in G₂, as shown in this study. It will be interesting to understand how these organelle checkpoint mechanisms communicate with one other and whether a similar checkpoint system is important for mammalian cell cycle progression to prevent the uncontrolled growth of tumors and cancer cells.

The strains and plasmids used in this study are listed in Tables S2 and S3, respectively. All strains are derivatives of YPH499 (Sikorski and Hieter, 1989).

Cells were grown in standard YPD medium [1% bacto yeast extract (BD Biosciences), 2% polypeptone (Wako Chemicals), 2% glucose (Wako Chemicals)], unless otherwise stated. To select transformants, SD medium [0.67% yeast nitrogen base without amino acids (BD Biosciences) and 2% glucose] supplemented with the amino acid selection marker, or YPD medium supplemented with 200 μg/ml geneticin (G418 sulfate; Sigma-Aldrich), 300 μg/ml hygromycin (Wako Chemicals) or 100 μg/ml clonNAT (Werner BioAgents), was used. Then, 1 μg/ml 5-fluoroorotic acid (5-FOA; Toronto Bioscience) was added to SD medium for counterselection of URA3 marker. Solid medium was prepared by adding 2% agar (Shoei, Tokyo, Japan). For sorbitol, echinocandin B (Ech B) and zymolyase treatments, 1 M sorbitol (Sigma-Aldrich), 8 μg/ml of Ech B, and 0.5 or 1.0 U/ml of zymolyase was added to the culture medium, respectively. For 1NM-PP1 treatment, 1NM-PP1 was dissolved in dimethyl sulfoxide (DMSO) (Wako Chemicals) and added to the medium at a final concentration of 12 μM; the concentration of DMSO in the medium did not exceed 0.1% (v/v).

Gene disruption of WSC1, SRO77, END3, BZZ1, RHO2, SMI1, VRP1, SHO1, STE50, STE11, PBS2, SSK1, SMP1, SKO1, HOT1, SLT2, BCK1, HOG1, RLM1, SWI6, KSS1 and FUS3 was conducted by polymerase chain reaction (PCR)-mediated gene disruption, as previously described (Ishihara et al., 2007; Sakumoto et al., 1999), to construct YOC2537, 3924, 3926, 3928, 3930, 4133, 4135, 4219, 4220, 4218, 4217, 4227, 4234, 4232, 4233, 4486, 4484, 4918, 4493, 4490, 4214 and 4215, respectively. Briefly, the Candida glabrata (Cg) LEU2 gene, which is compatible with S. cerevisiae LEU2, was amplified from the pBS-CgLEU2 plasmid with a pair of primers with flanking sequences derived from the upstream and downstream regions of the corresponding genes. The PCR products were transformed into FKS1 (YOC1001) or fks1-1154 (YOC1087) strains and the disruption and integration were confirmed by PCR. To supplement the growth of CWI pathway mutants during transformation, 1 M sorbitol (Sigma-Aldrich) was added to the selection medium. Deletions of GON7, HOG1 and SWI4 were performed as described previously (Ishihara et al., 2007; Sakumoto et al., 1999), but using the Candida glabrata (Cg) URA3 gene, which is compatible with the S. cerevisiae URA3, from the pBS-CgURA3 plasmid. The PCR product with flanking regions of GON7 was introduced into an FKS1/FKS1 (YOC3916) diploid strain, followed by standard sporulation and dissection to obtain FKS1 gon7Δ haploid strains, creating YOC4134. The PCR product with flanking regions of HOG1 and SWI4 was introduced into the FKS1 (YOC1001) or fks1-1154 (YOC1087) strains, creating YOC4478, YOC4479 and YOC4489. Gene disruption of FKH2 was conducted similarly, except that PCR amplification was performed using fkh2::KanMX of the BY-series Euroscarf collection (Giaever et al., 2002) as a template. The amplified cassette of fkh2::KanMX was introduced into the strains YOC1087, 4918, 4486 and 3928, and G418-resistant colonies were selected, resulting in YOC5115, 5119, 5137 and 5116, respectively. Gene disruption of WSC2, WSC3, MID2, MTL1 and SMK1 was conducted by PCR-mediated gene disruption with plasmid pFA6a-kanMX as a template. The PCR products were introduced into an fks1-1154 (YOC1087) strain, creating YOC5513, YOC5514, YOC5515, YOC5516 and YOC5524, respectively.

Double disruptions of MSB2 and HKR1, STE20 and CLA4, SSK2 and SSK22, MSN2 and MSN4, and MKK1 and MKK2 were constructed by performing two rounds of the described gene disruption protocol using the indicated markers, creating YOC4476, 4477, 4224, 4229 and 4483, respectively. Quintuple disruptions of SMP1, MSN2, MSN4, SKO1 and HOT1 were constructed by performing three rounds of PCR-mediated gene disruption using the plasmids pFA6a-kanMX, pFA6a-hphNT1 and pFA6a-natNT3 as a template. The PCR products were introduced into an fks1-1154 msn2Δ msn4Δ (YOC4229) strain, creating YOC5512.

For the generation of HOG1 (hog1-as, hog1-K52R, hog1-D144A and HOG1 F318S) and BCK1 (BCK1-20) point mutants, URA3 counterselection was used. hog1-as, hog1-K52R and hog1-D144A alleles were excised from pPW02, pPW28 and pPW51 (Westfall and Thorner, 2006; a gift from Dr Roelants, University of California, Berkeley, Berkeley, USA), introduced into YOC4479, and colonies were selected with 5-FOA, resulting in YOC4495, YOC4497 and YOC4499, respectively. For HOG1 F318S, the intergenomic region [380–960 base pairs (bp)] of HOG1 of YOC2888 and 3928 was first replaced with Cg-URA3. Then, pRS426-met3-HA-HOG1F318S (Yaakov et al., 2003; a gift from Dr Engelberg, The Hebrew University of Jerusalem, Jerusalem, Israel) was digested with PstI, which contains +184 bp to +1096 bp of HOG1, or the mutation sites of HOG1, and ligated to pUC19 (Yanisch-Perron et al., 1985), also digested with PstI. The resulting plasmid was digested with PstI to yield the intragenic HOG1 fragment for transformation. Colonies were selected against 5-FOA and further confirmed by PCR, resulting in YOC5153 and YOC5155. To supplement the growth of the hyperactive HOG1 mutant, HOG1 F318S, 10% sorbitol was added to the medium. To replace BCK1, YOC5143 (fks1-1154 bzz1Δ bck1:: KanMX) was constructed by replacing the genomic copy of BCK1 with a PCR-amplified bck1::KanMX cassette from the BY-series Euroscarf collection (Giaever et al., 2002), selecting G418-resistant colonies. pYO1884 (pRS316-BCK1-20) was then digested with XhoI/NotI and a 6.5-kilobase BCK1-20 fragment was ligated to pPS306, also digested with XhoI/NotI. The resulting plasmid was digested with MluI and transformed into YOC5143. The MluI site is located in the BCK1 promoter so that this plasmid can be integrated next to bck1::KanMX (Harrison et al., 2004). Transformants were selected on SD plates containing uracil because pYO1884 contains a URA3 marker, and the integration was confirmed by PCR, resulting in YOC5149. Detailed methods and sequences of oligonucleotides used in this study will be provided upon request.

To synchronize cells, centrifugal elutriation was performed as previously described (Suzuki et al., 2004), with minor modifications. Briefly, a 1-l culture of cells was prepared with 1.2–2.5×10⁷ cells/ml at 25°C. Cells were collected by centrifugation and the cell pellet was resuspended in 50 ml sterile water or YPD medium and briefly sonicated. The cells were selected by the elutriator equipped with an elutriation rotor (R5E; Hitachi), the pump speed was adjusted so the smallest cells passed through the elutriator, and the collected cells were concentrated by centrifugation. The concentration was adjusted to <1×10⁷ cells/ml, and then cells were incubated under the specific selected conditions.

To observe cells with mature buds, samples were fixed with 3.7% formaldehyde (Wako Chemicals) by gentle shaking for 30 min under the indicated conditions. Cells were collected by centrifugation, resuspended in 1 ml 10% PBS (Takara Bio Inc.) and stored at 4°C. The numbers of unbudded (no), small budded (small), and medium-to-large budded (mature) cells in the samples were counted (n>200 cells), and the percentage of cells with mature buds in the population was determined. Small buds were defined as a bud size of less than approximately one-third of the mother cell, and mature buds were defined as a bud size of more than approximately one-third of the mother cell. Cells with visibly elongated and abnormal buds were categorized as mature.

Microscopic observation was performed as previously described (Negishi et al., 2016; Suzuki et al., 2004). Briefly, samples were collected and fixed with 3.6–3.8% (v/v) formaldehyde (Wako Chemicals) by gentle shaking for 30 min. Cells were then resuspended in 1–2 ml 10% phosphate-buffered saline (PBS; Takara Bio Inc.) and stored for less than 3 days at 4°C.

The bud index of the samples under the indicated conditions and time points was evaluated by counting and categorizing cells (n>200) into ‘no’, ‘small’, and ‘medium-to-large’ buds, where a ‘small’ bud was defined as a bud size of less than approximately one-third of the mother cell and a ‘medium-to-large’ bud as a bud size of more than approximately one-third of the mother cell. Reproducibility was confirmed by repeating independent experiments, and one set of representative data is presented unless otherwise indicated.

Spindle morphology was visualized in the spheroplasts of cells acquired by zymolyase treatment followed by indirect immunofluorescence microscopy. Following immobilization of the spheroplasts on poly-L-lysine-coated slides, anti-tubulin 1/34 (anti-tubulin rat IgG2a monoclonal antibody, sc-53030, 1:100 dilution; Santa Cruz Biotechnology) and Alexa Fluor 564-labeled goat anti-rat IgG and IgM (1:100 dilution; Invitrogen) antibodies were used for tubulin staining. To visualize DNA, 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was used (final concentration 100–200 ng/ml). Cells with bipolar spindles were counted and the percentage in the population was determined (n>200) under the indicated conditions or at the indicated time points. Cells were observed using a standard fluorescent microscope equipped with a Leica DMRE with a 100× oil-immersion objective lens (Leica) or an AxioImager M1 with a 100× Plan-Apochromat oil-immersion objective lens (Carl Zeiss). Images were acquired with a CoolSNAP HQ cooled-CCD camera (Roper Scientific). Reproducibility was confirmed by repeating independent experiments, and one set of representative data is presented unless otherwise indicated.

RNA extraction and Northern blotting were performed as previously described (Negishi et al., 2016; Suzuki et al., 2004).

To examine Hog1 and phosphorylated Hog1 levels, cells were harvested and collected by centrifugation. The cells were immediately frozen in liquid nitrogen and boiled for 5 min in sodium dodecyl sulfate (SDS) sample buffer before electrophoresis. To examine Slt2 and phosphorylated Slt2p levels, protein extraction was performed as follows. Cells were washed once with distilled water and frozen at −80°C. Cell lysates were made by resuspending the pellets in ice-cold lysis buffer [100 mM NaCl, 5 mM NaF, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 0.1% SDS, 10% glycerol, 50 mM Tris-HCl, 1 mM phenylmethane sulfonyl fluoride, 1 mM sodium orthovanadate, 50 mM β-glycerol phosphate, 1 mM sodium pyrophosphate, and 25 µg/ml each of aprotinin, antipain, chymostatin, leupeptin, pepstatin, tosyl lysine chloromethyl ketone (TLCK) and tosyl phenylalanine chloromethyl ketone (TPCK)] and subjected to milling with glass beads with a Multi-Bead Shocker (Yasui Kikai). The samples were boiled for 5 min in SDS sample buffer before electrophoresis. Each protein sample was separated by SDS-polyacrylamide gel electrophoresis (PAGE).

Electrophoresis and western blotting were performed according to standard protocols. Briefly, 10% polyacrylamide gels were used and the separated protein extracts were transferred to a polyvinylidene fluoride membrane. Blotted membranes were blocked at room temperature in 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween (TBS-T). Hog1 and Slt2 were detected with anti-Hog1 polyclonal antibody y-215 (1:4000) (Santa Cruz Biotechnology) and anti-Slt2 polyclonal antibody y-244 (1:4000) (Santa Cruz Biotechnology), respectively, and activated phospho-Hog1 and activated phospho-Slt2 were detected with anti-phospho-p38 antibody (1:2000) (Cell Signaling Technology) and anti-phospho-p44/42 antibody (1:2000) (9101S, Cell Signaling Technology), respectively. The primary antibodies were dissolved in 5% bovine serum albumin in TBS-T. Following incubation with the primary antibodies, horseradish-peroxidase-conjugated antibodies for the appropriate IgG (Invitrogen) were used at a concentration of 1:2500 in TBS-T. Blots were detected using enhanced chemiluminescence (ECL Plus) reagent (GE Healthcare), and images of the membranes were acquired with a LAS-1000 Plus Luminescent Image Analyzer (Fuji Photo Film Co.).

Interactions among genes involved in the cell wall integrity checkpoint and cell wall construction genes involved in N-glycosylation, 1,3-β-glucan synthesis, and 1,6-β-glucan synthesis were detected as described previously (P<0.05) (Costanzo et al., 2016). Gene interaction networks were visualized with Cytoscape 3.4.0 (Shannon et al., 2003).

We thank David Engelberg, Haruo Saito and Jeremy Thorner for yeast strains, Charlie Boone for discussion, Ken-ichi Tominaga for his generous support and members of the Laboratory of Signal Transduction for helpful discussions.