Table 1 summarizes our findings regarding the participation of the HOG pathway in the Tn response. We assigned values of Tn resistance ranging from 10 for the wild type strain to 0 for the highly sensitive ire1D mutant. For comparison we included in our analysis the resistance values to NaCl which induces hyperosmotic stress. We divided our strains in three groups, those belonging to the SLN1 branch, those of the SHO1 branch and the Pbs2-Hog1 module.
To determine whether the Sln1 histidine kinase participates in the Tn response we expressed a thermosensitive protein (sln1(TS), which is inactivated at 37°C) in a strain carrying the hog1TAYA protein. This protein lacks the Thr-174 and Tyr-176 residues subjected to phosphorylation by Pbs2. It is important to recall that a strain expressing the hog1TAYA allele is highly sensitive to NaCl, but most importantly, partially resistant to Tn (Torres-Quiroz et al. 2010). In this strain, the inactivation of Sln1 does not induce the lethality otherwise induced by the constitutive activation of Hog1 wild type protein (Posas et al. 1996). When we analyzed growth of the sln1(TS)/hogTAYA strain and compared it with the growth of the hogTAYA strain we found that inactivation of Sln1 at 37°C caused a significant decrease of the Tn resistance (Table1). To analyze the contribution of the phosphorelay activity of Sln1 we followed the same strategy. We expressed, in the sln1(TS)/hog1TAYA strain, the sln1HQ allele (in which the phosphorylatable His-576 residue has been substituted by Gln); this protein is deficient in autophosphorylation and cannot transfer phosphate groups to its receiver domain (Fig. 1) (Posas et al. 1996). We found that the sensitivity of the sln1(TS) strain is complemented by the sln1HQ allele reaching a value near the wild type strain (Table 1). This result indicates that the unphosphorylated form of Sln1 is participating in the Tn response.
We also analyzed the contribution of the response regulator Ssk1. First, we confirmed the previous observation that a null ssk1D mutant shows sensitivity to Tn (Table1) (Torres-Quiroz et al. 2010). We then explored the contribution of the Asp-554 residue to the Tn response. The substitution of Asp-554 by an Ala residue (ssk1DA) prevents the Ypd1-dependent phosphorylation of Ssk1 (Fig. 1). We observed that the expression of the ssk1DA protein in the ssk1D strain, reverted the Tn sensitivity to the value near to that of the wild type strain. Finally, we determined the effect that elimination of the MAPKKKs Ssk2 and Ssk22 have in the Tn response. Elimination of Ssk2 significantly affected the growth in Tn medium, however elimination of Ssk22 had a much weaker effect. Elimination of both Ssk2 and Ssk22 moderately increased the sensitivity shown by the ssk2D mutant. This observation strongly suggests that Ssk2 plays a prominent role in the Tn response and disregards the participation of Ssk22.
In this work we also extended the studies regarding the participation of components of the SHO1 branch (Table 1). We confirmed that elimination of Sho1 did not affect growth in Tn and accordingly we found that elimination of the MAPKKK Ste11 had no effect either. Elimination of the adaptor protein Ste50 or the PAK-like kinase Ste20 barely induced a slight reduction in Tn resistance. The different effect that lack of these proteins had in the Tn response compared to the lack of Ste11 could be due to their participation in other transduction pathways.
Previous studies on the participation of components of the MAP kinase module in the response to inducers of ER stress, indicated that disruption of Hog1 and Psb2 caused high sensitivity to Tn (Bicknell et al. 2010; Torres-Quiroz et al. 2010). We confirmed those observations and found that the sensitivity of the pbs2D mutant was stronger than that of the hog1D mutant (Table 1). In addition, our quantitative studies showed that the expression of the unphosphorylated form of Hog1 (hog1TAYA) confers about three-fold resistance to Tn compared to the hog1 null mutant.
Then we analyzed the role of the scaffold domains of Pbs2, that have previously been described (Tatebayashi et al. 2003). To this end we constructed various Pbs2 mutants that compromise its association with Ssk2, Sho1 and Hog1. We also constructed a point mutation where the kinase activity of Pbs2 was eliminated (Fig. 2A). In these studies, we included a Hog1 version with the Pbs2BD domain deleted (Fig. 2B), which comprises the adjacent CD and PBD-2 sites required for Pbs2 binding (Murakami et al. 2008). All constructions were either integrated into their chromosomal loci, which places the mutated genes under the control of their endogenous promoter or cloned into the pYES2 vector and expressed under the control of the GAL1 promoter. In our experiments, we did not detect growth differences between strains carrying the integrated or the episomic copies.
Our results show that elimination of the Ssk2/22 binding domain (RSD-I, 5-56 aa) slightly reduced Tn resistance, while substitution of the Val54 residue for Gly, located within this domain (Fig. 2A), had no effect in the Tn resistance, which is comparable to the wild type strain (Table 1). This V54G mutation prevents binding of Ssk2/22 to the isolated RSD-I domain (Tatebayashi et al. 2003). Mutants lacking the Sho1 binding domain (RSD-II, 55-107 aa) showed a very light reduction of Tn resistance (Table 1).
We then analyzed the role of the Pbs2 kinase domain (KD), which has also a docking motif for Ssk2 (Tatebayashi et al. 2003); we found that a Pbs2 protein with the KD domain deleted (KD, 353-669 aa) still conferred significant resistance to Tn. Similarly, a strain carrying a Pbs2 protein devoid of kinase activity, pbs2KM (Lys-389 substituted by Met) (Fig. 2A), displayed mid resistance to Tn (Table1). It has been reported that simultaneous elimination of the KD domain and the substitution of Val-54 by Gly completely abrogates the interaction of Pbs2 with Ssk2 in the hyperosmotic system (Tatebayashi et al. 2003). In our assays we found that the double mutation KDD/V54G caused a strong Tn sensitivity; in this mutant the Tn resistance dropped to the level shown by the pbs2D null mutant (Table1).
To complement the Pbs2 domain dissection we analyzed the effect of the elimination of the HBD-1 (136-245 aa) domain of Pbs2 (Fig. 2A), which is a binding domain for Hog1 (Murakami et al. 2008). In our experiments, deletion of this domain caused a strong reduction on the Tn resistance (Table 1), and this effect was similar to the effect that elimination of the Hog1 Pbs2BD domain had in Tn resistance. The Hog1 Pbs2BD deletion includes the two binding domains described as Pbs2 docking motifs (Murakami et al. 2008). These results demonstrate that binding of Pbs2 and Hog1 is an essential requirement for the response to Tn.
Finally, to gain insight into the transcriptional effect that Tn treatment elicit in yeast, total RNA was extracted from cells exposed to Tn as indicated in the methods section and hybridized with an oligo-array representing 6529 ORFs. The result of the microarrays analysis is depicted in supplementary table S1. The log2 value of the differential expression between cells treated with Tn and not treated was calculated and used to generate heat maps of expressed (or repressed) genes belonging to three pathways annotated in KEGG: the MAPK (HOG) pathway, the protein processing in the ER pathway, and various types of N-Glycan biosynthesis pathway (Fig. 3). As expected, we detected that several genes of the UPR and N-Glycosylation pathways were induced under Tn treatment (Fig. 3B, C). Interestingly, Tn increased transcripts abundance of genes whose products participate in the SLN1 branch of the HOG pathway, namely SLN1, SSK2, SSK22, and YPD1 (Fig. 3A). In this assay expression of SSK1 was unchanged. It is also interesting to note that the expression of PBS2 was slightly increased while HOG1 was repressed. In this study we did not detect significant expression changes in components of the SHO1 branch (table S1).