Hog1p can be Phosphorylated and Activated in the ssk1Δste11Δ Double Mutant
In the HOG pathway, Ssk1p is considered as the activator of Ssk2p and Ssk22p 
. Early epistasis analysis placed Ssk2p and Ssk22p upstream of Pbs2p and downstream of Ssk1p 
. If Ssk1p is the sole activator of the Ssk2p and Ssk22p, the double mutant ssk1Δste11Δ
should be as osmosensitive as pbs2Δ
mutants and fail to phosphorylate Hog1p upon osmotic shock. However, some studies have found that expression of most osmoregulated genes are clearly induced or repressed in ssk1Δste11Δ
mutant under severe osmotic stress (0.5 M KCL and 1.0 M KCL) 
. The observations provided an interesting possibility that additional inputs into Pbs2 may exist 
To identify the alternative pathway, we constructed the double mutant ssk1Δste11Δ, and the triple mutant ste11Δssk2Δssk22Δ. We carried out the phosphorylation level of Hog1p in the mutant ssk1Δste11Δ and ste11Δssk2Δssk22Δ under a wide range of osmotic stress conditions (NaCl, KCl and sorbitol, from 0.2 M to 1.0 M). The results, including also measurements on the wide type strain, are shown in . We observed that the Hog1p was activated in the ssk1Δste11Δ mutant at 0.6 M sorbitol or a higher concentration (). However, Hog1p phosphorylation was not detected under mild osmotic stress (0.2 M and 0.4 M sorbitol/NaCl) in the double mutant (). In contrast, the phosphorylation of Hog1p could not be detected in the ste11Δssk2Δssk22Δ mutant in the wide range concentration of osmotic stress (NaCl, KCl and sorbitol, from 0.2 M to 1.0 M) ( C).
Hog1p phosphorylation level and growth phenotypes for the wild type (WT) and mutant yeast cells under various osmotic and salt stress conditions.
Under severe osmotic shock, for instance, 1.0 M sorbitol/NaCl, the phosphorylation of Hog1p peaked within 10 min and lasted for more than 60 min in the wild type strain (). In the ssk1Δste11Δ
mutant, although the level of phosphorylation of Hog1p reached was high, the duration was short. In the ssk1Δste11Δ
mutant, the phosphorylation of Hog1p disappeared within 20 min under 1.0 M sorbitol (). This result is consistent with the transcriptional profiles of osmoregulated genes in the strain ssk1Δste11Δ
. The expression of several osmoregulated genes (STL1
) in ssk1Δste11Δ
was induced at high level under 0.5 M KCl but the duration of the induction was shorter than that of the wide type strain 
Besides, the strain ssk1Δste11Δ exhibited much better growth than the hog1Δ mutant and the ste11Δssk2Δssk22Δ mutant under osmotic stress (). However, the growth of ssk1Δste11Δ mutant under osmotic stress depended greatly on the type of osmostressor. The mutant ssk1Δste11Δ show better osmoresistance under nonionic osmostressor (sorbitol) ( F) than under ionic stress even the Hog1p was similarly phosphorylated under ionic stress. The ssk1Δste11Δ cells grew better under KCL stress than under NaCL stress ( F).
Ssk2p can be Activated Independent of Ssk1p under Severe Osmotic Stress
As described above, the HOG pathway was activated in the ssk1Δste11Δ
mutant under osmotic stress but not in the ste11Δssk2Δssk22Δ
mutant, which indicated Ssk2p and Ssk22p may be activated independent of Ssk1p under osmotic stress. It has also been reported that the ssk1Δssk22Δsho1Δ
cells showed better resistance to 500 mM NaCl and 1.5 M sorbitol than ssk1Δ ssk2Δssk22Δsho1Δ
cells did 
To further analyze the alternate activation pathway independent of Ssk1p and Ste11p, we constructed two triple mutants: the ste11Δssk1Δssk2Δ mutant and ste11Δssk1Δssk22Δ mutant to analyze the phosphorylation state of Hog1p under osmotic stress. shows measurements of the phosphorylation level of Hog1p as well the growth phenotypes in our experiments with the mutant cells. The HOG pathway was activated in the absence of Ste11p, Ssk1p and Ssk22p () and was inactive if the STE11, SSK1 and SSK2 were deleted (). The Hog1p was significantly phosphorylated in the ste11Δssk1Δssk22Δ mutant under severe osmotic stress (higher than 0.6 M sorbitol). This implies that the MAPKKK Ssk2p can be activated in the absence of Ssk1p under severe osmotic stress. Moderate osmotic stress (concentration lower than 0.4 M sorbitol), on the other hand, could not lead to significant phosphorylation of Hog1p. The phosphorylation pattern of Hog1p under the stress in ste11Δssk1Δssk22Δ mutant in is similar to that of the ssk1Δ ste11Δ mutant shown in . In ste11Δssk1Δssk22Δ mutant, the phosphorylation of Hog1p peaked within 10 min and disappeared within 20 min under 1.0M sorbitol. The duration of the phosphorylated state of Hog1p in ste11Δssk1Δssk22Δ mutant was also shorter than wild type (). However, the response to the stress in the ste11Δssk1Δssk22Δ mutant was quick. The activation of Ssk22p, on the other hand, was totally dependent on Ssk1p. In ste11Δssk1Δssk2Δ mutant, we could not detect any phosphorylation of Hog1p under hyperosmotic stress (). Our results suggest that there may be an unidentified factor that activates Ssk2p under osmotic stress in addition to Ssk1p. Here we name the unidentified factor “X factor” temporarily. The growth of ste11Δssk1Δssk22Δ mutant was faster than that of the ste11Δssk1Δssk2Δ mutant ().
Ssk2p can be activated independent of Ssk1p under severe osmotic stress.
It has been reported that Ssk2p is specialized to promote actin cytoskeleton reassembly after osmotic shock 
. This function requires the kinase activity of Ssk2p 
. Osmotic stress induces a rapid disassembly of the actin cytoskeleton 
. Actin cytoskeleton disassembly induces Ssk2p to translocate from the cytosol to the septin cytoskeleton of the bud neck 
. Therefore, we asked whether actin disassembly would activate the Ssk2p to activate the HOG pathway. Lat B was used to induce rapid and complete disassembly of the actin cytoskeleton in strains BY4741 and ste11Δssk1Δ
. Within 20 min of Lat B treatment, neither strain displayed activation of Hog1p (). After 20 min incubation of both cells in 200 uM lat B, samples were fixed for Rd-phalloidin staining of actin structures. No actin structures were observed in the cells (). The results were in accordance with previous observation that activity of Hog1p activity is affected neither by actin-destabilizing drug latrunculin A, nor by actin-stabilizing drug jasplakinolide 
. These results indicate that X factor may not be the actin disassembly.
A Receiver Domain (Amino Acids 177~240) Near the N-terminus of SSK2 is Needed for the Activation of SSK2 Independent of SSK1
As observed above, Ssk2p can be activated without Ssk1p under osmotic stress, whereas the Ssk22p cannot. We carried out a sequence alignment analysis of the two proteins Ssk2p and Ssk22p. As shown in , the sequence comparison shows that Ssk2p and Ssk22p are quite similar. The similarity of the kinase domains of these two MAPKKKs is higher than that of the N-terminal noncatalytical domains. Ssk2p is larger than Ssk22p, mainly due to an extra N-terminal segment (1~176). There is a high variable N-terminal segment (177~240) in Ssk22p. Previous study has identified the Ssk1p binding domain (294~413) in Ssk2p 
. We assume that the binding site for the X factor is located in the near N-terminal region.
Comparison of protein sequences of Ssk2p and Ssk22p.
To determine the region in Ssk2p that is essential for its activation in the absence of Ssk1p, various segments near the N-terminus were deleted in Ssk2p. These constructs were transformed into the host strains ste11Δssk2Δssk22Δ and ste11Δssk1Δssk2Δ to test the activation of Hog1p, with results shown in . A mutant lacking the region (1~176) was able to activate the Hog1p in both the mutant hosts ste11Δssk2Δssk22Δ and ste11Δssk1Δssk2Δ. The mutant lacking segment of amino acid 1~240 could activate the Hog1p in the ste11Δssk2Δssk22Δ but not in the host ste11Δssk1Δssk22Δ. Besides, the mutant lacking the region of amino acid 177~239 could activate the HOG pathway in the ste11Δssk2Δssk22Δ but not in the host ste11Δssk1Δssk22Δ. The phenotype of Ssk2Δ(177~239) cells is similar to that of the Ssk2Δ(1~240) cells (). As the growth assay in show, the ssk2Δ(1~176) mutant in both hosts had no discernible effect on growth on high osmolarity media. The ssk2Δ(1~240) mutant and ssk2Δ(177~239) mutant in ste11Δssk1Δssk22Δ were osmosensitive, but not in the ste11Δssk2Δssk22Δ.
A receiver domain (amino acids 177~240) near the N-terminus of SSK2 is needed for the activation of SSK2 independent of SSK1.
These results suggest that the N- terminal portion of Ssk2p (amino acids 177–240) is indeed required for the activation of Ssk2p by the X factor under osmotic stress (). The segment is close to the Ssk1p BD (294~413) ( D). Previous research suggested that deletion of the N- terminal region of Ssk2p (amino acids 177–240) would not affect the binding of Ssk2p to either Ssk1p or actin 
Three MAPKKKs Involved in the HOG Pathway have Different Properties
Early research shows that the three MAPKKKs activate the Pbs2p then activate the HOG pathway under osmotic stress 
. They work functionally redundant to some extent 
. However, the three MAPKKKs have different activation patterns.
To study the activation patterns of the three MAPKKKs, we constructed the double mutant strains: ssk2Δssk22Δ, ste11Δssk2Δ and ste11Δssk22Δ and analyzed the phosphorylation of Hog1p under various levels of stress. Results of our experiments are presented in .
Three MAPKKKs involved in HOG pathway have different properties.
In the ssk2Δssk22Δ mutant that completely relies on Ste11p for Hog1p activation, the phosphorylation of Hog1p was weakly detected under 0.2 M sorbitol and exhibited a slower maximum response to the severe osmotic stress (1.0 M sorbitol) than the response of the wild type strain (). Under severe osmotic shock (1.0 M sorbitol), the phosphorylation of Hog1p peaked within 5 min in wild type strain (). In the ssk2Δssk22Δ mutant, Hog1p phosphorylation peaked at about 10 min under 1.0 M sorbitol.
Cells lacking STE11 and SSK2 (ste11Δssk2Δ mutant) did not respond to low osmolarity (up to 0.4 M sorbitol), and displayed much slower response at each osmolarity (Generally the peak falls at the 30 minutes point after osmotic shock ), and showed a much lower maximum response than the response of the wild type and other double mutants (). In the ste11Δssk2Δ mutant, Hog1p phosphorylation peaked at 30 min under 0.8 M and 1.0 M sorbitol.
In the ste11Δssk22Δ mutant which has a functional Ssk2p, cells responded as fast as wild type cells, with the maximum response that was similar to that of the wild type. But the duration of the response of the ste11Δssk22Δ mutant under severe osmotic stress (0.8 M–1.0M sorbitol) was shorter than that of the ssk2Δssk22Δ mutant and wild type strain (). However, as we discussed above, there are at least two inputs into the Ssk2p: Ssk1p and the X factor (). Therefore, the activation of Hog1p by Ssk2p should be divided into two parts. Pattern of Hog1p’s activation by the X factor can be considered similar to that of the mutant ste11Δssk1Δssk22Δ (). To test the activation pattern of Ssk1p, we transformed the mutant Ssk2p lacking the segment of amino acids 1~240 into the triple mutant ste11Δssk2Δssk22Δ. Phosphorylation of Hog1p was detected in the transgenic line under a range of osmotic stress at various time points (). Unexpectedly, the cells expressing the mutant ssk2Δ (1~240) lost the sensitivity to the mild osmotic stress (0.2 M sorbitol) (). Furthermore, the response is significantly attenuated under osmotic stress compared with that of the wild type Ssk2p. The strain ste11Δssk1Δ showed a quick response with a high amplitude.
Ssk2p Plays Essential Role in Salt Tolerance
Previous studies have demonstrated the redundant role of Ssk2p and Ssk22p. Actually, upon nonionic osmotic stress, the Ssk2p and Ssk22p can function equally well. However when subjected to the ionic osmotic stress, the double mutants display different tolerance.
The yeast cells which grow in the presence of high sodium concentrations (salt stress) face both an elevated external osmotic environment and an increasing amount of Na+
entering the cells 
. We have conducted a series of growth assay studies for the wild type and mutant cells under various levels of salt stress, with the results presented in . The mutant ssk2Δssk22Δ
showed no growth defect under severe osmotic stress (1.2 M sorbitol and 1.2 M KCL) (). However, the mutant ste11Δssk2Δ
showed poorer growth when exposed to the poison level of cation (0.8 M NaCL and 0.3 M LiCL), which indicates that Ssk2p and Ste11p are essential for salt-tolerance (). Actually, the mutant ste11Δssk2Δ
grows as well as the wild type strain even when being exposed to 1.2 M sorbitol or 1.2 M KCL ().
Ssk2p plays essential role in salt tolerance.
The mutant ste11Δssk1Δ also displayed severe growth defect upon sodium stress, even the phosphorylation level of Hog1p under osmotic stress caused by NaCL was similar or slightly higher than that caused by the sorbitol or KCL ( and ). The results imply that for salt tolerance, not only activation of Hog1p is required but MAPKKKs Ste11p and Ssk2p also play an important role. Although Ssk2p and Ssk22p are highly homologous, the Ssk2p shows better salt-tolerance than Ssk22p.
Furthermore, high level activation of Ssk2p is also required for the salt tolerance. As we discussed above, X factor can activate Ssk2p independent of Ssk1p and enhance the activation of Ssk2p by Ssk2p under osmotic stress. Here we found that the level of osmoresistance is slightly different between wild type Ssk2p cells and Ssk2Δ(1~240) cells (). Lacking the binding site (amino acid 177~240aa) for the X factor of Ssk2p would reduce the salt-resistance of the ste11Δssk22Δ cells (). The results indicate that the high level activation of Ssk2p is essential for saline-resistance.