Pka1 overexpression rescues the KCl-sensitive phenotype of the plb1∆ strain
It has been proposed that the cAMP/PKA pathway is a downstream target of Plb1 during the osmotic stress response induced by a high concentration of KCl (Yang et al. 2003). To clarify how Plb1 might affect the cAMP/PKA pathway, we first analyzed cell growth in the presence of a high concentration of KCl in a plb1∆ strain with Pka1 overexpression and in a pka1∆ strain with Plb1 overexpression. Pka1 overexpression rescued the growth defect of plb1∆ in the presence of 1.2 M KCl (Fig. 1a), whereas Plb1 overexpression did not recue the growth defect of pka1∆ (Fig. 1b). These results are compatible with the idea that the cAMP/PKA pathway is downstream of Plb1 in the context of responding to KCl stress.
S. pombe mutants lacking Plb1 and PKA are hypersensitive to hypertonic stress
We next analyzed the growth of single and double mutants under high-osmolarity stress. If Plb1 linearly affects the cAMP/PKA pathway, we expected that the plb1∆ cyr1∆ and plb1∆ pka1∆ double mutants would show the same KCl-sensitivity as the respective single mutants. Likewise, if cAMP only regulates PKA, we predicted that the cyr1∆ pka1∆ strain would exhibit the same sensitivity to hypertonic stress as the cyr1∆ and pka1∆ single mutants. The plb1∆ cyr1∆ and plb1∆ pka1∆ double mutants significantly exhibited the KCl-sensitive phenotype on a low concentration KCl (0.5 M), whereas the plb1∆, cyr1∆, and pka1∆ single mutants showed the KCl-sensitive phenotype on a high concentration of KCl (1.2 M). However, while the cyr1∆ pka1∆ strain grew normally on 0.5 M KCl, it showed the KCl-sensitive phenotype on 1.2 M KCl (Fig. 2a). The double mutants plb1∆ cyr1∆ and plb1∆ pka1∆ displayed the sorbitol-sensitive phenotype on 2 M sorbitol, while plb1∆, cyr1∆, pka1∆, and cyr1∆ pka1∆ mutants grew normally on the same plate (Fig. 2a). These results demonstrate that Plb1 and the cAMP/PKA pathway respond cooperatively to hypertonic stress.
Loss of functional Pka1 inhibits cell growth on media containing CaCl2 (Matsuo and Kawamukai 2017). We next investigated whether the single and double mutants are sensitive to CaCl2 and NaCl. While cyr1∆ and pka1∆ showed growth defects with 0.3 M CaCl2, wild-type and plb1∆ cells grew normally (Fig. S2). All the single and double mutants showed normal growth with 0.1 M CaCl2 or 0.2 M NaCl (Fig. S2), indicating that Plb1 is not involved in responding to CaCl2.
Git3 or Gpa2 overexpression rescues the growth defect of plb1∆ under KCl stress conditions (Yang et al. 2003). To better understand the relationship between Plb1 and the upstream component of the cAMP/PKA pathway, we analyzed the effect of the KCl stress on the plb1∆ git3∆ strain. This double mutant significantly exhibited the KCl-sensitive phenotype with a low concentration of KCl (0.5 M), but the plb1∆ and git3∆ single mutants grew normally on a plate containing 0.5 M KCl, only showing the KCl-sensitive phenotype with 1.2 M KCl (Fig. 2b). This indicates that Plb1 functions independently of the upstream cAMP/PKA pathway in the context of KCl stress.
Gain of functional Pka1 partially recues growth defects of plb1∆, cyr1∆, and cyr1∆ plb1∆ under hypertonic stress conditions
It has been shown that cgs1∆ rescued growth defects associated with the CaCl2- and TBZ-sensitive phenotypes of the cyr1∆ strain (Matsuo and Kawamukai 2017, Tanabe et al. 2019). These results suggest that gain of functional Pka1 would rescue the stress-sensitive phenotype of cyr1∆, and also of plb1∆ if Plb1 is indeed upstream of Pka1 in the context of hypertonic stress response. We investigated whether deleting cgs1 suppresses the growth defects of plb1∆, cyr1∆, and plb1∆ cyr1∆ upon KCl stress. If Plb1 is required for modulating Pka1 activity upon KCl stress, deleting cgs1 would completely rescue the hypertonic-stress-sensitive phenotype of plb1∆. As could be expected, we found that cgs1∆ significantly recued the KCl-sensitive phenotype of cyr1∆ with both 1.2 M and 1.5 M KCl (Fig. 3a). Additionally, cgs1∆ rescued the growth of plb1∆ with 1.2 M KCl but not with 1.5 M KCl (Fig. 3b). Intriguingly, cgs1∆ partially rescued the growth defect of plb1∆ cyr1∆ upon KCl stress and fully rescued the sorbitol-sensitive phenotype (Fig. 3b). These results indicate that while Pka1 activity is required for hypertonic stress response, Plb1 is not dispensable when responding to more serve hypertonic stress.
Loss of functional Rst2 rescues growth defects of plb1∆, pka1∆, and plb1∆ pka1∆ under hypertonic stress conditions
The transcription factor Rst2 is negatively regulated by Pka1 (Inamura et al. 2021, Takenaka et al. 2018). Loss of functional Rst2 rescues the phenotypes such as high expression of mug14 and ste11 mRNAs of the pka1∆ strain (Higuchi et al. 2002, Inamura et al. 2021). To investigate the role of Rst2 in hypertonic stress response, we next tested if rst2∆ rescues the growth defects of plb1∆, pka1∆, and plb1∆ pka1∆ under the hypertonic stress conditions. Our results showed that rst2∆ rescued the KCl-sensitive phenotype of plb1∆ with 1.0 M KCl but did not with 1.5 M KCl (Fig. 4a). Meanwhile, rst2∆ completely rescued the growth defect of pka1∆ on 1.5 M KCl (Fig. 4b). Finally, rst2∆ partially rescued the growth defects of plb1∆ pka1∆ on KCl-containing plates and fully rescued the growth defects on sorbitol-containing plates (Fig. 4c). These results indicate that Rst2 is the downstream target of the cAMP/PKA pathway in the context of responding to KCl stress, and that Plb1 is not dispensable when responding to higher levels of KCl.
Plb1 does not regulate Pka1 localization
We next analyzed the localization of Plb1 in the presence of KCl stress. We first made the plb1-GFP strain by tagging GFP at the C-terminus of Plb1, but we did not observe GFP fluorescence (data not shown). To overcome this problem, we next constructed a GFP-Plb1-expressing plasmid, in which Plb1 tagged with GFP at its N-terminus is expressed under the nmt41 promoter. GFP-Plb1 rescued the growth of the plb1∆ strain upon KCl stress, indicating that GFP-Plb1 is functional (Fig. S3). Plb1 localized at the endoplasmic reticulum (ER) and highly accumulated in vesicles such as the dots under normal conditions. Upon KCl stress, Plb1 localization to the ER did not change, but its localization to vesicles decreased (Fig. 5a).
Because the KCl-sensitive phenotype of plb1∆ is dependent on Pka1 activity, we next analyzed whether Pka1 localization is regulated by Plb1. Pka1-GFP mainly localizes in the nucleus and diffusely in the cytoplasm in wild-type cells (Matsuo et al. 2008). In the presence of inactive endogenous Pka1 in the cyr1∆ strain, Pka1-GFP only localized to the cytoplasm. In the context of constitutive Pka1 activation in cgs1∆ under normal conditions, Pka1-GFP localized to the nucleus and diffusely to the cytoplasm as observed in the wild-type strain ((Matsuo et al. 2008) and Fig. 5b). In plb1∆, Pka1-GFP exhibited the same localization as in wild-type and cgs1∆ cells. Under KCl stress, the localization of Pka1-GFP changed from the nucleus to the cytoplasm in wild-type cells, and still localized only in the cytoplasm in cyr1∆. In cgs1∆, the nuclear localization of Pka1-GFP decreased, and we observed accumulation as dots distributed through the cytoplasm. Under KCl stress conditions in plb1∆, Pka1-GFP changed its localization to the cytoplasm, as observed in wild-type cells (Fig. 5b). These results indicate that Plb1 does not regulate Pka1 localization, suggesting that Plb1 likely is not involved in the regulation of Pka1 activity.
We next tested whether deleting plb1 would affect Pka1 phosphorylation. Pka1 is phosphorylated at threonine 356 in cyr1∆, whereas this phosphorylation site is not detected in wild-type or cgs1∆ strains (Gupta et al. 2011a, McInnis et al. 2010). Under normal conditions, Pka1-13Myc was detected in a non-phosphorylated form in wild-type and cgs1∆ cells, while it was phosphorylated in the cyr1∆ strain (Fig. 5c). However, Pka1-13Myc was not phosphorylated under normal conditions in plb1∆ (Fig. 5c). This result indicates that Plb1 does not affect Pka1 phosphorylation. We attempted to analyze the phosphorylation profile of Pka1 under KCl stress conditions, but we were unable to detect Pka1 clearly, likely due to the instability of Pka1.
Glucose limitation induces growth defects in wild type and plb1∆ upon the KCl stress
Because the cAMP/PKA pathway is tightly regulated by extracellular glucose concentration (Byrne and Hoffman 1993, Inamura et al. 2021, Tanabe et al. 2020, Welton and Hoffman 2000), we next analyzed whether glucose limitation induces the KCl-sensitive phenotype. Wild type, plb1∆, and pka1∆ grew, but plb1∆ pka1∆ did not grow, on glucose-rich (3% glucose) medium with 0.3 M KCl (Fig. 6a). Additionally, plb1∆, pka1∆, and plb1∆ pka1∆ exhibited a KCl-hypersensitive phenotype on low-glucose (0.1% glucose) medium than on normal glucose (3% glucose) medium (Fig. 6a).
Next, we analyzed the localization of GFP-Plb1 under the glucose-limited conditions. Localization of GFP-Plb1 showed the same pattern, localization at the ER and in vesicles, in the glucose-rich (3% glucose) and glucose-limited (0.1% glucose) media, but the dots structure was decreased in the glucose-limited media (Fig. 6b), indicating that glucose levels affects Plb1 localization.
Gain of functional Pka1 rescues the KCl-sensitive phenotype of plb1∆ in glucose-limited media
We next tested whether gain of functional Pka1 affects growth during KCl stress under glucose-limited conditions. To do this, we compared the effect of cgs1 deletion in plb1∆ and plb1∆ cyr1∆ under glucose-limited conditions. While plb1∆ and plb1∆ cyr1∆ significantly exhibited the KCl-sensitive phenotype with 0.3 M KCl, plb1∆ cgs1∆ and plb1∆ cyr1∆ cgs1∆ grew on 0.5 M KCl under glucose-limited conditions (Fig. 7a). These results indicate that increased Pka1 activity overcomes sensitivity to KCl stress in plb1∆ under glucose-limited conditions.
We finally analyzed whether rst2∆ rescues the KCl-sensitive phenotype in the context of glucose limitation. The plb1∆ and plb1∆ pka1∆ strains exhibited the KCl-sensitive phenotype with 0.3 M KCl under glucose-limited conditions. Under the same conditions, the plb1∆ rst2∆ and plb1∆ pka1∆ rst2∆ strains grew, and the plb1∆ pka1∆ rst2∆ strain also grew with 0.5 M KCl under glucose-limited conditions (Fig. 7b). Therefore, loss of functional Rst2 rescued growth during KCl stress under glucose-limited conditions.