Abstract
The phospholipase B homolog Plb1 and the cAMP-dependent protein kinase (PKA) pathway are required by fission yeast, also known as to Schizosaccharomyces pombe, to grow under KCl-stress conditions. Here, we report the relative contributions of Plb1 and the cAMP/PKA pathway during the hypertonic stress response. We show that the plb1∆, cyr1∆, and pka1∆ single mutants are sensitive to high concentrations of KCl but insensitive to sorbitol-induced osmotic stress. In contrast, the plb1∆ cyr1∆ and plb1∆ pka1∆ double mutants are hypersensitive to KCl and sorbitol. The cyr1∆ pka1∆ double mutants showed the same phenotype of each single mutant. Growth inhibition due to hypertonic stress in the plb1∆, plb1∆ cyr1∆, and plb1∆ pka1∆ strains was partially rescued by cgs1 deletion—cgs1∆ has constitutively active Pka1—or by the deletion of transcription factor Rst2, which is negatively regulated by Pka1. Pka1-GFP localized in the nucleus and cytoplasm in plb1∆, whereas it is localized only in the cytoplasm in cyr1∆, indicating that Plb1 does not regulate Pka1 localization. Glucose limitation downregulates the PKA pathway, and it was accordingly observed that glucose limitation in plb1∆ further increased the strain’s sensitivity to KCl. Growth inhibition by KCl in plb1∆ under glucose-limited conditions was significantly rescued by cgs1∆ and slightly rescued by rst2∆. These findings indicate that, in fission yeast, Plb1 and the glucose-sensing cAMP/PKA pathway play a synergistic role in responding to hypertonic stress.
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Acknowledgements
The authors also thank all the members of the laboratory for helpful support and scientific advice. We would like to thank Editage (www.editage.com) for English language editing.
Funding
The authors thank the faculty of Life and Environmental Sciences in Shimane University for help in financial support for publication. This work was supported by a JSPS KAKENHI Grant Number JP18K05438 (to YM) and JP19K222831 (to MK).
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YM planned this study, designed the experiments, carried out the experiments, made the yeast strains, and analyzed the data; SM planned this study; MK analyzed the data and provided advice. YM wrote the original draft. YM and MK reviewed and edited the original draft.
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Fig. S1
Schematic diagram of the cAMP-dependent protein kinase pathway in S. pombe. Supplementary file1 (PPTX 54 KB)
Fig. S2
The plb1∆ strain normally grows in the presence of CaCl2 or NaCl. Wild-type (PR109), plb1∆ (YMP603), cyr1∆ (YMP28), plb1∆ cyr1∆ (YMP628), pka1∆ (YMP36), plb1∆ pka1∆ (YMP629), and cyr1∆ pka1∆ (YMP60) cells were spotted onto the YES, YES+0.1 M CaCl2, YES+0.3 M CaCl2, and YES+0.2 M NaCl plates and incubated at 30˚C for 3 to 5 days (YES for 3 days, YES+0.1 M CaCl2 for 4 days and the others for 5 days). Supplementary file2 (PPTX 220 KB)
Fig. S3
The GFP-Plb1 plasmid is functional. Wild-type (PR109) or plb1∆ (YMP603) cells harboring pREP41GFP (vector) or pREP41GFP-plb1 were cultured for 1 day on EMMU at 30˚C to induce expression from the nmt1 promoter. Cells were spotted onto EMMU in the presence or absence of KCl. The plates were incubated for 4 to 7 days at 30˚C (no stress for 4 days and KCl for 7 days). Supplementary file3 (PPTX 83 KB)
Fig. S4
Pka1-GFP localizes in the cytoplasm under KCl stress condition. Pka1-GFP (YMP19), plb1∆ Pka1-GFP (YMP893), cyr1∆ Pka1-GFP (YMP48), and cgs1∆ Pka1-GFP (YMP56) cells were cultured for 24 hours in the presence of 1.2 M KCl, stained by DAPI, and observed by fluorescent microscopy. Supplementary file4 (PPTX 107 KB)
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Matsuo, Y., Marcus, S. & Kawamukai, M. Synergistic roles of the phospholipase B homolog Plb1 and the cAMP-dependent protein kinase Pka1 in the hypertonic stress response of Schizosaccharomyces pombe. Curr Genet 68, 661–674 (2022). https://doi.org/10.1007/s00294-022-01253-z
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DOI: https://doi.org/10.1007/s00294-022-01253-z