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Expression of Mug14 is regulated by the transcription factor Rst2 through the cAMP-dependent protein kinase pathway in Schizosaccharomyces pombe

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Abstract

The cAMP-dependent protein kinase (Pka1) regulates many cellular events, including sexual development and glycogenesis, and response to the limitation of glucose, in Schizosaccharomyces pombe. Despite its importance in many cellular events, the targets of the cAMP/PKA pathway have not been fully investigated. Here, we demonstrate that the expression of mug14 is induced by downregulation of the cAMP/PKA pathway and limitation of glucose. This regulation is dependent on the function of Rst2, a transcription factor that regulates transition from mitosis to meiosis. The loss of the C2H2-type zinc finger domain in Rst2, termed Rst2 (C2H2∆), abolished the induction of Mug14 expression. Upon deletion of the stress starvation response element of the S. pombe (STREP: CCCCTC) sequence, which is a potential binding site of Rst2 on mug14, in the pka1∆ strain, its induction was abolished. The expression of Mug14 was significantly reduced and delayed by the limitation of glucose and also by nitrogen starvation in the rst2∆ strain. Mug14 is known to share a common function with Mde1 and Mta3 in the methionine salvage pathway, but the expression of mde1 and mta3 mRNAs was not enhanced by pka1 deletion and limitation of glucose. We conclude that the expression of Mug14 is upregulated by Rst2 under the control of the cAMP/PKA signaling pathway, which senses the limitation of glucose.

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Acknowledgements

FY21846 (mug14) was provided by the National Bio-Resource Project (NBRP), Japan. The authors also thank all the members of the laboratory for helpful support. 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 JP19K22283 (to MK) and JP18K05438 (to YM).

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Authors and Affiliations

  1. Graduate School of Natural Science and Technology, Shimane University, Matsue, 690-8504, Japan

    Shin-ich Inamura, Makoto Kawamukai & Yasuhiro Matsuo

  2. Department of Life Sciences, Faculty of Life and Environmental Sciences, Shimane University, Matsue, 690-8504, Japan

    Takuma Tanabe, Makoto Kawamukai & Yasuhiro Matsuo

  3. Institute of Agricultural and Life Sciences, Academic Assembly, Shimane University, Matsue, 690-8504, Japan

    Makoto Kawamukai & Yasuhiro Matsuo

Authors
  1. Shin-ich Inamura
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  2. Takuma Tanabe
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  3. Makoto Kawamukai
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  4. Yasuhiro Matsuo
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Contributions

SI planned this study, designed the experiments, carried out the experiments, made the yeast strains, and analyzed the data; TT planned this study and analyzed the data; MK analyzed the data and provided advice; YM planned this study, designed the experiments, made the plasmids and strains, carried out the experiments, and analyzed the data. YM wrote the original draft. SI, MK, TT, and YM reviewed and edited the original draft.

Corresponding author

Correspondence to Yasuhiro Matsuo.

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Communicated by Michael Polymenis.

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Supplementary Information

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294_2021_1194_MOESM1_ESM.pptx

Supplementary file1 (PPTX 54 KB). Fig. S1 Expression of Mug14-GFP is regulated by Rst2. a The pka1∆ rst2∆ Mug14-GFP (SIP15) strains harboring pREP81X-rst2 (wild-type) were cultured for 18 h in the presence or absence of thiamine and the expression of Mug14-GFP was analyzed by western blotting. The anti-PSTAIRE antibody was used as an internal loading control for Cdc2. b Expression levels of the Mug14-GFP protein were quantified and normalized to +thiamine. Experiments were performed three times: averages with S.D. are shown. Double asterisk (**) indicates P-value<0.01 for comparison with +thiamine.

294_2021_1194_MOESM2_ESM.pptx

Supplementary file2 (PPTX 164 KB). Fig. S2 Expression of Mug14-GFP is induced under conditions of glucose limitation. a Mug14-GFP (SIP1) cells were cultured in YES-glucose-limited medium. The cells were harvested at each time point by centrifugation. Mug14-GFP was detected with the anti-GFP antibody. The anti-PSTAIRE antibody was used as an internal loading control for Cdc2. b Expression levels of the Mug14-GFP protein were quantified and normalized to 0 h. Experiments were performed three times: averages with S.D. are shown. Asterisks (*) and double asterisks (**) indicate P-value <0.05 and <0.01 compared with 0 h. c The Mug14-GFP (SIP1) cells were cultured in YES-glucose-limited medium and observed by fluorescent microscopy. Scale bar: 10 µm. d Mug14-GFP (SIP1) and rst2∆ Mug14-GFP (SIP13) cells were cultured in YES, transferred into EMMLU-N (-nitrogen), and further cultured at 30 °C. The cells were harvested at each time point by centrifugation. Mug14-GFP was detected with the anti-GFP antibody. The anti-PSTAIRE antibody was used as an internal loading control for Cdc2. The bands were visualized by a longer exposure than in the experiment, the results of which are reported in Fig. 4. e Expression levels of the Mug14-GFP protein were quantified and normalized to the wild-type strain at 0 h. Experiments were performed three times: averages with S.D. are shown. Asterisks (*) and double asterisks (**) indicate P-value <0.05 and <0.01 compared with the wild-type strain at 0 h. NS indicates no significant difference with the wild-type at 0 h.

294_2021_1194_MOESM3_ESM.pptx

Supplementary file3 (PPTX 83 KB). Fig. S3 Expression of Mug14 is not affected by 2,2′-dipyridyl (Dip) and FeCl3 in the pka1∆ strain. a Mug14-GFP (SIP1) and pka1∆ Mug14-GFP (SIP2) cells were cultured in YES (control), transferred into YES with 250 µM Dip or 100 µM FeCl3, and cultured for 90 min at 30 °C. Mug14-GFP was detected with the anti-GFP antibody. The anti-PSTAIRE antibody was used as an internal loading control for Cdc2. b Expression levels of the Mug14-GFP protein were quantified and normalized to control in the wild-type strain. Experiments were performed three times: averages with S.D. are shown. NSs indicate no significant difference for comparison with each control. c Mug14-GFP (SIP1) and php4∆ Mug14-GFP (SIP137) cells were cultured in YES (control), transferred to YES glucose-limited medium (0.1% glucose), and cultured at 30 °C. The cells were harvested at each time point by centrifugation. Mug14-GFP was detected with the anti-GFP antibody. The anti-PSTAIRE antibody was used as an internal loading control for Cdc2. d Expression levels of the Mug14-GFP protein were quantified and normalized to control in the wild-type strain at 0 h. Experiments were performed three times: averages with S.D. are shown. Double asterisks (**) indicate P-value<0.01 for comparison with 0 h in each strain.

294_2021_1194_MOESM4_ESM.pptx

Supplementary file4 (PPTX 918 KB). Fig. S4 The deletion of mug14 does not affect the expression level of Ste11-GFP. a Ste11-GFP (SIP79), pka1∆ Ste11-GFP (SIP80), mug14∆ Ste11-GFP (SIP81), and pka1∆ mug14∆ Ste11-GFP (SIP82) cells were cultured in the EMMLU-N (1% glucose) liquid medium and observed by fluorescent microscopy at each time point. DAPI shows the nuclear staining. Scale bar: 10 µm. b Same strains as in Fig. S4a were cultured to the mid-log phase in YES at 30 °C. Ste11-GFP was detected with the anti-GFP antibody. The anti-PSTAIRE antibody was used as an internal loading control for Cdc2. c Expression levels of the Ste11-GFP protein were quantified and normalized to wild-type. Experiments were performed three times: averages with S.D. are shown. Double asterisks (**) and NS indicate P-value<0.01 and no significant difference for comparison with the wild-type strain.

294_2021_1194_MOESM5_ESM.pptx

Supplementary file5 (PPTX 535 KB). Fig. S5 The deletion of mug14 did not affect the phenotype of the pka1∆ strain. a Wild-type (PR109), pka1∆ (YMP36), mug14∆ (SIP17), and pka1∆ mug14∆ (SIP19) were cultured in the YES liquid medium and spotted onto YES in the presence or absence of 1.2 M KCl, 0.3 M CaCl2, 0.1 µM LatA, 18 µg/mL TBZ, 1.2 M sorbitol, and 10 µM camptothecin (CPT), and incubated for 3 or 5 days at 30 °C (YES, YES+0.1 µM LatA, YES+1.2 M sorbitol, and YES+10 µM CPT for 3 days; YES+1.2 M KCl, YES+0.3 M CaCl2, YES+18 µg/mL TBZ for 5 days). b Same strains as in Fig. S5a were cultured and spotted onto YES glucose-limiting medium in the presence or absence of 1.0 M KCl, 0.2 M CaCl2, 0.05 µM LatA, 18 µg/mL TBZ, 1.2 M sorbitol, and 5 µM CPT, and incubated for 3 or 5 days at 30 °C (YES, YES+0.05 µM LatA, YES+1.2 M sorbitol, and YES+5 µM CPT for 4 days; YES+1.0 M KCl, YES+0.2 M CaCl2, and YES+18 µg/mL TBZ for 5 days). c Wild-type (PR109) strain harboring pREP3X (vector) or pREP3X-mug14 were cultured in the EMMU liquid medium and spotted onto EMMU (3% glucose) in the presence or absence of 1.2 M KCl, 0.3 M CaCl2, or 0.1 µM LatA. All the plates were incubated for 4 days at 30 °C. d Wild-type (PR109) strains harboring pREP3X (vector) or pREP3X-mug14 were cultured for 18 h in the absence of thiamine. Total mRNA was prepared from each sample. cDNA was synthesized by reverse transcription. The expression level of mRNA was analyzed by quantitative PCR. The leu1 gene was used as a control. e Expression levels of the mug14 mRNA were quantified and normalized to wild-type. Experiments were performed three times: averages with S.D. are shown. Double asterisk (**) indicates P-value<0.01 for comparison with the wild-type strain. f Same strains as in Fig. S5a were cultured in the YES liquid medium and spotted onto YES in the presence or absence of 5 mM valproic acid (VPA), 40 µg/mL tamoxifen, or 30 mM sodium butyrate. All the plates were incubated for 3 days at 30 °C.

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Inamura, Si., Tanabe, T., Kawamukai, M. et al. Expression of Mug14 is regulated by the transcription factor Rst2 through the cAMP-dependent protein kinase pathway in Schizosaccharomyces pombe. Curr Genet 67, 807–821 (2021). https://doi.org/10.1007/s00294-021-01194-z

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  • DOI: https://doi.org/10.1007/s00294-021-01194-z

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