Abstract
In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 +) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 + expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 + expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 + and some of the regulation acts through pho7 +.
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Acknowledgments
This work was supported by the National Science Foundation grant MCB-1121714, and MCB-1412582, the Dennis M. Cook Endowed Gregor Mendel Chair in Genetics, the Villanova College of Liberal Arts and Sciences, and the Villanova Department of Biology. We also appreciate the suggested experiments from anonymous reviewers that improved the manuscript.
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Communicated by C. S. Hoffman.
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Supplemental Fig. 1 Measurement of phosphatase activity in a wild-type strain in varying phosphate and adenine conditions to demonstrate that de-repression of Pho1 activity in response to adenine starvation is not additive to phosphate starvation.
Supplemental Fig. 2 Transcript abundance of pho1 +, ade1 +, and pho84 +, after shifting from high to low adenine at time = 0 h, measured by quantitative PCR of reverse transcribed RNA. Transcripts were normalized to act1 +, and are presented as a ratio of expression. The average and standard deviation of triplicate measurements are given for each time point.
Supplemental Fig. 3 Measurement of phosphatase activity in mutants lacking ado1 +, an adenosine kinase, and asp1 +, the IP6 kinase. Phosphatase measurements suggest that Ado1 is a negative regulator of PHO1 expression, which is consistent with previous studies (Henry, et al. 2011, Lecoq, et al. 2001), and that Ado1 may be acting independently of all arms of the proposed pathway save for IP7, as the constitutive phenotype of ado1∆ is dependent on the presence of IP7.
Supplemental Fig. 4 Measurement of phosphatase activity in wild-type and pho7 + or snf5 + deletion strains and selected constitutive deletion strains. Deletion of pho7 + or snf5 + in strains deleted for aps1 + or csk1 + resulted in low phosphatase expression. Note that Pho1 expression may be more dependent on Pho7 than Snf5, suggesting that the loss of chromatin remodeling does not completely eliminate expression of PHO genes.
Supplemental Fig. 5 Measurement of phosphatase activity in strains with increased accumulation of cAMP. Mutants gpa2 R176H, cgs1-1, and cgs2-2 were generated, grown, and assayed as described in the Materials and Methods section (Hoffman and Winston 1991, Kao, et al. 2006). The cgs1-1 and cgs2-2 mutants have a slight constitutive appearance (higher level of phosphatase activity than wild-type in high adenine conditions) when grown on solid medium, but that qualitative appearance is not detectable using the quantitative liquid phosphatase assay used in this work. The discrepancy between the qualitative and quantitative results may indicate that factors in addition to cAMP are required for elevated Pho1 activity.
294_2014_466_MOESM1_ESM.pdf
Supplementary material 1 (PDF 92 kb) Supplemental Fig. 1 Measurement of phosphatase activity in a wild-type strain in varying phosphate and adenine conditions to demonstrate that de-repression of Pho1 activity in response to adenine starvation is not additive to phosphate starvation. Supplemental Fig. 2 Transcript abundance of pho1 +, ade1 +, and pho84 +, after shifting from high to low adenine at time = 0 hours, measured by quantitative PCR of reverse transcribed RNA. Transcripts were normalized to act1 +, and are presented as a ratio of expression. The average and standard deviation of triplicate measurements are given for each time point. Supplemental Fig. 3 Measurement of phosphatase activity in mutants lacking ado1 +, an adenosine kinase, and asp1 +, the IP6 kinase. Phosphatase measurements suggest that Ado1 is a negative regulator of PHO1 expression, which is consistent with previous studies (Henry, et al. 2011, Lecoq, et al. 2001), and that Ado1 may be acting independently of all arms of the proposed pathway save for IP7, as the constitutive phenotype of ado1Δ is dependent on the presence of IP7. Supplemental Fig. 4 Measurement of phosphatase activity in wild-type and pho7 + or snf5 + deletion strains and selected constitutive deletion strains. Deletion of pho7 + or snf5 + in strains deleted for aps1 + or csk1 + resulted in low phosphatase expression. Note that Pho1 expression may be more dependent on Pho7 than Snf5, suggesting that the loss of chromatin remodeling does not completely eliminate expression of PHO genes. Supplemental Fig. 5 Measurement of phosphatase activity in strains with increased accumulation of cAMP. Mutants gpa2 R176H, cgs1-1, and cgs2-2 were generated, grown, and assayed as described in the Materials and Methods section (Hoffman and Winston 1991, Kao, et al. 2006). The cgs1-1 and cgs2-2 mutants have a slight constitutive appearance (higher level of phosphatase activity than wild-type in high adenine conditions) when grown on solid medium, but that qualitative appearance is not detectable using the quantitative liquid phosphatase assay used in this work. The discrepancy between the qualitative and quantitative results may indicate that factors in addition to cAMP are required for elevated Pho1 activity.
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Estill, M., Kerwin-Iosue, C.L. & Wykoff, D.D. Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine. Curr Genet 61, 175–183 (2015). https://doi.org/10.1007/s00294-014-0466-6
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DOI: https://doi.org/10.1007/s00294-014-0466-6