Table 1.
Schizosaccharomyces pombe Strains used in this Study.
Table 2.
Primers used for semi-quantitative RT-PCR.
Fig 1.
Opposite regulations by the loss of Tor1 and Tor2 inhibition are distributed evenly across multiple TSSs.
The TSS distributions for isp5 (A), per1 (B), put4 (C), spbpb2b2.01 (D) and cat1 (E) are visualized by Integrative Genomics Viewer (IGV). The transcriptional start sites of isp5+, per1+, put4+, SPBPB2B2.01 and cat1+ identified by CAGE are 25bp, 627bp, 140bp, 352bp and 359bp from their respective translational start sites according to PomBase. The upper half of each image shows the distribution of the TPM (transcripts per million) value along the chromosomal position at a single base resolution. Ticks appear at 10bp intervals along the horizontal axis. The TPM range is adjusted to visualize most peaks, so that some peaks above a TPM range may be truncated. Two images in (B) show the TSS distributions in two separate chromosomal regions upstream of the per1+ gene with respective optimal TPM ranges. Two images in (C) show the TSS distribution in the same chromosomal region upstream of the put4+ gene with different TPM range (0–150 for left; 0–10 for right). The lower half of each image shows the locations of top peaks (green) and bottom peaks (blue), which basically represent narrow and broad peaks, by horizontal bars. The fold change in logarithmic scale (base 2) relative to the value in wild-type cells is shown below respective bars. Positive and negative values indicate upregulation and downregulation, respectively, compared to wild-type cells.
Fig 2.
The loss of Tor1 and Tor2 inhibition oppositely affect mRNA expression of several amino acid permeases.
Wild-type (wt) cells, Δtor1 cells and tor2-287 cells were grown overnight in EMM medium to early log phase, and then the cells were harvested. Total RNA was extracted and subjected to semi-quantitative RT-PCR for the indicated amino acid permeases. The values were obtained by the comparative CT method in comparison to those of act1, and then were normalized to those in wild-type cells (RQ: relative quantity). The values were averaged from three independent experiments and are shown. **P<0.01 and ***P<0.001 for Turkey’s test following one-way ANOVA for the comparisons with respective values of wild-type cells. ###P<0.001 for unpaired t-test for the planned comparisons with respective values of wild-type cells.
Fig 3.
The loss of Tor1 decreases, and Tor2 inhibition and nitrogen depletion increases, isp5+ promoter activity.
Wild-type (wt), tor2-287, Δtor1 and tor2-287Δtor1 cells harboring the reporter plasmid for the 1013bp region of isp5+ promoter (isp5-1013; pKB8527) were grown to exponential phase at 27°C without (A) or with (B) shift to 34°C for 2 hours. The medium was then replaced by EMM or nitrogen-depleted EMM (-NH4Cl) (left graphs in A and B), or by EMM containing DMSO (+DMSO), 0.2 μg/ml rapamycin (+Rap) or 2 μM Torin-1 (+Torin) (right graphs in A and B), and the cells were subjected to Renilla luciferase reporter assay. The bioluminescence was measured real-time as relative light unit (RLU) for 2 hours, and the peak RLU value was normalized to that in wild-type cells in control conditions (EMM or +DMSO). The values were averaged from three independent experiments, and are shown. Magnified parts of the graphs are shown in insets. *P<0.05, **P<0.01, ***P<0.001 for Tukey’s multiple comparison test following one-way ANOVA or unpaired t-test for the comparisons with vehicle conditions of respective genotypes. ###P<0.001 for unpaired t-test for the comparison with vehicle conditions in wild-type cells. ns, not significant.
Fig 4.
The GATAAG motif is necessary for isp5+ promoter activity and its activation by Tor2 inhibition and nitrogen depletion.
(A) The 1013-bp region (isp5-1013) and the 513bp region (isp5-513) of isp5+ promoter from the TSS were used to generate Renilla luciferase reporters (pKB8527 and pKB8632, respectively). The isp5-1013 promoter region contains two GATAAG motifs at designated locations (-645~-640bp and -124~-119bp), and the isp5-513 promoter region contains only one of these motifs proximal to the TSS. Both of these two GATAAG motifs (arrows) are on the complement strand of the isp5+ promoter. Wild-type cells harboring the respective reporter plasmids were grown to exponential phase at 27°C, and were assayed without or with nitrogen depletion (EMM and -NH4Cl, respectively) or without or with Torin-1 treatment (+DMSO and +Torin, respectively). The bioluminescence was measured and analyzed as described in Fig 3. The data were obtained from three independent experiments. **P<0.01, ***P<0.001 for unpaired t-test for the comparisons with respective vehicle conditions. (B) The 513bp region of isp5+ promoter (isp5-513) and the same promoter region with the GATAAG motif deleted (isp5-513Δ) were used to generate Renilla luciferase reporters (pKB8632 and pKB9112, respectively). For the left two graphs, wild-type (wt) cells harboring the respective reporter plasmids were grown to exponential phase, and were assayed without or with nitrogen depletion (EMM and -NH4Cl, respectively) or without or with Torin-1 treatment (+DMSO and +Torin, respectively). For the rightmost graph, wild-type (wt) cells and tor2-287 cells harboring the respective reporter plasmids were analyzed without stimulation (EMM). The bioluminescence was measured and analyzed as described in Fig 3. The data were obtained from three independent experiments. ***P<0.001 for unpaired t-test for respective vehicle conditions. ns, not significant.
Fig 5.
The loss of Tor1 decreases, and Tor2 inhibition and nitrogen depletion increases, the activity of the GATAAG reporter.
Wild-type (wt) cells, tor2-287 cells and Δtor1 cells harboring the luciferase reporter for the GATAAG motif (3xGATAAG reporter; pKB8742) were grown to exponential phase at 27°C without (A) or with (B) shift to 34°C for 2 hours. The cells were assayed without or with nitrogen depletion (EMM and -NH4Cl, respectively) or without or with rapamycin or Torin-1 treatment (+DMSO, +Rap, +Torin, respectively). The bioluminescence was measured and analyzed as described in Fig 3. The data were obtained from three independent experiments. Magnified parts of the graphs are shown in insets. *P<0.05, **P<0.01, ***P<0.001 for Tukey’s multiple comparison test following one-way ANOVA or unpaired t-test for the comparisons with vehicle conditions of respective genotypes. ##P<0.01, ###P<0.001 for unpaired t-test for the comparisons with vehicle conditions in wild-type cells. ns, not significant.
Fig 6.
Gaf1 is critical for the transcription driven by isp5+ promoter and the GATAAG motif.
(A) The loss of the basal activities of isp5+ promoter and the GATAAG reporter and their activation by nitrogen depletion and Torin-1 treatment in Δgaf1 cells. Wild-type (wt) cells and Δgaf1 cells harboring the reporter plasmids for isp5+ promoter (isp5-1013; pKB8527) or the GATAAG motif (3xGATAAG; pKB8742) were grown to exponential phase and assayed without or with nitrogen depletion (EMM or -NH4Cl, respectively) or without or with Torin-1 treatment (+DMSO or +Torin, respectively). The bioluminescence was measured and analyzed as described in Fig 3. The data were obtained from three independent experiments. ***P<0.001 for Tukey’s multiple comparison test following one-way ANOVA or unpaired t-test for the comparisons with vehicle conditions of respective genotypes. ns, not significant. (B) The loss of Gaf1 abolished the basal activity of isp5+ promoter and its activation by Tor2 inhibition. Wild-type (wt) cells, tor2-287 cells, Δgaf1 cells and tor2-287Δgaf1 cells harboring the reporter plasmid for isp5+ promoter (isp5-1013) were grown to exponential phase, and were assayed without or with nitrogen depletion (EMM or -NH4Cl, respectively) or without or with Torin-1 treatment (+DMSO or +Torin, respectively). The bioluminescence was measured and analyzed as described in Fig 3. The data were obtained from three independent experiments. Magnified parts of the graphs are shown in insets. *P<0.05, **P<0.01, ***P<0.001 for Tukey’s multiple comparison test following one-way ANOVA or unpaired t-test for the comparisons with vehicle conditions of respective genotypes. ###P<0.001 for unpaired t-test for the comparisons with vehicle values in wild-type cells. ns, not significant. (C) Reduced mRNA levels of isp5, per1, put4, but not cat1, in Δgaf1 cells. Wild-type (wt) cells and Δgaf1 cells were grown overnight in EMM media to early log phase. Total RNA was extracted and subjected to semi-quantitative RT-PCR analysis. The data were obtained from three independent experiments. ***P<0.001 for Tukey’s multiple comparison test following one-way ANOVA or unpaired t-test for the comparisons with vehicle conditions of respective genotypes. ns, not significant.
Fig 7.
Tor2 inhibition and nitrogen depletion induce nuclear localization of Gaf1.
(A) Nuclear localization of Gaf1 induced by nitrogen depletion and Tor inhibitors. Wild-type (wt) cells expressing Gaf1-YFP under its native promoter were grown to early log phase in EMM medium at 27°C. The cells were incubated without (EMM) or with nitrogen depletion (-NH4Cl), Torin-1 treatment (+Torin) or rapamycin treatment (+Rapamycin) for 5 or 15 min, and the fluorescent images of Gaf1-YFP were then acquired. Scale bar, 10 μm. (B) Quantification of nuclear localization of Gaf1 in the experiments shown in (A). The proportions of the cells which showed Gaf1 signals in the nucleus alone (Nuclear), in the cytosol alone (Cytoplasmic), or in both (Nuclear-Cytoplasmic) were determined. The averaged values from 3 independent experiments are shown. ***P<0.001 for Tukey’s multiple comparison test following one-way ANOVA for the comparisons with EMM condition at respective time points. (C) Intact nuclear localization of Gaf1 induced by nitrogen depletion in Δtor1 cells. Δtor1 cells expressing Gaf1-YFP under its native promoter were grown to early log phase in EMM media at 27°C. The cells were incubated before (EMM) or after nitrogen depletion (-NH4Cl) for 5 or 15 min, and the fluorescent images of Gaf1-YFP were then acquired. Scale bar, 10 μm. (D) Quantification of nuclear localization of Gaf1 in the experiments shown in (C). The proportions of the cells which showed Gaf1 signals in the nucleus alone, in the cytosol alone, or in both were determined. The averaged values from 3 independent experiments are shown. ***P<0.001 for Tukey’s multiple comparison test following one-way ANOVA for the comparisons with control conditions of respective genotypes. ns, not significant for unpaired t-test for the comparisons with corresponding values in wild-type cells. (E) Nuclear localization of Gaf1 induced by Tor2 inhibition. Wild-type (wt) cells and tor2-287 cells expressing Gaf1-YFP under its native promoter were grown to early log phase in EMM media at 27°C. A half of the cells were maintained at 27°C, and the other half were cultured at 34°C for 2 hours. The fluorescent images of Gaf1-YFP were then acquired. Scale bar, 10 μm. (F) Quantification of nuclear localization of Gaf1 in the experiments shown in (E). The proportions of the cells which showed Gaf1 signals in the nucleus alone, in the cytosol alone, or in both were determined. The averaged values from 3 independent experiments are shown. ***P<0.001 and ns, not significant for Tukey’s multiple comparison test following one-way ANOVA for the comparisons with the values at 27°C. #P<0.05, ###P<0.001 for Tukey’s multiple comparison test following one-way ANOVA for the comparison with corresponding values in wild-type cells.
Fig 8.
Tor2 inhibition and nitrogen depletion induce Gaf1 dephosphorylation.
(A) Immunoblot analysis of Gaf1 phosphorylation. To induce Gaf1-GFP expression, wild-type (wt) cells expressing Gaf1-GFP under the nmt41 promoter were grown to early log phase in EMM medium without thiamine at 27°C for 20 hours. Proteins were extracted and treated without or with λ-phosphatase (PPase) in the presence or absence of its inhibitors, as indicated in the graph. The resultant proteins were subjected to SDS-PAGE and immunoblot analyses with anti-GFP antibodies. Endogenous α-tubulin was detected as a loading control. (B) Gaf1 dephosphorylation induced by Torin1 treatment and nitrogen depletion. To induce Gaf1-GFP expression, wild-type (wt) cells expressing Gaf1-GFP under the control of the nmt41 promoter were grown to early log phase in EMM media without thiamine at 27°C for 20 hours. The cells were treated without or with rapamycin or Torin-1 (+DMSO, +Rap or +Torin, respectively) or without or with nitrogen depletion (EMM or -NH4Cl, respectively) for 15 min. The lysates of these cells were analyzed similarly to (A). (C) Gaf1 dephosphorylation induced by Tor2 inhibition. To induce Gaf1-GFP expression, wild-type (wt) cells and tor2-287 cells expressing Gaf1-GFP under the nmt41 promoter were grown to early log phase in EMM medium without thiamine at 27°C for 20 hours. The lysates of these cells were analyzed similarly to (A). (D) Intact Gaf1 dephosphorylation induced upon nitrogen depletion in Δtor1 cells. To induce Gaf1-GFP expression, wild-type (wt) cells and Δtor1 cells expressing Gaf1-GFP under the nmt41 promoter were grown to early log phase in EMM medium without thiamine at 27°C for 20 hours. The lysates of these cells were analyzed similarly to (A).