Transient Sequestration of TORC1 into Stress Granules during Heat Stress  Terunao Takahara, Tatsuya Maeda  Molecular Cell  Volume 47, Issue 2, Pages 242-252 (July 2012) DOI: 10.1016/j.molcel.2012.05.019 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Overexpression of Pbp1 Represses TORC1 Activation (A) Wild-type or TOR1L2134M cells were transformed with an empty vector (–) or with a plasmid overexpressing Pbp1 from the GPD promoter (+PBP1), and 10-fold serial dilutions were spotted onto the indicated plates. The low nitrogen plate contained 0.0625% of ammonium sulfate as the sole nitrogen source, which corresponds to one eighth of that in normal SC media. (B) TOR1 and TOR1L2134M cells expressing 3HA-Sch9 were transformed with an empty vector or with a plasmid that overexpresses Pbp1 (PBP1 OE), were cultured in SC media, and were then incubated in the low nitrogen condition for 1 hr. The phosphorylation status of Thr737 of Sch9 was analyzed by western blotting with an anti-phospho-T737-Sch9 antibody, and the total protein level of 3HA-Sch9 was assayed with an anti-HA antibody. (C) Cells expressing 3HA-Sch9 were transformed with an empty vector or with a plasmid that overexpresses Pbp1 (PBP1 OE). Logarithmically growing cells in SC media (Orig.) were nitrogen-starved for 40 min [–N(40′)] and were then stimulated with glutamine (+Gln) or ammonium sulfate (+AS) for the indicated times. (D) Pbp1 was overexpressed by adding galactose (2%) for 2 hr to cells expressing 3HA-Sch9 and carrying a plasmid containing GAL1 promoter-driven PBP1, and analyzed as in (C). (E) Phosphorylation of Atg13 was detected as the electrophoretic mobility retardation. The cells were transformed with an empty vector (–) or with a plasmid that overexpresses Pbp1 (+) together with a plasmid that expresses Atg13. The cells were cultured in YNB+Glc+Gln, and then starved for nitrogen (YNB+Glc) for 40 min followed by glutamine readdition for the indicated times. Atg13 was detected by western blotting using an anti-Atg13 antibody. (F) Cells expressing 3HA-Sch9 were transformed with an empty vector or with a plasmid that overexpresses Pbp1. The cells were cultured in YNB+Glc+Gln, and then starved for glucose (YNB+Gln) for 40 min followed by glucose readdition for the indicated times. See also Figure S1. Molecular Cell 2012 47, 242-252DOI: (10.1016/j.molcel.2012.05.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Overexpression of Pbp1 Forms SGs and Induces the Formation of Cytoplasmic foci of Kog1 (A and B) Cells expressing Pbp1-GFP and 3HA-Sch9 were transformed with an empty vector (–) or with a plasmid that overexpresses Flag-tagged full-length Pbp1 (Full), or C-terminally truncated Pbp1 variants (1–688) and (1–467). Pbp1-GFP localization in logarithmically growing cells in SD (+N) media, and after nitrogen starvation for 40 min (–N), were monitored with fluorescence microscopy (A). The phosphorylation status of Sch9, and the protein expression of 3HA-Sch9 and the Flag-Pbp1 variants were analyzed by immunoblotting (B). (C) Cells expressing both HA-Tor1 and Kog1-Myc were transformed with plasmids indicated in (A). Logarithmically growing cells were harvested and Flag-tagged Pbp1 in cell lysates was precipitated with an anti-Flag antibody. Coimmunoprecipitation of HA-Tor1 or Kog1-Myc was detected by immunoblotting with anti-HA and anti-Myc antibodies. (D) Cells expressing both Kog1-GFP and Pbp1-RFP were transformed with empty (–) or a plasmid that overexpresses PBP1 driven by the GPD promoter (Pbp1 OE). Kog1-GFP and Pbp1-RFP localization during the logarithmic growth phase were monitored under a fluorescence microscope. Percentages of cells with Kog1 foci (72%) or Pbp1 foci (41%). (E) Cells expressing both Pbp1-GFP and Pab1-RFP were transformed with a plasmid that overexpresses Pbp1 driven by the GAL1 promoter. Pbp1-GFP and Pab1-RFP localization were monitored after induction of Pbp1 by adding galactose for 3 hr. Percentages of cells with Pbp1 foci (53%) or Pab1 foci (28%). (F) Cells expressing both Kog1-GFP and Pab1-RFP were transformed with a plasmid that overexpresses Pbp1 driven by the GAL1 promoter. Kog1-GFP and Pab1-RFP localization were monitored as in (E). Percentages of cells with Kog1 foci (48%) or Pab1 foci (24%). See also Figure S2. Molecular Cell 2012 47, 242-252DOI: (10.1016/j.molcel.2012.05.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Heat Stress Induces Sequestration of TORC1 into SGs (A) Localization of Kog1-GFP and Pab1-RFP was monitored during the exponential growth phase (EXP), or upon heat stress (46°C) for 30 min. Cells were also treated with cycloheximide (+CHX; 100 μg/ml) for 2 min before heat stress. Arrowheads indicate representative foci in which Kog1-GFP colocalized with Pab1-RFP. (B) The Venn diagram showing colocalization of Kog1-GFP foci and Pab1-RFP foci derived from quantification of three independent assays (>50 cells each) after heat stress (46°C) for 30 min. Errors represent the SD. (C) Cells expressing Kog1-GFP were stained with the vacuolar membrane fluorescent dye FM4-64, and the localization of Kog1-GFP and the morphology of the vacuole (FM4–64) were examined during exponentially growing phase (EXP) and after heat stress (46°C) for 30 min. (D) Cells expressing Flag-Tor1 and Kog1-HA with or without Pab1-Myc were grown in SC media (heat stress; –), then heated at 46°C for 30 min (heat stress; +). Where indicated, cells were treated with cycloheximide (+CHX; 100 μg/ml) for 2 min before heat stress. Pab1-Myc in cell lysates was precipitated with an anti-Myc antibody, and coimmunoprecipitated proteins were analyzed by immunoblotting. (E) Cells expressing Kog1-GFP and Pab1-RFP were treated with heat (46°C) for 30 min, and then analyzed during the recovery phase at 30°C for 120 min under a fluorescence microscope. Arrowheads indicate representative foci in which Kog1-GFP colocalized with Pab1-RFP, and arrows at the 60 min time point indicate cells completely disassembled SGs. See also Figure S3. Molecular Cell 2012 47, 242-252DOI: (10.1016/j.molcel.2012.05.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Sequestration of TORC1 into SGs Delays Its Reactivation (A) Logarithmically growing cells were pretreated with cycloheximide (CHX; 100 μg/ml) for 1 min and were then heated at 46°C for 30 min. After removal of the remaining CHX, the cells were incubated in SC media at 30°C and phosphorylation of T737 of Sch9 was analyzed at the indicated times by immunoblotting. The asterisk indicates the unphosphorylated form of Sch9. (B) TORC1 was immunopurified from unstressed (–) or heat stress (+) conditions by using the anti-HA antibody, then kinase reactions were performed using GST-4EBP1 as the substrate. The phosphorylation levels of GST-4EBP1 were analyzed by immunoblotting with the anti-phospho-(T37/46)-4EBP1 antibody. (C) Cells expressing Ego1-Kog1-GFP and Pab1-RFP were analyzed during the exponential growth phase (EXP), and upon heat stress (46°C) for 30 min, under a fluorescence microscope. (D) Logarithmically growing Ego1-Kog1-expressing (fusion) and control (WT) cells were heated at 46°C for 30 min, and were then incubated at 30°C for 90 min. Aliquots were analyzed by immunoblotting at the indicated times. L.E., long exposure; S.E., short exposure. Relative phosphorylation of Thr737 of Sch9 at the 90 min time point was determined from three experiments with Image J (bar graph). Data are represented as mean ± SD. See also Figures S4 and S6. Molecular Cell 2012 47, 242-252DOI: (10.1016/j.molcel.2012.05.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 SG Disassembly Rates Control TORC1 Signaling during Recovery after Heat Stress (A) The control, pbp1Δ, and pub1Δ cells transformed with a plasmid expressing Pab1-GFP (Buchan et al., 2008) were analyzed during exponential phase (EXP), upon heat stress (46°C) for 30 min, or during the recovery phase at 30°C for 120 min under a fluorescence microscope. (B) The percentage of cells with Pab1-GFP foci at each time point from two independent experiments is shown (>250 cells were counted in each time point). Data are represented as mean ± SD. (C) The phosphorylation of Sch9 at each time point shown in A was analyzed by immunoblotting. L.E., long exposure; S.E., short exposure. See also Figure S5. Molecular Cell 2012 47, 242-252DOI: (10.1016/j.molcel.2012.05.019) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 SG-Mediated Inhibition of TORC1 Prevents Heat Stress-Induced Increase of Mutation Frequency (A) Logarithmically growing cells were or were not pretreated with cycloheximide (CHXpre; 100 μg/ml) for 2 min, followed by heat stress at 46°C for 30 min. As controls, CHX was added in unstressed cells (–Heat) for 30 min, or added at 15 min after heat stress and further incubated for 15 min at 46°C followed by incubation for 15 min at 30°C (CHXpost). After removal of the remaining CHX, cells were spread onto SC-Arg plates in the presence or absence of canavanine (60 μg/ml). Data are represented as mean ± SD from six experiments. ∗p < 0.05. (B) Wild-type cells were or were not pretreated with hippuristanol (Hipp; 100 μM) for 10 min, followed by heat stress at 46°C for 30 min, and were spread as in (A). Data are represented as mean ± SD from five experiments. ∗p < 0.05. (C) Wild-type cells were treated as in (A). The cells were further incubated in SC media in the presence or absence of rapamycin (25 nM) for 60 min at 30°C before spreading onto SC-Arg plates as in (A). Data are represented as mean ± SD from eight experiments. ∗p < 0.05. (D) Cells overexpressing PBP1 or DHH1 driven by the GPD promoter were untreated or subjected to heat stress at 46°C for 30 min, and spread onto SC-U-Arg plates as in (A). Data are represented as mean ± SD from five experiments. ∗p < 0.05; n.s., not significant. (E) Cells expressing wild-type SCH9 or a constitutively active allele, SCH92D3E, were treated as in (D). Data are represented as mean ± SD from five experiments. ∗p < 0.05. Molecular Cell 2012 47, 242-252DOI: (10.1016/j.molcel.2012.05.019) Copyright © 2012 Elsevier Inc. Terms and Conditions