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. 2018 Feb 15;29(4):510-522.
doi: 10.1091/mbc.E17-09-0553. Epub 2017 Dec 13.

Vacuole-mediated selective regulation of TORC1-Sch9 signaling following oxidative stress

Eigo Takeda  1 , Natsuko Jin  2 , Eisuke Itakura  1   3 , Shintaro Kira  4 , Yoshiaki Kamada  5   6 , Lois S Weisman  2   7 , Takeshi Noda  8   9 , Akira Matsuura  10   2   3
Affiliations

Vacuole-mediated selective regulation of TORC1-Sch9 signaling following oxidative stress

Eigo Takeda et al. Mol Biol Cell. .

Abstract

Target of rapamycin complex 1 (TORC1) is a central cellular signaling coordinator that allows eukaryotic cells to adapt to the environment. In the budding yeast, Saccharomyces cerevisiae, TORC1 senses nitrogen and various stressors and modulates proteosynthesis, nitrogen uptake and metabolism, stress responses, and autophagy. There is some indication that TORC1 may regulate these downstream pathways individually. However, the potential mechanisms for such differential regulation are unknown. Here we show that the serine/threonine protein kinase Sch9 branch of TORC1 signaling depends specifically on the integrity of the vacuolar membrane, and this dependency originates in changes in Sch9 localization reflected by phosphatidylinositol 3,5-bisphosphate. Moreover, oxidative stress induces the delocalization of Sch9 from vacuoles, contributing to the persistent inhibition of the Sch9 branch after stress. Thus, our results establish that regulation of the vacuolar localization of Sch9 serves as a selective switch for the Sch9 branch in divergent TORC1 signaling. We propose that the Sch9 branch integrates the intrinsic activity of TORC1 kinase and vacuolar status, which is monitored by the phospholipids of the vacuolar membrane, into the regulation of macromolecular synthesis.

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Figures

FIGURE 1:
FIGURE 1:
Signal outputs of the TORC1 pathway are differentially regulated in an input signal–dependent manner. Western blot analysis of the C-terminal fragment of Sch9-5HA (yet515), Npr1-13myc (yet610), Atg13 (yet562), and Par32-13myc (yet515) was performed. Cells in log phase were treated with SDC (control), SDC + 1 M NaCl (hyperosmotic stress), SD−N (nitrogen depletion), or SDC + 200 ng/ml rapamycin (TORC1 inhibitor) for 30 min, and the lysates were subjected to Western blotting.
FIGURE 2:
FIGURE 2:
The HOPS complex is specifically required for transmitting the TORC1 signal to the Sch9 branch. (A) Spot assay investigating growth recovery after rapamycin treatment in wild-type (WT) (BY4741), ∆vps41 (yet567), ∆ego1 (yet576), and ∆tco89 (yet701) cells. Cells in log phase were cultured in the presence or absence of 200 ng/ml rapamycin for 3 h at 30°C. The cell cultures were serially diluted 10-fold and then spotted onto YPD medium. Colonies were photographed after 1 d (YPD) or 2 d (Rap recovery). (B) Western blot analysis of C-terminal fragment of Sch9-5HA (yet515, 569, 577, and 729), Npr1-13myc (yet610, 618, 619, and 780), Atg13 (yet562, 574, 580, and 726), and Par32-13myc (yet515, 569, 577, and 729). Lysates of WT (yet515, 610, and 562), ∆vps41 (yet569, 618, and 574), ∆ego1 (yet577, 619, and 577), and ∆tco89 (yet729, 780, and 726) cells grown in SDC medium, or WT cells treated with 200 ng/ml rapamycin in SDC medium, were subjected to Western blotting. (C) Quantification of the band shift data of ∆vps41 and ∆ego1 cells for Sch9 from B (n = 5). To calculate the p value, we applied the Brunner-Munzel test. Error bars represent 95% confidence intervals. *p < 0.005. (D) Western blot analysis of C-terminal fragment of Sch9-5HA and Par32-13myc. WT (yet515), ∆vps41 (yet569), ∆ego1 (yet577), and ∆tco89 (yet729) cells under nitrogen starvation for 30 min were resuspended in SDC medium. Cells grown in SDC medium were collected at each time point, and cell lysates were subjected to Western blotting. (E) Western blot analysis of C-terminal fragment of Sch9-5HA. Lysates of VPS41 ∆gtr1 ∆gtr2 and ∆vps41 ∆gtr1 ∆gtr2 cells harboring empty vector (yet639, 755), GTR1WT GTR2WT (yet640, 756), GTR1GDP GTR2GTP (yet645, 761), or GTR1GTP GTR2GDP (yet647, 763) grown in SDC medium were subjected to Western blotting.
FIGURE 3:
FIGURE 3:
The HOPS complex is necessary for normal localization of Sch9. (A) Representative images of WT (yet120), ∆ego1 (yet691), and ∆tco89 (yet727) cells expressing Sch9-2GFP from the SCH9 promoter encoded on a centromeric plasmid. Cells at mid–log phase grown in SDC medium were stained with FM4-64 as a vacuolar marker. The signal intensities of Sch9-2GFP and FM4-64 along the indicated lines were measured by softWoRx software. Scale bar = 5 µm. (B) Represen­tative images of WT (yet120) and ∆vps41 (yet732) cells expressing Sch9-2GFP. Cells at mid–log phase grown in SDC medium were stained with CMAC as a vacuolar marker. Scale bar = 5 µm. (C) Represen­tative images of WT (SKY374-A) and ∆vps41 (yet665) cells expressing Tor1-GFP. Cells at mid–log phase grown in SDC medium were stained with CMAC as a vacuolar marker. Scale bar = 5 µm.
FIGURE 4:
FIGURE 4:
Disruption of HOPS complex decreases TORC1 output to the Sch9 branch by inducing Sch9 delocalization from vacuolar membranes. (A) Representative images of WT (yet234) and ∆vps41 (yet606) cells expressing GFP-FYVE-Sch9. Cells were analyzed during mid–log phase in SDC (+ uracil) medium. Scale bar = 5 µm. (B) Western blot analysis of the C-terminal fragment of Sch9-5HA. Lysates of WT (yet628, 629), and ∆vps41 (yet661, 662) cells in SDC (+ uracil) medium and/or WT cells treated with 200 ng/ml rapamycin + SDC (+ uracil) medium were subjected to Western blotting. GFP-Sch9 (yet628, 661) or GFP-FYVE-Sch9 (yet629, 662) were expressed from the endogenous SCH9 promoter. (C) Spot assay investigating growth recovery after rapamycin treatment in VPS41 GFPSCH9 (yet36), VPS41 GFP-FYVESCH9 (yet234), ∆vps41 GFPSCH9 (yet605), and ∆vps41 GFP-FYVESCH9 (yet606) cells. Cells in mid–log phase were cultured in the presence of absence of 200 ng/ml rapamycin for 3 h. The cell cultures were serially diluted 10-fold and then spotted onto YPD medium. Colonies were photographed after 1 d (YPD) or 3 d (Rap recovery).
FIGURE 5:
FIGURE 5:
Oxidative stress alters Sch9 localization and inhibits TORC1-Sch9 signaling. (A) Western blot analysis of the C-terminal fragment of Sch9-5HA (yet515) and Atg13 (yet562). Cells in mid–log phase were treated with SDC (control), SDC + 2 mM H2O2 (oxidative stress), SDC at 42°C (heat stress), SD−N (nitrogen depletion), or SDC + 200 ng/ml rapamycin (TORC1 inhibitor) for 30 min, and the lysates were subjected to Western blotting. (B) Western blot analysis of the C-terminal fragment of Sch9-5HA (yet515), Npr1-13myc (yet610), and Atg13 (yet562). Cells in mid–log phase were treated with 2 mM H2O2 in SDC, and the lysates were subjected to Western blotting at each time point. (C) Quantification of the band shift data of Npr1 and Sch9 from B (Npr1: n = 5, Sch9: n = 3). Error bars represent 95% confidence intervals. (D) Representative images of control or oxidative stress-induced (treated with 2 mM H2O2 for 30 min) cells expressing Sch9-2GFP and Ego3-3mCherry (yet765) during mid–log phase in SDC medium. Ego3-3mCherry was used as a vacuolar marker. Scale bar = 5 µm. (E) Quantification of the ratio of Sch9 fluorescence intensity of vacuolar membranes to that of the cytoplasm. Related to D. A ratio of 1.00 means no enrichment on vacuolar membranes. To calculate the p value, we applied the Brunner-Munzel test. *p < 0.005.
FIGURE 6:
FIGURE 6:
The localization of Sch9 is responsible for TORC1-dependent Sch9 phosphorylation on oxidative stress. (A) Representative images of control and oxidative stress-induced (treated with 2 mM H2O2 for 30 min) cells (yet234) expressing GFP-FYVE-Sch9 during mid–log phase in SDC (+ uracil) medium. Scale bar = 5 µm. (B) Western blot analysis of the C-terminal fragment of Sch9-5HA. Cells expressing Sch9-5HA (yet628) or FYVE-Sch9-5HA (yet629) were exposed to oxidative stress (2 mM H2O2) or exposed to oxidative stress and treated with rapamycin (200 nM) during mid–log phase in SDC medium, and the lysates were subjected to Western blotting at each time point. (C) Quantification of the band shift data of Sch9 and FYVE-Sch9 from B (n = 3). Error bars represent 95% confidence intervals. (D) Representative images of control and oxidative stress-induced (treated with 2 mM H2O2 for 30 min) cells (yet857) expressing Vac8-GFP-cSch9-5HA (cSch9 = C-terminal 116 amino acids of Sch9) during mid–log phase in SDC (+ uracil) medium. Scale bar = 5 µm. (E) Western blot analysis of Vac8-cSch9-5HA. Cells expressing Vac8-cSch9-5HA (yet859) were exposed to oxidative stress (2 mM H2O2) or exposed to oxidative stress and treated with rapamycin (200 nM) during mid–log phase in SDC medium, and the lysates were subjected to Western blotting at each time point. (F) Quantification of the band shift data of Vac8-cSch9 from E (n = 3). Error bars represent 95% confidence intervals.
FIGURE 7:
FIGURE 7:
Subcellular localization of PI(3,5)P2 localization is altered in HOPS mutants and under oxidative stress conditions. (A) Representative images of WT (yet681), ∆vps41 (yet709), and ∆vac7 (yet782) cells expressing GFP-Atg18 at mid–log phase in SDC (+ uracil) medium. Scale bar = 5 µm. (B) Representative images of control and oxidative stress-induced (treated with 2 mM H2O2 for 30 min) cells (yet777) expressing GFP-Atg18 and Ego3-3mCherry during mid–log phase in SDC (+ uracil) medium. Scale bar = 5 µm. (C) Quantification of the ratio of Atg18 fluorescence intensity of vacuolar membranes to that of the cytoplasm. Related to B. (D) Representative images of control and oxidative stress-induced (treated with 2 mM H2O2 for 30 min) cells (yet793) expressing Fab1-GFP and Ego3-3mCherry during mid–log phase in SDC (+ uracil) medium. Scale bar = 5 µm. (E) Quantification of the ratio of Fab1 fluorescence intensity of vacuolar membranes to that of the cytoplasm. Related to D. In C and E, we applied the Brunner-Munzel test to calculate p values. *p < 0.005. (F) Representative images of control and oxidative stress-induced (treated with 2 mM H2O2 for 30 min) cells (yet838) expressing GFP-FYVE and mCherry-Atg18 during mid–log phase in SDC (+ uracil) medium. Scale bar = 5 µm.
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