Tripartite suppression of fission yeast TORC1 signaling by the GATOR1-Sea3 complex, the TSC complex, and Gcn2 kinase
- Department of Cellular Physiology, Niigata University Graduate School of Medical and Dental Sciences, Japan
- Division of Biological Science, Nara Institute of Science and Technology, Japan
- Faculty of Biology, Universitas Gadjah Mada, Indonesia
- Department of Microbiology and Molecular Genetics, University of California, Davis, United States
Figures
Figure 1 with 1 supplement
Identification of GATOR2 components as GATOR1-binding proteins.
(A) Affinity purification of the GATOR complex in fission yeast. The GATOR1 complex was purified from cells co-expressing FLAG-Npr2 and Iml1-myc, as well as those co-expressing FLAG-Npr3 and Iml1-myc…
Figure 1—figure supplement 1
The GATOR1 subunits Iml1, Npr2, and Npr3 are localized to vacuolar membranes.
(A–C) The indicated GFP-tagged strains were grown in EMM at 30°C for microscopy. Vacuolar membranes were visualized by the fluorescent dye FM4-64. Z-axial images were collected, and mid-section …
Figure 2
Sea3-GATOR1 attenuates TORC1 signaling via the Gtr1–Gtr2 Rag GTPases.
(A–E) Wild-type and the indicated mutant strains were grown in liquid EMM medium and their serial dilutions were spotted onto the plates. (A) Sea3 is required for normal cell growth. The indicated …
Figure 3 with 3 supplements
Sea3 connects the GATOR2 components to GATOR1.
(A–C) Sea3 mediates the physical interaction of other GATOR2 proteins with GATOR1. Crude lysates (input) were prepared from cells expressing one of the GATOR2 components tagged with myc, with (+) or …
Figure 3—figure supplement 1
The GATOR2 components are tethered to the vacuolar surface by GATOR1 complex through Sea3.
(A–E) Wild-type and the indicated mutant cells expressing fluorescence-tagged Sea2, Sea4, Seh1, or Sea3 were grown in EMM at 30°C for microscopy. Vacuolar membranes were visualized by the …
Figure 3—figure supplement 2
Sea3 is dispensable for the stability and vacuolar localization of GATOR1.
(A) The protein level of the GATOR1 components is not affected by the absence of Sea3. Wild-type and sea3∆ cells expressing myc-tagged Iml1, Npr2, or Npr3 were grown in EMM at 30°C for …
Figure 3—figure supplement 3
A conserved arginine in Npr2 is required for the GATOR1 function.
(A, C) The indicated strains were spotted onto solid YES medium with (Rapamycin) or without (–) 100 ng/ml rapamycin. Normal cell growth requires Arg98 of Npr2, but not Arg854 of Iml1. The expression …
Figure 4 with 1 supplement
The Gcn2 signaling pathway regulates autophagy induction upon amino acid starvation.
(A) Nitrogen starvation-induced autophagy is reduced but not completely prevented in cells defective in the TSC complex (tsc2∆), GATOR1 (iml1∆), or both (tsc2∆ iml1∆). Autophagy was monitored by …
Figure 4—figure supplement 1
The Gcn2 signaling pathway is essential for autophagy induction upon amino acid starvation.
(A) The processing of GFP-Pgk1 depends on autophagy. Wild-type and the indicated mutant strains expressing GFP-tagged Pgk1 were grown in EMM at 30°C, followed by shifting to the same medium without …
Figure 5 with 1 supplement
The Gcn2 signaling pathway facilitates amino acid starvation-induced autophagy by attenuating TORC1 signaling.
(A) Inactivation of TORC1 upon leucine starvation is impaired in cells lacking Gcn2 or Fil1 and the mutant carrying the non-phosphorylated form of eIF2α. TORC1 activity was monitored by detecting …
Figure 5—figure supplement 1
The Gcn2 signaling pathway is not necessary for autophagy induction upon nitrogen starvation.
(A, B) Wild-type and the indicated mutant cells expressing Pgk1-GFP were grown in EMM at 30°C and shifted to the same medium without nitrogen source (–N). Cells were collected at the indicated time …
Figure 6 with 3 supplements
The Gcn2 signaling pathway negatively regulates TORC1 upon nitrogen starvation in the absence of the TSC complex and GATOR1.
(A–C) Gcn2, Tsc2, and Iml1 promote autophagy induction in parallel upon nitrogen starvation. Autophagy was monitored by detecting the GFP moiety processed from Pgk1-GFP. The indicated strains were …
Figure 6—figure supplement 1
The absence of the GATOR1 and TSC complexes during nitrogen starvation up-regulates phosphorylation of eIF2α.
(A–C) Cells lacking Iml1, Tsc2, or autophagic degradation exhibit highly phosphorylated eIF2α during nitrogen starvation. The indicated strains were grown in EMM at 30°C, followed by shifting to the …
Figure 6—figure supplement 2
Gcn2, GATOR1, and the TSC complex redundantly regulate TORC1.
(A, B) The kinetics of autophagy induction in cells expressing Iml1, Gcn2, or Tsc2 as a main TORC1 inhibitor. The indicated strains were grown in EMM at 30°C, followed by shifting to the same medium …
Figure 6—figure supplement 3
TORC1 inhibitors are involved in cell survival and sexual differentiation during nitrogen starvation.
(A) Iml1 and Tsc2 are involved in cell survival during long-term starvation. The indicated strains cultured in EMM at 30°C were shifted to the same medium without nitrogen source and incubated at …
-
Figure 6—figure supplement 3—source data 1
Source data for Figure 6—figure supplement 3B.
- https://cdn.elifesciences.org/articles/60969/elife-60969-fig6-figsupp3-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Schizosaccharomyces pombe) | Fission yeast strains used in this study | See Supplementary file 1 | See Supplementary file 1 | |
| Antibody | Anti-phospho-p70 S6K (Thr389) (mouse monoclonal) | Cell Signaling Technology | Cat# 9206; AB_2285392 | (1:4000) |
| Antibody | Anti-Psk1 (rabbit polyclonal) | DOI:10.7554/eLife.30880 | N/A | (1:5000) |
| Antibody | Anti-Spc1 (rabbit polyclonal) | DOI:10.1128/MCB.23.15.5132-5142.2003 | N/A | (1:10,000) |
| Antibody | Anti-FLAG (mouse monoclonal) | Sigma–Aldrich | Cat# F3165; RRID: AB_259529 | (1:4000) |
| Antibody | Anti-c-myc (mouse monoclonal) | Covance | Cat# MMS150P; RRID: AB_291322 | (1:4000) |
| Antibody | Anti-c-myc (rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-789; RRID: AB_631274 | (1:4000) |
| Antibody | Anti-GFP (mouse monoclonal) | Takara | Cat# 632380; RRID: AB_10013427 | (1:10,000) |
| Antibody | Anti-histone H3 (rabbit polyclonal) | Abcam | Cat# ab1791; RRID: AB_302613 | (1:10,000) |
| Antibody | Anti-phospho-eIF2alpha (Ser51) (rabbit polyclonal) | Cell Signaling Technology | Cat# 9721; RRID: AB_330951 | (1:2000) |
| Antibody | Anti-DDDDK (rabbit polyclonal) | MBL | Cat# PM020; RRID: AB_591224 | (1:10,000) |
| Antibody | Anti-mouse IgG, Peroxidase conjugated (goat polyclonal) | Merck Millipore | Cat# AP124P; RRID: AB_90456 | (1:10,000) |
| Antibody | Anti-rabbit IgG, Peroxidase conjugated (goat polyclonal) | Jackson ImmunoResearch | Cat# 111-035-003; RRID: AB_2313567 | (1:10,000) |
| Antibody | Anti-rabbit IgG, Peroxidase conjugated (goat polyclonal) | Promega | Cat# W4011; RRID: AB_430833 | (1:2000) |
| Antibody | Anti-mouse IgG Peroxidase conjugated (goat polyclonal) | Promega | Cat# W4021; RRID: AB_430834 | (1:2000) |
| Chemical compound, drug | R-5000 Rapamycin | LC Laboratories | Cat# 53123-88-9 | |
| Chemical compound, drug | Caffeine, anhydrous | Nacalai Tesque | Cat# 06712–42 | |
| Chemical compound, drug | Caffeine | Wako | Cat# 033–06791 | |
| Chemical compound, drug | SynaptoRed C2 (FM4-64) | Biotium | Cat# 70021 | |
| Chemical compound, drug | ViVidFluor Neuro Red (FM4-64) | FUJIFILM | Cat# 222–02121 | |
| Chemical compound, drug | L-Methioninesulfoximine | Nacalai Tesque | Cat# 21730–74 | |
| Chemical compound, drug | 3-Amino-1,2,4-triazole | Wako | Cat# 014–10911 | |
| Commercial assay or kit | SuperSignal West Pico Chemiluminescent Substrate | Thermo Scientific | Cat# 34080 | |
| Commercial assay or kit | SilverQuest staining kit | Invitrogen | Cat# LC6070 | |
| Commercial assay or kit | EzWestLumi plus | Atto | Cat# WSE-7120 | |
| Commercial assay or kit | Clarity Max Western ECL Substrate | Bio-Rad | Cat# 1705062 |
Additional files
-
Supplementary file 1
Fission yeast strains used in this study.
- https://cdn.elifesciences.org/articles/60969/elife-60969-supp1-v1.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/60969/elife-60969-transrepform-v1.docx
