Snf1/AMPK fine-tunes TORC1 signaling in response to glucose starvation
- Department of Biology, University of Fribourg, Switzerland
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, Italy
- Institute of Biochemistry and Molecular Biology, Faculty of Medicine, University of Freiburg, Germany
- Functional Biology, KU Leuven, Belgium
- Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Germany
Figures
Figure 1 with 1 supplement
Snf1 is required for proper downregulation of TORC1 in glucose-starved cells.
(A, B) Wild-type (WT), snf1Δ, and snf1as (analog-sensitive) cells were grown exponentially (Exp) and starved for glucose for 10 min (-C) in the absence (-; DMSO vehicle control) or presence (+) of 2NM-PP1. Phosphorylation of the bona fide TORC1 target residue Thr737 in Sch9 and of Thr210 in Snf1 was detected by immunoblot analyses of whole cell extracts using phospho-specific antibodies against the respective phospho-residues. Anti-Sch9 and anti-His6 antibodies served to detect the levels of Sch9 and Snf1, respectively (A). Notably, binding of 2NM-PP1 to the catalytic cleft of Snf1as inhibits its kinase activity and, at the same time, prevents the dephosphorylation of phosphorylated Thr210 (pThr210) in Snf1 (Chandrashekarappa et al., 2013). The mean TORC1 activities (i.e. Sch9-pThr737/Sch9) were quantified, normalized relative to exponentially growing WT cells (set to 1.0), and shown in the bar diagram in (B) (n=6; + SEM; unpaired Student’s t-test, ***≤0.0005). (C, D) Analog-sensitive snf1as cells were treated as in (A), but harvested at the times indicated following glucose starvation (-C). The respective relative TORC1 activities were assessed as in (B), but cross-normalized (for each of the two sets of blots) to the same sample from exponentially growing cells (lane 1; Exp), and are shown in (D) (n=4; + SEM; unpaired Student’s t-test, *p≤0.05, **p≤0.005). (E) WT and reg1∆ cells were grown exponentially and assayed for their mean relative TORC1 activities (Sch9-pThr737/Sch9) and Snf1-Thr210 phosphorylation levels (Snf1-pThr210/Snf1), each normalized to WT cells (set to 1.0; n=4; ± SEM). In unpaired Student’s tests, both values in reg1∆ cells were significantly different from the ones in WT cells (***p≤0.0005, ****p≤0.00005). (F) Exponentially growing cells (of the indicated genotype) were 10-fold serially diluted, spotted on synthetic complete medium containing, or not (control), 3 nM rapamycin, and grown for 3 days at 30 °C (n=3). The online version of this article includes the following source data for Figure 1—source data 1, quantification of blots for graphs shown in (B, D and E); Figure 1—source data 2, uncropped blots shown in (A, C and E); Uncropped dropspots shown in (F); Figure 1—source data 3, raw blots shown in (A, C and E) and raw dropspots shown in (F).
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Figure 1—source data 1
Quantification of blots for graphs shown in B, D, E.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig1-data1-v2.xlsx
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Figure 1—source data 2
Uncropped blots shown in A, C, E and uncropped dropspots shown in F.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig1-data2-v2.pdf
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Figure 1—source data 3
Raw blots shown in A, C, E and raw dropspots shown in F.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig1-data3-v2.zip
Figure 1—figure supplement 1
Specificity of 2NM-PP1 and differential Snf1 activation upon nitrogen and carbon starvation.
(A) 2NM-PP1 treatment inhibits Snf1as activity in vivo. WT and ATP-analogue-sensitive snf1as cells expressing a plasmid-encoded synthetic reporter of Snf1 activity that is based on a rat ACC1 peptide (ACC1-GFP; Deroover et al., 2016) were grown exponentially (Exp) and then starved for 2, 5, and 15 min for glucose (-C) and treated with vehicle (-; DMSO) or 2NM-PP1 (+). Immunoblot analyses were performed as in Figure 1A, except that anti-GFP and anti-ACC1-pSer79 were additionally used to detect the levels of ACC1-GFP and the phosphorylation state of the Snf1/AMPK target residue in ACC1-GFP that corresponds to Ser79 in rat ACC1 (n=3). (B, C) Activation of Snf1 following glucose or nitrogen starvation. Wild-type cells (as in (A)) were grown exponentially (Exp), or starved for nitrogen (-N; 10 min), or glucose (-C; 10 min) and subjected to immunoblot analyses (B). The mean relative TORC1 (Sch9-pThr737/Sch9) and Snf1 activities (i.e. Snf1-pThr210/Snf1 and ACC1-GFP-pSer79/ACC1-GFP) were quantified, normalized relative to exponentially growing WT cells (set to 1.0), and shown in the bar diagrams in (C) (n=3; + SEM; unpaired Student’s t-test, *≤0.05, **p≤0.005, ***p≤0.0005, ****p≤0.00005). The online version of this article includes the following source data for Figure 1—figure supplement 1—source data 1, quantification of blots for graphs shown in (C); in Figure 1—figure supplement 1—source data 2, uncropped blots shown in (A and B); Figure 1—figure supplement 1—source data 3, raw blots shown in (A and B).
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Figure 1—figure supplement 1—source data 1
Quantification of blots for graphs shown in C.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig1-figsupp1-data1-v2.xlsx
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Figure 1—figure supplement 1—source data 2
Uncropped blots shown in A, B.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig1-figsupp1-data2-v2.pdf
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Figure 1—figure supplement 1—source data 3
Raw blots shown in A, B.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig1-figsupp1-data3-v2.zip
Figure 2
Snf1 prevents transient TORC1 restimulation by glutamine in glucose- and glutamine-starved cells.
(A–F) Exponentially growing snf1as cells were starved for 20 min for nitrogen and glucose (-N, -C) and then restimulated for the times indicated, in the absence (DMSO) or presence of 2NM-PP1, with 2% glucose (Glc) (A, B), 2% glucose and 3.3 mM glutamine (Gln) (C, D), or 3.3 mM glutamine (E, F). Immunoblot analyses were performed as in Figure 1A and the relative TORC1 activities in (A), (C), and (E), were quantified as in Figure 1B and are shown in (B), (D), and (F), respectively (n=3; + SEM; unpaired Student’s t-test, *p≤0.05, ***p≤0.0005). The online version of this article includes the following source data for Figure 2—source data 1, quantification of blots for graphs shown in (B, D and F); Figure 2—source data 2, uncropped blots shown in (A, C and E); Figure 2—source data 3, raw blots shown in (A, C and E).
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Figure 2—source data 1
Quantification of blots for graphs shown in B, D and F.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig2-data1-v2.xlsx
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Figure 2—source data 2
Uncropped blots shown in A, C and E.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig2-data2-v2.pdf
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Figure 2—source data 3
Raw blots shown in A, C and E.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig2-data3-v2.zip
Figure 3 with 1 supplement
Quantitative phosphoproteomic analyses for the identification of potential Snf1 target sites.
(A) Quantitative MS-based proteomics workflow (SILAC n=5, OBIKA n=5). (B) Histogram of the number of identified phosphosites in the SILAC analysis after each filtering step and the relative amount of phosphosites attributed to classes I and II among the significant ones. (C) Proportional Venn diagram highlighting the commonly identified phosphosites in the current and two recent Snf1 phosphoproteomic studies. (D) Heatmap of phosphosite kinetics. Normalized SILAC ratios (treated versus untreated) of class I and class II sites (highlighted in B) were log2 transformed and z normalized prior unsupervised hierarchical clustering of the rows tree using Euclidean distance as matrix. Five major clusters a-e, each highlighted by a different color, are observed. (E) Motif analyses of Snf1 phosphosites identified by in vivo SILAC and OBIKA experiments as outlined in (A). (F) Protein-protein interaction network comprising 57 interconnected proteins out of the 98 where at least one Snf1-dependent phosphosite was shortlisted by the intersection of the SILAC and OBIKA analyses. The network was generated with Cytoscape using the STRING plugin, setting the confidence (score) cut-off at 0.25. Edge thickness represents the score value of each interaction. (G) Schematic model representing components of the Snf1/AMPK and TORC1 signaling pathways that contain Snf1-regulated phosphosites. Phosphosites attributed to class II are highlighted with a bold-pink outline. The three Snf1 β-subunits are represented with a dashed outline on the left. Solid and dashed arrows refer to direct and indirect activating interactions, respectively. Dashed lines with bars refer to potential (question marks) inhibitory interactions that are experimentally addressed in the indicated figures in this study. Source data for this figure are provided in Supplementary file 2.
Figure 3—figure supplement 1
Specific motif analyses of Snf1 phosphosites and role of Lst4 in TORC1 reactivation in glucose-starved, Snf1-inhibited cells.
(A, B) Motif analyses of Snf1 phosphosites identified by both in vivo SILAC and OBIKA experiments and only responding after 5 min (A) and 15 min (B). (C) Lst4 does not mediate TORC1 reactivation in glucose-starved, Snf1-inhibited cells. Exponentially growing snf1as or snf1as lst4∆ cells (Exp) were starved for glucose (-C; 10 min) in the presence of vehicle (-; DMSO) or 2NM-PP1 (+), and analyzed as in Figure 1A. The mean TORC1 activities (i.e. Sch9-pThr737/Sch9) were quantified, normalized relative to exponentially growing snf1as cells (set to 1.0), and shown below the Sch9 input blot (n=3; ± SEM). The online version of this article includes the following source data for Figure 3—figure supplement 1—source data 1, quantification of blots for values shown in (C); Figure 3—figure supplement 1—source data 2, uncropped blots shown in (C); Figure 3—figure supplement 1—source data 3, raw blots shown in (C).
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Figure 3—figure supplement 1—source data 1
Quantification of blots for values shown in C.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig3-figsupp1-data1-v2.xlsx
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Figure 3—figure supplement 1—source data 2
Uncropped blots shown in C.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig3-figsupp1-data2-v2.pdf
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Figure 3—figure supplement 1—source data 3
Raw blots shown in C.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig3-figsupp1-data3-v2.zip
Figure 4
Snf1 weakens the Pib2-Kog1 association by phosphorylating Pib2-Ser268,309.
(A) Schematic representation of the structure of Pib2 with the N-terminal TORC1-inhibitory (NID), Kog1-binding (Kog1-BD), phosphatidylinositol-3-phosphate (PI3P) -binding Fab1-YOTB-Vac1-EEA1 (FYVE), and C-terminal TORC1-activatory (CAD) domains (Hatakeyama, 2021). The residues Ser268 and Ser309 in Pib2 were both identified as potential Snf1 targets (P) in vivo (SILAC), while only Ser309 was recovered in our highly multiplexed on-beads in vitro kinase assays (OBIKA) with Snf1. (B, C) Snf1 controls the phosphorylation of Ser268 and Ser309 in Pib2 in vivo. Phosphorylation levels of Pib2-Ser268 (B) and Pib2-Ser309 (C) in untreated (DMSO) and 2NM-PP1-treated snf1as cells that were grown exponentially (Exp) and limited for glucose (0.05%) for the times indicated. Mean values were extracted from the SILAC experiment (Supplementary file 2 and Figure 3) and normalized to the ones in exponentially growing cells (set to 1.0) (n=3; + SD; unpaired Student’s t-test, *FDR ≤ 0.05). (D) Snf1 phosphorylates Ser268 and Ser309 in Pib2 in vitro. Snf1 (WT) and kinase-inactive Snf1T210A (TA) were purified from yeast and used in protein kinase assays with [γ-32P]-ATP and a bacterially-expressed fragment of Pib2 (Pib2221-635) lacking the N-terminal 220 amino acids that, according to our in vivo proteomics analyses, did not contain a potential Snf1 target residue. In parallel protein kinase assays, we also used the respective Pib2S268A, Pib2S309A, and Pib2S268A/S309A (Pib2SASA) mutant fragments as substrates. Substrate phosphorylation was detected by autoradiography (32P, lower panel) and Sypro Ruby (SyR) staining is shown as loading control for the Snf1 variants (upper panel) and the His6-tagged Pib2 fragments that were partially degraded and ran in more than 1 band (panel in the middle). The mean phosphorylation of Pib2S268A, Pib2S309A, and Pib2S268A/S309A (Pib2SASA) fragments by wild-type Snf1 (i.e. 32P signal/Sypro Ruby [SyR] substrate input level [including the indicated faster migrating proteolytic forms]; nd, not detected) was assessed relative to the one of the Pib2WT fragment (set to 1.0; n=3; ± SEM; unpaired Student’s t-test, ***p≤0.0005, ****p≤0.00005). (E) The Snf1 complex binds Pib2. The binding affinity between bacterially purified, titrated Pib2221-635 and yeast purified Snf1 complex (containing the C-terminally GFP-tagged Snf4 γ-subunit) was assessed by microscale thermophoresis. The dissociation constant (KD; 95% profile likelihood = 136–1098 nM, n=3; ± SEM) was calculated using a nonlinear asymmetric sigmoidal regression. (F, G) Expression of the phosphomimetic Pib2S268E,S309E allele, like loss of Pib2, rescues the TORC1 inactivation defect in glucose-starved, Snf1-compromised cells. Exponentially growing snf1as, snf1as pib2∆, snf1as pib2S268A,S309A (pib2SASA), and snf1as pib2S268E,S309E (pib2SESE) cells were grown exponentially (Exp) and then starved for 10 min for glucose (-C) in the absence (-; DMSO) or the presence (+) of 2NM-PP1. Immunoblot analyses of Sch9-pThr737 and Sch9 (left blots) and of Snf1as-pThr210 and Snf1as (right blots) were carried out as in Figure 1A (F). The mean relative TORC1 activities in the four strains were quantified as in Figure 1B and normalized to the values in exponentially growing snf1as cells (set to 1.0; n=4; + SEM; unpaired Student’s t-test, *p≤0.05, **p≤0.005, ***p≤0.0005) (G). (H) The phosphomimetic Pib2S268E,S309E is compromised for TORC1-binding. Kog1-HA3-expressing cells co-expressing Pib2-myc13 (WT), Pib2S368A,S309A-myc13 (SASA), Pib2S368E,S309E-myc13 (SESE), or untagged Pib2 (-) were grown exponentially. Lysates (input) containing 60 mM glutamine and anti-myc immunoprecipitates (IP: anti-myc) were analyzed by immunoblotting with anti-HA and anti-myc antibodies, respectively. The mean relative amount of Kog1-HA3 that was immunoprecipitated with Pib2-myc13 and its variants was determined and normalized to the one between Kog1-HA3 and Pib2-myc13 (set to 1.0; n=4; + SEM; unpaired Student’s t-test, *p≤0.05; nd, not detected). (I) Pib2-myc13 alleles are adequately expressed. Expression of Pib2-myc13 variants (as in F) was probed by immunoblot analysis in extracts of exponentially growing cells using anti-myc antibodies. Values were quantified relative to Adh1 levels (detected with anti-Adh1 antibodies) and normalized to the respective Pib2-myc13/Adh1 ratio in cells expressing the WT Pib2-myc13 (n=3; ± SEM; unpaired Student’s t-test; nd, not detected). The online version of this article includes the following source data for Figure 4—source data 1, data for the graph shown in (B, C and E) and quantifications of the blots in (D, H and I) and for the graph shown in (G); Figure 4—source data 2, uncropped blots, gels and autoradiographies shown in (D, F, H and I); Figure 4—source data 3, raw blots, gels and autoradiographies shown in (D, F, H and I).
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Figure 4—source data 1
Data for the graph shown in B, C and E and quantifications of the blots in D, H and I and for the graph shown in G.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig4-data1-v2.xlsx
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Figure 4—source data 2
Uncropped blots, gels and autoradiographies shown in D, F, H and I.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig4-data2-v2.pdf
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Figure 4—source data 3
Raw blots, gels and autoradiographies shown in D, F, H and I.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig4-data3-v2.zip
Figure 5 with 1 supplement
Snf1 phosphorylates Sch9-Ser288 to antagonize Sch9-Thr737 phosphorylation.
(A) Schematic representation of the structure of Sch9 with the C2 domain, the kinase domain including the T-loop, the turn motif, and the hydrophobic motif (HM). The position of the potential Snf1 target residue (i.e. phosphorylated [P] Ser288) and one of the 5 C-terminal TORC1 target residues (Urban et al., 2007; i.e. phosphorylated [P] Thr737 in the HM motif that is used to probe TORC1 activity here) are indicated. (B) Phosphoproteomic analyses identify Sch9-Ser288 as a potential Snf1 target. Mean phosphorylation levels of Sch9-Ser288 in untreated (DMSO) and 2NM-PP1-treated snf1as cells, grown exponentially (Exp) and limited for glucose (0.05% Glc) for the times indicated, were extracted from the SILAC experiment (Supplementary file 2 and Figure 3) and normalized to the ones in exponentially growing cells (set to 1.0; n=3; + SD; unpaired Student’s t-test, *FDR ≤0.05). (C) Snf1 phosphorylates Ser288 in Sch9 in vitro. Snf1 (WT) and kinase-inactive Snf1T210A (TA) were purified from yeast and used in protein kinase assays with [γ-32P]-ATP and the N-terminal fragment of Sch9 (encompassing the N-terminal 394 amino acids; Sch91-394) as substrate (WT). In parallel protein kinase assays, we also used the respective Sch91-394 fragment harboring the Ser288-to-Ala mutation as substrate (S288A). Substrate phosphorylation was detected by autoradiography (32P, lower panel) and Sypro Ruby (SyR) staining is shown as loading control for the Snf1 variants (upper panel) and the Sch9 fragments (panel in the middle). The mean phosphorylation of the Sch9S288A mutant fragment by wild-type Snf1 (i.e. 32P signal/Sypro Ruby [SyR] substrate input level; nd, not detected) was assessed relative to the one of the Sch9WT fragment (set to 1.0, n=3; ± SEM; unpaired Student’s t-test, **p≤0.05; nd, not detected). (D) Snf1 controls Sch9-Ser288 phosphorylation in vivo. Cells (i.e. snf1as, snf1as sch9S288A, and snf1as sch9S288E) were grown exponentially on 2% glucose (Exp) and then starved for glucose (-C) in the absence (DMSO) or presence of 2NM-PP1 for the times indicated (panels on the left). In parallel, cells were grown exponentially (Exp) on low glucose media (0.05%; panels on the right) and then treated with vehicle (DMSO) or 2-NMPP1 for the times indicated. The levels of Ser288 phosphorylation in Sch9 (Sch9-pSer288) and total levels of Sch9 were assayed by immunoblot analyses using anti-Sch9-pSer288 and anti-Sch9 antibodies, respectively (n=3). (E) Snf1 binds Sch9. The binding affinity between Sch91-394 (purified from yeast) titrated against the Snf1 complex (also purified from yeast and containing the C-terminally GFP-tagged Snf4 γ-subunit) was assessed by microscale thermophoresis. The dissociation constant (KD; 95% profile likelihood = 3.2–19.1 nM; n=3; ± SEM) was calculated using a nonlinear asymmetric sigmoidal regression. (F, G) The phosphomimetic Sch9S288E allele compromises proper phosphorylation of the C-terminal Thr737 residue in Sch9. snf1as, snf1as sch9S288A (sch9SA), and snf1as sch9S288E (sch9SE) cells were grown exponentially (Exp) and then starved for 10 min for glucose (-C) in the absence (-; DMSO) or the presence (+) of 2NM-PP1. Immunoblot analyses of Sch9-pThr737, Sch9, Snf1-pThr210, and Snf1 were carried out as in Figure 1A (F). The mean relative TORC1 activities in the three strains were quantified as in Figure 1B and normalized to the values in exponentially growing snf1as cells (set to 1.0; n=6; + SEM; unpaired Student’s t-test, *p≤0.05, **p≤0.005) (G). The online version of this article includes the following source data for Figure 5—source data 1, data for the graph shown in (B and E) and quantifications of the blots in (C) and for the graph shown in (G); Figure 5—source data 2, uncropped blots, gels and autoradiographies shown in (C, D and F); Figure 5—source data 3, raw blots, gels and autoradiographies shown in (C, D and F).
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Figure 5—source data 1
Data for the graph shown in B and E and quantifications of the blots in C and for the graph shown in G.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig5-data1-v2.xlsx
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Figure 5—source data 2
Uncropped blots, gels and autoradiographies shown in C, D and F.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig5-data2-v2.pdf
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Figure 5—source data 3
Raw blots, gels and autoradiographies shown in C, D and F.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig5-data3-v2.zip
Figure 5—figure supplement 1
Snf1 in vitro kinase assays.
The assays were performed as in Figure 5C, except for the use of cold ATP and the inclusion of a full-length kinase-dead Sch9 variant (Sch9KD: KD) as substrate. In addition, phosphorylation of Sch9-Ser288 and the levels of Sch9KD or Sch91-394 variants were assayed by immunoblot analyses using specific anti-Sch9-pSer288 (left blot) and anti-Sch9 (right blot) antibodies, respectively (n=2). Nomenclature: wild-type Snf1 complex: WT; kinase-inactive Snf1T210A complex: TA; N-terminal wild-type fragment of Sch9 encompassing the N-terminal 394 amino acids (Sch91-394): WT; and Sch91-394 harboring the Ser288-to-Ala mutation: SA. The online version of this article includes the following source data for Figure 5—figure supplement 1—source data 1, uncropped blots; Figure 5—figure supplement 1—source data 2, raw blots. .
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Figure 5—figure supplement 1—source data 1
Uncropped blots.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig5-figsupp1-data1-v2.pdf
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Figure 5—figure supplement 1—source data 2
Raw blots.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig5-figsupp1-data2-v2.zip
Figure 6
Physiological effects of Sch9S288E and Pib2S268E,S309E are additive.
(A, B) Exponentially growing (E) snf1as cells expressing the indicated combinations of wild type Pib2 (Pib2 variant: WT), Pib2S268A,S309A (Pib2 variant: SASA), and Pib2S268E,S309E (Pib2 variant: SESE) and wild type Sch9 (Sch9 variant: WT), Sch9288A (Sch9 variant: SA), and Sch9S288E (Sch9 variant: SE) were starved for glucose (10 min; -C) in the absence (-; DMSO; upper panels) and presence of 2NM-PP1 (+; lower panels). Immunoblot analyses were performed as in Figure 1A and the mean relative TORC1 activities (i.e. Sch9-pThr737/Sch9) were quantified, normalized relative to exponentially growing snf1as cells (set to 1.0), and shown in the bar diagram in (B) (n=4; + SEM; unpaired Student’s t-test, *p≤0.05, **p≤0.005, ***p≤0.0005). (C) Growth on media containing low nitrogen levels activates Snf1-Thr210 phosphorylation and decreases TORC1 activity. WT cells were grown exponentially on SC or low nitrogen medium and assayed for their mean relative TORC1 activities (Sch9-pThr737/Sch9) and Snf1-Thr210 phosphorylation levels (Snf1-pThr210/Snf1), each normalized to the values of cells grown on SC (set to 1.0; n=10; ± SEM). In unpaired Student’s tests, both values in cells growing on low nitrogen medium were significantly different form the ones growing on SC (**p≤0.005, ****p≤0.005). (D, E) Combined expression of Sch9S288E and Pib2S268E,S309E causes reduced Sch9-Thr737 phosphorylation (D) and enhanced rapamycin sensitivity (E). In (D), indicated strains were grown exponentially on low nitrogen medium and assayed for their mean TORC1 activities as in (C), which were normalized to the value of WT cells (set to 1.0; n=4; ± SEM; unpaired Student’s t-test, **p≤0.005). In (E), cells with the indicated genotypes were grown exponentially in SD, then 10-fold serially diluted, spotted on control plates (SD) or 2.5 nM rapamycin-containing plates, and grown for 3 days at 30 °C (n=4). The online version of this article includes the following source data for Figure 6—source data 1, quantifications of the blots in (C and D) and for graph shown in (B); Figure 6—source data 2, uncropped blots shown in (A, C and D); uncropped dropspots shown in (E); Figure 6—source data 3, raw blots shown in (A, C and D); raw dropspots shown in (E).
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Figure 6—source data 1
Quantifications of the blots in C and D and for graph shown in B.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig6-data1-v2.xlsx
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Figure 6—source data 2
Uncropped blots shown in A, C and D; uncropped dropspots shown in E.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig6-data2-v2.pdf
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Figure 6—source data 3
Raw blots shown in A and C.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig6-data3-v2.zip
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Figure 6—source data 4
Raw blots shown in D; raw dropspots shown in E.
- https://cdn.elifesciences.org/articles/84319/elife-84319-fig6-data4-v2.zip
Additional files
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Supplementary file 1
Strains, plasmids and oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/84319/elife-84319-supp1-v2.docx
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Supplementary file 2
Significantly in vivo and in vitro regulated phosphosites.
- https://cdn.elifesciences.org/articles/84319/elife-84319-supp2-v2.xlsx
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MDAR checklist
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Snf1/AMPK fine-tunes TORC1 signaling in response to glucose starvation
eLife 12:e84319.
https://doi.org/10.7554/eLife.84319
