Figure 1. Snf1 is required for proper downregulation of TORC1 in glucose-starved cells.

(A, B) Wild-type (WT), snf1Δ, and snf1^as (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 Snf1^as 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 snf1^as 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).

Figure 1—figure supplement 1. Specificity of 2NM-PP1 and differential Snf1 activation upon nitrogen and carbon starvation.

(A) 2NM-PP1 treatment inhibits Snf1^as activity in vivo. WT and ATP-analogue-sensitive snf1^as 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).