Fig. 5. Perturbations to Acc1 and FASN/Fas1 downregulate mTORC1 independently of key upstream regulators.
a, Immunoblots with lysates from the indicated yeast strains analysed as in Fig. 2a (n = 3 independent experiments). b, Immunoblots of the lysates of the indicated yeast strains assayed for TORC1 activity as in Fig. 2a. c, The level of phosphorylation at T737 of Sch9 (p-Sch9T737/Sch9) was used to assay the TORC1 activity levels in b (n = 6 independent experiments). d,e, Constitutive activation of Acc1 (acc1S1157A) does not alter vacuolar morphology or the subcellular localization of GFP–Tor1 (d) or GFP-Gtr1 (e). Vacuoles were stained with FM4-64. Scale bars, 5 μm; n = 3 independent experiments. f,g, Expression of the GTP-locked Gtr1Q65L allele does not suppress the TORC1 inhibition mediated by the Acc1S1157A allele. g, Levels of TORC1 activity (p-Sch9T737/Sch9; n = 6 independent experiments). h,i, FASN inhibition downregulates mTORC1 activity independently of the Rags. h, RagA/B-knockout and WT HEK293FT were treated with 25 μM Fasnall for the indicated times and their lysates were immunoblotted. i, Levels of mTORC1 activity (p-S6KT389/S6K) normalized to the respective DMSO-treated control (n = 4 independent experiments). j, RagA/B-knockout, RagC/D-knockout and WT HEK293FT cells were treated with 50 μM cerulenin for the indicated times (n = 4 independent experiments). k,l, RagA/B-knockout and WT HEK293FT were treated as in h but with FASN knockdown. Levels of mTORC1 activity (p-S6KT389/S6K) normalized to the respective DMSO-treated control (n = 4 independent experiments). m,n, FASN inhibition downregulates mTORC1 activity independently of the TSC complex. m, TSC1-knockout and WT HEK293FT cells were treated with 25 μM Fasnall (30 min) or amino-acid-starvation medium (AA; 1 h). n, Levels of mTORC1 activity (p-S6KT389/S6K) normalized to the respective DMSO-treated control (nFasnall = 6 (left) and nAA = 3 independent experiments (right)). o,p, TSC1-knockout and WT HEK293FT cells were treated with cerulenin (50 μM, 4 h) as in m,n. p, Levels of mTORC1 activity (p-S6KT389/S6K) normalized to the respective DMSO-treated control (n = 3 independent experiments). q,r, TSC1-knockout and WT HEK293FT cells were treated as in m,n but with FASN knockdown. Levels of mTORC1 activity (p-S6KT389/S6K) normalized to the respective control knockdown (n = 4 independent experiments). c,g,i,l,n,p,r, Data are the mean ± s.e.m. *P < 0.05; **P < 0.005; ***P < 0.0005; and NS, not significant. Ceru, cerulenin; and KO, knockout. Source numerical data and unprocessed blots are provided.