Extended Data Fig. 2: Carboxy-terminal tagging of Acc1S1157A restrains its capacity to stimulate Mal-CoA levels and inhibit TORC1 in yeast.
From: Malonyl-CoA is a conserved endogenous ATP-competitive mTORC1 inhibitor
a,b, Carboxy-terminal GFP tagging of endogenous Acc1S1157A significantly reduces Mal-CoA levels. See Fig. 2a,b for details. a, Ponceau staining as loading control. b, Quantification of relative Mal-CoA levels (GFP/Ponceau signal); n = 3 independent experiments. c, Carboxy-terminal GFP tagging of endogenous Acc1S1157A partially reverts its ability to render cells rapamycin- and cerulenin-sensitive. In control experiments, C-terminal GFP tagging of endogenous Fas1 rendered WT cells cerulenin-sensitive and further enhanced the cerulenin sensitivity of acc1S1157A cells, while marginally affecting the rapamycin-sensitivity of the respective cells. Spot assays with the indicated yeast genotypes and rapamycin and/or cerulenin concentrations performed as in Fig. 1b; n = 3 independent experiments. d,e, Carboxy-terminal GFP tagging of endogenous Acc1S1157A suppresses its ability to inhibit TORC1 in exponentially growing and Gln-restimulated cells. d, TORC1 activities assessed as in Fig. 2d. e, Quantification of relative TORC1 activity (p-Sch9Thr737/Sch9). d, The samples derive from the same experiment and gels/blots were processed in parallel; n = 3 independent experiments. Data in graphs are shown as the mean ± s.e.m. *P < 0.05, **P < 0.005. Source numerical data and unprocessed blots are provided.
