Extended Data Fig. 1. Perturbations to Acc1 influence Mal-CoA levels and TORC1 activity in yeast cells.
a,b, Mal-CoA levels in WT and hyperactive acc1S1157A cells are significantly reduced by introduction of the hypomorphic acc1E392K mutation. Mal-CoA levels were assessed by GFP blots in cells grown at 24 °C (a) and quantified (b) as in Fig. 2a,b, respectively; n = 3 independent experiments. c,d, Pharmacogenetic interactions of rapamycin and cerulenin indicate a potential inhibitory role of Mal-CoA in TORC1 signalling. c, gtr1Δ and acc1S1157Agtr1Δ mutant strains containing an empty vector or expressing plasmid-encoded wild-type GTR1, gtr1Q65L or gtr1S20L were spotted and cultured on plates with the indicated concentrations of rapamycin and/or cerulenin. d, The respective gtr2Δ and acc1S1157Agtr2Δ mutant strains expressing plasmid-encoded wild-type GTR2, gtr2S23L or gtr2Q66L are shown; n = 3 independent experiments. e, The acc1E392K mutation causes temperature-sensitive growth even when combined with the acc1S1157A mutation. Exponentially growing cells with the indicated genotypes were spotted on plates (tenfold serial dilutions) and cultured for 3 d at 24 °C or 37 °C; n = 3 independent experiments. f,g, Mutation of E392K in acc1S1157A suppresses the TORC1-inhibitory effect of acc1S1157A. Immunoblots (f) and quantifications of TORC1 activity (p-Sch9Thr737/Sch9; g) were carried out as in Fig. 2d,e, respectively. f, The samples derive from the same experiment and gels/blots were processed in parallel; n = 4 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.