Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae
Fig 1
BCATs and KIC activate TORC1 signaling.
(a) Branched-chain amino acid metabolism in S. cerevisiae. (b) TORC1 kinase activity of WT cells is stimulated by addition of leucine, α-ketoisocaproate (KIC), or dimethyl α-ketoglutarate (d-KG) following leucine starvation. Cells were grown in SC-his-ura-lys+gln to OD600nm~1, and protein extracts were prepared from the following conditions: no treatment (control), incubation with 200 nM rapamycin for 30 min (+rapa), 2 hr leucine starvation, or addition of 2 mM leucine, KIC, isovaleraldehyde (IVA), isoamyl alcohol (IAA), isoleucine, α-keto-β-methylvalerate (K-ile), valine, α-ketoisovalerate (K-val), glutamate, or d-KG, for 1 hr to cells that have been leucine-starved for 2 hr. (b, d-f) TORC1 activity was assessed by monitoring the phosphorylation status of Sch9 Thr737 and the overall protein levels of Sch9 by Western blot with the anti-phospho-Thr737-Sch9 (Sch9-P) and anti-732-743-Sch9 (Sch9) antibodies, respectively. Where shown, Sch9 phosphorylation was normalized to Sch9 levels and expressed as an average percentage of WT Sch9 phosphorylation from three independent experiments, with error bars depicting the standard deviation from the mean. (c) bat1 bat2 mutant strains are rapamycin (rapa) sensitive and recover poorly from rapamycin-induced growth arrest. Strains were grown to OD600nm ~1 in SC-his-ura-lys+gln (top panel) or YPD (bottom panel). To test recovery from rapamycin-induced growth arrest, strains were incubated for 6 hr with 200 nM rapamycin, washed, serially diluted 5-fold and plated on drug-free media as indicated. Aliquots of untreated cultures were similarly diluted and plated to media +/- rapamycin. Plates were incubated for 2 to 5 days as indicated. (d) TORC1 kinase activity is not reduced by aro10 thi3 mutation. (e) TORC1 activity is reduced in bat1 bat2 double mutants compared with bat1 or bat2 single mutants. (f) Leucine or KIC addition to leucine-starved bat1 bat2 mutants stimulates TORC1 activity, although at a reduced level compared to WT. All experiments were performed in triplicate except (b), (e), and (f), which were performed in duplicate.