Supplementary Figure 3: Identification and characterization of Palmitoylcarnitine (PalmC).
From: Decrease in plasma membrane tension triggers PtdIns(4,5)P2 phase separation to inactivate TORC2
(a) Principle of the high-throughput screen leading to the discovery of PalmC. A small molecule library was screened at 10µM on the WT and bypass strains, and compounds having a differential effect on the growth of both strains were selected for further characterization. (b) Comparison between the Normalized Percentage of Inhibition of growth (NPI) of the tested compounds towards the WT and the bypass strain. (c) Structure of PalmC. (d) The TORC2 bypass strain is resistant to PalmC. Strains were grown overnight at 30 °C, diluted to OD600=0.1, regrown into exponential phase and diluted to OD600=0.3. 3μL of 1/5 serial dilutions were then spotted onto plates with or without 20μM PalmC. This experiment was repeated three times with similar results. (e) Mutations affecting the HOG and/or CWI pathways do not alter the TORC2 response to PalmC. The indicated strains were tested for Ypk1 T662 phosphorylation at the indicated time points after PalmC treatment. (f) PalmC does not activate the HOG1 pathway. Hog1 phosphorylation was monitored at the indicated time points after PalmC treatment or hyper-osmotic shock using an anti-phospho-p38 antibody. (g) PalmC and hyper-osmotic shock affect PM tension in an additive manner. Phosphorylation of Ypk1 T662 was monitored in cells treated with a low concentration of PalmC, a low-magnitude hyper-osmotic shock, or a combination of both treatments, at the indicated time points. (h) The effect of PalmC on TORC2 does not require exocytosis. Phosphorylation of Ypk1 T662 was monitored at the indicated time points following PalmC treatment in WT cells or in sec18ts cells deficient for exocytosis27. (i) The effect of PalmC on TORC2 does not require endocytosis. Phosphorylation of Ypk1 T662 was monitored at the indicated time points following PalmC treatment in LatA-treated cells. All blots are representative of at least two independent experiments, and unprocessed scans are shown in Supplementary Figure 7.
