Research Paper Volume 11, Issue 19 pp 8418—8432

mTORC1-Sch9 regulates hydrogen sulfide production through the transsulfuration pathway

Figure 1. Deletion of SCH9 decreased H2S production in different yeast strains. (A) WT and Δsch9 cells in the TB50a background were transformed with pRS316-SCH9 or empty vector and inoculated into 1L of SDC medium at initial OD600nm=0.005. H2S production was monitored using lead acetate strips at indicated times (Upper 3 panels) after inoculation. The level of Sch9 protein and actin loading control were determined by Western blotting as shown in the lower 2 panels. (B) Millimeters of darkening of the lead acetate strips inserted into the headspace of the culture flask shown in panel A normalized by OD600nm. (C) Methylene blue assays of H2S produced by WT and Δsch9 cells in BY4741 or BY4742 background. Note that there is spontaneous oxidation of methylene blue when H2S is absent which gave negative readings for methylene reduction (red and blue dash lines). (D) Intracellular H2S production in WT and Δsch9 cells in BY4741 or BY4742 background monitored by H2S fluorescent with probe WSP-1. (* p<0.05; ** p<0.01; *** p<0.005). (E) H2S production by WT and Δsch9 cells in BY4742 background assayed by using lead acetate strips which were replaced every 24 hours under caloric restriction conditions (CR, medium containing 0.5% glucose) or no restriction (NR, medium containing 2% glucose).