Fig. 5: SESAME-catalyzed H3pT11 prevent the nuclear export of Sir2 and autophagy-mediated Sir2 degradation.
From: Metabolic regulation of telomere silencing by SESAME complex-catalyzed H3T11 phosphorylation
a Representative fluorescence images showing the distribution of Sir2-2xGFP (green) in WT and H3T11A cells expressing Sir2-2xGFP from the native SIR2 locus. The nucleus DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI) as shown in blue. There was more Sir2 diffuse throughout the cell in H3T11A mutant compared with WT. Arrows indicate the nucleus-localized Sir2 in WT and cytoplasm-localized Sir2 H3T11A mutant, respectively. Bar, 10 μm. b Quantification of Sir2-2xGFP localization in cells displayed in Fig. 5a, c, respectively. The bar graphs represent the percentages of cells exhibiting Sir2-2xGFP localized in the nucleus (Nucleus only), or exported to the cytoplasm (Nucleus + Cyto). Data show mean ± SE from at least three biological independent experiments, with ∼350 cells counted for each strain per experiment. c Knockdown of CRM1 expression retained most Sir2 in the nucleus. WT TetO7 and TetO7-CRM1 cells grown in YPD medium were treated with 6.25 μg/ml doxycycline for 1.5 h followed by fluorescence microscopy. Bar, 10 μm. d Knockdown of CRM1 expression partly rescued rapamycin-reduced Sir2. WT TetO7 and TetO7-CRM1 cells grown in YPD medium were treated with 6.25 μg/ml doxycycline and rapamycin for 0–1.5 h. e The global Sir2 protein levels in H3T11A mutant were significantly lower than those in WT when treated with rapamycin. f, g Rapamycin-reduced Sir2 in H3T11A mutant was partly rescued by PMSF (f) and CQ (g) treatments. h Western blots analysis of Sir2 and H3pT11 in WT and H3T11A mutant when aged for 0–6 days in YPD medium. i Western blots showing deletion of ATG2 rescued the reduced Sir2 in H3T11A and pyk1-ts mutants. For WT, atg2Δ, pyk1-ts, and pyk1-ts atg2Δ mutants, these four strains were treated at 37 °C for 2 h. For Fig. 5b, d, e, h, i, the quantitative data represent means ± SE; n = 3 biological independent experiments. Statistical significance was tested using two-sided Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001. For Fig. 5f, g, shown are the typical example of three biological independent experiments.
