Figure S4. Calcium signaling is required... | Rockefeller University Press

Figure S4.

$Calcium signaling is required for glucose starvation–induced autophagy and the interaction between Atg11 and Bmh1/2 and Snf1-Bmh2 interaction is independent of Atg1 or Atg11. (A and B) Yeast cells expressing jGCaMP7f were treated with DMSO, 10 mM EGTA, or 20 mM EGTA for 1 h, and then cultured in nitrogen starvation medium (SD-N) (A) or glucose starvation medium (SD-G) (B) in the presence of DMSO, 10 mM EGTA, or 20 mM EGTA. Cells were observed using a fluorescence-inverted microscope (Olympus IX83). Images were captured at 4-s intervals using time-lapse microscopy and shown in 24 fps movies. ImageJ software was used to calculate the relative fluorescence intensity of cells to reflect cytoplasmic calcium signaling. The data are representative of three independent experiments. (C) Wild-type (WT) or atg1∆ yeast cells expressing GFP-Atg8 were treated with or without 20 mM EGTA for 1 h. Subsequently, they were treated with rapamycin for 4 h, with or without 20 mM EGTA. Autophagic activity was assessed by Western blotting to detect cleavage of GFP-Atg8, with Pgk1 serving as a loading control. The data are representative of three independent experiments. (D) Cells expressing Cmd1-3✕HA-AID were treated with 0.5 mM IAA for 2 h, and the expression levels of Cmd1 protein were detected by using Anti-HA antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (E) Cells expressing GFP-Atg8 in WT, atg1∆, or Cmd1-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in SD-N or SD-G in the presence of IAA for 4 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (F) Cells from E were quantified for the vacuolar localization of GFP-Atg8. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. (G and H) Co-expressing HA-Atg11 and Bmh1-3✕FLAG(G) or Bmh2-3✕FLAG(H) yeast strains were treated with or without 20 mM EGTA for 1 h, and then cultured in nutrient-rich medium, nutrient-rich medium with rapamycin, nitrogen starvation medium, or glucose starvation medium in the presence or absence of EGTA for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (I) Cells co-expressing Bmh2-3✕FLAG and HA-Snf1 were grown to the log-growth phase and then subjected to glucose starvation for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (J) Cells co-expressing Bmh2-3✕FLAG and HA-Atg11 in WT, atg1∆, or atg11∆ yeast strains were cultured in a glucose starvation medium for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (K) Cells co-expressing Bmh2-3✕FLAG and HA-Atg11 in WT or snf1∆ yeast strains were grown to the log-growth phase and then subjected to glucose starvation for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (L) Wild-type (BY4741) or atg11∆ atg17∆ yeast cells expressing Snf1-GFP were cultured in glucose starvation medium for 1 h. Cell lysates were analyzed by gel filtration chromatography using a Superose 6 10/300 GL column. Each fraction was detected by immunoblotting using the indicated antibodies. The red dashed box represents PAS fractions. The data are representative of three independent experiments. Source data are available for this figure: SourceData FS4.$

Calcium signaling is required for glucose starvation–induced autophagy and the interaction between Atg11 and Bmh1/2 and Snf1-Bmh2 interaction is independent of Atg1 or Atg11. (A and B) Yeast cells expressing jGCaMP7f were treated with DMSO, 10 mM EGTA, or 20 mM EGTA for 1 h, and then cultured in nitrogen starvation medium (SD-N) (A) or glucose starvation medium (SD-G) (B) in the presence of DMSO, 10 mM EGTA, or 20 mM EGTA. Cells were observed using a fluorescence-inverted microscope (Olympus IX83). Images were captured at 4-s intervals using time-lapse microscopy and shown in 24 fps movies. ImageJ software was used to calculate the relative fluorescence intensity of cells to reflect cytoplasmic calcium signaling. The data are representative of three independent experiments. (C) Wild-type (WT) or atg1∆ yeast cells expressing GFP-Atg8 were treated with or without 20 mM EGTA for 1 h. Subsequently, they were treated with rapamycin for 4 h, with or without 20 mM EGTA. Autophagic activity was assessed by Western blotting to detect cleavage of GFP-Atg8, with Pgk1 serving as a loading control. The data are representative of three independent experiments. (D) Cells expressing Cmd1-3✕HA-AID were treated with 0.5 mM IAA for 2 h, and the expression levels of Cmd1 protein were detected by using Anti-HA antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (E) Cells expressing GFP-Atg8 in WT, atg1∆, or Cmd1-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in SD-N or SD-G in the presence of IAA for 4 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (F) Cells from E were quantified for the vacuolar localization of GFP-Atg8. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. (G and H) Co-expressing HA-Atg11 and Bmh1-3✕FLAG(G) or Bmh2-3✕FLAG(H) yeast strains were treated with or without 20 mM EGTA for 1 h, and then cultured in nutrient-rich medium, nutrient-rich medium with rapamycin, nitrogen starvation medium, or glucose starvation medium in the presence or absence of EGTA for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (I) Cells co-expressing Bmh2-3✕FLAG and HA-Snf1 were grown to the log-growth phase and then subjected to glucose starvation for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (J) Cells co-expressing Bmh2-3✕FLAG and HA-Atg11 in WT, atg1∆, or atg11∆ yeast strains were cultured in a glucose starvation medium for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (K) Cells co-expressing Bmh2-3✕FLAG and HA-Atg11 in WT or snf1∆ yeast strains were grown to the log-growth phase and then subjected to glucose starvation for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (L) Wild-type (BY4741) or atg11∆ atg17∆ yeast cells expressing Snf1-GFP were cultured in glucose starvation medium for 1 h. Cell lysates were analyzed by gel filtration chromatography using a Superose 6 10/300 GL column. Each fraction was detected by immunoblotting using the indicated antibodies. The red dashed box represents PAS fractions. The data are representative of three independent experiments. Source data are available for this figure: SourceData FS4.