Figure 8. Glc7-mediated Snf1 binding... | Rockefeller University Press

Figure 8.

$Glc7-mediated Snf1 binding with Bmh1/2 is required for glucose starvation–induced autophagy. (A) Flowchart illustrating the process of identifying candidate Snf1-binding partners among Bmh1-associated proteins under glucose starvation conditions. (B) Representative proteins identified by LC-MS from the experiment described in A. (C) Cells co-expressing Bmh1-3✕FLAG and Sip1-3✕HA were cultured to the log-growth phase and then subjected to glucose starvation for 15 min or 30 min. 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. (D) Cells co-expressing Bmh1-3✕FLAG and HA-Snf1 in WT and Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in nutrient-rich medium or glucose starvation medium in the presence of IAA for 0.5 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. (E) Cells expressing GFP-Atg8 and Vph1-Cherry in wild-type (WT), atg1∆, or Glc7-3✕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. Autophagic activity was analyzed by Western blot for GFP-Atg8 cleavage. The data are representative of three independent experiments. (F) Wild-type or Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in glucose starvation medium in the presence of IAA 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. (G) Cells expressing Atg1-GFP in WT or Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then were cultured in SD-N or SD-G in the presence of IAA for 1 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (H) Cells from G were quantified for the number of cells with Atg1 puncta. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. ***P < 0.001; NS, not significant; two-tailed Student’s t tests were used. (I) Cells expressing Atg1-3✕FLAG in WT or Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in glucose or nitrogen starvation medium in the presence of IAA for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-p-T226-Atg1 antibody. The data are representative of three independent experiments. Source data are available for this figure: SourceData F8.$

Glc7-mediated Snf1 binding with Bmh1/2 is required for glucose starvation–induced autophagy. (A) Flowchart illustrating the process of identifying candidate Snf1-binding partners among Bmh1-associated proteins under glucose starvation conditions. (B) Representative proteins identified by LC-MS from the experiment described in A. (C) Cells co-expressing Bmh1-3✕FLAG and Sip1-3✕HA were cultured to the log-growth phase and then subjected to glucose starvation for 15 min or 30 min. 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. (D) Cells co-expressing Bmh1-3✕FLAG and HA-Snf1 in WT and Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in nutrient-rich medium or glucose starvation medium in the presence of IAA for 0.5 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. (E) Cells expressing GFP-Atg8 and Vph1-Cherry in wild-type (WT), atg1∆, or Glc7-3✕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. Autophagic activity was analyzed by Western blot for GFP-Atg8 cleavage. The data are representative of three independent experiments. (F) Wild-type or Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in glucose starvation medium in the presence of IAA 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. (G) Cells expressing Atg1-GFP in WT or Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then were cultured in SD-N or SD-G in the presence of IAA for 1 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (H) Cells from G were quantified for the number of cells with Atg1 puncta. n = 300 cells were pooled from three independent experiments. Data are presented as means ± SD. ***P < 0.001; NS, not significant; two-tailed Student’s t tests were used. (I) Cells expressing Atg1-3✕FLAG in WT or Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in glucose or nitrogen starvation medium in the presence of IAA for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-p-T226-Atg1 antibody. The data are representative of three independent experiments. Source data are available for this figure: SourceData F8.