Figure 7. Bmh1/2 is required for Snf1... | Rockefeller University Press

Figure 7.

$Bmh1/2 is required for Snf1 recruitment to the PAS by regulating the association of Atg11/Atg1 with Snf1 under glucose starvation conditions. (A) Cells co-expressing Bmh1-3✕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. (B) Cells co-expressing Bmh1-3✕FLAG and HA-Snf1 in WT, atg1∆, or atg11∆ yeast strains were cultured in SD-G 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. (C) Cells co-expressing Bmh1-3✕FLAG and HA-Atg11 in WT or snf1∆ yeast strain 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. (D) WT and bmh1∆ Bmh2-3✕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 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. (E) Cells co-expressing Atg1-3✕FLAG and Snf1-GFP in wild-type and bmh1∆ Bmh2-3✕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 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-GFP antibody. The data are representative of three independent experiments. (F) Gel filtration chromatography experiment using a Superose 6 10/300 GL column for proteins lysed from the indicated yeast strain after 1 h of glucose starvation. 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) Wild-type (BY4741) or bmh1∆ Bmh2-3✕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. (H) Snf1 levels in the PAS fractions from G were quantified. Data are presented as means ± SD (n = 3). ***P < 0.001; **P < 0.01; two-tailed Student’s t tests were used. Source data are available for this figure: SourceData F7.$

Bmh1/2 is required for Snf1 recruitment to the PAS by regulating the association of Atg11/Atg1 with Snf1 under glucose starvation conditions. (A) Cells co-expressing Bmh1-3✕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. (B) Cells co-expressing Bmh1-3✕FLAG and HA-Snf1 in WT, atg1∆, or atg11∆ yeast strains were cultured in SD-G 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. (C) Cells co-expressing Bmh1-3✕FLAG and HA-Atg11 in WT or snf1∆ yeast strain 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. (D) WT and bmh1∆ Bmh2-3✕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 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. (E) Cells co-expressing Atg1-3✕FLAG and Snf1-GFP in wild-type and bmh1∆ Bmh2-3✕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 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-GFP antibody. The data are representative of three independent experiments. (F) Gel filtration chromatography experiment using a Superose 6 10/300 GL column for proteins lysed from the indicated yeast strain after 1 h of glucose starvation. 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) Wild-type (BY4741) or bmh1∆ Bmh2-3✕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. (H) Snf1 levels in the PAS fractions from G were quantified. Data are presented as means ± SD (n = 3). ***P < 0.001; **P < 0.01; two-tailed Student’s t tests were used. Source data are available for this figure: SourceData F7.