Figure S5.
Glc7 is required for the binding Bmh1/2 with Snf1 and Atg1 puncta formation under glucose starvation conditions, and overexpression of Bmh1 or Bmh2 does not affect nitrogen starvation-induced autophagy. (A) Cells co-expressing Bmh2-3✕FLAG and Sip1-3✕HA were grown 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. (B) Glc7-3✕HA-AID yeast strains were treated with 0.5 mM IAA for 2 h, and the expression levels of Glc7 protein were detected using anti-HA antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (C) The indicated yeast strains, either untreated or treated with IAA for 3 days, were plated in fourfold serial dilution onto YPD plates and incubated at 30°C. (D) Cells co-expressing Bmh2-3✕FLAG and HA-Snf1 in WT or Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then were 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 co-expressing Glc7-3✕HA and Snf1-GFP were treated with glucose starvation for 0 h or 1 h. Cell lysates were immunoprecipitated with anti-GFP agarose beads and analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (F and G) Cells co-expressing Glc7-3✕HA and Bmh1-3✕FLAG (F) or Bmh2-3✕FLAG(G) were treated with glucose starvation for 0 h or 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (H) Cells expressing GFP-Snf1 in WT or Glc7-3✕HA-AID yeast strains were treated with DMSO or IAA for 2 h, and then cultured in glucose starvation medium in the absence or presence of IAA for 0.5 h or 1 h. The activity of Snf1 was detected by immunoblotting with anti-phospho-AMPKα (Thr172) antibody. The data are representative of three independent experiments. (I) Cells co-expressing an empty vector or 3✕HA-Bmh2 with the Cup1 promoter and GFP-Atg8 in a wild-type strain were treated with or without CuSO4 for 2 h, and then cultured in glucose starvation medium in the absence or presence of CuSO4 for 0, 1, 2, or 4 h. Autophagic activity were analyzed by Western blot for the cleavage of GFP-Atg8. Pgk1 served as a loading control. (K and M) Cells co-expressing an empty vector or 3✕HA-Bmh1(K)/2(M) with the Cup1 promoter and GFP-Atg8 in a wild-type strain were treated with or without CuSO4 for 2 h, and then cultured in nitrogen starvation medium in the absence or presence of CuSO4 for 0, 1, 2, or 4 h. Autophagic activity were analyzed by Western blot for the cleavage of GFP-Atg8. Pgk1 served as a loading control. (J, L, and N) Quantification of the ratio of free GFP/GFP+GFP-Atg8 from I, K, and M (n = 3). Data are presented as means ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. (O) Atg1-3✕FLAG Cells expressing an empty vector or 3✕HA-Bmh2 with the Cup1 promoter were treated with or without CuSO4 for 2 h and then cultured in glucose starvation medium in the absence or presence of CuSO4 for 0 or 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-p-T226-Atg1 antibody. (P) The phosphorylation level of Atg1 T226 from O was quantified by calculating the ratio of p-Atg1-T226/FLAG-Atg1 (n = 3). Data are presented as mean ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. Source data are available for this figure: SourceData FS5.

Glc7 is required for the binding Bmh1/2 with Snf1 and Atg1 puncta formation under glucose starvation conditions, and overexpression of Bmh1 or Bmh2 does not affect nitrogen starvation-induced autophagy. (A) Cells co-expressing Bmh2-3✕FLAG and Sip1-3✕HA were grown 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. (B) Glc7-3✕HA-AID yeast strains were treated with 0.5 mM IAA for 2 h, and the expression levels of Glc7 protein were detected using anti-HA antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (C) The indicated yeast strains, either untreated or treated with IAA for 3 days, were plated in fourfold serial dilution onto YPD plates and incubated at 30°C. (D) Cells co-expressing Bmh2-3✕FLAG and HA-Snf1 in WT or Glc7-3✕HA-AID yeast strains were treated with IAA for 2 h and then were 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 co-expressing Glc7-3✕HA and Snf1-GFP were treated with glucose starvation for 0 h or 1 h. Cell lysates were immunoprecipitated with anti-GFP agarose beads and analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (F and G) Cells co-expressing Glc7-3✕HA and Bmh1-3✕FLAG (F) or Bmh2-3✕FLAG(G) were treated with glucose starvation for 0 h or 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (H) Cells expressing GFP-Snf1 in WT or Glc7-3✕HA-AID yeast strains were treated with DMSO or IAA for 2 h, and then cultured in glucose starvation medium in the absence or presence of IAA for 0.5 h or 1 h. The activity of Snf1 was detected by immunoblotting with anti-phospho-AMPKα (Thr172) antibody. The data are representative of three independent experiments. (I) Cells co-expressing an empty vector or 3✕HA-Bmh2 with the Cup1 promoter and GFP-Atg8 in a wild-type strain were treated with or without CuSO4 for 2 h, and then cultured in glucose starvation medium in the absence or presence of CuSO4 for 0, 1, 2, or 4 h. Autophagic activity were analyzed by Western blot for the cleavage of GFP-Atg8. Pgk1 served as a loading control. (K and M) Cells co-expressing an empty vector or 3✕HA-Bmh1(K)/2(M) with the Cup1 promoter and GFP-Atg8 in a wild-type strain were treated with or without CuSO4 for 2 h, and then cultured in nitrogen starvation medium in the absence or presence of CuSO4 for 0, 1, 2, or 4 h. Autophagic activity were analyzed by Western blot for the cleavage of GFP-Atg8. Pgk1 served as a loading control. (J, L, and N) Quantification of the ratio of free GFP/GFP+GFP-Atg8 from I, K, and M (n = 3). Data are presented as means ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. (O) Atg1-3✕FLAG Cells expressing an empty vector or 3✕HA-Bmh2 with the Cup1 promoter were treated with or without CuSO4 for 2 h and then cultured in glucose starvation medium in the absence or presence of CuSO4 for 0 or 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-p-T226-Atg1 antibody. (P) The phosphorylation level of Atg1 T226 from O was quantified by calculating the ratio of p-Atg1-T226/FLAG-Atg1 (n = 3). Data are presented as mean ± SD. ***P < 0.001; ns, no significance; two-tailed Student’s t tests were used. Source data are available for this figure: SourceData FS5.

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