Figure 2.
Bmh1/2 regulate glucose starvation–induced autophagy by governing the PAS recruitment and activation of Atg1. (A)bmh1∆ cells expressing Bmh2-3✕HA-AID were treated with 0.5 mM IAA for the indicated periods and the levels of Bmh2 protein were detected by using Anti-HA antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (B) The indicated yeast strains, either untreated or treated with IAA for 3 days, were plated in fourfold serial dilution onto YPD at 30°C. (C) Cells co-expressing GFP-Atg8 and Vph1-Cherry in wild-type or bmh1∆ Bmh2-3✕HA-AID yeast strains were treated with DMSO or IAA for 2 h and then cultured in SD-N, SD-G, or placed under rapamycin treatment in the absence or presence of IAA for the indicated periods. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (D) Cells from C 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. (E) Cells from C were analyzed by Western blot for the cleavage of GFP-Atg8. Pgk1 served as a loading control. The data are representative of three independent experiments. (F) Cells expressing Atg11, Atg17, or Atg1 fused with GFP tag in wild-type or bmh1∆ Bmh2-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in nitrogen starvation medium (SD-N) or glucose starvation medium (SD-G) in the presence of IAA for 1 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (G) Cells from F were quantified for the number of cells with the indicated ATG protein 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. (H) Cells expressing Atg1-3✕FLAG in WT or bmh1∆ Bmh2-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in SD-N, SD-G, or rapamycin treatment 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 F2.

Bmh1/2 regulate glucose starvation–induced autophagy by governing the PAS recruitment and activation of Atg1. (A) bmh1∆ cells expressing Bmh2-3✕HA-AID were treated with 0.5 mM IAA for the indicated periods and the levels of Bmh2 protein were detected by using Anti-HA antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (B) The indicated yeast strains, either untreated or treated with IAA for 3 days, were plated in fourfold serial dilution onto YPD at 30°C. (C) Cells co-expressing GFP-Atg8 and Vph1-Cherry in wild-type or bmh1∆ Bmh2-3✕HA-AID yeast strains were treated with DMSO or IAA for 2 h and then cultured in SD-N, SD-G, or placed under rapamycin treatment in the absence or presence of IAA for the indicated periods. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (D) Cells from C 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. (E) Cells from C were analyzed by Western blot for the cleavage of GFP-Atg8. Pgk1 served as a loading control. The data are representative of three independent experiments. (F) Cells expressing Atg11, Atg17, or Atg1 fused with GFP tag in wild-type or bmh1∆ Bmh2-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in nitrogen starvation medium (SD-N) or glucose starvation medium (SD-G) in the presence of IAA for 1 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (G) Cells from F were quantified for the number of cells with the indicated ATG protein 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. (H) Cells expressing Atg1-3✕FLAG in WT or bmh1∆ Bmh2-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in SD-N, SD-G, or rapamycin treatment 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 F2.

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