Figure S1.
The deletion of BMH1 or BMH2 had no effect on autophagy or the Cvt pathway, confirming the specific binding of Bmh1/2 with Atg11. (A) Cells co-expressing GFP-Atg8 and Vph1-mCherry in WT, bmh1∆, or bmh2∆ yeast strains were cultured in SD-N, SD-G, or placed under rapamycin treatment, for the indicated time periods. The samples 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. (B) WT, atg11∆, bmh1∆, or bmh2∆ yeast strains were cultured in a nutrient-rich medium. The samples were analyzed by Western blot for the maturation of PrApe1. Pgk1 served as a loading control. The data are representative of three independent experiments. (C) Growth phenotypes of bmh1∆ Bmh2-3×HA-AID. Serial dilutions of wild-type (BY4741), 9✕MYC-Tir1, bmh1∆, bmh2∆, bmh1∆ Bmh2-3×HA-AID yeast strains were plated in fourfold serial dilution onto YPD plates with or without HU and MMS at the indicated concentrations. The plates were incubated at 30°C or 37°C for 2 days, and spotting assays were performed to assess the growth phenotypes. (D) WT, atg11∆, or bmh1∆ Bmh2-3✕HA-AID yeast strains were grown to the early log-growth phase and then treated with IAA or DMSO for 2 h. The samples were analyzed by Western blot for the maturation of PrApe1. Pgk1 served as a loading control. The data are representative of three independent experiments. (E) Cells expressing3×FLAG-Snf1 in WT or bmh1∆ Bmh2-3✕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. The activity of Snf1 was detected by immunoblotting with anti-phospho-AMPKα (Thr172) antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (F) WT, cox6∆ (positive control), bmh1∆, bmh2∆, or bmh1∆ Bmh2-3✕HA-AID yeast strains were treated with DMSO or IAA for 2 h, and then cultured in nutrient-rich medium or glucose starvation medium in the absence or presence of IAA for 0.5 h. Cells were harvested and oxygen consumption was measured using Oroboros O2K. n = 3 independent experiments were quantified. Data are presented as means ± SD. ***P < 0.001; NS, not significant; two-tailed Student’s t tests were used. (G)atg1∆ cells expressing empty vector, Atg1-3✕FLAG, or Atg1-3✕FLAG T226A were grown to the early log-growth phase and then treated with rapamycin 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. (H) Cells expressing Atg1-3✕FLAG in WT, atg11∆, atg13∆, or atg17∆ yeast strains were grown to the early log-growth phase, and treated with rapamycin or glucose starvation 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. (I–N) Cells co-expressing Bmh1-3✕FLAG or Bmh2-3✕FLAG with HA-Atg1 (I and J), Atg17-6✕HA (K and L), or Atg13-6✕HA (M and N) were cultured in nutrient-rich medium or SD-G for 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. (O) Cells co-expressing Atg29-2✕GFP or Atg31-2✕GFP with Bmh1-3✕FLAG or Bmh2-3✕FLAG were cultured in SD-G for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-GFP antibody. The data are representative of three independent experiments. Source data are available for this figure: SourceData FS1.

The deletion of BMH1 or BMH2 had no effect on autophagy or the Cvt pathway, confirming the specific binding of Bmh1/2 with Atg11. (A) Cells co-expressing GFP-Atg8 and Vph1-mCherry in WT, bmh1∆, or bmh2∆ yeast strains were cultured in SD-N, SD-G, or placed under rapamycin treatment, for the indicated time periods. The samples 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. (B) WT, atg11∆, bmh1∆, or bmh2∆ yeast strains were cultured in a nutrient-rich medium. The samples were analyzed by Western blot for the maturation of PrApe1. Pgk1 served as a loading control. The data are representative of three independent experiments. (C) Growth phenotypes of bmh1∆ Bmh2-3×HA-AID. Serial dilutions of wild-type (BY4741), 9✕MYC-Tir1, bmh1∆, bmh2∆, bmh1∆ Bmh2-3×HA-AID yeast strains were plated in fourfold serial dilution onto YPD plates with or without HU and MMS at the indicated concentrations. The plates were incubated at 30°C or 37°C for 2 days, and spotting assays were performed to assess the growth phenotypes. (D) WT, atg11∆, or bmh1∆ Bmh2-3✕HA-AID yeast strains were grown to the early log-growth phase and then treated with IAA or DMSO for 2 h. The samples were analyzed by Western blot for the maturation of PrApe1. Pgk1 served as a loading control. The data are representative of three independent experiments. (E) Cells expressing3×FLAG-Snf1 in WT or bmh1∆ Bmh2-3✕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. The activity of Snf1 was detected by immunoblotting with anti-phospho-AMPKα (Thr172) antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (F) WT, cox6∆ (positive control), bmh1∆, bmh2∆, or bmh1∆ Bmh2-3✕HA-AID yeast strains were treated with DMSO or IAA for 2 h, and then cultured in nutrient-rich medium or glucose starvation medium in the absence or presence of IAA for 0.5 h. Cells were harvested and oxygen consumption was measured using Oroboros O2K. n = 3 independent experiments were quantified. Data are presented as means ± SD. ***P < 0.001; NS, not significant; two-tailed Student’s t tests were used. (G)atg1∆ cells expressing empty vector, Atg1-3✕FLAG, or Atg1-3✕FLAG T226A were grown to the early log-growth phase and then treated with rapamycin 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. (H) Cells expressing Atg1-3✕FLAG in WT, atg11∆, atg13∆, or atg17∆ yeast strains were grown to the early log-growth phase, and treated with rapamycin or glucose starvation 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. (I–N) Cells co-expressing Bmh1-3✕FLAG or Bmh2-3✕FLAG with HA-Atg1 (I and J), Atg17-6✕HA (K and L), or Atg13-6✕HA (M and N) were cultured in nutrient-rich medium or SD-G for 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. (O) Cells co-expressing Atg29-2✕GFP or Atg31-2✕GFP with Bmh1-3✕FLAG or Bmh2-3✕FLAG were cultured in SD-G for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-GFP antibody. The data are representative of three independent experiments. Source data are available for this figure: SourceData FS1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal