Figure 3.
Rck2 is required for the binding of Atg11-Bmh1/2 and glucose starvation–induced autophagy. (A) Schematic representation of the Atg11 domains and the deletions. CC1∆, CC2∆, CC3∆, and CC4∆ correspond to the deletion of amino acids at positions 272–321, 536–576, 627–858, and 859–1,178, respectively. (B) Cells co-expressing an empty vector, WT HA-Atg11, HA-Atg11-CC1∆, HA-Atg11-CC2∆, HA-Atg11-CC3∆, or HA-Atg11-CC4∆ with Bmh1-3✕FLAG in the atg11∆ strain were 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. (C) Cells co-expressing WT HA-Atg11 and Bmh1-3✕FLAG in the atg11∆ strains were cultured in SD-G for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads. Bmh1-associated proteins were then treated with or without lambda protein phosphatase (λPPase) and analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (D) In vitro Ni-NTA pulldowns were performed using His-Bmh1with GST-Atg11 CC4 purified from E. coli. Protein samples were separated by SDS-PAGE and detected using Coomassie blue staining. The data are representative of three independent experiments. (E) Cells co-expressing HA-Atg11 and Bmh1-3✕FLAG in the WT or rck2∆ yeast strain were cultured in full medium, SD-N, or SD-G 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. (F) Cells co-expressing HA-Atg11 and Rck2-3✕FLAG were 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. (G) Cells expressing GFP-Atg8 and Vph1-mCherry in WT, atg1∆, or rck2∆ yeast strains were cultured in SD-N or SD-G for 4 h. 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. (H) Cells expressing Atg1-GFP in WT, atg11∆, rck2∆, or snf1∆ yeast strains were cultured in SD-N or SD-G for 1 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (I) Cells from (H) were quantified for the number of cells with Atg1-GFP 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. (J) Cells expressing Atg1-3✕FLAG in WT or rck2∆ yeast strains were were grown to the Log growth phase and then cultured in glucose or nitrogen starvation medium 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 F3.

Rck2 is required for the binding of Atg11-Bmh1/2 and glucose starvation–induced autophagy. (A) Schematic representation of the Atg11 domains and the deletions. CC1∆, CC2∆, CC3∆, and CC4∆ correspond to the deletion of amino acids at positions 272–321, 536–576, 627–858, and 859–1,178, respectively. (B) Cells co-expressing an empty vector, WT HA-Atg11, HA-Atg11-CC1∆, HA-Atg11-CC2∆, HA-Atg11-CC3∆, or HA-Atg11-CC4∆ with Bmh1-3✕FLAG in the atg11∆ strain were 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. (C) Cells co-expressing WT HA-Atg11 and Bmh1-3✕FLAG in the atg11∆ strains were cultured in SD-G for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads. Bmh1-associated proteins were then treated with or without lambda protein phosphatase (λPPase) and analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (D) In vitro Ni-NTA pulldowns were performed using His-Bmh1with GST-Atg11 CC4 purified from E. coli. Protein samples were separated by SDS-PAGE and detected using Coomassie blue staining. The data are representative of three independent experiments. (E) Cells co-expressing HA-Atg11 and Bmh1-3✕FLAG in the WT or rck2∆ yeast strain were cultured in full medium, SD-N, or SD-G 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. (F) Cells co-expressing HA-Atg11 and Rck2-3✕FLAG were 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. (G) Cells expressing GFP-Atg8 and Vph1-mCherry in WT, atg1∆, or rck2∆ yeast strains were cultured in SD-N or SD-G for 4 h. 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. (H) Cells expressing Atg1-GFP in WT, atg11∆, rck2∆, or snf1∆ yeast strains were cultured in SD-N or SD-G for 1 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (I) Cells from (H) were quantified for the number of cells with Atg1-GFP 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. (J) Cells expressing Atg1-3✕FLAG in WT or rck2∆ yeast strains were were grown to the Log growth phase and then cultured in glucose or nitrogen starvation medium 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 F3.

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