Figure S2.
The association of Atg11 with Bmh2 depends on its CC4 domain, and Rck2 is required specifically for glucose starvation–induced autophagy. (A) Cells co-expressing an empty vector, WT HA-Atg11, HA-Atg11 CC1∆, HA-Atg11 CC2∆, HA-Atg11 CC3∆, or HA-Atg11 CC4∆ with Bmh2-3✕FLAG in the atg11∆ strains 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. (B) Cells co-expressing WT HA-Atg11 and Bmh2-3✕FLAG in the atg11∆ strains were cultured in SD-G for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads. Bmh2-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. (C) In vitro Ni-NTA pulldowns were performed using His-Bmh2 with GST-Atg11 CC4 purified from E. coli. Protein samples were separated by SDS-PAGE and then detected using Coomassie blue staining. The data are representative of three independent experiments. (D) Atg11-associated protein kinases were found in the SGD database. (E) Cells co-expressing HA-Atg11 and Bmh2-3✕FLAG in the WT or rck2∆ yeast strains were cultured in nutrient-rich medium, SD-N, or 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. (F and G) Cells coexpressing GFP-Atg8 and Vph1-mCherry in WT, atg1∆, or rck2∆ yeast strains were cultured in SD-N or SD-G for 4 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (H) Cells from F and G 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. (I) Cells expressing HA-Snf1 in WT or rck2∆ yeast strains were grown to the early log-growth phase and then were subjected to glucose starvation for 0.5 h. The activity of Snf1 was detected by immunoblotting with anti-phospho-AMPKα (Thr172) antibody. The data are representative of three independent experiments. (J) WT, atg1∆, or rck2∆ yeast strains were grown to the log-growth phase. 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. Source data are available for this figure: SourceData FS2.

The association of Atg11 with Bmh2 depends on its CC4 domain, and Rck2 is required specifically for glucose starvation–induced autophagy. (A) Cells co-expressing an empty vector, WT HA-Atg11, HA-Atg11 CC1∆, HA-Atg11 CC2∆, HA-Atg11 CC3∆, or HA-Atg11 CC4∆ with Bmh2-3✕FLAG in the atg11∆ strains 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. (B) Cells co-expressing WT HA-Atg11 and Bmh2-3✕FLAG in the atg11∆ strains were cultured in SD-G for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads. Bmh2-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. (C) In vitro Ni-NTA pulldowns were performed using His-Bmh2 with GST-Atg11 CC4 purified from E. coli. Protein samples were separated by SDS-PAGE and then detected using Coomassie blue staining. The data are representative of three independent experiments. (D) Atg11-associated protein kinases were found in the SGD database. (E) Cells co-expressing HA-Atg11 and Bmh2-3✕FLAG in the WT or rck2∆ yeast strains were cultured in nutrient-rich medium, SD-N, or 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. (F and G) Cells coexpressing GFP-Atg8 and Vph1-mCherry in WT, atg1∆, or rck2∆ yeast strains were cultured in SD-N or SD-G for 4 h. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (H) Cells from F and G 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. (I) Cells expressing HA-Snf1 in WT or rck2∆ yeast strains were grown to the early log-growth phase and then were subjected to glucose starvation for 0.5 h. The activity of Snf1 was detected by immunoblotting with anti-phospho-AMPKα (Thr172) antibody. The data are representative of three independent experiments. (J) WT, atg1∆, or rck2∆ yeast strains were grown to the log-growth phase. 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. Source data are available for this figure: SourceData FS2.

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