Figure 4.
Rck2-mediated Atg11 phosphorylation is required for glucose starvation–induced autophagy by regulating Bmh1/2-Atg11 binding. (A) In vitro kinase assays were performed using the Atg11 CC4 domain purified from E. coli as substrates and WT or KD Rck2-3✕FLAG purified from glucose-starved yeast cells as a protein kinase. Phosphorylation of the Atg11 CC4 domain was detected using anti-thioP antibody. The data are representative of three independent experiments. (B) In vitro kinase assays were performed using Atg11 CC4 or Atg11 CC4 2A(T1114A-S1119A) purified from E. coli as substrates with Rck2-3✕FLAG purified from glucose-starved yeast cells as a protein kinase. Phosphorylation of Atg11 CC4 were detected using anti-thioP antibody. The data are representative of three independent experiments. (C)atg11∆ cells co-expressing Bmh1-3✕FLAG with HA-Atg11 WT or 2A were cultured in 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. (D) In vitro GST pulldowns were performed using GST-Bmh1 purified from E. coli with glucose-starved yeast lysates expressing HA-Atg11 WT or 2A. Protein samples were separated by SDS-PAGE and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (E) In vitro phosphorylation assays were performed using GST-Atg11 CC4 WT or 2A purified from E. coli as substrates, with WT or KD Rck2-3✕FLAG purified from glucose-starved yeast cells as a protein kinase. After that, in vitro Ni-NTA pulldowns were performed using His-Bmh1 protein purified from E. coli with the samples of in vitro phosphorylation reaction. Protein samples were separated by SDS-PAGE and then detected using Coomassie blue staining. The data are representative of three independent experiments. (F and G) Cells expressing GFP-Atg8 and Vph1-Cherry in atg11∆, HA-Atg11 WT, HA-Atg11-CC4∆, or HA-Atg11 2A strains were cultured in SD-N(F) or SD-G(G) for 4 h. Autophagic activity was analyzed by Western blot for GFP-Atg8 cleavage. The data are representative of three independent experiments. (H) Cell expressing Atg1-GFP in atg11∆, HA-Atg11 WT, HA-Atg11-CC4∆, or HA-Atg11 2A 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)atg11∆ cells expressing HA-Atg11 WT or 2A with Atg1-3✕FLAG were cultured in SD-G or SD-N for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-p-T226-Atg1 antibody. (K) the phosphorylation level of Atg1 from J was quantified. Data are presented as means ± SD (n = 3). ***P < 0.001; NS, not significant; two-tailed Student’s t tests were used. Source data are available for this figure: SourceData F4.

Rck2-mediated Atg11 phosphorylation is required for glucose starvation–induced autophagy by regulating Bmh1/2-Atg11 binding. (A) In vitro kinase assays were performed using the Atg11 CC4 domain purified from E. coli as substrates and WT or KD Rck2-3✕FLAG purified from glucose-starved yeast cells as a protein kinase. Phosphorylation of the Atg11 CC4 domain was detected using anti-thioP antibody. The data are representative of three independent experiments. (B) In vitro kinase assays were performed using Atg11 CC4 or Atg11 CC4 2A(T1114A-S1119A) purified from E. coli as substrates with Rck2-3✕FLAG purified from glucose-starved yeast cells as a protein kinase. Phosphorylation of Atg11 CC4 were detected using anti-thioP antibody. The data are representative of three independent experiments. (C)atg11∆ cells co-expressing Bmh1-3✕FLAG with HA-Atg11 WT or 2A were cultured in 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. (D) In vitro GST pulldowns were performed using GST-Bmh1 purified from E. coli with glucose-starved yeast lysates expressing HA-Atg11 WT or 2A. Protein samples were separated by SDS-PAGE and then analyzed by Western blot using anti-HA antibody. The data are representative of three independent experiments. (E) In vitro phosphorylation assays were performed using GST-Atg11 CC4 WT or 2A purified from E. coli as substrates, with WT or KD Rck2-3✕FLAG purified from glucose-starved yeast cells as a protein kinase. After that, in vitro Ni-NTA pulldowns were performed using His-Bmh1 protein purified from E. coli with the samples of in vitro phosphorylation reaction. Protein samples were separated by SDS-PAGE and then detected using Coomassie blue staining. The data are representative of three independent experiments. (F and G) Cells expressing GFP-Atg8 and Vph1-Cherry in atg11∆, HA-Atg11 WT, HA-Atg11-CC4∆, or HA-Atg11 2A strains were cultured in SD-N(F) or SD-G(G) for 4 h. Autophagic activity was analyzed by Western blot for GFP-Atg8 cleavage. The data are representative of three independent experiments. (H) Cell expressing Atg1-GFP in atg11∆, HA-Atg11 WT, HA-Atg11-CC4∆, or HA-Atg11 2A 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)atg11∆ cells expressing HA-Atg11 WT or 2A with Atg1-3✕FLAG were cultured in SD-G or SD-N for 1 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and analyzed by Western blot using anti-p-T226-Atg1 antibody. (K) the phosphorylation level of Atg1 from J was quantified. Data are presented as means ± SD (n = 3). ***P < 0.001; NS, not significant; two-tailed Student’s t tests were used. Source data are available for this figure: SourceData F4.

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