Phosphorylation of T1114 and S1119 residues on Atg11 are required for Atg11-Bmh2 binding and the MAPK signaling pathway is not involved in the activation of Rck2 and glucose starvation–induced autophagy. (A) LC-MS/MS identified phosphorylated peptides of Atg11 CC4 by Rck2. (B) In vitro kinase assays were performed using Atg11 CC4 WT, T1114A, S1119A, or T1120A purified from E. coli as substrates with Rck2-3✕FLAG purified from glucose-starved yeast cells as a protein kinase. The phosphorylation levels of Atg11 CC4 and its variants were detected using anti-thioP antibody. The data are representative of three independent experiments. (C)atg11∆ cells co-expressing Bmh2-3✕FLAG with HA-Atg11 WT or 2A 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. (D) In vitro GST pulldowns were performed using GST- Bmh2 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 Atg11 CC4 or 2A purified from E. coli as substrates and with WT or KD Rck2-3✕FLAG purified from glucose-starved yeast cells as a protein kinase. In vitro Ni-NTA pulldowns were then performed using His-Bmh2 purified from E. coli with the samples from the 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)atg11∆ yeast cells co-expressing GFP-Atg8 and empty vector, HA-Atg11, or HA-Atg11 T1114E-S1119E were subjected to glucose starvation (SD-G) for 0 or 4 h. The autophagic activity was assessed by Western blotting to detect cleavage of GFP-Atg8, with Pgk1 serving as a loading control. (G) Quantification of the ratio of free GFP/GFP+GFP-Atg8 from F and presented as mean ± SD (n = 3). ***P < 0.001; **P < 0.01; two-tailed Student’s t tests were used. (H–J) Yeast cells expressing Hog1-6✕HA were cultured to early log phase and subsequently subjected to rapamycin treatment (H), nitrogen starvation (SD-N) (I), glucose starvation (SD-G) (J), or 0.4 M NaCl treatment for the indicated time periods. The activation of Hog1 was assessed using anti-phospho-p38 MAPK antibody (#4511; Cell Signaling Technology). The data are representative of three independent experiments. (K–M) Wild-type (WT), atg1∆, hog1∆, or pbs2∆ yeast cells were cultured to early log phase and subsequently subjected to rapamycin treatment (K), nitrogen starvation (SD-N) (L), or glucose starvation (SD-G) (M) for 0 or 4 h. The autophagic activity was detected by Western blots using anti-GFP antibody. Pgk1 served as a loading control. The data are representative of three independent experiments. (N) In vitro kinase assays were performed using GST-Atg11 CC4 purified from E. coli as substrates and Rck2-3✕FLAG purified yeast wild-type or hog1∆ cell cultured under full medium, glucose starvation, nitrogen starvation, or rapamycin-treated conditions as a protein kinase. Phosphorylation levels of GST-Atg11 CC4 were detected using anti-thioP antibody. The data are representative of three independent experiments. (O) Wild-type yeast expressing cytosolic-anchored Ca2+ fluorescence probe jGCaMP7f plasmid were grown to the early log-growth phase, and then subjected to nitrogen starvation (SD-N), glucose starvation (SD-G), or treated with 0.2 M CaCl2. Cells were observed using a fluorescence inverted microscope (Olympus IX83). Images were captured at 4-s intervals using time-lapse microscopy and shown in 24 fps movies. ImageJ software was used to calculate the relative fluorescence intensity of cells to reflect cytoplasmic calcium signaling. The data are representative of three independent experiments. Source data are available for this figure: SourceData FS3.
