Glucose starvation triggers the elevation of cytoplasmic calcium and activates Rck2. (A) In vitro phosphorylation assays were performed using Atg11 CC4 purified from E. coli as substrates and Rck2-3✕FLAG purified from nutrient-rich, nitrogen starvation, or glucose starvation-treated yeast cells as a protein kinase. Phosphorylation levels of Atg11 CC4 and its variants were detected using anti-thioP antibody. The data are representative of three independent experiments. (B) Wild-type yeast expressing the cytosolic-anchored Ca²⁺ fluorescence probe jGCaMP7f plasmid were grown to the early log-growth phase and then subjected to nitrogen starvation, glucose starvation, or treated with 0.2 M CaCl₂. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (C) Cytosolic relative fluorescence intensity from B calculated by ImageJ software. n = 60 cells were pooled from three independent experiments. Data are presented as means ± SD. ***P < 0.001; two-tailed Student’s t tests were used. (D) Cells co-expressing Rck2-3✕FLAG with Atg11-3✕HA were subjected to nitrogen or glucose starvation for 0.5 h. Cell lysates were immunoprecipitated with anti-FLAG agarose beads and then analyzed by Western blot using anti-HA antibody. (E) The results from D were quantified. Data are presented as means ± SD (n = 3). **P < 0.01; two-tailed Student’s t tests were used. (F) Cells co-expressing Rck2-GFP, Atg17-2✕mCherry, and Vph1-BFP were grown to the early log-growth phase and then subjected to nitrogen starvation or glucose starvation for 0.5 h. Images of cells were obtained using a spinning disk confocal microscope. Scale bar, 2 µm. (G) Cells from F were quantified for the number of cells in which Rck2-GFP colocalized with Atg17-2×mCherry puncta. n = 300 cells were pooled from three independent experiments. Data are shown as mean ± SD. ***P < 0.001; two-tailed Student’s t tests were used. (H) Yeast cells expressing jGCaMP7f were treated with DMSO, 10 mM EGTA, or 20 mM EGTA for 1 h, and then cultured in nitrogen starvation medium (SD-N) or glucose starvation medium (SD-G) in the presence of DMSO, 10 mM EGTA, or 20 mM EGTA. Images of cells were obtained using an inverted fluorescence microscope. Scale bar, 2 µm. (I and J) Wild-type (WT) or atg1∆ yeast strains expressing GFP-Atg8 were treated with or without 20 mM EGTA for 1 h. Subsequently, they were cultured for 4 h in SD-N medium (I) or SD-G medium (J), with or without 20 mM EGTA. The autophagic activity was assessed by Western blotting to detect cleavage of GFP-Atg8, with Pgk1 serving as a loading control. The data are representative of three independent experiments. (K) Cells expressing GFP-Atg8 in WT, atg1∆, or Cmd1-3✕HA-AID yeast strains were treated with IAA for 2 h and then cultured in SD-N or SD-G in the presence of IAA for 4 h. The autophagic activity was analyzed by Western blot for GFP-Atg8 cleavage. Pgk1 served as a loading control. (L and M) The cleavage of GFP-Atg8 from (k) was quantified and presented as mean ± SD (n = 3). ***P < 0.001; NS, no significance; two-tailed Student’s t tests were used. (N) In vitro kinase assays were performed using GST-Atg11 CC4, purified from E. coli, as substrates, and Rck2-3✕FLAG, purified from yeast cells treated with nutrient-rich, glucose starvation, nitrogen starvation, or rapamycin, with or without 20 mM EGTA treatment, as a protein kinase. Phosphorylation levels of GST-Atg11 CC4 were assessed using an anti-thioP antibody. The data are representative of three independent experiments. Source data are available for this figure: SourceData F5.