Fig. 1

Phosphorylation pattern of full length Sch9 in glucose-rich and glucose-starved conditions. a Domain structure and phosphorylation sites of the AGC family kinase Sch9. TORC1 targets residues within the C-terminal domain (CT): T723 and S726 are located in a region, which resembles the turn motif (TM) of AGC kinases, T737 is located in the conserved hydrophobic motif (HM), which serves as the docking site for PDK, S758 and S765 are located in HM-like regions^{46, 49}. These 5 TORC1-dependent sites were changed to alanines in the Sch9-5A mutant or to glutamate/aspartate in the Sch9-2D3E mutant²⁴. PDK targets residue T570 within the activation loop²⁴. SNF1 targets poorly characterized residues within a region of unknown function^{60, 61}. b–e Glucose-grown Δsch9 cells expressing Sch9-FLAG, Sch9-5A-FLAG, Sch9-T570A-FLAG or Sch9-T570A/5A-FLAG were treated with cycloheximide (+CHX), rapamycin (+rapa), or were starved 10 min for glucose as indicated. Total protein extracts were resolved on phos-tag or Tris-Tricine (PAGE) gels and were subsequently analyzed via immunoblotting with α-FLAG or α-phospho-T570 (α-T570-Pi) antibodies. If indicated, total extract was treated with lambda phosphatase (λP) prior to the analysis. Sch9 species prominent on glucose (S0, S1, S3) are indicated in black, species prominent after starvation are indicated in blue (S2, S4, S2**). f Cartoon summary of Sch9 phosphorylation. In glucose-rich conditions Sch9 is distributed between S0, S1, and S3. In glucose-starved conditions Sch9 is distributed between S4 and S2. Sch9-T570A/5 A is non-phosphorylated in glucose-rich conditions (S0) and becomes phosphorylated at a single residue upon starvation (S2**). The kinases responsible for the respective phosphorylation events are indicated. PDK-dependent phosphorylation, blue circle; TORC1-dependent phosphorylation; red circles; SNF1-dependent phosphorylation, yellow circle; residues mutated to alanines are indicated by red asterisks. For more details compare Results section