Atg1 is hyperphosphorylated in ptc2Δ ptc3Δ strains. (A) Atg1 phosphorylation in WT, atg13Δ, ptc2Δ ptc3Δ, and atg13Δ ptc2Δ ptc3Δ strains after 4-h rapamycin or MMS treatment. Samples were blotted with anti-FLAG antibody for FLAG-Atg1 detection, Pgk1, and GFP. (B) Atg1 phosphorylation in WT, ptc2Δ ptc3Δ, atg11Δ, and atg11Δ ptc2Δ ptc3Δ strains after 4-h rapamycin treatment. (C) Atg1 phosphorylation in ptc2Δ ptc3Δ mutant strains that carry an atg1-T226A kinase-defective allele 4 h after rapamycin treatment. Numbers below lanes indicate the GFP-Atg8 cleavage for the corresponding lane.