PP2A is required for autophagy induction after the inactivation of TORC1.

<p>(A) Exponentially growing cells of strains BY4741 (wild type; WT), SCU3086 (<i>pph21</i>Δ) and SCU3087 (<i>pph22</i>Δ) harboring plasmid pSCU1998 (pGFP-ATG8) were treated with 200 ng/ml rapamycin for 3 h. Whole cell extracts were subjected to western blotting using an anti-GFP antibody. Cyclin-dependent kinase (CDK) was detected as the loading control using an anti-CDK antibody. All western blotting experiments were performed at least twice independently to confirm reproducibility of the results. Free GFP processed from GFP-tagged protein was measured using ImageJ software, and quantified by calculating the ratio of cleaved free GFP versus uncleaved full-length protein. The average was determined for each sample of two independent experiments and relative values normalized against the value in control cells are shown. (B) Cells of strains BY4741 (wild type; WT), SCU3088 (<i>pph3</i>Δ) and SCU2142 (<i>sit4</i>Δ) harboring plasmid pSCU1998 (pGFP-ATG8) were treated with rapamycin for 3 h. Whole cell extracts were subjected to western blotting. Pgk1 was detected as the loading control using an anti-Pgk1 antibody. (C) Cells of strains SCU893 (wild type, isogenic to W303) and SCU2422 (<i>pph21</i>Δ <i>pph22</i>Δ; <i>pp2a</i>Δ) harboring a plasmid pSCU1998 were treated with rapamycin for 3 h. Whole cell extracts were subjected to western blotting using an anti-GFP antibody. (D) Ape1 processing assays indicated that autophagy induction after rapamycin treatment is repressed in <i>pp2a</i>Δ cells. Cells of strains SCU3720 (<i>atg11</i>Δ) and SCU3736 (<i>pph21</i>Δ <i>pph22</i>Δ <i>atg11</i>Δ) were treated with rapamycin for 6 h. Whole cell extracts were subjected to western blotting and pre-Ape1 (preApe1) and mature Ape1 (mApe1) were detected using an anti-Ape1 antibody. Mature Ape1 processed from pre-Ape1 was measured using ImageJ software, and quantified by calculating the ratio of mature Ape1 versus pre-Ape1. Relative values normalized against the value in control cells are shown. (E, F) Cells of strains SCU893 (wild-type) and SCU2422 (<i>pph21</i>Δ <i>pph22</i>Δ) harboring a plasmid pSCU2260 expressing Rosella (pH-sensitive GFP fused to RFP) were treated with rapamycin for 18 h (+Rap). Rapamycin-untreated cells were used as the control (Ctrl). Representative images of cells are shown (E). Scale bars, 5 μm. GFP and RFP signals in the cytoplasm and vacuole were quantified in cells and the Rosella values (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166636#sec002" target="_blank">Materials and Methods</a>”) were calculated and shown along with statistical analysis in (F). Numbers above the bars are sample sizes. The error bars indicate SEM. n.s., non-significant; *, P < 0.01 (Fisher’s exact test).