Orchestrated Action of PP2A Antagonizes Atg13 Phosphorylation and Promotes Autophagy after the Inactivation of TORC1

Abstract

Target of rapamycin complex 1 (TORC1) phosphorylates autophagy-related Atg13 and represses autophagy under nutrient-rich conditions. However, when TORC1 becomes inactive upon nutrient depletion or treatment with the TORC1 inhibitor rapamycin, Atg13 dephosphorylation occurs rapidly, and autophagy is induced. At present, the phosphatases involved in Atg13 dephosphorylation remain unknown. Here, we show that two protein phosphatase 2A (PP2A) phosphatases, PP2A-Cdc55 and PP2A-Rts1, which are activated by inactivation of TORC1, are required for sufficient Atg13 dephosphorylation and autophagy induction after TORC1 inactivation in budding yeast. After rapamycin treatment, dephosphorylation of Atg13, activation of Atg1 kinase, pre-autophagosomal structure (PAS) formation and autophagy induction are all impaired in PP2A-deleted cells. Conversely, overexpression of non-phosphorylatable Atg13 suppressed defects in autophagy in PP2A mutant. This study revealed that the orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1.

Fig 1. PP2A is required for autophagy induction after the inactivation of TORC1.

(A) Exponentially growing cells of strains BY4741 (wild type; WT), SCU3086 (pph21Δ) and SCU3087 (pph22Δ) 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 (pph3Δ) and SCU2142 (sit4Δ) 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 (pph21Δ pph22Δ; pp2aΔ) 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 pp2aΔ cells. Cells of strains SCU3720 (atg11Δ) and SCU3736 (pph21Δ pph22Δ atg11Δ) 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 (pph21Δ pph22Δ) 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 “Materials and Methods”) 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).

Fig 2. Both PP2A-Cdc55 and PP2A-Rts1 are involved in autophagy induction.

(A) Cells of strains SCU893 (wild-type), SCU4221 (cdc55Δ) and SCU4223 (rts1Δ) harboring plasmid pSCU1998 (pGFP-ATG8) were treated with rapamycin for 3 h. (B) Cells of strains SCU893 (wild-type) and SCU4225 (cdc55Δ rts1Δ) harboring plasmid pSCU1998 were treated with rapamycin for 3 h. Whole cell extracts were subjected to western blotting using the anti-GFP antibody. (C) Cells of strains SCU3720 (atg11Δ) and SCU4069 (cdc55Δ rts1Δ atg11Δ) were treated with rapamycin for 6 h. Whole cell extracts were subjected to western blotting using the anti-Ape1 antibody.

Fig 3. PP2A is required for sufficient Atg13 dephosphorylation after the inactivation of TORC1.

(A) Cells of strains SCU893 (wild type, isogenic to W303a) and SCU2422 (pph21Δ pph22Δ) harboring plasmid pSCU1984 (pATG13) were treated with rapamycin for 15 min. Whole cell extracts were subjected to western blotting using an anti-Atg13 antibody. For detection of phosphorylation statuses of Atg13, 7.5% acrylamide gels were used. Phosphorylated Atg13 was measured using ImageJ software, and quantified by calculating the ratio of phosphorylated Atg13 versus dephosphorylated Atg13. Relative values normalized against the value in control cells are shown. P-Atg13, phosphorylated Atg13. Asterisks depict non-specific bands. (B) Cells of strains SCU893 (wild type) and SCU4225 (cdc55Δ rts1Δ) harboring plasmid pSCU1984 (pATG13) were treated with rapamycin for 15 min. Whole cell extracts were subjected to western blotting as for panel (A).

Fig 4. PP2A is required for Atg1 activation after the inactivation of TORC1.

(A) Cells of strains SCU893 (wild type) and SCU2422 (pph21Δ pph22Δ) were treated with rapamycin for 15 min. Whole cell extracts were subjected to western blotting using an anti-Atg1 antibody. Phosphorylated Atg1 was measured using ImageJ software, and quantified by calculating the ratio of phosphorylated Atg1 versus dephosphorylated Atg1. Relative values normalized against the value in control cells are shown. P-Atg1, phosphorylated Atg1. (B) Cells of strains SCU893 (wild type) and SCU4225 (cdc55Δ rts1Δ) were treated with rapamycin for 15 min. Whole cell extracts were subjected to western blotting as for panel (A).

Fig 5. Characterization of rapamycin-induced Atg8 and Atg1 puncta.

(A, B) Cells of strains SCU893 (wild type) and SCU2422 (pph21Δ pph22Δ) harboring plasmid pSCU1998 (pGFP-ATG8) or pSCU2138 (pATG1-GFP) in combination with pSCU2148 (pRFP-APE1) were treated with rapamycin for 1 h. GFP puncta that are colocalized and non-colocalized with RFP-Ape1 puncta were counted and are expressed as percentages. For examination of PAS formation, more than 100 cells with Atg8-marked puncta were counted and were scored. Microscope observations were performed at least twice independently to confirm reproducibility of the results. Data are shown as means ± errors. *, P < 0.01 (Fisher’s exact test). (C, D) Cell images with Atg8 and Atg1 puncta colocalized with or without Ape1 after rapamycin treatment are shown. Scale bars, 5 μm.

Fig 6. Overexpression of non-phosphorylatable Atg13 recovers autophagy induction after rapamycin treatment in pp2aΔ cells.

(A) Cells of strains SCU893 (wild-type) and SCU4154 (pph21Δ pph22Δ) harboring plasmid an pSCU154 (empty vector; EV), pSCU1986 (pGAL1-ATG13; ATG13) or pSCU1987 (pGAL1-ATG13-8SA; 8SA) in combination with pSCU1978 (pGFP-ATG8) cultured in raffinose-base media were added with 2% galactose for 3 h. Whole cell extracts were subjected to western blotting using an anti-GFP antibody. (B) In nutrient-rich conditions, TORC1 phosphorylates Atg13 and represses PP2A, promoting Atg13 phosphorylation. Whereas, in starvation conditions, TORC1 is inactivated, and activated PP2A mediates Atg13 dephosphorylation, promoting Atg13 dephosphorylation.