TORC1 regulates autophagy induction in response to proteotoxic stress in yeast and human cells

Misfolded and aggregated proteins are eliminated to maintain protein homeostasis. Autophagy contributes to the removal of protein aggregates. However, if and how proteotoxic stress induces autophagy is poorly understood. Here we show that proteotoxic stress after treatment with azetidine-2-carboxylic acid (AZC), a toxic proline analog, induces autophagy in budding yeast. AZC treatment attenuated target of rapamycin complex 1 (TORC1) activity, resulting in the dephosphorylation of Atg13, a key factor of autophagy. By contrast, AZC treatment did not affect target of rapamycin complex 2 (TORC2). Proteotoxic stress also induced TORC1 inactivation and autophagy in fission yeast and human cells. This study suggested that TORC1 is a conserved key factor to cope with proteotoxic stress in eukaryotic cells

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

<div><p>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.</p></div

Overexpression of non-phosphorylatable Atg13 recovers autophagy induction after rapamycin treatment in <i>pp2a</i>Δ cells.

<p>(A) Cells of strains SCU893 (wild-type) and SCU4154 (<i>pph21</i>Δ <i>pph22</i>Δ) harboring plasmid an pSCU154 (empty vector; EV), pSCU1986 (pGAL1-ATG13; <i>ATG13</i>) or pSCU1987 (pGAL1-ATG13-8SA; <i>8SA</i>) 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.</p

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).</p

Characterization of rapamycin-induced Atg8 and Atg1 puncta.

<p>(A, B) Cells of strains SCU893 (wild type) and SCU2422 (<i>pph21</i>Δ <i>pph22</i>Δ) 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.</p

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

<p>(A) Cells of strains SCU893 (wild type) and SCU2422 (<i>pph21</i>Δ <i>pph22</i>Δ) 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 (<i>cdc55Δ rts1</i>Δ) were treated with rapamycin for 15 min. Whole cell extracts were subjected to western blotting as for panel (A).</p

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

<p>(A) Cells of strains SCU893 (wild-type), SCU4221 (<i>cdc55</i>Δ) and SCU4223 (<i>rts1</i>Δ) harboring plasmid pSCU1998 (pGFP-ATG8) were treated with rapamycin for 3 h. (B) Cells of strains SCU893 (wild-type) and SCU4225 (<i>cdc55</i>Δ <i>rts1</i>Δ) 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 (<i>atg11</i>Δ) and SCU4069 (<i>cdc55</i>Δ <i>rts1</i>Δ <i>atg11</i>Δ) were treated with rapamycin for 6 h. Whole cell extracts were subjected to western blotting using the anti-Ape1 antibody.</p