TORC1 directly phosphorylates Atg13 in vitro. (A) Detection of immunoprecipitated TORC1. ^FlagKog1 was immunoprecipitated, and wild-type ^HATor1 (WT) and a kinase-dead D2294E mutant (KD) contained in the immunocomplex were detected. Results of a control experiment using untagged Kog1 are shown in lane 1. α, anti. (B) In vitro TORC1 kinase assay. TORC1 isolated as for panel A was used in an in vitro kinase assay using 4 μg of recombinant trigger factor (TF)-fused Atg13 (wild type [lanes 1 to 3] or Atg13-8SA [lanes 4 to 6]) or TF (lane 7) as substrates. The results of autoradiography (kinase assay) and Coomassie blue staining (CBB) are shown. (C) TORC1 kinase assay using various Atg13 constructs. TORC1 assay was performed using the indicated TF-Atg13 proteins. TF-Atg13-4SAb and TF-Atg13-7SAb migrated faster than the wild type, presumably because they were C-terminally processed in E. coli cells.

Atg13-8SA induces autophagy in normally grown cells. (A) Pho8Δ60-expressing (OND88) and YYK757 (atg2Δ) cells harboring the indicated p416GAL1[ATG13] plasmids were grown in SCRaf and shifted to SCRafGal. Alkaline phosphatase (ALP) activity (U, solid line) and cell growth (OD₆₀₀, broken line) were monitored. (B) Pho8Δ60-expressing cells harboring the p306GAL1[ATG13-8SA] plasmid were subjected to the ALP assay before or after a 4-h incubation with SCRafGal or a 4-h incubation with nitrogen-depleted medium [SRaf(−N)]. The ALP activity of each sample is shown. Error bars represent the standard deviations [SD] from three independent experiments. (C) Accumulation of autophagic bodies. Cells used for panel A were incubated in SCRafGal in the presence of 1 mM PMSF (to inhibit degradation of autophagic bodies) for 6 h. Autophagic bodies were visualized by phase-contrast microscopy. (D) Electron microscopy of cells used for panel B. Bar, 1 μm.

Autophagy induction by Atg13 constructs. (A) Pho8Δ60-expressing cells harboring the indicated p416GAL1[ATG13] plasmids were subjected to the ALP assay before or after a 4-h incubation with SCRafGal. Error bars represent the SD from at least three independent experiments. (B) Atg13 proteins were detected by immunoblotting. (C) Summary of in vitro TORC1 assay and ALP assay using various Atg13 constructs. The shaded box represents the Atg1-binding site.

Determination and prediction of Atg13 phosphorylation sites. (A and B) Product ion mass spectra of phosphopeptides 426-YSSSFGNIRRHpSpSVK-440 (A) and 643-SSIpSPRpSIDSISSSFIK-659 (B), obtained from the His₆-tagged Atg13 protein. In these spectra, multicharged ion peaks, [M + nH]ⁿ⁺, on ESI mass spectra were converted to single-charged ion peaks, [M + H]⁺ (protonated molecules). Only monoisotopic ion peaks are shown. Isotopic ion peaks are not shown. (C) Phosphorylation sites of Atg13 mapped by mass spectrometry. Four Ser residues determined by MS/MS analysis (top) and the putative phosphorylation sites similar to the determined Ser sites (bottom) are shown. (D) Immunoblot of Atg13. YEPD-grown cells (OND88) expressing the indicated Atg13 protein were treated with rapamycin (Rapa) (0.2 μg/ml) for 1 h. The phosphorylation state of Atg13 was assessed by immunoblotting.

Atg13-8SA promotes Atg1 complex formation, Atg1 activation, and PAS organization. (A) Formation of the Atg1 complex. Cells (YYK412) harboring the indicated p416GAL1[ATG13] plasmid grown in SCRaf were incubated in SCRafGal for 2 h to induce Atg13 protein. Atg13 and Atg17 coimmunoprecipitated with in ^HAAtg1 were detected by immunoblotting. The catalytic activity of Atg1 kinase was also examined using myelin basic protein (MBP) as a substrate. The asterisk denotes a nonspecific band in the total cell lysates that was recognized by the anti-HA ascites. α, anti. (B) PAS assembly of Atg proteins. Cells (TMK625, atg11Δ) grown in SCRaf were shifted to SCRafGal for 1 h to induce Atg13. GFP-Atg17 and mCherry-Atg8 were observed by fluorescence microscopy.

Expression of Atg13-8SA mimics TORC1 inactivation to induce autophagy. (A) Cells grown in SCRaf were treated with 0.2 μg/ml rapamycin for 4 h, incubated in SCRafGal for 4 h, or incubated in SCRafGal for 2 h before a 2-h treatment with 0.2 μg/ml rapamycin (Rapa). Atg1 complex formation and Atg1 kinase activity were examined as for Fig. 3A. α, anti. (B) Pho8Δ60-expressing cells harboring the indicated p306GAL1[ATG13] plasmids were treated as for panel A and subjected to the ALP assay. Error bars represent the SD from three independent experiments.

Effect of Atg13-8SA on other Atg proteins. (A) Atg13-8SA expression does not significantly affect Atg protein expression. Atg protein levels in cells used for panel A were assessed by immunoblotting. The asterisk denotes the phosphatidylethanolamine-conjugated form of Atg8. (B) Requirement for ATG genes for autophagy induced by Atg13-8SA expression. Cells harboring the indicated mutations carrying the p416GAL1[ATG13-8SA] plasmid were subjected to the ALP assay before or after a 4-h incubation with SCRafGal to induce Atg13-8SA expression. Error bars represent the SD form three independent experiments.