ULK1 phosphorylates Atg13 in vitro. (A and B) ULK1-FLAG (Wt) and kinase-dead ULK1^K46N-FLAG mutant (KD) were immunoprecipitated from HEK293T cells cultured in the starvation medium for 1 h. The resulting precipitates were subjected to immunoblot analysis (A) or ULK1 kinase assay (B) using GST-Atg13 and GST (negative control) as substrates.

Atg13 localizes to the isolation membrane upon autophagy induction. (A) NIH3T3 cells stably expressing GFP-Atg13 were cultured in regular DMEM (complete) or amino acid-free DMEM without FBS (starvation) for 3 h. They were then cultured in fresh complete medium for an additional 1 h (Starvation→Complete). (B) NIH3T3 cells stably expressing GFP-Atg13 were cultured in starvation medium for 2 h. The cells were then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660-conjugated secondary antibody. Scale bars, (A and B) 20 μm.

Atg13 forms a complex with ULK1 and FIP200, and is partially dephosphorylated during starvation. (A) HEK293T cells were cotransfected with FLAG-Atg13 and either HA-ULK1 or HA-FIP200. Cell lysates were then subjected to immunoprecipitation (IP) using an anti-FLAG antibody. The resulting precipitates were examined by immunoblot analysis with the indicated antibodies. (B and C) HEK293T cells were subjected to 1-h starvation or rapamycin (100 ng/ml) treatment, and their lysates were immunoprecipitated with anti-ULK1 (B), anti-Atg13 antibody (C) or preimmune rabbit serum (Pre), and analyzed by immunoblot analysis using the indicated antibodies. (D) HEK293T cells, HeLa cells, and MEFs were cultured in complete or starvation medium for the indicated periods. Parts of cell lysates (time 0) were incubated with λ-phosphatase in the presence or absence of phosphatase inhibitors for 15 min and then subjected to immunoblot analysis using anti-Atg13 antibody. Asterisks indicate nonspecific immunoreactive bands. (E) Atg13 is dephosphorylated in FIP200^−/− MEFs. FIP200^+/+ and FIP200^−/− MEFs were cultured in complete or starvation medium for 60 min. Cell lysates were then analyzed by immunoblotting using anti-Atg13 antibody. (F) Atg13 is dephosphorylated in the absence of ULK1. HeLa cells treated with siRNA against ULK1 were analyzed by immunoblotting using anti-Atg13 antibody.

mTORC1 interacts with the ∼3-MDa complex under nutrient-rich conditions. (A) HEK293T cells were transfected with an empty vector or ULK1-FLAG. Cells were then cultured in complete or starvation medium as indicated. In the restimulation experiments (replenishment), starved cells were additionally cultured in fresh complete medium for the indicated times. Cell lysates were subjected to immunoprecipitation (IP) using M2 agarose beads. The resulting precipitates were examined by immunoblot analysis with the indicated antibodies. (B) HEK293T cells were cotransfected with FLAG-raptor and HA-ULK1 and analyzed as described in A (30-min replenishment condition). (C) NIH-3T3 cells were cultured in starvation medium for 180 min. In the replenishment experiments, starved cells were cultured in fresh complete medium for additional 30 min. S100 fractions were separated as described in Figure 4A. The shift of the raptor-positive fractions is indicated. Asterisks indicate the nonspecific immunoreactive bands. (D) HEK293T cells transfected with FLAG-tagged ULK1, Atg13, and FIP200 were subjected to 3-h starvation and 30-min replenishment treatments. Cell lysates were immunoprecipitated with anti-FLAG antibody, and coprecipitation of endogenous raptor was analyzed using anti-raptor antibody. (E) HEK293T cells were transfected with FLAG-tagged ULK1 mutants, and interaction with raptor and Atg13 was determined.

TOR phosphorylates ULK1 and Atg13. (A and B) Endogenous mTOR was immunoprecipitated from HEK293T cells cultured in complete medium with anti-mTOR antibody as described in Materials and Methods. As a control, normal mouse IgG was used for immunoprecipitation. The resulting precipitates were subjected to mTOR kinase assay using FLAG-tagged ULK1^K46N (a kinase-dead mutant) (A) or Atg13 (B) as the substrate. GST-PRAS40 and GST-4E-BP1 were used as positive controls, and GST alone was used as a negative control. (C) HEK293T cells were cultured in starvation medium for the indicated times. Total cell lysates were analyzed by immunoblot analysis. (D) HEK293T cells were transfected with an empty vector or ULK1-FLAG. Cells were then starved for 180 min, followed by culturing in fresh complete medium for the indicated times. Cell lysates were analyzed as described in Figure 5A. (E) NIH3T3 cells were cultured in complete or starvation medium for 180 min. In the replenishment experiments, starved cells were pretreated with 100 ng/ml rapamycin or vehicle (DMSO) for 30 min before replenishment and then cultured in fresh complete medium for an additional 30 min with and without rapamycin.

Atg13 constitutively forms a ∼3-MDa complex including ULK1 and FIP200. (A) MEFs were cultured in complete or starvation medium for 60 min. S100 fractions of the cell lysates (the supernatant obtained after centrifugation at 100,000× g) was separated by size exclusion chromatography on a Superose 6 column. Each fraction was analyzed by immunoblotting with anti-Atg13, anti-ULK1, and anti-FIP200 antibodies. Positions of the molecular mass standards (in kDa) are shown. V, void fraction. Asterisks indicate nonspecific immunoreactive bands. (B) Lysates of wild-type and FIP200^−/− MEFs cultured in complete medium were analyzed as described in A. Asterisks indicate nonspecific immunoreactive bands. (C and D) HeLa cells were treated with the control or Atg13 siRNA. Total cell lysates were subjected to immunoblot analysis using the indicated antibodies (C). The S100 fractions were then analyzed by gel filtration as in A (D). Asterisks indicate the nonspecific immunoreactive bands found in human cells. (E) HEK293T cells were cotransfected with FLAG-FIP200, HA-ULK1, and Atg13. Cell lysates were subjected to immunoprecipitation (IP) using anti-FLAG antibody and examined by immunoblot (IB) analysis with anti-Atg13, anti-HA, and anti-FLAG antibodies. Both endogenous and exogenous Atg13 were detected with the anti-Atg13 antibody.

Atg13 is essential for autophagy. (A and B) HeLa cells were transfected with two different Atg13 siRNA sets or control siRNA. After 2 d, cells were once again transfected with the same siRNA and cultured for an additional 3 d. Cells were then incubated in regular DMEM or starvation medium in the presence or absence of E64d (50 μM) and pepstatin A (50 μg/ml) for 2 h (A) or in the absence of these inhibitors for 4 h (B). Total lysates were analyzed by immunoblotting using anti-Atg13, anti-LC3, anti-p62, and anti-β-actin antibodies. An asterisk indicates nonspecific immunoreactive bands. (C) HeLa cells stably expressing GFP-LC3 were treated with Atg13 or control siRNA. After 4 d, cells were incubated in complete or starvation medium for 4 h then GFP-LC3 signals were observed by fluorescence microscopy. Scale bar, 20 μm. (D) HeLa cells treated with or without Atg13 siRNA (Atg13_#2) were starved for 2 h with E64d (50 μM) and pepstatinA (50 μg/ml) and then subjected to conventional electron microscopic analysis. Autophagic vacuoles (autophagosomes + autolysosomes) are indicated by arrowheads. Bar, 1 μm. The ratio of the total area of autophagic vacuoles to total cytoplasmic area was determined by morphometric analysis.