Volume 31, Issue 6, Pages 734-746 (December 2014) AMBRA1 Interplay with Cullin E3 Ubiquitin Ligases Regulates Autophagy Dynamics  Manuela Antonioli, Federica Albiero, Francesca Nazio, Tiziana Vescovo, Ariel Basulto Perdomo, Marco Corazzari, Claudia Marsella, Pierluca Piselli, Christine Gretzmeier, Jörn Dengjel, Francesco Cecconi, Mauro Piacentini, Gian Maria Fimia  Developmental Cell  Volume 31, Issue 6, Pages 734-746 (December 2014) DOI: 10.1016/j.devcel.2014.11.013 Copyright © 2014 Elsevier Inc. Terms and Conditions

Developmental Cell 2014 31, 734-746DOI: (10.1016/j.devcel.2014.11.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 AMBRA1 Interaction with Cullin E3 Ubiquitin Ligases (A) Immunoblotting analysis of anti-Flag immunoprecipitation (IP) and corresponding total extracts from Flag-AMBRA1-expressing 2FTGH cells using the indicated antibodies. GFP-expressing cells (−) were used as a negative control. (B) Immunoblotting analysis of anti-Flag IP and corresponding total extracts from Flag-AMBRA1-expressing 293T cells transfected with expression vectors for seven Myc-tagged Cullins (1, 2, 3, 4A, 4B, 5, and 7) using an anti-Flag and anti-Myc antibodies. (C) Characterization of AMBRA1/Cullin complexes at endogenous level. Immunoblotting analysis of anti-AMBRA1 IP and corresponding total extracts from 2FTGH cells using anti-DDB1, anti-Elongin B (Elo B), anti-CUL4A, and anti-CUL7 antibodies. IgG, immunoglobulin G. (D) Immunoblotting analysis of anti-BECLIN1 IP and corresponding total extracts from 293T cells using anti-DDB1, AMBRA1, and BECLIN1 antibodies. (E) Immunoblotting analysis of anti-TRAF6 IP and corresponding total extracts from 293T cells using anti-DDB1, AMBRA1 and TRAF6 antibodies. An unrelated IgG was used as negative control. See also Figure S1. Developmental Cell 2014 31, 734-746DOI: (10.1016/j.devcel.2014.11.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Regulation of AMBRA1 Protein Stability during Autophagy (A) Immunoblotting analysis of AMBRA1 levels in 2FTGH cells treated with proteasome inhibitor (MG132) or lysosome (Bafilomycin A1) (Baf A1) inhibitors. h, hours. (B) Flag-AMBRA1-expressing 2FTGH cells were treated, or not, with MG132 and subjected to immunoprecipitation (IP) in denaturating condition using anti-Flag antibody. AMBRA1 ubiquitination was evaluated using an anti-Ubiquitin (Ub) antibody. (C) Immunoblotting analysis of AMBRA1, VPS34, BECLIN 1, and LC3 levels in 2FTGH cells incubated with the amino-acid-free medium EBSS for 1, 2, 4, or 6 hr. Error bars indicate mean ± SD. (D) Immunoblotting analysis of ectopically expressed Flag-AMBRA1 in 2FTGH cells starved for 1, 2, 4, or 6 hr. Error bars indicate mean ± SD. ∗p ≤ 0.05. (E) Real-time PCR analysis of AMBRA1 mRNA levels in 2FTGH cells incubated in EBSS medium for 0.5, 1, 2, 4, or 6 hr. Data are expressed as mean ± SD, n = 5 independent experiments. a.u., arbitrary units. Error bars indicate mean ± SD. ∗p ≤ 0.05. (F) Immunoblotting analysis of AMBRA1 levels in 2FTGH cells starved for 1, 2, or 4 hr in the presence or absence of MG132. (G) 293T cells were transfected with a vector encoding a 6xHis-tag ubiquitin and starved for 1, 2, or 4 hr. Protein extracts, prepared in urea buffer, were subjected to Ni-NTA purification and ubiquitinated AMBRA1 levels were evaluated by immunoblotting analysis. See also Figure S2. Developmental Cell 2014 31, 734-746DOI: (10.1016/j.devcel.2014.11.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 DDB1 and Cullin 4 Ubiquitinate AMBRA1 (A) Upper panel: immunoblotting analysis of SUGT1, Elongin B (Elo B), or DDB1 levels in 2FTGH cells transfected with indicated RNAi oligonucleotides. Lower panel: immunoblotting analysis of AMBRA1 levels in silenced cells upon 0 or 4 hr starvation. (B) DDB1-silenced 293T cells were transfected with HA-tag ubiquitin vector, subjected to anti-HA immunoprecipitation (IP) in denaturating conditions, and analyzed by immunoblotting to evaluate AMBRA1 ubiquitination using an anti-AMBRA1 antibody. (C) In vitro AMBRA1 ubiquitination assay using immunopurified Myc-tag Cullin 4A or Myc-tag Cullin 4B, Flag AMBRA1, and recombinant HA ubiquitin and E2 ubiquitin-conjugating enzyme (UbcH5C). Reactions were performed in the presence or absence of E1 ubiquitin-activating enzyme (Ube1) to verify ubiquitination reaction specificity. AMBRA1 ubiquitination was evaluated upon IP by immunoblotting using an anti-HA antibody. (D) Flag-AMBRA1-expressing 2FTGH cells were starved for 0.5, 1, 2, or 4 hr; subjected to IP using an anti-Flag antibody; and analyzed by immunoblotting using anti-DDB1 and anti-Flag antibodies. Corresponding total extracts were also probed with these antibodies and an anti-GAPDH as a loading control. To immunoprecipitate equal amounts of AMBRA1 in cells with different AMBRA1 levels, proteins were immunoprecipitated using a nonsaturating quantity of Flag antibody. (E) 2FTGH cells expressing a dominant-negative ULK1 mutant with a Lys46-to-Ile substitution (ULK1 K46I) were starved for 0, 1, and 2 hr. Protein extracts were subjected to IP using an anti-AMBRA1 antibody and analyzed by immunoblotting together with corresponding total extracts using anti-DDB1 and anti-AMBRA1 antibodies. Lower panel: Band density ratio of immunoprecipitated DDB1 relative to immunoprecipitated AMBRA1. Data are expressed as mean ± SD; where not indicated, statistical significance refers to the respective control (∗p ≤ 0.05; ∗∗p ≤ 0.005). See also Figure S3. Developmental Cell 2014 31, 734-746DOI: (10.1016/j.devcel.2014.11.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Regulation of Autophagy by DDB1 (A) Representative fluorescence images of GFP-RFP-LC3-expressing 2FTGH cells transfected with DDB1 RNAi oligonucleotides (iDDB1a and iDDB1b) or unrelated oligonucleotides as negative control (iCtr). GFP and RFP puncta per cell were counted in fed and starved (4 hr) conditions in three independent experiments (see graph). Scale bar, 15 μm. (B) 2FTGH cells stably expressing Flag mATG16L were transfected with DDB1 RNAi oligonucleotides, and autophagy was induced by starving cells for 0, 1, 2, 4, 6, or 8 hr (h). ATG16-positive dots per cell were counted to evaluate the rate of autophagosome formation. Representative fluorescence images are accompanied by a graph reporting data from three independent experiments. Scale bar, 12 μm. (C) Flag-mATG16L-expressing 2FTGH cells were infected with retroviral vectors encoding GFP (Ctr), Flag-AMBRA1 FL, or Flag-AMBRA1 ΔWD40 and starved for 0, 1, 2, 4, 6, or 8 hr. The analysis of autophagy induction was evaluated as described in (B). Scale bar, 10 μm. Data in all panels are expressed as mean ± SD of triplicate samples with 50 cells analyzed per sample (∗∗∗p < 0.001). See also Figure S4. Developmental Cell 2014 31, 734-746DOI: (10.1016/j.devcel.2014.11.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Cullin 5 Regulates Autophagy by Modulating DEPTOR Stability (A) Representative fluorescence images of GFP-RFP-LC3-expressing 2FTGH cells transfected with Elongin B (iELO1a and iELO1b) or SUGT1 (iSUGT1a and iSUGT1b) RNAi oligonucleotides. Unrelated oligonucleotides were used as negative control (iCtr). GFP and RFP puncta per cell were counted in fed and starved (4 hr) conditions in three independent experiments (see graph). Scale bar, 15 μm. Data are expressed as mean ± SD with 50 cells analyzed per sample (∗∗∗p < 0.001). (B) Elongin B expression was downregulated by RNAi, and cells were nutrient starved for the indicated times. Protein extracts were analyzed by immunoblotting for the expression of DEPTOR, phospho-Ser757 ULK1 (pS757 ULK1), ULK1, phospho-p70 S6K1, p70 S6K1, 4EBP1, AMBRA1, LC3, and Elongin B. GAPDH was analyzed as a loading control. The graph reports the quantification of DEPTOR levels. h, hours. (C) RNAi-silenced cells for DEPTOR expression were nutrient starved for the indicated times. pS757 ULK1, ULK1, LC3, AMBRA1, and DEPTOR protein levels were analyzed by immunoblotting. GAPDH was analyzed as a loading control. Quantification of DEPTOR levels is shown in the graph. Data in (B) and (C) are expressed as mean ± SD (n = 3). Where not indicated, the statistical significance refers to the respective control (∗p ≤ 0.05; ∗∗p ≤ 0.005; ∗∗∗p ≤ 0.0005). (D) Upper panels: representative fluorescence images of RFP-LC3-expressing 2FTGH cells transfected with Elongin B (ELOBa) or DEPTOR RNAi oligonucleotides either separately or in combination. RFP puncta per cell were counted to evaluate autophagy levels in three independent experiments as shown in the lower panel. Scale bar, 18 μm. Data are expressed as mean ± SD of triplicate samples, with 50 cells analyzed per sample (∗∗∗p < 0.001). (E) Flag DEPTOR encoding vector (1 μg) was transfected in 293T cells alone or in combination with Myc CUL2 or Myc CUL5 plasmids (7.5 μg). DEPTOR and Cullins protein levels were analyzed by immunoblotting using anti-Flag and anti-Myc antibodies, respectively. GAPDH was analyzed as a loading control. (F) In vitro AMBRA1 ubiquitination assay using immunopurified Myc-tag Cullin 5A and Flag-DEPTOR together with recombinant HA-ubiquitin and E2 ubiquitin-conjugating enzyme (UbcH5C). Reactions were performed in the presence or absence of E1 ubiquitin-activating enzyme (Ube1) to verify ubiquitination reaction specificity. DEPTOR ubiquitination were evaluated upon IP by immunoblotting using an anti-HA antibody. See also Figure S5. Developmental Cell 2014 31, 734-746DOI: (10.1016/j.devcel.2014.11.013) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 AMBRA1 Allows DEPTOR Protein Stabilization during Autophagy by Negatively Regulating Cullin 5 Activity (A) Immunoblotting analysis of anti-Flag immunoprecipitation (IP) and corresponding total extracts from Flag-AMBRA1-expressing cells starved for the indicated times using anti-Elongin B (Elo B) and anti-Flag antibodies. To immunoprecipitate equal amounts of AMBRA1 in cells with different AMBRA1 levels, proteins were immunoprecipitated using a nonsaturating quantity of Flag antibody. Total extracts were also probed with DEPTOR and GAPDH antibodies. (B) Wild-type and AMBRA1gt/gt MEFs were cultured in fed or starved conditions for the indicated times. Protein extracts were probed by immunoblotting to analyze the expression levels of pS757 ULK1, ULK1, and DEPTOR proteins. GAPDH was analyzed as a loading control. (C) ULK1/ULK2 DKO MEFs were cultured in the starved condition for the indicated times. Protein extracts were analyzed by immunoblotting to analyze the expression of DEPTOR, Elongin B, AMBRA1, and DDB1. GAPDH was analyzed as a loading control. (D) 293T cells were transfected with Flag DEPTOR encoding vector (1 μg) in combination with plasmids encoding Myc CUL2 or Myc CUL5 (7 μg), and AMBRA1 (2 μg), as indicated. DEPTOR, Cullins, and AMBRA1 protein levels were analyzed by immunoblotting using anti-Flag, anti-Myc, and anti-AMBRA1 antibodies, respectively. GAPDH was analyzed as a loading control. (E) 293T cells were transfected with increasing amounts of AMBRA1-encoding vector (2.5 μg and 5 μg). The amount of Cullin 5-Elongin B interaction was evaluated by analyzing Elongin B levels copurified with Myc-tag CUL5. (F) The in vitro ubiquitination assay of DEPTOR described in Figure 5E was repeated in the presence or absence of immunopurified AMBRA1. See also Figure S6. Developmental Cell 2014 31, 734-746DOI: (10.1016/j.devcel.2014.11.013) Copyright © 2014 Elsevier Inc. Terms and Conditions