Yeast two-hybrid screening
pGBKT7-AMBRA1 was generated by cloning the C-terminal region (aa 533–1,269) of AMBRA1 into the EcoRI and SalI sites of pGBKT7 (Takara Bio Inc.). A human brain cDNA library cloned in pACT2 (Takara Bio Inc.) was screened by cotransformation with the pGBKT7-AMBRA1 into AH109 yeast strain. Positive clones were selected based on their growth on Trp, Leu, Ade, and His dropout media (Takara Bio Inc.) containing 5 mM 3-amino-1,2,4-triazole (3AT; Sigma-Aldrich). Recovery of the plasmids and β-galactosidase (βgal) assay were performed following the manufacturer’s directions (Matchmaker two-hybrid system protocol; Takara Bio Inc.).
The human fibrosarcoma 2F, HEK293, and ETNA cells were cultured in DME (Sigma-Aldrich) supplemented with 10% FCS (Sigma-Aldrich), 2 mM l-glutamine, and 1% penicillin/streptomycin solution at 37°C (2F and HEK293) or 33°C (ETNA) under 5% CO2. For autophagy induction, cells were treated with 0.2–2 µM rapamycin (Sigma-Aldrich) or cultured for 4 h in Earle’s balanced salt solution (Invitrogen). When indicated, cells were incubated in complete or starvation medium in the presence of 1 or 10 µM nocodazole (Sigma-Aldrich), 10 µg/ml E64d together with 10 µg/ml pepstatin A (Sigma-Aldrich), 200 nM Wortmannin (Sigma-Aldrich), 10 mM 3-methyl adenine, and 10 µM JNK inhibitor SP600125 (Sigma-Aldrich) for 4 h before the assay. For EGFR internalization assay, cells were grown overnight in DME plus 0.1% FCS and treated with 100 ng/ml EGF (PeproTech) for 10, 20, and 40 min before analysis. For lysosome staining, 50 nM Lysotracker green (Invitrogen) was added to cells 30 min before analysis. 2F and 293 cells were transiently transfected with expression vectors using Lipofectamine 2000 (Invitrogen) as indicated by the supplier. 2F cells stably expressing ER-GFP protein were obtained by culturing pCMV/Myc/ER-GFP–transfected cells in the presence of 0.4 mg/ml G418 (Invitrogen) for 2 wk.
For retroviral expression, all constructs were cloned in pCLPCX vector. BECLIN 1, GFP-LC3, GFP-p40Phox, and FL, F1, F2, and F3 AMBRA1 vectors were previously described (Fimia et al., 2007
). GFP-DFCP1 plasmid was provided by N.T. Ktistakis (Babraham Institute, Cambridge, England, UK; Axe et al., 2008
). ULK1 constructs were provided by S.A. Tooze (London Research Institute, London, England, UK; Chan et al., 2009
). The human DLC1 was cloned in frame with HA tag. AMBRA1 mutants TAT1 and TAT2 were generated by using the site-directed mutagenesis kit (Agilent Technologies) and cloned in frame with a Myc or Flag tag. mCherryLC3 was generated by replacing, in pCLPCX-GFP-LC3 plasmid, the GFP sequence with mCherry amplified by PCR from the pmCherry-N1 vector (Takara Bio Inc.). mCherryAMBRA1 was generated by subcloning mCherry sequence at the 5′ end of AMBRA1 in pCLPCX-AMBRA1. pCMV Myc ER-GFP was purchased from Invitrogen. The sequences of all PCR-amplified cDNAs were verified by DNA sequencing analysis.
Retrovirus generation and infection
15 µg retroviral vectors was cotransfected with 5 µg expression plasmid for the vesicular stomatitis virus G protein into 293 gp/bsr cell line by using the calcium phosphate method. 48 h later, the supernatant containing the retroviral particles was recovered and supplemented with 4 µg/ml polybrene. 2F or ETNA cells were infected by incubation with retroviral containing supernatant for 6–8 h.
The following primary antibodies were used in this study: rabbit anti-Myc tag (Millipore), mouse anti-HA tag (Sigma-Aldrich), mouse anti-Flag tag (WB and EM analyses; Sigma-Aldrich), mouse anti-DLC1 (BD), mouse anti-DIC (Santa Cruz Biotechnology, Inc.), rabbit anti-ULK1 (Sigma-Aldrich), rabbit and goat anti–BECLIN 1 (WB and immunofluorescence analyses, respectively; Santa Cruz Biotechnology, Inc.), rabbit anti–BECLIN 1 (WB analysis; Cell Signaling Technology), rabbit anti-VPS34 (Invitrogen), rabbit anti-LC3 (Cell Signaling Technology), rabbit anti-AMBRA1 (WB analysis; Strategic Diagnostic, Inc.), rabbit anti-AMBRA1 (IF and EM analysis; Covalab), rabbit anti-AMBRA1 CT (IP and EM analysis; ProSci, Inc.), mouse anti-ERp57 (Stressgen), mouse anti-LAMP1 and anti-EEA1 (Abcam), mouse anti-GOLGIN (Invitrogen), mouse anti–complex V α subunit (Invitrogen), mouse anti–α-tubulin (Sigma-Aldrich), mouse anti-EGFR (Millipore), and rabbit anti-calreticulin (Stressgen).
Autophagy was measured as described previously (Fimia et al., 2007
). In brief, starvation was induced by incubating cells in Earle’s balanced salt solution medium (Sigma-Aldrich) for 4–5 h. For immunodetection of LC3 puncta, cells were grown on coverslips and fixed with 4% PFA in PBS, washed three times, and directly examined by confocal microscopy. The results indicate the percentage of GFP-LC3–positive cells with GFP-LC3 punctate dots or the numbers of GFP-LC3 punctate dots per cell. A minimum of 50–100 cells per sample was counted for triplicate samples per condition per experiment. To quantify the development of AVOs, cells were detached by trypsin digestion, washed with PBS, stained with 1 µg/ml Acridine orange (Sigma-Aldrich) for 15 min, and analyzed using a flow cytometer (FACScan; BD) and CellQuest software (BD). Autophagy experiments in vivo were performed using C57BL/6 mice. 5-mo-old male mice from the same littermate were kept without food but with water for 24 and 48 h or fed ad libitum before sacrifice and tissue analysis.
For electron microscopy, cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 45 min at 4°C, rinsed in cacodylate buffer, postfixed in 1% OsO4 in cacodylate buffer, dehydrated, and embedded in epon. Ultrathin sections were briefly contrasted with uranyl acetate and photographed with an electron microscope (CM900; Carl Zeiss, Inc.).
IP and WB assays
In IP experiments, cells or tissues were lysed in HEMG buffer (25 mM Hepes, pH 8.0, 100 mM NaCl, 25 mM MgCl2
, 0.5% Triton X-100, 0.1 mM EDTA, and 10% glycerol) plus protease and phosphatase inhibitors (protease inhibitor cocktail plus 1 mM sodium fluoride, 1 mM sodium orthovanadate, and 1 mM sodium molibdate; Sigma-Aldrich). For analysis of the BECLIN 1–dynein complex, IP was performed with a different lysis buffer (40 mM Hepes, pH 7.4, 2 mM EDTA, 10 mM glycerophosphate, and 0.3% CHAPS plus protease and phosphatase inhibitors) and a wash buffer (40 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 mM glycerophosphate, and 0.3% CHAPS) as described previously (Hosokawa et al., 2009
). 1–3 mg lysates was incubated at 4°C for 30 min. After a centrifugation at 4°C for 10 min at 13,000 g
to remove insoluble debris, equal amounts of protein were incubated with 20 µl monoclonal anti-cMyc or anti-Flag antibody conjugated with protein A agarose beads (Takara Bio Inc. and Sigma-Aldrich, respectively) with rotation at 4°C for 4 h or with 30 µl monoclonal anti-HA antibody conjugated with protein A agarose (Sigma-Aldrich) or 2 µg anti-DIC antibody overnight at 4°C, or 2 µg anti-AMBRA1 antibody (CT; ProSci, Inc.) followed by 60-min incubation with 30 µl protein A/G–Sepharose beads (Roche). The beads were collected by centrifugation and washed four times with HEMG buffer. Proteins bound to the beads were eluted with 50 µl SDS-PAGE sample buffer and boiled at 95°C for 10 min. WB analyses were performed using 5% vol/vol of the whole extracts and 30% vol/vol of eluted proteins. In AMBRA–tubulin interaction experiments, immunocomplexes were eluted by incubation with a Flag peptide (Sigma-Aldrich) for 30 min at RT to prevent the presence of immunoglobulins in the gel.
Proteins were separated on NuPAGE Bis-Tris gel (Invitrogen) and electroblotted onto nitrocellulose (Protran; Schleicher & Schuell) or PVDF (Millipore) membranes. Blots were incubated with primary antibodies in 5% nonfat dry milk in TBS plus 0.1% Tween-20 overnight at 4°C. Detection was achieved using horseradish peroxidase–conjugated secondary antibody (Bio-Rad Laboratories) and visualized with ECL plus (GE Healthcare). Note that endogenous AMBRA1 usually migrates as a doublet band, whereas the overexpressed AMBRA1 shows additional lower molecular weight bands.
To better visualize AMBRA1 mobility shift induced by phosphorylation, protein samples were resolved on SDS-PAGE gels (Anderson et al., 1973
). For the λ-phosphatase assay, cells were lysed in HEMG and incubated with 400 U λ-phosphatase (New England BioLabs, Inc.) for 45 min at 30°C. Reactions were stopped by addition of 4× SDS sample buffer and boiled for 10 min.
Confocal and immunogold analysis
For confocal analysis, cells were grown on coverslips and fixed with 4% PFA in PBS followed by permeabilization with 0.1% Triton X-100 in PBS or, when indicated, fixed with ice-cold methanol or with 4% PFA in PBS followed by permeabilization with ice-cold methanol. Primary antibodies were incubated for 1 h at RT and visualized by means of Cy3- and Cy2-conjugated secondary antibodies. (Jackson ImmunoResearch Laboratories, Inc.). Coverslips were mounted in antifade (SlowFade; Invitrogen) and examined under a confocal microscope (TCS SP2; Leica) equipped with a 63× 1.40–0.60 NA HCX Plan Apo oil λBL objective at RT. For colocalization analysis, both confocal (Leica) and ImageJ software (National Institutes of Health) were used. Regarding the confocal software analysis, the local intensity distribution of both fluorophores across a line scan drawn on a single optical slice was plotted in a graph. Regarding ImageJ software analysis, the pixels of two 8-bit images (red and green channels of each image) are considered colocalized if their intensities are higher than the threshold of their channels (set at 50) and if the ratio of their intensity is higher than the ratio setting value (set at 50%). Colocalization was assessed by calculating the Pearson’s correlation coefficient r of at least 10 cells analyzed in two independent experiments. The Pearson’s correlation coefficient was expressed as mean ± SD. The fluorescence intensity of each fluorochrome was simultaneously analyzed and plotted.
For electron microscopy, 2F cells were fixed in 2% freshly depolymerised PFA and 0.2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 1 h at 4°C. Samples were rinsed in buffer, partially dehydrated, and embedded in London Resin white (LR White; Agar Scientific Ltd.). As shown in Fig. S3 J, to preserve membrane structures, the fixed cells were rinsed in the same buffer, treated with 0.25% tannic acid in buffer for 1 h at 4°C, and rinsed in 50 mM ammonium chloride in buffer. The samples were postfixed for 30 min in 2% uranyl acetate in buffer, partially dehydrated, and embedded under UV light at −20°C in London Resin gold (LR White; Agar Scientific Ltd.). Ultrathin sections were processed for the immunogold technique. Grids were preincubated with 10% normal goat serum in 10 mM PBS containing 1% BSA and 0.13% NaN3 (medium A) for 15 min at RT. Sections were incubated with anti-AMBRA1 or anti-Flag antibodies diluted in medium A overnight at 4°C. After rinsing in medium A containing 0.01% Tween-20 (Merck), sections were incubated with the appropriate secondary antibodies conjugated to 15-nm colloidal gold (British Biocell International) diluted 1:30 in medium A containing fish gelatin for 1 h at RT. After incubation in medium A for 15 min at RT, a second immunolabeling was performed using a rabbit polyclonal anti-DLC1 or anti-calreticulin, as primary antibodies and goat anti–rabbit IgG conjugated to 5-nm colloidal gold as secondary antibody. Grids were thoroughly rinsed in distilled water, contrasted with aqueous 2% uranyl acetate for 20 min, and photographed in an electron microscope (EM 900 Carl Zeiss, Inc.).
RNAi was performed using the following oligonucleotides from Invitrogen: DLC1, DLC2, ROADBLOCK1, TCTEX1, and ULK1 oligo. AMBRA1 siRNA oligonucleotides were described previously (Fimia et al., 2007
). 2 × 105 cells/well were transfected with 100 pmol siRNA in 6-well plates by Lipofectamine 2000 (Invitrogen) as indicated by the supplier. Transfection was repeated on two consecutive days to increase transfection efficiency. RNA decrease was checked by real-time PCR and WB analysis 48 h after transfection.
RNA was prepared with Trizol reagent (Invitrogen). cDNA synthesis was generated using the reverse transcription kit (Promega) according to manufacturer recommendations. Real-time PCR reactions were performed with the LightCycler (Roche). The LightCycler FastStart DNA Master SYBR green I (Roche) was used to produce fluorescent-labeled PCR products during repetitive cycling of the amplification reaction as previously described (Fimia et al., 2007
). Primer sets for all amplicons were designed using the Primer Express software system (version 1.0; Applied Biosystems): AMBRA1
(forward), 5′-AACCCTCCACTGCGAGTTGA-3′ and (reverse) 5′-TCTACCTGTTCCGTGGTTCTCC-3′; L34
(forward), 5′-GTCCCGAACCCCTGGTAATAGA-3′ and (reverse) 5′-GGCCCTGCTGACATGTTTCTT-3′; DLC1
(forward), 5′-CCCCCACCTCAGGTAACCAT-3′ and (reverse) 5′-GCCTGAGTAGCGCACTCCAC-3′; DLC2
(forward), 5′-GACTCGCCTCCGTGAAGTGTC-3′ and (reverse) 5′-GGCGCAGTCAACGGCAT-3′; ROADBLOCK1
(forward), 5′-GAGCCAGAAGGGAGTGCAGG-3′ and (reverse) 5′-TGAGGCTGGCATACTGGGTG-3′; TCTEX1
(forward), 5′-GGGACAGCTCTACTGACGGGA-3′ and (reverse) 5′-AAGGCCATAGGCTGGACTGC-3′; and ULK1
(forward), 5′-AAGGCCATAGGCTGGACTGC-3′ and (reverse) 5′-AAGGCCATAGGCTGGACTGC-3′. L34
mRNA level was used as an internal control. β-Actin
levels were used as additional controls to confirm significant decreases.
Bidimensional gel electrophoresis analysis
Proteins were precipitated using a clean up kit (Ettan 2-D; GE Healthcare) following the manufacturer’s instructions and were subsequently resuspended in urea buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1% sulfobetaine SB3-10, 1% amidosulfobetaine ASB14, and 50 mM DTT).
For the first dimension of protein separation, isoelectric focusing was performed using 7-cm immobilized nonlinear pH gradient strips, 3–10, and linear pH gradient strips, 4–7 (GE Healthcare), on an electrophoresis unit (IPGphor II; GE Healthcare). 30 µg proteins was loaded by passive in-gel rehydration for 12 h then run using a program in which the voltage was set for 90 min at 30 V, 30 min at 100 V, 30 min at 200 V, voltage gradient 15 min up to 500 V, 30 min at 500 V, voltage gradient 15 min up to 1,000 V, 30 min at 1,000 V, voltage gradient 60 min up to 5,000 V, and 180 min at 5,000 V.
Before the second dimension of electrophoresis, IPG gel strips were equilibrated for 15 min at RT in 1% DTT to reduce the proteins, and sulfhydryl groups were subsequently derivatized using 4% iodoacetamide (both solutions were prepared in 50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 2% bromophenol blue) for 15 min. Strips were transferred to 1.0-mm-thick 8%, 10%, and 15% (wt/vol) polyacrylamide minigels for AMBRA1, DIC, and DLC1 detection, respectively, and the second-dimension gels were run at 120 V for 2 h. The 2D gels were transferred to nitrocellulose filters as described for WB.
In vitro mixed beads kinase assay
HEK293 cells were transiently transfected with Myc-tagged wild-type or K46I ULK1 or with Myc-tagged AMBRA1 cDNAs, and the ULK1-transfected ones were nutrient starved for 2 h. Protein extracts were subjected to IP with an anti-Myc antibody, and a mixed beads kinase assay was performed as described previously (Chan et al., 2009
). In brief, wild-type Myc-ULKI– and K46I Myc-ULK1–immunopurified proteins were washed four times with HEMG buffer and once with kinase reaction buffer (20 mM Hepes, pH 7.5, 20 mM MgCl2
, 25 mM β-glycerophosphate, 2 mM DTT, and 100 µM sodium orthovanadate) before being combined together with Myc-AMBRA1–immunopurified proteins and incubated in a 20-µl final volume of kinase reaction buffer containing 5 µCi γ-[32
P]ATP (PerkinElmer) at 30°C for 30 min. Reactions were stopped by addition of 4× SDS sample buffer and boiled for 10 min. [32
P]-labeled reaction products were resolved on SDS-PAGE and analyzed by autoradiography using a phosphoimager scanner (Typhoon; GE Healthcare).
Velocity sedimentation by sucrose gradient
2F cells were treated with 5 µM taxol (Sigma-Aldrich) for 4 h. Cell lysates were suspended in a buffer containing 0.25 M sucrose, 10 mM Hepes, and 1 mM EDTA plus a protease inhibitor cocktail. Cell suspension was homogenized by 100 strokes in a dounce potter homogenizer and centrifuged for 10 min at 600 g to obtain a postnuclear supernatant. The postnuclear supernatant was recentrifuged for 15 min at 11,000 g to obtain a postmitochondrial supernatant. The postmitochondrial supernatant was layered onto a discontinuous four-step gradient consisting of 2 ml each of 2.0 M, 1.3 M, 1.0 M, and 0.6 M sucrose in 10 mM Hepes. Centrifugation was performed using a rotor (SW41 Ti; Beckman Coulter) at 27,000 g for 18 h, and 0.4-ml fractions were manually collected and checked for density.
All experiments were performed at least three times. Excel (Microsoft) was used for statistical analysis. Statistical significance was determined using the Student’s t test. P ≤ 0.05 was considered significant.
Online supplemental material
Fig. S1 shows the colocalization of AMBRA1 and DLC1 in normal and starvation conditions by immunogold analysis. Fig. S2 shows that AMBRA1 is phosphorylated during autophagy in an ULK1-dependent manner both in vitro and in vivo. In particular, ULK1 K46I dominant negative prevents AMBRA1 phosphorylation after autophagy induction, and the dissociation of AMBRA1 from the dynein motor complex during autophagy is independent of JNK and PI3K activity. Fig. S3 shows a detailed analysis of AMBRA1 localization in different subcellular compartments, thus demonstrating the translocation of AMBRA1 to the ER after autophagy induction. It also shows that AMBRA1 partially colocalizes with the PI3P-binding protein DFCP1 upon autophagy induction. Fig. S4 shows the modulation of autophagy levels by down-regulation of different DLCs. Moreover, the interaction between AMBRA1 and DLC2 is reported. Fig. S5 shows that AMBRA1 is not required for the endosomal sorting of EGF receptor and that ULK1 is required for the activity of constitutive proautophagic AMBRA1 mutants. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201002100/DC1