Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy
2011; Springer Nature; Volume: 30; Issue: 7 Linguagem: Inglês
10.1038/emboj.2011.49
ISSN1460-2075
AutoresFlavie Strappazzon, Matteo Vietri Rudan, Silvia Campello, Francesca Nazio, Fulvio Florenzano, Gian María Fimia, Mauro Piacentini, Beth Levine, Francesco Cecconi,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoArticle25 February 2011free access Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy Flavie Strappazzon Flavie Strappazzon Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Matteo Vietri-Rudan Matteo Vietri-Rudan Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Biology, Dulbecco Telethon Institute, University of Rome 'Tor Vergata', Rome, Italy Search for more papers by this author Silvia Campello Silvia Campello Department of Cell Physiology and Metabolism, University of Geneva Medical School, Geneva, Switzerland Search for more papers by this author Francesca Nazio Francesca Nazio Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Biology, Dulbecco Telethon Institute, University of Rome 'Tor Vergata', Rome, Italy Search for more papers by this author Fulvio Florenzano Fulvio Florenzano Confocal Microscopy Unit, CNR-S.Lucia Foundation, Rome, Italy Search for more papers by this author Gian Maria Fimia Gian Maria Fimia National Institute for Infectious Diseases IRCCS 'L.Spallanzani', Rome, Italy Search for more papers by this author Mauro Piacentini Mauro Piacentini National Institute for Infectious Diseases IRCCS 'L.Spallanzani', Rome, Italy Department of Biology, University of Rome 'Tor Vergata', Rome, Italy Search for more papers by this author Beth Levine Beth Levine Howard Hughes Medical Institute and UT Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Francesco Cecconi Corresponding Author Francesco Cecconi Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Biology, Dulbecco Telethon Institute, University of Rome 'Tor Vergata', Rome, Italy Search for more papers by this author Flavie Strappazzon Flavie Strappazzon Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Matteo Vietri-Rudan Matteo Vietri-Rudan Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Biology, Dulbecco Telethon Institute, University of Rome 'Tor Vergata', Rome, Italy Search for more papers by this author Silvia Campello Silvia Campello Department of Cell Physiology and Metabolism, University of Geneva Medical School, Geneva, Switzerland Search for more papers by this author Francesca Nazio Francesca Nazio Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Biology, Dulbecco Telethon Institute, University of Rome 'Tor Vergata', Rome, Italy Search for more papers by this author Fulvio Florenzano Fulvio Florenzano Confocal Microscopy Unit, CNR-S.Lucia Foundation, Rome, Italy Search for more papers by this author Gian Maria Fimia Gian Maria Fimia National Institute for Infectious Diseases IRCCS 'L.Spallanzani', Rome, Italy Search for more papers by this author Mauro Piacentini Mauro Piacentini National Institute for Infectious Diseases IRCCS 'L.Spallanzani', Rome, Italy Department of Biology, University of Rome 'Tor Vergata', Rome, Italy Search for more papers by this author Beth Levine Beth Levine Howard Hughes Medical Institute and UT Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Francesco Cecconi Corresponding Author Francesco Cecconi Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Department of Biology, Dulbecco Telethon Institute, University of Rome 'Tor Vergata', Rome, Italy Search for more papers by this author Author Information Flavie Strappazzon1, Matteo Vietri-Rudan1,2, Silvia Campello3, Francesca Nazio1,2, Fulvio Florenzano4, Gian Maria Fimia5, Mauro Piacentini5,6, Beth Levine7 and Francesco Cecconi 1,2 1Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy 2Department of Biology, Dulbecco Telethon Institute, University of Rome 'Tor Vergata', Rome, Italy 3Department of Cell Physiology and Metabolism, University of Geneva Medical School, Geneva, Switzerland 4Confocal Microscopy Unit, CNR-S.Lucia Foundation, Rome, Italy 5National Institute for Infectious Diseases IRCCS 'L.Spallanzani', Rome, Italy 6Department of Biology, University of Rome 'Tor Vergata', Rome, Italy 7Howard Hughes Medical Institute and UT Southwestern Medical Center at Dallas, Dallas, TX, USA *Corresponding author. Department of Biology, Dulbecco Telethon Institute, University of Rome 'Tor Vergata', Via della Ricerca Scientifica, Rome 00133, Italy. Tel.: +39 067 259 4230; Fax: +39 067 259 4222; E-mail: [email protected] The EMBO Journal (2011)30:1195-1208https://doi.org/10.1038/emboj.2011.49 There is a Have you seen? (April 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info BECLIN 1 is a central player in macroautophagy. AMBRA1, a BECLIN 1-interacting protein, positively regulates the BECLIN 1-dependent programme of autophagy. In this study, we show that AMBRA1 binds preferentially the mitochondrial pool of the antiapoptotic factor BCL-2, and that this interaction is disrupted following autophagy induction. Further, AMBRA1 can compete with both mitochondrial and endoplasmic reticulum-resident BCL-2 (mito-BCL-2 and ER-BCL-2, respectively) to bind BECLIN 1. Moreover, after autophagy induction, AMBRA1 is recruited to BECLIN 1. Altogether, these results indicate that, in normal conditions, a pool of AMBRA1 binds preferentially mito-BCL-2; after autophagy induction, AMBRA1 is released from BCL-2, consistent with its ability to promote BECLIN 1 activity. In addition, we found that the binding between AMBRA1 and mito-BCL-2 is reduced during apoptosis. Thus, a dynamic interaction exists between AMBRA1 and BCL-2 at the mitochondria that could regulate both BECLIN 1-dependent autophagy and apoptosis. Introduction Autophagy has a crucial role in many health and disease processes. This catabolic process in eukaryotic cells is involved in the degradation of cellular components through the lysosome machinery. During this process, portions of cytoplasm are sequestered by double-membraned vesicles, the autophagosomes, and degraded after fusion with lysosomes for subsequent recycling. BECLIN 1, the mammalian ortholog of yeast Atg6, has an evolutionarily conserved role in macroautophagy. Several BECLIN 1-interacting proteins have been reported as regulating this function, both positively (e.g., hAtg14, Bif-1, UVRAG; Takahashi et al, 2007, 2009; Itakura et al, 2008; Liang et al, 2008; Sun et al, 2009) and negatively (e.g., Rubicon, Bcl-2 family members; Pattingre et al, 2005; Levine et al, 2008; Matsunaga et al, 2009). AMBRA1 is a positive regulator of the BECLIN 1-dependent programme of autophagy (Fimia et al, 2007). Its functional deficiency in mouse embryos leads to neuroepithelial hyperplasia associated with autophagy impairment and excessive apoptotic cell death (Cecconi et al, 2007). Further, we demonstrated that autophagosome formation is primed by AMBRA1 release from the cytoskeleton: When autophagy is induced, AMBRA1 is released from dynein in an ULK1-dependent manner, and re-localizes to the endoplasmic reticulum (ER), thus enabling autophagosome nucleation. Therefore, AMBRA1 can be considered as a crucial factor in regulating autophagy initiation (Di Bartolomeo et al, 2010). A complex relationship between autophagy and cell death exists (Levine et al, 2008; Djavaheri-Mergny et al, 2010). Currently, the well-known antiapoptotic factor BCL-2 is a key player in this context. The pool of BCL-2 resident at the ER (ER-BCL-2) is able, indeed, with the contribution of NAF-1 (nutrient-deprivation autophagy factor-1), to negatively regulate the BECLIN 1-dependent autophagic programme (Chang et al, 2010). In contrast, the mitochondrial pool of BCL-2 (mito-BCL-2) is shown to exert only an antiapoptotic function (Pattingre et al, 2005; Levine et al, 2008) even though mitochondria have been recently demonstrated to be selected sites for autophagosome formation (Hailey et al, 2010). BCL-2-regulated apoptotic cell death has been extensively studied. Overexpression of BCL-2 promotes a protective effect against a wide range of inducers of apoptosis. This antiapoptotic action derives from the fact that BCL-2 neutralizes proapoptotic BCL-2 family members, preventing mitochondrial membrane permeabilization and consequent cell death (Adams and Cory, 2007). As previous studies have demonstrated that Beclin 1/Vps34-mediated autophagy is negatively regulated through a direct interaction between BECLIN 1 and ER-BCL-2, we investigated whether the antiapoptotic BCL-2 protein and AMBRA1 could bind each other. In this context, we found that AMBRA1 is a new partner of BCL-2 in mammalian cells and that their binding is independent of BECLIN 1. More importantly, by targeting BCL-2 either to mitochondria (mito-BCL-2) or ER (ER-BCL-2), we demonstrated that mito-BCL-2 is able to bind AMBRA1 and that this binding is disrupted after both autophagy and apoptosis induction. Our findings indicate that AMBRA1 and BCL-2 bind BECLIN 1 on the same site. AMBRA1 can thus compete with mito- and ER-BCL-2 to bind BECLIN 1. In addition, we demonstrated that, after autophagy induction, AMBRA1/BECLIN 1 interaction increases both in mitochondrial and microsomal fractions, whereas the AMBRA1–mito-BCL-2 interaction is disrupted. Altogether, these results lead us to propose a model in which, under normal conditions, a pool of AMBRA1 is docked by BCL-2 at the mitochondria, inhibiting its autophagic function; after autophagy induction, this mitochondrial pool of AMBRA1 separates from mito-BCL-2 and increases its binding to BECLIN 1 in order to favour the autophagic programme. Results AMBRA1 is a new partner of the antiapoptotic and antiautophagic factor BCL-2 AMBRA1 interacts with BECLIN 1 and its associated kinase Vps34, and favours BECLIN 1/Vps34 functional interaction (Fimia et al, 2007). This interaction, which requires the F2 fragment of AMBRA1 (Figure 1A), is crucial in the autophagic process; taken together, AMBRA1, BECLIN 1 and Vps34 have been defined as the autophagy core-complex (He and Levine, 2010). In contrast, BECLIN 1 is negatively regulated by a direct interaction with the antiapoptotic factor BCL-2. Therefore, we investigated the existence of a putative interaction between the antiapoptotic protein BCL-2 and AMBRA1 in a co-immunoprecipitation experiment. BCL-2 and AMBRA1 were found to be associated in HEK293 cells coexpressing both AMBRA1 and BCL-2 (Figure 1B, lane 2). Next, we wanted to find the AMBRA1 domain responsible for this binding. To this end, we transfected cells with cDNAs encoding various AMBRA1 portions (Figure 1A): FL (full length), F1 (amino acids 1–532), F2 (amino acids 533-750) and F3 (amino acids 767–1269). As shown in Figure 1B (lanes 3 and 5), F1 and F3 fragments (the N-terminal and C-terminal part of the protein, respectively) are sufficient to bind BCL-2, whereas the central region (F2), which is known to bind BECLIN 1 (Fimia et al, 2007), shows no interaction. Similar results were obtained following reciprocal co-immunoprecipitation experiments (Supplementary Figure S1). BCL-2 co-immunoprecipitates with AMBRA1 (lane 2) and its F1 and F3 fragments (lanes 3 and 5). To confirm these biochemical results, we performed a confocal microscopy analysis in HeLa cells expressing detectable endogenous levels of both AMBRA1 and BCL-2. As illustrated in Figure 1C, endogenous AMBRA1 and BCL-2 showed a partial colocalization. In order to give two quantitative outputs for this colocalization, we used Pearson (rp) and Spearman (rs) statistics. Both tests produce values in the range (−1, 1), 0 indicating that there is no discernable correlation and −1 and +1 meaning strong negative and positive correlations, respectively. We obtained an rp=0.38 and an rs=0.39 confirming that AMBRA1 and BCL-2 colocalize in mammalian cells. This analysis indicates also that the interaction between the two proteins is partial. To confirm the result, we analysed the interaction between endogenous AMBRA1 and BCL-2 in HeLa cells. As illustrated in Figure 1D, we found that endogenous AMBRA1 binds specifically endogenous BCL-2. To strengthen our results on this interaction, we performed a Förster resonance energy transfer experiment to measure AMBRA1–BCL-2 proteins proximity within the cells. GFP and mCherry are two fluorescent proteins which use as an FRET pair that has been recently validated for reproducible quantitative determination of the energy transfer efficiency, both in vivo and in vitro (Albertazzi et al 2009). In order to study the interaction of BCL-2 and AMBRA1 proteins, they were fused with the GFP and mCherry fluorescent proteins and co-transfected in HEK293 cells. On a morphological ground, visual inspection of the fluorescence expression patterns in the histological material showed that BCL-2–GFP and AMBRA1–mCherry appeared to be coexpressed in structures mainly resembling mitochondria and ER. The colocalization of the two proteins was almost complete. The average FRET efficiency values measured was 11.14%±2.1 (see Figure 1E). In a previous paper, the FRET behaviour of two tandem mCherry–EGFP fusion proteins (which differed for the distance, short linker and long linker) has been investigated (Albertazzi et al 2009). A FRET efficiency value of 0.41 for the short linker and of 0.29 for the long linker was achieved for the mCherry–EGFP tandem proteins. Comparison of our AMBRA1–BCL-2 FRET value with that of the mCherry–EGFP tandem construct suggests that AMBRA1 and BCL-2 are in proximity in the interacting complex. Figure 1.AMBRA1 interacts with the antiapoptotic factor BCL-2 in mammalian cells. A scheme of AMBRA1 with its WD40 domains is illustrated in (A). The binding site with BECLIN 1 is also reported on AMBRA1. HEK293 cells were co-transfected with vectors encoding FLAG–BCL-2 and myc–AMBRA1 FL (full length), myc-β-galactosidase (βGal) as a negative control or myc–AMBRA1 mutants F1, F2 or F3. (B) Protein extracts were immunoprecipitated using an myc antibody. Purified complexes and corresponding total extracts were analysed by western blot (WB) using an anti-BCL-2 antibody (B). Asterisks point to the molecular weight of proteins corresponding to the original AMBRA1 fragments. (C) Partial colocalization between endogenous AMBRA1 and BCL-2 in HeLa cells. HeLa cells grown in normal media were stained by anti-AMBRA1 (red), anti-BCL-2 (green) antibodies. The merge of the two fluorescence signals is shown in the right panel. Scale bar, 6 μm. (D) Endogenous AMBRA1 co-immunoprecipitates with endogenous BCL-2 in HeLa cells. HeLa cells were lysed and protein extracts were then immunoprecipitated using an anti-AMBRA1 antibody or preimmune IgG as a negative control. Purified complexes and corresponding total extracts were analysed by WB using anti-AMBRA1 and anti-BCL-2 antibodies. (E) Confocal microscope images of FRET acceptor photobleaching assay of HEK293 cells co-transfected with BCL-2–GFP and AMBRA1–mCherry. A bleached region of the cytosol is indicated by the white rectangle. (eI) BCL-2–GFP (donor) channel before the bleach. (eII) BCL-2–GFP (donor) channel after the bleach. (eIII) Pseudo-coloured image showing an FRET efficiency values map. (eIV) AMBRA1–mCherry (acceptor) channel before the bleach. (eV) AMBRA1–mCherry (acceptor) channel after the bleach. (eVI) Merge of BCL-2–GFP and AMBRA1–mCherry channels after the bleach. N indicates a close-by nuclear area. Scale bar, 5 μm. Download figure Download PowerPoint BECLIN 1 is not required for AMBRA1–BCL-2 interaction The F2 fragment of AMBRA1 is required for the interaction between AMBRA1 and BECLIN 1 (Fimia et al, 2007), but not for its binding to BCL-2. This suggests that BECLIN 1 is not necessary for this latter association. To confirm this hypothesis, we performed co-immunoprecipitation experiments using a mutant of BCL-2 unable to bind BECLIN 1. This mutant (EEE-BCL-2) possesses simultaneous glutamine substitutions at three phosphorylation sites (T69, S70 and S87), and mimics multi-phosphorylations that occur following autophagy induction (Wei et al, 2008). This impedes the interaction of BCL-2 with BECLIN 1 (Figure 2A and B). Vectors coding for AMBRA1 and for human-BCL-2 (h-BCL-2) or for the EEE-BCL-2 mutant were overexpressed in HEK293 cells; then, co-immunoprecipitations of AMBRA1 and BCL-2 were performed in normal conditions. As shown in Figure 2C, AMBRA1 is able to bind h-BCL-2 as well as the BCL-2 mutant (EEE-BCL-2) that cannot bind BECLIN 1. Figure 2.The interaction between AMBRA1 and BCL-2 does not require BECLIN 1. (A) Simultaneous glutamine substitutions at the three phosphorylation site on BCL-2 (EEE-BCL-2 mutant) induce a disruption of the BECLIN 1/BCL-2 complex (Pattingre et al, 2005). (B) BECLIN 1 co-immunoprecipitates with hBCL-2 but not with EEE-BCL-2 mutant in HEK293 cells. HEK293 cells were co-transfected with vectors encoding FLAG–BECLIN 1, and myc-h-BCL-2 or the EEE-BCL-2 mutant. Protein extracts were immunoprecipitated using an anti-FLAG antibody. Purified complexes and corresponding total extracts were analysed by WB using anti-BECLIN 1 and anti-BCL-2 antibodies. (C) AMBRA1 co-immunoprecipitates with both hBCL-2 and EEE-BCL-2 mutants in HEK293 cells. HEK293 cells were co-transfected with vectors encoding myc–AMBRA1, and myc-h-BCL-2 or the EEE-BCL-2 mutant. Protein extracts were immunoprecipitated using an anti-AMBRA1 antibody. Purified complexes and corresponding total extracts were analysed by WB using anti-AMBRA1 and anti-BCL-2 antibodies. Download figure Download PowerPoint Mitochondrial BCL-2 preferentially binds AMBRA1 and this interaction is disrupted after autophagy induction BCL-2 is predominantly found on the outer mitochondrial and ER membranes (Germain and Shore, 2003), these subcellular localizations being related to BCL-2 function (Lithgow et al, 1994). To gain an insight into the functional significance of the interaction between AMBRA1 and BCL-2, we evaluated whether AMBRA1 differentially binds the ER resident or the mitochondrial pool of BCL-2. To this end, two BCL-2 mutants with restricted subcellular localization were used in co-immunoprecipitation experiments with AMBRA1. In the first mutant, the C-terminal hydrophobic sequence of BCL-2 is exchanged for an equivalent sequence from modified ActA, which binds specifically to the cytoplasmic face of mitochondrial outer membranes (Pistor et al, 1994). The other mutant possesses a C-terminal sequence exchanged for a sequence from cytochrome b5 (DNA encoding the analogous 35 amino-acid sequence of the ER-specific isoform of rat hepatic cytochrome b5 (Mitoma and Ito, 1992), resulting in ER-specific localization (Zhu et al, 1996)). As shown in Figure 3A, myc–AMBRA1 significantly co-immunoprecipitates with mito-targeted BCL-2 (lane 3), whereas only a small amount of ER-targeted BCL-2 is found to bind AMBRA1 (lane 1). These results were confirmed by reversing the order of the co-immunoprecipitation (Supplementary Figure S2a). Knowing that the interaction between BCL-2 and BECLIN 1 is differential in normal conditions versus starvation conditions (Pattingre et al, 2005), we decided to analyse the interaction between AMBRA1 and mito-BCL-2 on autophagy induction. When cells were shifted for 4 h in an amino acid-free medium (EBSS medium), the interaction between AMBRA1 and mito-BCL-2 decreased, whereas no difference was observed in the binding between ER-BCL-2 and myc–AMBRA1. The reduction of p62/A170/SQSTM1, a ubiquitin-binding protein degraded by autophagy (Ichimura et al, 2008), was used as a marker to confirm autophagy induction (Figure 3A, lower panel). To control for the specificity of the co-immunoprecipitation between AMBRA1 and mito-BCL-2, negative controls using the myc antibody were performed in HEK293 cells, which do not overexpress myc–AMBRA1 or BCL-2 (Supplementary Figure S2B–D). The interaction between AMBRA1 and mito-BCL-2 can also be disrupted, at a lower extent, using rapamycin as an autophagy inducer (Figure 3B). During autophagy, phosphorylation of BCL-2 by JNK1 kinase abrogates the binding between BECLIN 1 and BCL-2. Thus, we also tested whether inhibition of JNK1 would be responsible for the AMBRA1–BCL-2 binding regulation. However, as illustrated in Figure 3B, the JNK1 kinase inhibitor SP600125 does not impair the release of AMBRA1 from mito-BCL-2 after autophagy induction. Next, we observed binding between endogenous AMBRA1 and mito-BCL-2. To this end, we isolated mitochondrial fractions from cells grown in normal conditions or for 4 h in EBSS. We performed an immunoprecipitation of AMBRA1 in the mitochondrial fractions, and we were able to appreciate the binding with endogenous BCL-2 in normal conditions. In contrast, this binding was disrupted after autophagy induction (Figure 3C). Of note, when checking by densitometric analysis, we can observe a binding between endogenous AMBRA1 and BECLIN 1 in these mitochondrial fractions in normal conditions, with this binding being increased following autophagy induction (Supplementary Figure S2E). To consolidate these data, we decided to carry out a mitochondrial crosslink before performing a subcellular fractionation experiment in HEK293 cells grown in normal or starvation conditions. As illustrated in Figure 3D, when cells are not fixed, BCL-2 can be detected as a 26-kDa band (appropriate molecular weight) in normal condition, and this band decreases following autophagy induction. However, after cell fixation, this band is not detectable, whereas a band of ∼170 kDa is detectable. The 170-kDa band may corresponds most likely to a complex including at least endogenous AMBRA1 and BCL-2, thus confirming the interaction. Next, we set out to establish whether this dynamic interaction could be monitored by fluorescence microscopy. As illustrated in Figure 4A, mito-BCL-2 colocalizes perfectly with the mitotracker staining, as expected, and there is a significant colocalization between myc–AMBRA1 and mito-BCL-2 in normal conditions, whereas some cells showed only a slight colocalization between ER-BCL-2 and AMBRA1 (arrows in Supplementary Figure S3A, B). We performed the same type of staining at the endogenous level to confirm this colocalization (see Supplementary Figure S4). By performing statistical colocalization analysis, we found that AMBRA1 shows a very good colocalization with mito-BCL-2, as the rp and rs values are 0.7 and 0.8, respectively (Supplementary Figure S3C). When cells are shifted to EBSS a clear decrease in AMBRA1 and mito-BCL-2 colocalization is observed (Figure 4B, starvation lane). Furthermore, the percentage of cells where myc–AMBRA1 colocalizes either with mito-BCL-2 or ER-BCL-2 was determined. As illustrated in Figure 4C, 70% cells showing colocalization between AMBRA1 and mito-BCL-2 are found in normal conditions, whereas only 40% cells show the same colocalization after autophagy induction. Such a reduction of colocalization is not observed in the case of AMBRA1–ER-BCL-2 binding, where we found ∼30% of colocalizing cells in both conditions. Of note, this colocalization seems to be due to a short 'proximity' between the stained compartments, rather than an overlap, as illustrated in Supplementary Figure S3B. Figure 3.Mitochondrial-BCL-2 preferentially binds AMBRA1, and this binding is disrupted after autophagy induction. (A) HEK293 cells were co-transfected with vectors encoding either ER-BCL-2 or mito-BCL-2 and myc-AMBRA1. Cells were grown either in normal media or in EBSS media for 4 h. Protein extracts were immunoprecipitated using an myc antibody. Purified complexes and corresponding total extracts were analysed by WB, using an anti-BCL-2 antibody or an myc antibody. Autophagy induction was controlled in total extracts by using an anti-p62 antibody. (B) Inhibition of JNK1 does not regulate the AMBRA1–mito-BCL-2 binding. HEK293 cells were co-transfected with vectors encoding myc–AMBRA1 and mito-BCL-2 or left untransfected, as a negative control. Cells were treated with rapamycin to induce autophagy or shifted in EBSS media plus the JNK1 kinase inhibitor for 4 h. Then, protein extracts were immunoprecipitated using an myc antibody. Purified complexes and corresponding total extracts were analysed by WB, by using anti-BCL-2 and myc antibodies. (C) HEK293 cells were grown in normal or in EBSS media for 4 h. Mitochondrial fractions were then isolated by centrifugation and the quality of the fractions controlled by WB by using MnSOD and GAPDH antibodies. Endogenous mitochondrial AMBRA1 was next precipitated using anti-AMBRA1 polyclonal antibody in both conditions or with the preimmune IgG as a negative control. Purified complexes were analysed by WB, by using anti-BCL-2 and anti-AMBRA1 antibodies. (D) HEK293 cells were grown in normal or in EBSS media for 4 h and then fixed 10 min with PFA 0.5% or left without fixation. Mitochondrial fractions were isolated by centrifugation and the quality of the fractions controlled by WB by using MnSOD and GAPDH antibodies. Endogenous mitochondrial AMBRA1 was next precipitated using anti-AMBRA1 polyclonal antibody in both conditions or with preimmune IgG, as a negative control. Purified complexes were analysed by WB, by using anti-BCL-2 antibody. BCL-2 was detected at 26 kDa when cells were not fixed, whereas this band disappears and a band at ∼170 kDa is detectable upon fixation. Download figure Download PowerPoint Figure 4.AMBRA1 dynamic colocalization with mito-BCL-2 and the mitochondrial network. (A, B) HEK293 cells co-transfected with vectors encoding mito-BCL-2 and myc-AMBRA1, grown either in normal media (A) or in EBSS (B) for 4 h and stained with an anti-BCL-2 antibody (green), an anti-myc–AMBRA1 antibody (blu) and Mitotracker (red). The merge of the two or three fluorescence signals are shown in the bottom panels, as indicated. Scale bar, 6 μm. White arrows point to strong triple colocalization areas. (C) AMBRA1–mito-BCL-2 colocalization decreases after autophagy induction. Quantification of cells showing colocalization between AMBRA1 and ER- or mito-BCL-2 in normal conditions and after autophagy induction is shown. Results are expressed as percentage of cells (±s.d.) showing colocalization between AMBRA1 and ER- or mito-BCL-2. Each point value represents the mean±s.d. of triplicate wells from three independent experiments. Statistical analysis was performed by analysis of variance (one-way ANOVA). *P<0.05 versus mito-BCL-2 in normal conditions. Download figure Download PowerPoint AMBRA1 partially localizes at mitochondria in normal conditions AMBRA1 is a cytoplasmic protein showing a diffuse signal in normal conditions, mostly overlapping with dynein light chains. After autophagy induction, a fraction of AMBRA1 translocates to the perinuclear region of the cell and more precisely to the ER (Di Bartolomeo et al, 2010). The binding of AMBRA1 with mito-BCL-2 led us to check whether there is a subcellular colocalization of AMBRA1 with mitochondria. As illustrated in Figure 5A, we found, indeed, that AMBRA1 presents a partial colocalization with mitochondria, as the rp and rs are 0.61 and 0.70, respectively. Of note, these quantitative results are similar to those obtained with the AMBRA1/mito-BCL-2 colocalization analysis. Figure 5.AMBRA1 partially colocalizes with the mitochondrial network. (A) HeLa cells grown in normal media were stained with Mitotracker (red) and an anti-AMBRA1 antibody (green). The merge of the two fluorescence signals is shown in the right panels. Scale bar, 6 μm. The Pearson correlation coefficient rp and Spearman correlation coefficient rs are indicated on the scatter plot. (B) Immunogold analysis of whole cells and purified mitochondria from HeLa cells. Fifteen nm particles label the AMBRA1 protein (red arrows). Scale bar, 0.2 μm. M, mitochondria. Download figure Download PowerPoint In order to confirm our observation, we performed an immunogold assay using an anti-AMBRA1 antibody on HeLa cells grown in normal conditions and on their purified mitochondria (Figure 5B and Supplementary Figure S5). Altogether, these results indicate that a pool of AMBRA1 (corresponding to 49±10% of the total protein) can localize at the mitochondria in normal conditions. Of note, after autophagy induction, this mitochondrial pool of AMBRA1 seems to be slightly reduced (see Supplementary Figure S6A). These results led us to hypothesize that mito-BCL-2 should anchor/inhibit AMBRA1 at the mitochondria in order to block its participation in the BECLIN 1-dependent autophagy programme. Thus, we decided to evaluate the effect of mito-BCL-2 on autophagy induced by AMBRA1. We overexpressed AMBRA1 in the presence of a control plasmid (pCDNA3), or either with mito-BCL-2 or WT-BCL-2. LC3 protein conversion was used as a marker of autophagy induction (Mizushima et al, 2004, 2010). As shown in Figure 6, overexpression of mito-BCL-2 is sufficient to dramatically reduce AMBRA1-induced autophagy. However, the F2 fragment of AMBRA1, which is known to bind BECLIN 1 (Fimia et al, 2007), but not BCL-2 (see Figure 1A and B), is able to evade from mito-BCL-2 inhibition but not from ER-BCL-2 effect. Finally, in order to evaluate the autophagic flux (autophagosome on-rate versus off-rate), we treated cells wi
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