The substitution of Proline 168 favors Bax oligomerization and stimulates its interaction with LUVs and mitochondria

Biochimica et Biophysica Acta 1859 (2017) 1144–1155

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The substitution of Proline 168 favors Bax oligomerization and stimulates its interaction with LUVs and mitochondria Lilit Simonyan a, Alexandre Légiot a, Ioan Lascu a, Grégory Durand b, Marie-France Giraud a, Cécile Gonzalez a, Stéphen Manon a,⁎ a

Institut de Biochimie et de Génétique Cellulaires, UMR5095, CNRS et Université de Bordeaux, CS61390, 1 Rue Camille Saint-Saëns, 33000 Bordeaux, France Institut des Biomolécules Max Mousseron, UMR 5247, CNRS, Université de Montpellier, ENSCM, et Université d'Avignon et des Pays de Vaucluse, BP 21239, 301 rue Baruch de Spinoza, 84916 Avignon cedex 9, France

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a r t i c l e

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Article history: Received 19 October 2016 Received in revised form 28 February 2017 Accepted 14 March 2017 Available online 16 March 2017 Keywords: Bax Mitochondria LUV Cell-free expression Cell-free assays

a b s t r a c t Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant BaxP168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria. © 2017 Elsevier B.V. All rights reserved.

1. Introduction The relocation of the pro-apoptotic protein Bax from the cytosol to the outer mitochondrial membrane is one of the crucial steps along the implementation of apoptosis ([1–4] for reviews). Bax is thought to be mainly cytosolic and monomeric in non-apoptotic cells, and to form membrane-inserted oligomers in apoptotic cells [5]. The NMR resolution of the cytosolic structure of Bax has been very useful to determine which Bax residues and domains support conformational changes during the relocation [6]. Indeed, the replacement of strategically localized residues has provided mechanistic insights on Bax relocalization and insertion into the mitochondrial outer membrane (MOM) and its permeabilization to apoptogenic factors, namely cytochrome c ([7–16], for a non-exhaustive list of examples). The mechanisms causing the release of apoptogenic factors have been largely debated, but there is now a large consensus to consider that the initial steps of apoptosis are associated to the selective permeabilization of the MOM by a Bax formed/regulated pore [17–19]. Mitochondria swelling consecutive to the opening of the so-called Permeability Transition Pore (mPTP) may later occur ([20,21] for a review), but this mechanism is more likely associated to necrotic cell death following, for example, ischemia/reperfusion [22,23].

⁎ Corresponding author. E-mail address: [email protected] (S. Manon).

http://dx.doi.org/10.1016/j.bbamem.2017.03.010 0005-2736/© 2017 Elsevier B.V. All rights reserved.

Although the existence of a Bax-related pore has been postulated very early after the discovery of the protein, the presence of a largesized pore in the MOM of apoptotic cells, termed MAC (Mitochondria Apoptotic Channel) has been demonstrated later, through electrophysiological experiments [24]. Furthermore, the fact that this pore was related to Bax has been established through the facts that it appeared (1) in the MOM of yeast cells expressing human Bax [24], and (2) in the MOM of isolated mitochondria incubated in the presence of Bax oligomers but not of Bax monomers [17]. Until recently, the analogy with bacterial toxins was the main support to the hypothesis that Bax was inserted through its two amphipathic helices α5 and α6 [25]. This implied that the pore was of proteinaceous nature, with the ‘walls’ formed of hairpins of these two helices [19]. Recent progresses have been obtained on the structure of Bax after it has been inserted. Crystallographic data of BaxΔC (deprived of the C-terminal hydrophobic helix α9), activated by a BH3 domain, showed that the hairpin could ‘open’ so that α5 and α6 formed an elongated dimeric amphipathic structure that could remain lain on the membrane [18,26]. This structure further suggested that Bax oligomers could be formed through the association and the insertion of several dimers [27]. This model is consistent with many observations, including electrophysiological data showing that the MAC exhibited several conductance substates [28], that could correspond to different oligomers containing a variable number of dimers. Recent microscopy data showed, indeed, that Bax actually forms a large ‘hole’ in the MOM [29, 30] with Bax located on the walls [31].

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However, the unknown behavior of the hydrophobic helix α9 remains a striking limitation to this model. For technical reasons, this helix was absent from the structural data of active Bax. Being highly hydrophobic, it is generally considered that this helix is spontaneously inserted in the membrane, as soon as the structural changes of Bax allows α9 to move away from the core of the protein. For long, the analogy of Bax-α9 with the C-terminal helices of anti-apoptotic Bcl-2 and Bcl-xL, which are necessary and sufficient for their membrane localization [32], led to consider that the membrane insertion of α9 was an obvious process. It has been shown that helix α9 alone is able to be inserted [33] and to permeabilize synthetic membranes [34]. However, the inversion of the C-terminal helices of Bax and Bcl-xL led to chimeras that do not have the expected behavior of interchangeable helices. Indeed, while the protein BaxΔC fused to the C-terminal α-helix of BclxL was spontaneously inserted into isolated mitochondria, the protein Bcl-xLΔC fused to the C-terminal α-helix of Bax was not [35]. This showed that, although the C-terminal α-helix of Bax might be able to be spontaneously located to mitochondria, it was not sufficient to drive the membrane localization of Bax, in contrast to the C-terminal α-helix of Bcl-xL. Previously, it had been shown that Bax-α9 did not drive full-length Bax insertion, unless the S184 residue was deleted [36]. In parallel, a number of experiments showing the existence of Bax-induced permeabilization of artificial or biological membranes were in fact done with BaxΔC, that is easier to produce, purify and manipulate than full-length Bax [37–39], suggesting that Bax-α9 is neither required for membrane localization, at least in synthetic systems. These observations disputed the too simple view that Bax-α9 was a bona fide membrane anchor. Still, a full Bax activation seems to be associated to a movement of α9. As cited above, the deletion of S184, that is expected to destabilize the interaction of the C-terminal end with the core of the protein, is sufficient to make the protein constitutively associated to the membrane [6,36], and also to remove regulations, such as the ability to interact with the mitochondrial receptor Tom22 [40–42]. The substitution T174D, that introduces a negative charge facing the negative charge of Glu69 located in the BH3 domain, strongly stimulated the ability of Bax to be relocated and to permeabilize MOM in yeast [12]. This suggests that the repulsive effect moving α9 away from the hydrophobic groove formed by the BH domains was sufficient to activate Bax. In the same study, the substitution of the conserved P168, that is located in the short loop between α8 and α9, by a smaller and more mobile residue was studied. A mutant P168A exhibited a dramatically increased capacity to be relocated and inserted into the MOM, and to promote the release of cytochrome c. This was not a specificity of yeast, since a mutant P168V expressed in human glioblastoma cells had the same behavior [10]. However, other data disputed these observations. In Hela cells overexpressing FLAG-Bax, substitutions P168A/G/E led to proteins that remained cytosolic following an apoptotic stimulus [11]. A similar observation was done with a YFP-Bax fusion protein carrying a mutation P168A [43]. Schinzel et al. [11] observed that Bax could interact with the peptidyl-prolyl cis-trans-isomerase Pin1, in a way that depended on the phosphorylation of residue T167, suggesting that a cistrans isomerisation of P168 might regulate the conformation of the α8α9 loop, thus playing a crucial role in Bax interaction with mitochondria. The replacement of P168 by residues less able to acquire the cis conformation would thus prevent Bax activation. A recent study reported that a mutant P168G could form stable dimers that remain cytosolic and were less efficient to permeabilize membranes [44]. These somewhat contradictory data pushed us to investigate more directly the consequences of the P168A substitution on Bax ability to interact with membranes. Wild-Type and P168A mutants were produced through a cell-free method and biochemical properties were studied in solution and in the presence of isolated mitochondria or liposomes. We observed that the substitution P168A was sufficient to promote a conformational change favoring Bax activation. However, it did not differ

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significantly from wild-type Bax in its capacity to permeabilize liposomes, but was slightly more efficient to permeabilize mitochondria. 2. Experimental procedures 2.1. Expression of Bax in yeast cDNA of full-length and untagged human Bax was cloned in the pYES3C/T plasmid, as described previously [10]. Substitution of the P168 residue was done with the Quickchange method and the cDNA was sequenced to verify that no other mutation was introduced. The plasmid was introduced in the W303-1B strain (matα, ade1, his3, leu2, trp1, ura3). Yeast cells were grown aerobically in a synthetic medium supplemented with lactate (0.17% Yeast Nitrogen Base (Difco), 0.5% ammonium sulfate, 0.1% potassium phosphate, 0.2% Drop Mix, 0.01% auxotrophic markers, 2% DL-lactate, pH 5.5). The induction of Bax expression was done by adding 0.5% galactose and was extended overnight (~14 h). 2.2. Yeast mitochondria isolation Mitochondria were isolated from yeast cells expressing Bax or not, as described previously. Briefly, washed yeast cells were preincubated in the presence of 0.5 M β-mercaptoethanol for 15 min and washed twice with 0.5 M KCl. Cells were then suspended (0.1 g of dry weight/ mL) in a 20 mM phosphate buffer (pH 6.8) containing 1.35 M sorbitol and 1 mg/mL zymolyase 20 T. Digestion was extended for 20–35 min (and followed by observing cells under a microscope). 30 min was usually sufficient for a N 90% transformation of cells to spheroplasts. Spheroplasts were washed twice in a 10 mM maleate-tris buffer (pH 6.8) containing 1.1 M sorbitol 0.4 M mannitol, 1 mM EGTA, 0.1% BSA. Cells were resuspended in a 10 mM maleate-tris buffer containing 0.6 mannitol, 1 mM EGTA, 0.2% BSA, and homogeneized during 3 × 3 s. in a Waring Blendor. Cell debris was removed by a 15 min., 900× g centrifugation, and mitochondria-enriched fraction was recovered after a 15 min., 17,000×g centrifugation. Mitochondria were resuspended and gently homogeneized in the same buffer without BSA (10 mM maleate-tris buffer (pH 6.8) containing 0.6 M mannitol, 1 mM EGTA (MR buffer)), and the same cycle of centrifugation was done to recover the mitochondria pellet. Yeast cellular extracts were done with 10 mL cultures at 2 O.D. units. Cells were centrifuged and washed in the MR buffer supplemented with anti-proteases inhibitors cocktail (Complete, Roche), and resuspended in 0.5 mL in the same buffer. Glass beads (0.45–0.5 mm mesh) were added and the suspension were vortexed for 6 × 30 s, with 30 s pauses on ice. Cell debris and beads were eliminated by a 5 min centrifugation at 900 × g, and the supernatant was used for immunoprecipitation assays. 2.3. HCT-116 BaxKO mitochondria isolation Human colorectal cancer HCT-116 BaxKO cells were grown, and mitochondria were isolated as described previously [45]. 2.4. Cytochromes spectra Mitochondria were suspended at 3–5 mg/mL in the MR buffer, and poured into the two cuvettes of a double-beam double-wavelength spectrophotometer (Cary 4000, Varian). The reference was oxidized with potassium ferricyanide and the sample was reduced with sodium dithionite. Difference spectra were acquired between 650 and 500 nm. The content of cytochromes c + c1 and b were calculated from the values at 550 minus 540, and 561 minus 575, respectively, and graphically corrected for the variations of isobestic points [45]. The molar extinction coefficients were 18,000 M−1·cm−1 for both.

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2.5. Western-blotting For mitochondria analyzes, proteins were precipitated with 0.3 M trichloroacetic acid (TCA), washed twice with acetone, solubilized in the Laemmli buffer containing 2% β-mercaptoethanol, and heated at 70 °C for 15 min. Depending on the experiments, 5–100 μg purified Bax or mitochondrial proteins were loaded on 12.5% acrylamide SDSPAGE, and transferred onto 0.2 μm PVDF membranes (ImmobilonPSQ, Millipore) or 0.2 μm nitrocellulose membranes (Protran, Amersham). Membranes were saturated with 5% milk in PBS-Tween 20. Primary antibodies were added overnight at 4 °C, and secondary antibodies were added for 45 min at room temperature. Antibodies were as follows: rabbit polyclonal anti-human Bax N20 (Santa-Cruz Biotechnology) 1/5000 dilution, mouse monoclonal anti-human Bax 2D2 (Santa-Cruz Biotechnology) 1/5000 dilution, mouse monoclonal antiyeast porin (Novex) 1/50,000 dilution, mouse monoclonal anti-human porin (Mitosciences), 1/10,000 dilution, rabbit polyclonal anti-yeast cytochrome c (custom antibody, Millegen) 1/5000 dilution, mouse monoclonal anti-pigeon cytochrome c (R&D Systems) 1/5000 dilution, rabbit polyclonal anti-hexahistidine tag (Bethyl Laboratories) 1/5000 dilution, peroxidase-coupled goat anti-rabbit IgG (Jackson Immunoresearch) 1/ 10,000 dilution, peroxidase coupled goat anti-mouse IgG (Jackson Immunoresearch) 1/10,000 dilution. Peroxidase activity was revealed by ECL (Luminata Forte, Millipore), recorded with a digital camera (GBox, Syngene) and analyzed with Image J (https://imagej.nih.gov/ij/). 2.6. Cell-free transcription-translation and purification of Bax Human Bax cDNA was cloned in a pIVEX 2.4 plasmid (Roche), in phase with a hexahistidine Tag and a cleavage site by Factor Xa on the N-terminal side. The in vitro synthesis was done in a small volume dialysis chamber (100 μL), separated from a feeding reservoir (1700 μL) by a dialysis membrane (MW 10,000). The system was routinely set up in an inverted microcentrifuge tube. Both chambers contain 0.1 M Hepes (pH 8.0), 1 mM EDTA, 0.05% NaN3, 2% PEG 8000, 151 mM Potassium Acetate, 7.1 mM Magnesium Acetate, 0.1 mg/mL Folinic acid, 2 mM DTT, 1 mM (each) NTP mix, 0.5 mM (each) aminoacid mix, 1 mM (each) RDEWCM mix, 20 mM PEP, 20 mM acetylphosphate, protease inhibitors cocktail (Complete, Roche). Depending on the experiments (see results), 0.01% Brij-58 (Sigma) or 0.05% F8-TAC (degree of polymerization ~ 5; MW ~ 1355 g/mol) [46] were added. The dialysis chamber was added with components for the synthesis: 35% (w/v) S30 E.coli BL21DE3 lysate [47], 35% S30 buffer (10 mM tris-acetate, pH 8.2, 14 mM Magnesium Acetate, 60 mM Potassium Acetate, 0.5 mM DTT), 0.04 mg/mL Pyruvate Kinase (Sigma), 15 μg/mL pIVEX-Bax plasmid, 0.5 mg/mL tRNAs mix (Roche), 6 units/mL T7 RNA polymerase [48], 3 units/mL RNAGUARD (Amersham). The synthesis was done for 20 h at 30 °C under gentle agitation. The reaction mix was centrifuged for 15 min. at 20,000×g. Although a significant proportion of the synthesized protein was found in the pellet, only the supernatant was used in the next steps. It should be noted that, even when they were added in the production step, the detergent Brij-58 and the fluorinated surfactant F8-TAC were omitted from the buffers in all the subsequent steps. The supernatant was diluted five times in the equilibration buffer (25 mM Hepes, pH 7.4, containing 500 mM NaCl, 40 mM imidazole and a protease inhibitors cocktail (Complete EDTA free, Roche)). It was applied to a Ni-Sepharose 6 FastFlow 1 mL column (GE Healthcare), preloaded with Ni2+ ions. The circuit was closed and connected to a peristaltic pump, and the samples were passed overnight through the column, at 4 °C. The column was connected to an Äkta purifier FPLC system and was washed with 20 volumes of equilibration buffer. Non-specifically bound material was washed off by increasing imidazole concentration up to 50 mM, 85 mM and 109 mM, by mixing (v/v) 95%, 90% and 85% of equilibration buffer with 5%, 10%, and 15% of elution buffer (7 mL each) respectively. Bound proteins were eluted using 100% elution buffer (25 mM Hepes,

pH 7.4, 500 mM NaCl, 500 mM imidazole) in 4–5 fractions of 1 mL each. Each fraction was analyzed by SDS-PAGE and Coomassie staining. Fractions containing Bax were pooled and injected to HiPrep 26/10 desalting column (GE healthcare) and desalted against 25 mM Hepes, pH 7.4, 200 mM NaCl, 1 mM EDTA (buffer GF). The fractions containing purified Bax were collected and protein concentration was determined by the bicinchoninic acid (BCA)-assay (Thermo Scientific). Bax produced by this method still had the N-terminal His6 tag, that could be removed by a cleavage by Factor Xa, with a 50% loss of yield (data not shown). For most experiments presented below, the His6 tag was still present. 2.7. Recombinant tBid production Recombinant human His6-Bid was produced in E. coli strain BL21DE3 transformed with the expression plasmid pET23d-His6-Bid, purified, and cleaved with caspase-8 as described by Kudla et al. [49]. 2.8. Circular dichroism Far UV spectra (200–250 nm) were measured on a Jasco J-810 CD spectropolarimeter. The acquisition was carried out at 25 °C with a 1 mm optical path length cell under nitrogen atmosphere. The protein concentration was 25–75 μg/mL in 200 μL fractions obtained after the purification. 10 scans were collected from 250 to 200 nm at 1 nm intervals, then averaged and baseline-corrected by subtracting blank buffer. They were analyzed using the K2D3 software (http://cbdm-01.zdv. uni-mainz.de/~andrade/k2d3/) [50]. 2.9. Bis-ANS binding The hydrophobic molecule 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS) binds to non-polar domains of proteins in aqueous solution, resulting in the increase of fluorescence emission. The protein solution (10–50 μg/mL) was added with 16.7 μM bis-ANS. Excitation wavelength was set at 380 nm, and emission spectra was recorded between 400 and 550 nm in a Hitachi F-7000 spectrofluorimeter. 2.10. Mitochondria permeabilization assay Mitochondria were isolated from the wild-type strain W303-1B not expressing Bax or from Bax-KO human colorectal cancer cell line HCT116. 200 μg of mitochondrial proteins were suspended in 100 μL of permeabilization buffer (10 mM Hepes, pH 7.4, 250 mM sucrose, 80 mM KCl, 2 mM magnesium acetate, 1 mM potassium phosphate, 1 mM ATP, 0.08 mM ADP, 5 mM succinate) (PMB buffer). 0.3 to 1 μg purified Bax was added, and the incubation was extended for 1 h at 30 °C. Mitochondria were centrifuged (25,000×g, 10 min., 4 °C). The pellets were resuspended in 100 μL of water, and both the pellet and supernatant were analyzed for Bax and cytochrome c content by Western-blotting after a separation by SDS-PAGE. 2.11. LUVs preparation Large Unilamellar Vesicles (LUVs) were prepared as described previously [51] from a mixture containing 4 mg of lipids (Avanti Polar Lipids) with the following molar ratio: 46.5% phosphatidylcholine (PC), 28.5% phosphatidylethanolamine (PE), 9% phosphatidylinositol (PI), 9% phosphatidylserine (PS), 7% cardiolipin (CL). Chloroform was evaporated under argon for 3 h at room temperature. The dry lipid film was resuspended with 1 mL LUV buffer (10 mM Hepes, pH 7.4, 0.2 mM EDTA, 200 mM KCl, 5 mM MgCl2), containing 12.5 mM of the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and 45 mM of the quencher p-xylene-bis-pyridinium bromide (DPX). The lipid suspension was sonicated in a water bath for 3 min and extruded through 400 nm pore size Nucleopore Track-Etched membrane in a

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Fig. 1. The substitution P168A activates Bax. (A) Structure of monomeric, soluble and inactive Bax (Suzuki et al. [6]; PDB number 1F16) showing the 9 helices, and highlighting the position of P168 in the short loop between helices α8 and α9. Homology domains BH1 (loop between helices α4 and α5), BH2 (helices α7 and α8) and BH3 (helix α2) form a hydrophobic groove in which helix α9 is laid. (B) Bax localization. Western-blots of BaxWT and Bax-P168A were done on whole cells and isolated mitochondria, following a 14 hours-expression in yeast. Mitochondrial porin was used as a loading control. Antibodies: anti-yeast Porin (Novex, 1/50,000), anti-human BaxN20 (Santa-Cruz, 1/5000). (C) Bax-induced release of cytochrome c. Typical redox spectra of suspensions of mitochondria isolated from yeast cells expressing BaxWT or Bax-P168A, highlighting the content of cytochromes c + c1 (A550nm minus A540nm; red) and b (A561nm minus A575nm; blue). (D) Bax-P168A membrane insertion. Carbonate treatment of mitochondria did not remove Bax-P168A, showing that it was actually inserted in the membrane. B, C and D are representative data of 8 independent experiments. Similar results had already been published previously [12]. (E) Cytochrome c release induced by different substitutions of P168. Mitochondria were isolated from yeast expressing the indicated mutants of Bax overnight. The ratios cytochrome c + c1/cytochrome b was calculated. (F) Bax-P168A decreases yeast viability. Yeast cells expressing BaxWT or Bax-P168A for 24 h were counted and plated on glucose-containing plates (turning off Bax expression). After a 3-days incubation, the number of growing colonies relative to the number of plated cells was counted.

Liposofast extruder (Avestin). LUVs were applied on a 10 mL SephadexG50 gravity flow column. 1 mL fractions were collected and the LUVs containing fractions were identified by their cloudy appearance. The fractions were stored at 4 °C in the dark and LUVs were used within one week. Dynamic light scattering (DLS) was performed on a DynaPro NanoStar (Wyatt). 5 μL LUVs were diluted in 95 μL LUV buffer for DLS measurements. The LUV suspension was found to be highly homogenous, around 400 nm. This was important since it has been shown that membrane curvature is a factor regulating Bax activity [52]. For proteins, DLS of the solutions was measured at different dilutions, to obtain low-noise signals. Particle size was calculated with the embedded

Dynamics software. Values are indicative, but permit a comparison between the different species. 2.12. LUV permeabilization ANTS release assay was performed at 30 °C using 96-well plates. 5–10 μL of LUVs were added with 100 to 200 ng of Bax, in the LUV buffer (100 μL final volume). Fluorescence was measured every 1 or 2 min during 60– 90 min in a microplate fluorescence reader Clariostar (BMG Labtech). The excitation and emission wavelengths were set to 355 nm and 520 nm, respectively. The 0% of fluorescence was measured on LUVs alone, and the 100% was measured after adding 0.5% CHAPS to LUVs.

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Fig. 2. In vitro production, purification and characterization of His6-Bax. (A) (Top) After the production in the presence of 0.01% Brij-58, the mixes were centrifuged (20,000×g; 15 min.), and the proteins from pellets and supernatants were separated by SDS-PAGE and stained with Coomassie Blue. Although the mutant His6-Bax-P168A was slightly more present in the insoluble pellet than His6-BaxWT, a significant proportion of both proteins remained soluble, allowing further purification. (Bottom) Fractions eluted from the Ni-sepharose column. S: supernatant of the 20,000×g centrifugation; F: Flow-through; 3–36: Fractions obtained from the elution with imidazole, having a significant absorbance at 260 nm. Fractions 30–36 (for BaxWT) or 29–35 (for Bax-P168A) were kept for further purification steps. (B) CD spectra were acquired as described in the Experimental Procedures. (Top) Proteins produced in the absence of surfactants. (Middle) Proteins produced in the presence of Brij-58 (40 μg/mL). (Bottom) Proteins produced in the presence of F8-TAC (25 μg/mL). Calculations with the K2D3 software resulted in N80% of α-helical structure for both mutants under both conditions. (C) Immediately after purification, BaxWT and Bax-P168A solutions were analyzed by size-exclusion chromatography on a HiLoad Superdex 200 PG column (GE Healthcare). Bax monomer and dimer were identified by comparison to myoglobin (17 kDa) and ovalbumin (44 kDa).

2.13. LUV flotation assay

3. Results

30% and 80% (w/w) Histodenz (Sigma) stock solutions were prepared in LUV buffer. 100 μL of Bax solution (~ 5 μg) were mixed with 100 μL of LUVs, completed to 1 mL with LUV buffer, and incubated for 1–2 h at 4 °C. 750 μL of the 80% Histodenz solution were added and thoroughly mixed with 750 μL of the proteinLUV sample in Thinwall centrifuge tubes (Beckman Coulter 50.2ti rotor). 1.5 mL of the 30% Histodenz solution were overlaid, and 1.5 mL LUV buffer was added as the top layer. Tubes were centrifuged overnight at 30,000 × g at 4 °C in a 50.2ti swinging bucket rotor in a Beckman L-60 ultracentrifuge. The gradients were fractionated in 500 μL aliquots, precipitated with 0.3 M TCA, and analyzed by western blotting against Bax.

3.1. The P168A mutant of Bax interacts more with MOM than the wild-type protein

2.14. Statistical analyzes Unpaired, paired and one-sample t-tests were done with tools included in GraphPad Prism 6 software.

The heterologous expression of human Bax in yeast has proved to be a powerful tool to measure how Bax is able to interact with a membrane, namely the outer mitochondrial membrane. Indeed, when expressed in yeast, wild-type Bax does not interact with mitochondria, like in non-apoptotic mammalian cells. As a matter of fact, it does not significantly permeabilize the outer mitochondrial membrane. The introduction of mutations at strategical positions in Bax sequence may induce a mitochondrial relocation and a permeabilization of the MOM, providing crucial information about which domains of the protein are involved in the conformational changes associated to its activation. The residue P168 is located in the short loop between helices α8 and α9 and may confer some rigidity to this loop, which is thought to help maintaining helix α9 in the groove formed by the BH domains (Fig. 1A). However, the ability of P168 to acquire a cis conformation opens the possibility that a cis-trans isomerisation might be responsible for a

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Fig. 3. Recognition of Bax conformation by 6A7 and 2D2 monoclonal antibodies. (A) Bax immunoprecipitation from cellular extracts. 1 mg of cellular extracts from yeast cells expressing BaxWT or Bax-P168A were diluted in 0.5 mL of RB buffer and added with 50 μL of IP 10× buffer (Sigma). After a 40 min incubation at 4 °C, 2 μg of 2D2 or 6A7 antibodies (both from Sigma) were added and the immunoprecipitation was done overnight. 10 μL of the suspension were taken for Input. 50 μL of Protein A-agarose beads (Sigma) were added and the incubation was extended for 4 h. Beads were washed 5 times with IP buffer 1× and once with IP buffer 0.1×. 25 μL Laemmli buffer was added, samples were heated at 90 °C for 5 min and the eluate was recovered by a 2 min centrifugation. Inputs and eluates were resolved on SDS-PAGE, blotted, and probed with the N20 polyclonal antibody (Santa-Cruz, 1/5000). Blots are representative of 5 independent experiments. (B) Purified Bax immunoprecipitation. 4.3 μg of pure BaxWT or Bax-P168A (produced in the presence of 0.01% Brij-58) were diluted in 107 μL buffer GF, in the absence or in the presence of 0.7% β-octylglucoside. 1 μg 2D2 or 6A7 antibodies were added, and the immunoprecipitation and sample recovery were performed as in (A). Blots are representative of 3 independent experiments. (C) Quantification of the 6A7/2D2 ratios. Western-blots were revealed by ECL and a digital camera, and quantified with Image J. Each color corresponds to experiments ran in parallel. Note that the blot shown in (A) was revealed with a film and was thus not included in the quantification in (C). Paired t-tests indicated significant differences between BaxWT and Bax-P168A (*p = 0.05, **p b 0.05).

large-amplitude movement of helix α9, a process that is unlikely to occur with another residue such as alanine ([53], for a review). This is particularly relevant to Bax, which might be a target of cancer-linked peptidyl-prolyl isomerase Pin1 [11,54]. We have previously accumulated a considerable amount of data showing that, when expressed in yeast, the mutant P168A has a greater mitochondrial localization than wild-type Bax (Fig. 1B), this being associated to a dramatic increase in the release of cytochrome c (Fig. 1CE) [10,12]. This is associated to a loss of viability of yeast cells, that does not occur with BaxWT (Fig. 1F). Strikingly, Bax-P168A could not be released from MOM by a sodium carbonate treatment, and required a treatment with Triton X-100, showing that it is actually inserted in the lipid bilayer (Fig. 1D). These data raised the question to know if this behavior of the mutant Bax-P168A was linked to changes in its interaction with cellular partners: indeed, some of them are conserved in yeast, such as TOM components [40] or protein kinases [13,16]. Alternatively, this behavior could reflect changes in the intrinsic ability of the mutant P168A to interact spontaneously with membranes, independently from the cellular context. Experiments with purified recombinant proteins were therefore undertaken, to test if this later possibility could be supported by in vitro assays. 3.2. Cell-free Bax production and purification While wild-type Bax can be produced by expression in E. coli, namely as a fusion protein with intein [6], the mutant P168A could not be produced

with a yield and a stability compatible with biochemical and functional studies. This might likely be due to its above depicted ability to interact with membranes, which impaired bacteria viability (data not shown). To circumvent this technical limitation, in vitro synthesis was used. The protein was first produced in the absence of any added detergent or surfactant. Although both variants of Bax, wild-type and P168A mutant, could be produced and purified with a reasonable yield, the rapid formation of aggregates in the solution did not allow getting reliable data (not shown). Consequently, we tested the effect of the inclusion of small concentrations of detergents or fluorinated surfactants during the production. The later have been found to be suitable alternatives to the use of detergents to maintain membrane proteins in solution [46]. Two molecules were found to be efficient: the detergent Brij-58 (0.01%) and the fluorinated surfactant F8-TAC (0.05%). To prevent side effects of these molecules on Bax properties, they were omitted from the purification steps, so that, assuming a homogenous dilution process, their final concentration in purified Bax solutions was 300-fold lower than initially added. Even in the presence of the detergent Brij-58, a significant proportion of wild-type Bax was found in the non-soluble pellet and, as expected, this proportion was higher for the P168A mutant (Fig. 2A). However, after the insoluble fraction was discarded, the purification of the His6tagged protein by affinity FPLC on Ni2+-NTA allowed to obtain both proteins with a high degree of purity (Fig. 2A). After purification, a 100 μLdialysis chamber allowed to produce in average ~100 μg protein with a higher than 98% purity, estimated by Coomassie Blue staining.

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Fig. 4. Biochemical characterization of Bax in solution. (A) Bis-ANS binding to Bax. Solutions (10–25 μg/mL) of BaxWT or Bax-P168A (produced in the presence of 0.01% Brij-58) were incubated for 5 min in the presence of bis-ANS (16.7 μM). Excitation was set at 380 nm and emission was measured between 400 and 550 nm. (Top) Whole emission spectra. (Bottom) Difference of fluorescence emission between 485 and 435 nm for different protein preparations. Identical colors represent protein preparations done in parallel. The grey points correspond to a preparation made in the presence of F8-TAC (0.05%), and the others were made in the presence of Brij-58 (0.01%). Paired t-test indicated a significant difference between the two proteins. (B) DLS measurement of protein preparation in solution. DLS of protein solutions was measured at a concentration of 40 μg/mL. Diameters values are indicative, assuming perfectly spherical particles. The experiment is representative of 3 independent experiments. (C) Bax oligomers. Solutions of BaxWT or Bax-P168A (produced in the presence of 0.01% Brij-58) were stored for 72 h at 4 °C, loaded on SDS-PAGE and analyzed by Western-blot with anti-human BaxN20 (Santa-Cruz). The two lanes are from the same gel and the same membrane, where non-relevant experiments were masked. The experiment is representative of 5 independent experiments.

3.3. Characterization of the P168A mutant in solution In non-apoptotic cells, Bax is essentially cytosolic, or loosely associated to intracellular membranes. This suggests that it behaves mostly like a soluble protein, although having some hydrophobic surfaces that could be exposed to membranes. The NMR structure of monomeric BaxWT has been determined, and showed that the protein is organized in 9 α-helices connected by short loops [6] (see Fig. 1A). Since the P168A mutation was expected to favor movements of the hydrophobic C-terminal α-helix, we determined if in vitro produced BaxWT and BaxP168A were similarly, and correctly, folded. Circular dichroism measurements showed that both proteins were similarly folded, whether they were produced in the presence of the detergent Brij-58 or of the fluorinated surfactant F8-TAC (Fig. 2B). Calculations with the software K2D3 predicted a α-helix content above 80% for all types of protein preparations (wild-type/mutant, no surfactant/Brij-58/F8TAC), suggesting that they were reasonably folded as a succession of α-helices. In spite of the presence of Brij-58 or F8-TAC, we observed that a significant proportion of Bax was spontaneously dimeric, as observed on gel filtration experiments. However, immediately after the purification, there was no significant difference between BaxWT and Bax-P168A (Fig. 2C).

The activity of Bax is related to conformational changes that affect different domains of the protein. It has been established that the movement of the N-terminal end of Bax is a reporter of the active conformation. Specifically, the monoclonal antibody 6A7 is able to recognize active Bax but not inactive Bax [55,56], a property that has largely been used in immunofluorescence experiments, to detect active Bax in mitochondria. Immunoprecipitation assays by the two monoclonal antibodies 2D2 and 6A7 have been done from cellular extracts of yeast cells expressing BaxWT or BaxP168A and on the two proteins produced in the presence of Brij-58 (Fig. 3). In both assays, the proportion of 6A7-immunoprecipitable Bax was higher for Bax-P168A than for BaxWT, showing that the mutant has acquired a more active conformation. As a positive control, a treatment of BaxWT with the detergent β-octylglucoside converted the protein into the 6A7-reactive conformation. A difference of conformation between BaxWT and Bax-P168A was also revealed through the binding of bis-ANS. The fluorescence emission of this hydrophobic probe increases when it binds to the non-polar domains of proteins [57]. We observed that Bax-P168A bound more bisANS than BaxWT, suggesting that it exposed larger non-polar domains (Fig. 4A). Additional observations supported the view that the mutant Bax-P168A had a spontaneous tendency to dimerize and oligomerize. DLS measurements of Bax solutions stored for at least 24 h showed that a higher proportion of Bax-P168A than BaxWT was present in

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Fig. 5. Interaction of Bax with LUV by flotation assay. (A) 5 μg of purified BaxWT or Bax-P168A (produced in the presence of 0.01% Brij-58) were mixed or not with 0.5 μg of caspase 8treated Bid or with 0.7% β-octylglucoside in 250 μL buffer GF. The mix was then added to 750 μL of LUV, and incubated at room temperature for 2 h on a slow-motion rotating wheel. 750 μL of this solution was mixed with an equal volume of 80% Histodenz solution in LUV buffer, and added in a ultracentrifuge tube (Beckman 50.2Ti rotor). 1.5 mL of a 30% Histodenz solution in LUV buffer, and 1.5 mL of LUV buffer alone were gently layered on the top of the mix. The gradients were centrifuged at 100,000×g for 15 h at 4 °C. Fractions of 0.5 mL were collected, precipitated with 50 μL TCA 3 M and solubilized in 25 μL Laemmli Buffer. 10 μL of each sample was resolved on SDS-PAGE, blotted, and revealed with anti BaxN20 antibody. (B) Quantification of the interaction of BaxWT and Bax-P168A with LUVs. After densitometry of gels similar to that shown in (A) (without treatment), the % of BaxWT and Bax-P168A present in the four top fractions was calculated. Only Bax at the size of the monomer was taken into account. Paired t-test indicated a significant difference between the two proteins (p b 0.05).

large particles, that likely corresponded to oligomers (Fig. 4B). Consistently, we observed that Bax-P168A was indeed more prone to form stable oligomers, after several days of storage at 4 °C (Fig. 4C). 3.4. Bax interaction with LUVs We first investigated if Bax could be spontaneously associated to LUVs. LUVs and purified Bax were mixed to 80%-Histodenz (1:1 v/v; 1 mL in total). The mix was covered by a 30%-Histodenz solution (1.5 mL) and LUV buffer (1.5 mL). After a 14-hours centrifugation, free proteins and aggregates are localized in high-density fractions at the bottom of the tubes, while low-density LUVs are at the top. When produced in the presence of Brij-58, BaxWT was poorly present in low-density fractions (Fig. 5). The addition of tBid, that favors the conformational change leading to Bax activation and oligomerization

[49,58], increased the presence of BaxWT in low-density fractions, as expected. Conversely, Bax-P168A was spontaneously present in lowdensity fractions, and poorly sensitive to the presence of tBid. As a matter of fact, in spite of the presence of SDS in the gels, Bax-P168A remained spontaneously more present under the form of dimers and oligomers, even though tBid and the detergent β-octylglucoside increased their formation for both BaxWT and Bax-P168A (Fig. 5). 3.5. LUVs permeabilization by Bax Bax-induced permeabilization of LUVs was measured as the increase of ANTS fluorescence following its release from LUVs and from binding to DPX [51,52]. We first tested the proteins produced in the presence of Brij-58 (Fig. 6A). We observed that both BaxWT and Bax-P168A were able to induce a permeabilization of the LUVs. Although the mutant

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had a tendency to be more rapid, the differences between the two proteins were not sufficient to conclude that the mutant was more active than the wild-type. Proteins were then produced in the presence of F8-TAC, instead of Brij-58. Fluorinated surfactants have less conformational effects on pro-

teins than detergents [59], and therefore expected to have a lesser tendency to favor the active conformation of Bax. Indeed, we observed that both BaxWT and Bax-P168A permeabilized LUVs much more slowly than when they were produced in the presence of Brij-58 (Fig. 6B), but a higher extent of permeabilization was reached. Here again, BaxP168A appeared to be slightly more efficient than BaxWT, but the difference between the two proteins remained weak and not significant. Taken altogether (Fig. 6C), these experiments showed a slight tendency of Bax-P168A to be more active than BaxWT but the precision of the measurement did not make it possible to conclude that there was a significant difference between the two proteins. The addition of tBid activated the maximal ratio of permeabilization caused by the two proteins, at a similar extent (Fig. 6D). 3.6. Bax effect on isolated mitochondria Both proteins were tested for their ability to bind to mitochondria, and to induce the release of cytochrome c. To avoid interferences with endogenous Bax, two types of mitochondria devoid of Bax were used: yeast mitochondria and mitochondria isolated from Bax-KO human colorectal cancer cells HCT-116, where Bax is the only active pro-apoptotic protein [60,61]. Bax concentration was optimized on yeast mitochondria, that are easier to obtain with an intact outer membrane than mitochondria from mammalian cell cultures: we found that 4.5 μg BaxP168A/mg mitochondrial protein was able to promote a large release of cytochrome c, while the same amount of BaxWT was without effect (Fig. 7AB). A concentration of 5 μg Bax/mg mitochondria was then used in subsequent assays. For both types of mitochondria (yeast and HCT-116 BaxKO), we observed a higher binding of Bax-P168A than BaxWT, that was associated to a higher release of cytochrome c (Fig. 7CD). 4. Discussion We first report a method that allows to produce highly purified Bax in an in vitro system. Biochemical tests showed that the protein is functional: indeed, BaxWT can be activated by tBid, as shown through liposomes binding (Fig. 5A) and liposomes permeabilization (Fig. 6D). In parallel, we produced a mutant Bax-P168A which, from our previous studies, we expected to have a greater activity [10,12] (Fig. 1). Indeed, we found that the mutant Bax-P168A exhibited a greater tendency to associate with LUVs (Fig. 5) and mitochondria (Fig. 7), and to permeabilize mitochondria to cytochrome c (Fig. 7). However, when the permeabilization of LUVs was measured, the difference between the mutant and wild-type Bax was not significant (Fig. 6). This might be due to the fact that ANTS, a small molecule (~300 Da), is likely more easily released than cytochrome c (~12,000 Da): consequently, even if Bax does not reach the ultimate conformation allowing the stabilization of a large pore permeable to cytochrome c [17], an intermediate state might be

Fig. 6. Permeabilization of ANTS-loaded LUV by Bax. (A,B) Time-course of ANTS release. 200 ng purified BaxWT or Bax-P168A, produced in the presence of 0.01% Brij-58 (A) or 0.05% F8-TAC (B), were added to LUV buffer (80 μL final volume). At time 0, 20 μL ANTSloaded LUV were added and the fluorescence was followed in a fluorescence plate reader (Clariostar). Control experiments were done with Brij-58 or F8-TAC at the concentration used during the in vitro synthesis (0.01% and 0.05%, respectively). At the end of the assay, 100% fluorescence was set by adding 0.5% CHAPS, to fully permeabilize LUVs. In (B), circles and squares represent different assays ran in parallel. (C) Quantification of ANTS release. Maximal release was calculated by doing a non-linear regression of ANTS release curves (assuming a second order kinetics). Ratio Bax-P168A/ BaxWT were calculated on experiments ran in parallel (parallel proteins production and purification, same LUV preparation, same assay plate). A one sample t-test showed that the ratio between the release induced by Bax-P168A on the release induced by BaxWT was not significantly different from 1, for Brij-58 preparations (p N 0.05), nor for F8-TAC preparations (p N 0.1). (D) Stimulation of BaxWT and Bax-P168A by caspase 8-treated Bid. Same conditions as in (A). Where indicated, caspase 8-treated Bid was added before Bax (0.1 μg/μg Bax). The experiment was done in duplicate and triplicate on two different preparations for each Bax protein, with similar results.

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Fig. 7. Binding and permeabilization of mitochondria. (A,B) 200 μg yeast W303-1B mitochondria were suspended in the presence of the indicated amount of BaxWT or Bax-P168A, in PMB buffer (100 μL final volume). Samples were incubated at 30 °C for 1 h under gentle mixing, then centrifuged at 25000×g for 10 min. The pellet was resuspended in 100 μL water, and both pellet and supernatant were precipitated with 10 μL TCA 3M. The pellets were washed twice with acetone and resolubilized in Laemmli buffer for SDS-PAGE, Western-Blotting and probing with the following antibodies: anti-yeast Porin (Novex, 1/50,000), anti-human BaxN20 (Santa-Cruz, 1/5000), anti-yeast cytochrome c (Millegen, 1/5000). Cytochrome c was quantified by densitometry. The experiment is representative of two independent experiments. (C) 200 μg yeast W303-1B or human colorectal cancer cells HCT-116 BaxKO mitochondria were suspended and incubated as in (A) in the presence of 1 μg BaxWT or Bax-P168A and analyzed with the same antibodies for yeast and the following antibodies for human mitochondria: anti pigeon cytochrome c (R&D Systems, 1/5000), anti-human BaxN20 (Santa-Cruz, 1/5000), anti-human porin (Mitosciences, 1/10,000). The experiment is representative of 3 independent experiments on yeast mitochondria and 2 independent experiments on HCT-116 BaxKO mitochondria. (D) Quantification of cytochrome c release. Western-Blots against cytochrome were quantified and the ratio between released (supernatant) and total (pellet + supernatant) cytochrome c was calculated for each experiment. Paired t-test on the experiment with yeast mitochondria indicated a significant difference (p b 0.01).

sufficient to release ANTS [28]. This suggests that the ANTS/DPXdequenching method might not have enough resolution to depict small differences of Bax activity. Other methods, like the dequenching of selfquenched calcein have been only used with Bax α9-helix alone, but not with the full-length protein [34]. Anyway, although larger than ANTS, calcein (~700 Da) remains much smaller than cytochrome c. Consequently, measuring the release of cytochrome c from mitochondria might still be the most adequate method to test Bax activity. The consequence of the mutation of residue P168 on Bax conformation remains an interesting issue. This proline is located in the small loop between helices α8 and α9, and we had considered that replacing it by a less rigid residue would allow Bax activation through helping hydrophobic helix α9 to move away from the core of the protein, thus exposing hydrophobic domains. This is precisely what was observed when mutants of P168 were expressed in yeast [12] and in human glioblastoma [10]. However, opposite conclusions were made when P168 mutants of YFP-Bax [43] or FLAG-Bax [11] were expressed in Hela cells. Furthermore, a recent study demonstrated that a mutant Bax-P168G formed stable dimers [44]. We observed that, immediately following purification, the mutant Bax-P168A did not have a significant tendency to form more dimers than BaxWT (Fig. 2C). However, after 24 h at 4 °C, Bax-P168A formed more oligomers (Fig. 4BC), associated to a greater exposure of hydrophobic domains (Fig. 4A). Taken together, these data suggest that P168 substitution may stabilize inactive dimers in a soluble environment [44], but may favor the formation of active oligomers in the presence of mitochondria ([10,12], this paper). It may be

noted that it is unlikely that the difference may come from the nature of the substituting residue since we found that all three mutant P168A, G or V exhibited the same high ability to release cytochrome c in yeast (Fig. 1E). This implied that (a) mitochondrial factor(s) common to yeast and human mitochondria (but absent from liposomes) may participate to the oligomerization/activation process of Bax-P168A. Interestingly, we have previously reported that inhibiting the interaction between Bax and the ubiquitous mitochondrial receptor Tom22 did not decrease Bax translocation to mitochondria, but decreased its capacity to be further oligomerized and activated [62]. The behavior of Bax-P168A mutant is relevant to the recent models of Bax interaction with the membrane, based on structural data [19, 27]. It is proposed that, instead of being inserted in the membrane, like it was thought before, helices α5 and α6 might lay flat on the membrane [18,26]. However, the structural model did not contain helix α9 and its position in the final active structure is not yet fully defined. It might be hypothesized that, once Bax is interacting with the membrane, α9 insertion could stabilize the pore. This is supported by experiments showing that α9 participates to an interface of dimerization involved in the expansion to high-sized oligomers, starting from the initial dimers [63,64]. The greater activity of mutant P168A is compatible with this hypothesis. Also, the fact that, in vitro or under conditions where it is strongly overexpressed, Bax deprived of the α9 helix may also induce membrane permeabilization [37–39] is compatible with this hypothesis: indeed, the insertion of α9 helix might be dispensable when a sufficiently high amount of Bax is present to destabilize the membrane.

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The role of helix α9 may also be discussed in terms of the retrotranslocation process. It has been shown that, once it is present at the mitochondrial membrane, Bax can be retrotranslocated, and this process is greatly stimulated by anti-apoptotic proteins such as Bcl-xL [65–67]. A similar process occurred when Bax and Bcl-xL were coexpressed in yeast [45] and, interestingly, Bax-P168A could not be retrotranslocated (Supplementary Fig. S6 in [45]). Since the capacity of this mutant to release cytochrome c was still largely inhibited by BclxL [12], the absence of retrotranslocation is probably not related to a defective interaction between Bax-P168A and Bcl-xL, but most likely to a thermodynamic limitation preventing membrane release once α9 is inserted. Independently of the methods used to measure Bax activity, the cellfree production method has a clear advantage over classical expression in E. coli. Indeed, in our experiments, we have introduced small amounts of the detergent Brij-58 or the fluorinated surfactant F8-TAC, that were not included in further purification steps. This was sufficient to maintain BaxWT and Bax-P168A in solution long enough to overcome Bax tendency to form aggregates when these molecules were omitted, namely for the mutant protein. A drawback of detergent or surfactant inclusion could be the promotion of the constitutive activation of Bax, like it had been shown when detergents such as β-octylglucoside or Triton X100 were included in purification buffers [55,68]: as a matter of fact, we observed that β-octylglucoside induced the conversion of BaxWT produced in the presence of Brij-58 towards the 6A7-reactive conformation (Fig. 4), indirectly showing that Brij-58 alone did not induce this conversion. In our experiments, Brij-58 and F8-TAC were only included at the production step, meaning that they were diluted at least 300 times at the final steps (assuming a homogenous dilution). However, it is likely that a fraction of these molecules remained associated to Bax, thus counteracting its proneness to aggregate. But, contrary to βoctylglucoside, they did not convert Bax towards its fully active conformation, allowing us to discriminate the behavior of the two variants. Approaches described herein will therefore be applied to further structure/function studies on other Bax mutants of interest, such as substitutions of Ser184 [16,69]. They might also be used to study Bax behavior in response to the interaction with its partners, such as other Bcl-2 family members, to small ligands, such as prostaglandins [70], or to post-translational modifications such as phosphorylation [16,68,71]. Conflicts of interest The authors declare that they have no conflict of interest with the contents of this article. Authors contributions LS conducted most of the experiments. AL participated to the experiments with LUVs. IL did the CD and bis-ANS binding experiments with LS. GD synthesized F8-TAC. MFG and CG set up the cell free production with LS. SM participated to the experiments with mitochondria and wrote the manuscript, that was corrected by the other authors. Transparency Document The Transparency document associated with this article can be found, in the online version. Acknowledgments This work was supported by grants from the Agence Nationale de la Recherche (Project “Phosbax”) (ANR-12-ISV8-0002-01), the Centre National de la Recherche Scientifique, and the Université de Bordeaux. The authors wish to thank Dr Thibaud Renault (Charité Hospital, Berlin) and Dr Manuel Rojo (IBGC, Bordeaux) for helpful advices.

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