The membrane insertion of the pro-apoptotic protein Bax is a Tom22-dependent multi-step process: a study in nanodiscs

Abstract

Membrane insertion of the pro-apoptotic protein Bax was investigated by setting up cell-free synthesis of full-length Bax in the presence of pre-formed nanodiscs. While Bax was spontaneously poorly inserted in nanodiscs, co-synthesis with the mitochondrial receptor Tom22 stimulated Bax membrane insertion. The initial interaction of Bax with the lipid bilayer exposed the hydrophobic GALLL motif in Hα1 leading to Bax precipitation through hydrophobic interactions. The same motif was recognized by Tom22, triggering conformational changes leading to the extrusion and the ensuing membrane insertion of the C-terminal hydrophobic Hα9. Tom22 was also required for Bax-membrane insertion after Bax was activated either by BH3-activators or by its release from Bcl-xL by WEHI-539. The effect of Tom22 was impaired by D¹⁵⁴Y substitution in Bax-Hα7 and T¹⁷⁴P substitution in Bax-Hα9, which are found in several tumors. Conversely, a R⁹E substitution promoted a spontaneous insertion of Bax in nanodiscs, in the absence of Tom22. Both Tom22-activated Bax and BaxR⁹E alone permeabilized liposomes to dextran-10kDa and formed ~5-nm-diameter pores in nanodiscs. The concerted regulation of Bax membrane insertion by Tom22 and BH3-activators is discussed.

Introduction

The mitochondrial apoptotic pathway involves proteins of the Bcl-2 family, including anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, multi-domain pro-apoptotic proteins Bax, Bak, and Bok, and pro-apoptotic BH3-only proteins, such as Bid and Bim ([1, 2] for recent reviews). In healthy cells, Bax is inactive in the cytosol or loosely bound to intracellular membranes. It becomes active following apoptotic signals that induce a series of events culminating in the formation of a pore that permeabilizes the mitochondrial outer membrane (MOM) to apoptogenic factors.

Soluble, inactive Bax is structured in 9 α-helices, organized around a hairpin formed by Hα5 and Hα6 [3] (Fig. S1). The C-terminal Hα9 is hydrophobic, like the C-terminal membrane anchors of anti-apoptotic proteins Bcl-2 and Bcl-xL [4, 5]. However, Bax-Hα9 does not drive the MOM-insertion of Bcl-xL while the C-terminal α-helix of Bcl-xL drives the MOM-insertion of Bax [3, 6], suggesting that this helix alone is not sufficient to target and insert the protein into the MOM. Indeed, Bax Hα9 is stabilized in a hydrophobic groove formed by the Bcl-2 Homology domains, owing to the presence of hydrophobic residues on its core-facing side and hydroxyl residues on its outside-facing side [3]. The only hydroxyl residue on the core-facing side, S¹⁸⁴, is facing D⁹⁸ in Hα4, and both residues are close enough to generate a hydrogen bond, which stabilizes Hα9 in the hydrophobic groove. Indeed, the deletion of S¹⁸⁴, or its substitution by a non-hydroxylated residue, promotes the spontaneous insertion of Bax in both reconstituted and cellular models [3, 7]. Hα9 extrusion from the hydrophobic groove thus appears as a crucial step towards Bax membrane insertion. The BH3-only protein Bim binds at a site close to Hα1, α1-α2 loop and Hα6 (the so-called “non-canonical” binding site) [8], resulting in long-range conformational changes that may ultimately lead to the extrusion and the insertion of Hα9 [9]. Accordingly, mutagenesis-induced conformational changes in the α1-α2 loop prevent Hα9 extrusion [10]. Anti-apoptotic proteins competing with Bim-binding might also block these long-range conformational changes [11].

The N-terminal extremity of Bax is also involved in the regulation of its mitochondrial localization. Indeed, the absence of the 19 N-terminal residues generates a natural variant, called BaxΨ, having a constitutive mitochondrial localization [12], which was found in low-grade glioblastomas displaying high level of apoptosis [13]. Bax-Hα1 (residues 16–35) fused to RFP compete with the mitochondrial addressing of BaxΨ, suggesting that a receptor for Hα1 is involved in Bax mitochondrial localization [14]. Hα1 was further shown to interact with the mitochondrial receptor Tom22, a component of the TOM complex (Translocase of Outer Membrane) [15]. Tom22 down-regulation decreases Bax mitochondrial localization in apoptotic mammalian cells [15, 16] and following heterologous expression in yeast [15, 17] and in Drosophila [18]. However, because other studies did not report any significant role of TOM components on Bax localization and function neither in mammalian cells [19] nor in yeast [20], the potential role of Tom22 in Bax mitochondrial localization remains debated. It is noteworthy that the inactivation of Tom22 strongly impairs cell viability [15], limiting the usefulness of cellular studies, and likely explaining the different results and conclusions obtained by different investigators.

Another TOM subunit, namely Tom20, is involved in the mitochondrial localization of Bcl-2 [21, 22] and Metaxins 1 and 2, components of the SAM complex (Sorting and Assembly Machinery), are involved in the mitochondrial localization and activation of Bak [23, 24]. It therefore appears that the role of MOM receptors in the targeting, activation, and regulation of Bcl-2 family members deserves further investigations ([25, 26] for reviews).

Membrane models provide a reliable approach for the stepwise dissection of the molecular aspects of the regulation of Bax membrane insertion. Among these models, nanodiscs are small planar lipid bilayers, stabilized by a dimer of Membrane Scaffold Protein (MSP) that masks the hydrophobic tails ([27] for review). They are homogenous in size, more stable than liposomes and bicelles, and the addition of tags to MSP facilitates their purification by affinity chromatography. The reconstitution of membrane proteins in nanodiscs is a successful approach not only to resolve the structure of membrane proteins but also to gain insight into mechanistic aspects ([28, 29] for reviews). A cryoEM study of Bax activated by Bid-BH3 at low pH, and inserted into nanodiscs, showed the occurrence of a small pore (~3.5 nm in diameter) [30]. On this basis, molecular dynamics simulations supported that the reorganization of a limited number of lipid molecules around Bax could generate a toroidal pore also involving Hα5 and Hα6 [31]. We have set up a more direct approach by running the cell-free synthesis of Bax in the presence of pre-formed nanodiscs [32]. We used full-length BaxWT and a mutant BaxP¹⁶⁸A, which was previously shown to be spontaneously membrane-inserted in glioblastoma cells [33], yeast cells [34], and liposomes [35]. However, both variants largely precipitated in the presence of nanodiscs and therefore failed to be inserted [32]. The precipitated fraction, containing pure Bax, was used to co-form nanodiscs: interestingly, TEM studies showed that nanodiscs containing BaxP¹⁶⁸A had a larger size than those containing BaxWT, and that some of them displayed a pore having a diameter close to 5 nm [32]. However, the objects were heterogeneous, precluding structural investigations. Subsequently, we observed that the cell-free co-synthesis of Bax with its anti-apoptotic partner Bcl-xL prevented its precipitation and stimulated its insertion into nanodiscs [36]. This urged us to further investigate the mechanisms underlying Bax interaction with nanodiscs and the possible role of Tom22, by setting up a minimal system involving the co-synthesis of Bax and Tom22 in the presence of nanodiscs. By using several Bax mutants, we showed that Tom22 interacted with Bax Hα1 to promote a conformational change resulting in the extrusion and the membrane insertion of Bax Hα9. Tom22 was also required for Bax-membrane insertion following Bax activation by BH3-activators or by its interaction with Bcl-xL then release by WEHI-539. Furthermore, Tom22 stimulated the ability of Bax to form a pore. We propose a model that emphasizes the concerted action of Tom22 and BH3-activators in Bax targeting, insertion and pore formation.

Results

The co-synthesis of Tom22 prevents Bax precipitation and stimulates Bax insertion into nanodiscs

As reported previously [32], the presence of nanodiscs induced the precipitation of a large fraction of Bax (Fig. S2), while nanodiscs remained in the supernatant (Fig. 1A). The fraction of Bax that was not precipitated still failed to interact with nanodiscs, as shown by its detection in the unbound fraction of Ni-NTA-sepharose affinity chromatography to immobilize the His7-tagged nanodisc scaffold protein MSP1E3D1 (Fig. 1C).

Fig. 1. Tom22 co-synthesis prevented Bax-precipitation and promoted its insertion into nanodiscs.

A BaxWT was synthesized alone or co-synthesized with Tom22 in the presence of 3.5 µM nanodiscs (ND). The ratio of piVEX plasmids encoding the two proteins was 2 to 1 (typically 15 ng and 7.5 ng for Bax and Tom22, respectively). After the synthesis, the reaction mix was weighted and centrifuged at 20,000 × g for 15 min. The pellet was re-suspended in the same volume of buffer B (25 mM Hepes/Na pH 7.4, 200 mM NaCl, 0.1 mM EDTA). Aliquots from both the pellet (p) and supernatant (sn) were mixed with an equal volume of Laemmli buffer 2×, and analyzed by SDS-PAGE and western blot against Bax and Tom22. Note that Tom22 was visible under the forms of a monomer and a dimer. For the sake of clarity, only the monomer will be displayed in following experiments. Blots are representative of at least eight independent experiments. B Quantitative analysis of the effect of the co-synthesis of Tom22 on nanodisc-induced Bax precipitation. Non-saturated western blots similar to (A) were quantified with Image J and the % of Bax in the pellets was calculated. Each point represents an independent synthesis assay. (The % of Bax in pellet was 53.7 ± 15.4 (s.d.) and 17.7 ± 15.6 (s.d.), for “-Tom22” and “+Tom22”, respectively; ****unpaired t-test, p < 0.0001). C The remaining supernatant (typically 85 µL) was mixed with 100 µL of Ni-NTA-agarose (Qiagen) pre-washed in buffer B, and incubated for 2 h at 4 °C under gentle agitation. The mixture was centrifuged (2000 × g, 30 s) and the unbound fraction (lanes u) was collected. The resin was then washed 3 times with 500 µL buffer W (20 mM Tris/HCl pH 8.0, 500 mM NaCl). An identical volume as the unbound fraction of buffer E (20 mM Tris/HCl pH 8.0, 200 mM NaCl, 300 mM imidazole) was added to recover Ni²⁺-bound nanodiscs) (lanes b). An identical volume of the unbound (u) and bound (b) fractions were mixed with an identical volume of Laemmli buffer 2×, and analyzed by SDS-PAGE and western blot against Bax, Tom22, and His7-MSP1E3D1. Blots are representative of at least 8 independent experiments. D Quantification of Bax bound to nanodiscs (b). Non-saturated western blots similar to (C) were quantified with Image J and the % of Bax in fraction b was calculated. Each point represents an independent synthesis assay. (The % of Bax associated to nanodiscs were 17.2 ± 16.8 (s.d.), 58.8 ± 14.8 (s.d.) and 42.5 ± 16.7 (s.d.) for “-Tom22”, “+Tom22” and “+Tom22(1–52)”, respectively; **unpaired t-test, p < 0.01). E Purified nanodiscs in the fraction “b” of (C) were dialyzed against buffer D (20 mM Tris/HCl pH 8.0, 200 mM NaCl) to remove imidazole. They were then added with 100 mM Na₂CO₃, pH 10.0, and incubated for 15 min. Nanodiscs were then re-purified on Ni-NTA, as in (C). Blots are representative of 3 independent experiments. F–H Same experiments as in (A, C, E) with Tom22(1–52) instead of full-length Tom22. Blots are representative of 3 independent experiments.

It has been reported that the mitochondrial receptor Tom22 was involved in Bax targeting to the MOM [15]. Strikingly, we observed that the co-synthesis of Tom22 with Bax prevented Bax precipitation (Fig. 1A, B) and promoted the association of Bax to nanodiscs (Fig. 1C, D).

To discriminate between loose protein/lipid interaction and membrane-inserted proteins, nanodiscs were submitted to an alkaline treatment to remove peripheral proteins. This treatment did not alter the stability of nanodiscs (Fig. S3A) and efficiently removed Kras, a known peripheral protein [37] (Fig. S3B). In the presence of Tom22, a large fraction of Bax remained associated to nanodiscs, showing that the co-synthesis with Tom22 promoted the membrane insertion of Bax (Fig. 1E).

The alkaline treatment showed that Tom22 was itself inserted into nanodiscs (Fig. 1E). We previously demonstrated that the co-synthesis of Bcl-xL, but not of Bcl-xLΔC, stimulated Bax insertion into nanodiscs [36]. We then asked if the insertion of Tom22 was also required for Bax insertion. Bax was co-synthesized with the cytosolic N-terminal domain of Tom22 (residues 1–52), that contains the negatively charged residues involved in the sorting of mitochondria-targeted proteins [38]. Like full-length Tom22, Tom22(1–52) prevented Bax precipitation (Fig. 1F), and stimulated Bax association to nanodiscs (Fig. 1D, G) and Bax insertion (Fig. 1H). We concluded that, contrary to Bcl-xL, the membrane insertion of Tom22 was not required to promote Bax insertion. This showed that the stimulating effect of Tom22 on Bax insertion did not involve the co-insertion of the two proteins, but rather promoted a conformational change of Bax enabling its membrane insertion.

Importantly, the effects of Tom22 were specific: the co-synthesis of Bax with Tom20 did not prevent Bax precipitation nor promoted Bax insertion (Fig. S3C, D), in agreement with the observation that the down-regulation of Tom20 did not change Bax mitochondrial localization and function in cellulo [15].

Bax precipitation and Tom22 effects depend on the exposure of the hydrophobic GALLL motif in Bax Hα1

The interaction of soluble Bax with liposomes induces the exposure of the N-terminal epitope recognized by the 6A7 antibody [39], which is masked in soluble, inactive Bax, contrary to the 2D2 antibody, which recognizes all Bax conformations [40]. Bax was then synthesized in the absence of nanodiscs, then added to nanodiscs: like liposomes, nanodiscs increased the exposure of the 6A7 epitope, to the same extent as the constitutively active mutant BaxR⁹E ([41]; see also below) in which the 6A7 epitope was constitutively exposed (Fig. 2A).

Fig. 2. The GALLL motif in Hα1 was responsible for nanodisc-induced Bax precipitation and interacted with Tom22.

A BaxWT and the constitutively active mutant BaxR⁹E were synthesized in the absence of nanodiscs. Reaction mixes were centrifuged (20,000 × g, 15 min) and equal volumes of the supernatants were added or not with 3.5 µM nanodiscs. 1 µg anti-Bax 6A7 or 2D2 antibodies (Santa-Cruz Biotechnology) were added and samples were incubated overnight at 4 °C under gentle agitation. 50 µL Protein-G sepharose beads (Cytiva) were washed with buffer IP (20 mM Tris/HCl pH 8.0, 200 mM NaCl) and were added for 3 h. The suspensions were then washed 5 times with 200 µL of buffer WIP (25 mM Tris/HCl pH 7.4, 500 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol) and twice with the same buffer diluted 10 times. Bound proteins were recovered by adding 30 µL of Laemmli buffer (without β-mercaptoethanol) and heating at 65 °C for 15 min. Samples were analyzed by SDS-PAGE and western blot against Bax, with a distinct antibody from the ones used for immunoprecipitations (Abcam). The 2D2 antibody did not immunoprecipitate the Bax R⁹E mutant. The figure is representative of 3 independent experiments. B BaxWT and BaxΨ (deleted of residues 1–19) were synthesized in the absence or in the presence of nanodiscs and analyzed like in Fig. 1A. Western blots were done with the Abcam antibody of which the undisclosed epitope is away from the N-terminus. Blots are representative of 4 independent experiments. C Supernatants from BaxΨ in (B) were analyzed as in Fig. 1C, showing the absence of BaxΨ-association to nanodiscs. The blot is representative of 2 independent experiments. D BaxΨ was co-synthesized with Tom22 in the presence of nanodiscs, and samples were analyzed like in (B). Blots are representative of 5 independent experiments. E Supernatants from (D) were analyzed like in (C), showing the partial association of BaxΨ to nanodiscs. Blots are representative of 3 independent experiments. F Representation of the Hα1 of Bax in the 1F16 structure [3]. Hydrophilic residues are in blue and hydrophobic residues are in red. G Same experiment as in Fig. 1A with BaxWT, BaxA²⁴R, and BaxL²⁶Q, showing the absence of precipitation of BaxA²⁴R and BaxL²⁶Q. Blots are representative of 2 independent experiments. H Same experiment as in Fig. 1C with BaxA²⁴R showing its absence of association to nanodiscs. Blots are representative of 2 independent experiments.

This antibody recognizes the epitope ¹³PTSSEQI¹⁹ of Bax only when it moves away from the core of the protein [42]. It is located within the ART sequence (Apoptotic Regulation of Targeting; residues 1–19), of which the full deletion stimulated Bax mitochondrial localization and its ability to release cytochrome c [12, 14]. We then compared the cell-free synthesis of BaxΨ (carrying a deletion of residues 1–19) to that of full-length Bax (BaxWT). Contrary to BaxWT, BaxΨ precipitated in the absence of nanodiscs (Fig. 2B), suggesting that ART deletion led to the exposure of a hydrophobic domain of the protein leading to its precipitation, mirroring the effect of nanodiscs on BaxWT. This was consistent with the hypothesis that the interaction of BaxWT with nanodiscs induced a movement of the ART sequence resulting in the exposure of hydrophobic domains leading to Bax precipitation. When synthesized in the presence of nanodiscs, the behavior of BaxΨ was the same as BaxWT: it precipitated (Fig. 2B) and the small fraction remaining in the supernatant was not inserted (Fig. 2C), unless co-synthesized with Tom22 (Fig. 2D, E). This effect of Tom22 explains why BaxΨ was always spontaneously inserted in the MOM when expressed in mammalian and yeast cells [12, 14].

ART deletion exposes Hα1, and substitutions in this helix decrease the mitochondrial localization of both BaxΨ and BaxWT [14]. Hα1 contains a succession of hydrophilic and hydrophobic residues, with a longer motif of five hydrophobic residues ²³GALLL²⁷, thus displaying a hydrophobic face, and a hydrophilic face that is interrupted by a hydrophobic helix turn (Fig. 2F). Since the exposure of the hydrophobic GALLL motif might be responsible for Bax precipitation, we replaced A²⁴ or L²⁶ by hydrophilic residues R and Q, respectively. Contrary to BaxWT, mutants BaxA²⁴R and BaxL²⁶Q did not precipitate when synthesized in the presence of nanodiscs (Fig. 2G), showing that the hydrophobic GALLL motif was indeed involved in intermolecular interactions driving Bax molecules to precipitate following Hα1 exposure. Interestingly, when co-expressed with Tom22, BaxA²⁴R was not inserted (Fig. 2H) and BaxL²⁶Q displayed an intermediate behavior between BaxWT and BaxA²⁴R (Fig. S4). These data emphasized the role of the GALLL motif in Bax/Tom22 interaction, in agreement with the observation that Tom22 bound to GALLL-containing peptides [15].

Bax activated by BH3 domains requires Tom22 for membrane insertion

In cellulo, the interaction between Bax and Tom22 only occurs after apoptosis has been triggered, suggesting that an initial conformational change of Bax is required [15]. BH3-only proteins Bid and Bim are the main activators of Bax. However, we observed that cBid, Bid-BH3 and Bim-BH3 peptides and the Bax-activator BTSA1 did not prevent nanodisc-induced Bax precipitation (Fig. S5A, B), nor stimulated Bax insertion into nanodiscs (Fig. 3A, B). As expected, the co-synthesis of Tom22 prevented Bax precipitation (Fig. S5C) and restored Bax insertion in the presence of BH3 activators (Fig. S5D).

Fig. 3. Tom22-dependent insertion of Bax activated by BH3-domains.

A Same experiments as in Fig. 1C (without Tom22), except that 10 µg caspase-8-cleaved Bid (cBid) was included in the reaction mix. We determined that the maximal concentration of Bax produced in the cell-free system was 0.5 mg/mL, i.e. 50 µg of Bax in the 100 µL-reaction mix. We therefore set up a cBid to Bax ratio of ~1 to 5 (considering that the sizes of the two proteins are close to each other) [69]. Blot is representative of 2 independent experiments. B Same experiment as in Fig. 1C (without Tom22) in the presence of 1 µM Bim-BH3 (PEIWIAQELRRIGDEFNAYYA), 1 µM Bid-BH3 (ESQEDIIRNIARHLAQVGDSMDRSIPPG) (Genecust), or 1 µM BTSA1 (Medchem) [83]. The concentration refers to the volume of the whole mix (reaction mix + feeding mix) because the sizes of the three molecules are below the cut-off of the dialysis membrane. Blot is representative of 2 independent experiments. C Bax and Bcl-xL were co-synthesized in the presence of nanodiscs made with His7-tagged MSP1E3D1, purified on Ni-NTA, and dialyzed against ND buffer (200 mM NaCl, 10 mM Hepes/Na, pH 7.4) [36]. Tom22 was synthesized in the presence of nanodiscs made with StrepII-tagged MSP1E3D1, purified on Strep-Tactin (IBA), and dialyzed against ND buffer. Bax/Bcl-xL-containing His7-nanodiscs were mixed with the same amount of StrepII-nanodiscs, either empty or containing Tom22. 250 nM of WEHI-539 were added and the mixture was incubated overnight at 4 °C. StrepII-nanodiscs were purified again on Strep-Tactin and the unbound and bound fractions were analyzed. Blot is representative of 3 independent experiments. D Schematic representation of the results of (C). E Bax and Bcl-xL were co-synthesized in the presence of nanodiscs, as in (C). Nanodiscs were purified and added to HEK293FT mitochondria, preincubated in the absence or in the presence of an anti-Tom22 antibody and of WEHI-539. After an overnight incubation at 4 °C, mitochondria were analyzed for the content of cytochrome c, with VDAC as a control. F Quantification of Cytochrome c/VDAC ratios from (F), made on two independent mitochondria preparations from HEK293FT cells (circles) and two independent mitochondria preparations from HCT116 BaxKO cells (triangles). Values were 0.93 ± 0.11 (s.d.) and 0.60 ± 0.14 (s.d.) for “+abTom22” and “-abTom22”, respectively; *p < 0.05. G Schematic representation of experiments in (E, F).

In cancer cells, the overexpression of anti-apoptotic proteins, such as Bcl-xL, prevents Bax activation. This inhibition is relieved by BH3-only proteins, and by BH3-mimetic compounds ([43] for review), releasing Bax under a primed conformation [44]. We set up a nanodisc-to-nanodisc transfer experiment to appraise if Tom22 was still required when Bax was primed by its interaction with Bcl-xL then released by the BH3-mimetic WEHI-539. Bax and Bcl-xL were co-synthesized and co-inserted in nanodiscs formed with His7-tagged MSP1E3D1 [36]. These nanodiscs were purified and incubated with nanodiscs formed with StrepII-tagged MSP1E3D1, either empty or containing Tom22. The addition of WEHI-539 was expected to break the interaction between Bax and Bcl-xL, therefore releasing Bax under a primed conformation. The presence of Bax was then measured in purified StrepII-tagged nanodiscs (Fig. 3C). We observed that Bax was translocated to StrepII-nanodiscs when Tom22 was present, but not to empty StrepII-nanodiscs. This showed that the presence of Tom22 was required for Bax insertion after it was primed through its interaction with Bcl-xL followed by its release by WEHI-539 (Fig. 3D).

We then investigated if the role of Tom22 in MOM-permeabilization could be observed. Bax and Bcl-xL were co-synthesized in the presence of nanodiscs, and purified as above. They were incubated in the presence of mitochondria isolated from HEK-293 cells, and preincubated or not with an anti-Tom22 antibody, with or without WEHI-539. Mitochondria were then analyzed for cytochrome c content. As expected, in the absence of WEHI-539, no permeabilization could be observed (Fig. 3E, F). In the presence of WEHI-539, and in the absence of anti-Tom22 antibody, a decrease of mitochondrial cytochrome c content was observed, showing that Bax was able to permeabilize MOM. On the contrary, in the presence of anti-Tom22 antibody, no decrease of mitochondrial cytochrome c was observed (Fig. 3E, F), suggesting that Tom22 was required for Bax ability to form a cytochrome c permeable pore (Fig. 3G). Similar results were obtained with mitochondria isolated from HCT116 Bax-KO cells (Fig. 3F).

These data showed that, even after Bax had been activated by its interaction with its partners, either directly by Bim or Bid, or indirectly by its interaction with Bcl-xL then release by WEHI-539, the interaction with Tom22 was still required for its insertion into nanodiscs and for its ability to permeabilize MOM to cytochrome c.

Tom22 triggers a conformational change leading to the extrusion of Bax Hα9

Our data suggest that a two-step process initiates Bax insertion into nanodiscs: (i) the interaction of Bax with the lipid bilayer induces a movement of ART exposing GALLL motif in Hα1 and (ii) Tom22 interacts with the exposed GALLL motif, thus triggering conformational changes leading to Bax membrane insertion. We next investigated the nature of these conformational changes.

Because the stability of the soluble conformation of Bax depends on the embedding of its C-terminal hydrophobic Hα9 in the hydrophobic groove, Bax membrane insertion first requires the extrusion of Hα9 from the groove [3]. To investigate the effect of Tom22 on Hα9 mobility, we measured the exposure of a single cysteine residue introduced at position 177. Like BaxWT, the triple mutant BaxC⁶²S/C¹²⁶S/V¹⁷⁷C was dependent on Tom22 for its insertion into nanodiscs (Fig. 4A). We next measured the accessibility of the V¹⁷⁷C residue to NEM-PEG in the different conformations stabilized by our assay (Fig. 4B). In the absence of nanodiscs, V¹⁷⁷C was accessible to NEM-PEG. When synthesized in the presence of nanodiscs, V¹⁷⁷C residue of the soluble fraction of Bax remained exposed, regardless of the co-synthesis of Tom22. On the contrary, V¹⁷⁷C of nanodisc-inserted Bax (in the presence of Tom22) was no longer accessible, showing that Hα9 was inserted into the lipid bilayer.

Fig. 4. Effect of nanodiscs and Tom22 on the exposure and insertion of Hα9.

A Same experiment as in Fig. 1C with the mutant BaxC⁶²S/C¹²⁶S/V¹⁷⁷C, in which the two endogenous cysteine residues in positions 62 and 126 have been replaced by serine, and a cysteine has been introduced in Hα9. Blot is representative of 3 independent experiments. B Fractions from (A) were incubated for 1 min in the presence of 0.2 mM NEM-PEG (5 kDa) and the reaction was stopped by adding 20 mM NEM and a 10-min additional incubation. The lane “-ND” is a synthesis without ND and Tom22. Samples were then analyzed by SDS-PAGE and western blot against Bax. Blot is representative of 3 independent experiments. C, E Same experiments as in Fig. 1A on mutants BaxΔα9 and BaxT¹⁷⁴P, showing that Tom22 did not prevent their precipitation. Blots are representative of 3 independent experiments. D, F Same experiments as in Fig. 1C on mutants BaxΔα9 and BaxT¹⁷⁴P showing that Tom22 did not promote their insertion. Blots are representative of three independent experiments.

We next showed that the insertion of Hα9 was a prerequisite for Tom22-induced membrane insertion of Bax, and the protection against nanodisc-induced precipitation. For this, we analyzed Bax deprived of Hα9 (BaxΔα9, residues 1-169). Like full-length Bax, and contrary to BaxΨ, BaxΔα9 was soluble in the absence of nanodiscs and precipitated in their presence (Fig. 4C). However, its precipitation was not prevented by the co-synthesis with Tom22 (Fig. 4C). As expected, BaxΔα9 was not inserted in nanodiscs (Fig. 4D). These data confirmed that the rescue of Bax precipitation by Tom22 was actually related to the Hα9-dependent insertion of Bax.

According to the Cosmic database (https://cancer.sanger.ac.uk/cosmic), a T¹⁷⁴P substitution in Hα9 is frequently found in squamous cell carcinomas of the upper digestive track [45]. We constructed a mutant carrying this substitution, thus keeping Hα9 length, but breaking its helical conformation. The co-synthesis of Tom22 did not significantly rescue the precipitation of BaxT¹⁷⁴P (Fig. 4E) and did not stimulate its poor membrane insertion (Fig. 4F). This discarded the possibility that a side effect linked to the full deletion of Hα9 was responsible for nanodisc-induced BaxΔα9 precipitation, and showed that defective Bax insertion might be involved in defective apoptosis in cancer cells containing the T¹⁷⁴P substitution.

These data established that the effects of Tom22, both on the prevention of Bax precipitation and the stimulation of Bax insertion into nanodiscs, strictly depended on the presence of a Hα9 able to insert into the bilayer. This was consistent with a model where, following the initial interaction of Bax with the lipid bilayer exposing the GALLL motif in Hα1 (without any conformational change at the C-terminus at this stage), the subsequent interaction of Tom22 with the GALLL motif promoted the extrusion of Hα9 followed by its insertion. This later step could not occur in BaxΔα9 and BaxT¹⁷⁴P mutants, resulting in their precipitation caused by the hydrophobic interaction between GALLL motifs.

Involvement of Bax R⁹ and D¹⁵⁴ in Tom22-induced Hα9 insertion

We have previously reported that soluble, inactive Bax might be stabilized by an electrostatic interaction between residues R⁹ in the ART and D¹⁵⁴ in Hα7: indeed, single charge inversions of either one of these residues (R⁹E or D¹⁵⁴K) resulted in their constitutive mitochondrial localization and permeabilization of the MOM to cytochrome c, while the double-substituted mutant R⁹E/D¹⁵⁴K, in which the electrostatic interaction was restored, displayed the same soluble, inactive conformation as BaxWT [41]. We therefore tested the active mutant R⁹E and the inactive revertant R⁹E/D¹⁵⁴K in our set-up.

Contrary to BaxWT, BaxR⁹E did not precipitate when synthesized in the presence of nanodiscs (Fig. 5A) and, strikingly, was spontaneously associated to nanodiscs (Fig. 5B). As expected, this was reversed by the introduction of the D¹⁵⁴K mutation (Fig. 5A, B). Next, the R⁹E substitution was introduced in the triple mutant BaxC⁶²S/C¹²⁶S/V¹⁷⁷C described above. NEM-PEG labeling showed that Hα9 was actually inserted in the lipid bilayer (Fig. 5C). A BaxR⁹E/Δα9 mutant showed that the deletion of Hα9 impaired both the protective effect of the R⁹E mutation on Bax precipitation (Fig. 5D) and its association to nanodiscs (Fig. 5E). These data showed that the BaxR⁹E mutant synthesized alone displayed the same behavior as BaxWT co-synthesized with Tom22, leading to the spontaneous insertion of Hα9.

Fig. 5. Effects of substitutions on R⁹ and D¹⁵⁴ residues.

A, B Same experiments as in Figs. 1A and 1C on mutants BaxR⁹E and BaxR⁹E/D¹⁵⁴K in the absence of Tom22, showing that BaxR⁹E was spontaneously inserted, and that the mutation D¹⁵⁴K reversed the spontaneous insertion. Blot is representative of 3 independent experiments. C Same experiment as in Fig. 3B on mutant BaxR⁹E/C⁶²S/C¹²⁶S/V¹⁷⁷C synthesized in the absence of Tom22, showing the spontaneous insertion of Hα9. Blot is representative of 3 independent experiments. D, E The mutant BaxR⁹E/Δα9 was synthesized in the absence of Tom22 and analyzed as in Fig. 1A, C, showing that the deletion of Hα9 impaired the absence of precipitation and prevented the insertion induced by the mutation R⁹E. Blots are representative of 3 independent experiments. F, G Same experiments as in Fig. 1A, C on mutant BaxD¹⁵⁴Y, showing that Tom22 prevented its precipitation but did not promote its insertion. Blots are representative of 3 independent experiments.

The presence of a D¹⁵⁴Y substitution in some tumors has been reported in Uniprot (https://www.uniprot.org/uniprotkb/Q07812/variant-viewer) and in the Spanish database Intogen (https://www.intogen.org/search?gene=BAX). Like for BaxWT, the co-synthesis of Tom22 protected BaxD¹⁵⁴Y against nanodisc-induced precipitation (Fig. 5F). However, contrary to BaxWT, Tom22 did not promote BaxD¹⁵⁴Y insertion (Fig. 5G). From these data, we concluded that the substitution D¹⁵⁴Y did not impair the interaction of Bax with Tom22 and the associated rescue of Bax precipitation, but rather prevented Tom22-induced conformational change leading to Bax insertion. Like for the mutant T¹⁷⁴P described above, this behavior might relate to a defective apoptotic process in cancer cells displaying the D¹⁵⁴Y substitution.

Pore-forming activity of Bax inserted through Tom22-mediated conformational change

Cell-free synthesis was done in the presence of liposomes loaded with Dextran-FITC (10 kDa). An anti-FITC antibody was added to quench the fluorescence of released Dextran-FITC, and the fluorescence decay was followed during the synthesis of Bax, alone or co-synthesized with Tom22(1–52). The co-synthesis with Tom22(1–52) increased the rate of fluorescence decay, compared to BaxWT alone (Fig. 6A, B). This suggested that Tom22(1–52) not only stimulated the insertion of BaxWT but further stimulated its ability to form pores permeable to a molecule having a size similar to cytochrome c (12.5 kDa). BaxR⁹E synthesized alone without Tom22 also exhibited a higher rate of permeabilization, consistent with the above experiments showing that the R⁹E substitution promoted the spontaneous insertion of Hα9. These experiments showed that both BaxWT co-synthesized with Tom22(1–52) and BaxR⁹E synthesized alone stimulated the formation of pores large enough to release cytochrome c.

Fig. 6. Effect of Tom22 and of the mutation BaxR⁹E on Bax pore-forming capacity.

A BaxWT was synthesized alone or co-synthesized with Tom22(1–52), and the mutant BaxR⁹E was synthesized alone. Twenty microliters liposomes loaded with Dextran-FITC (10 kDa) (Sigma) and 4 µg of a fluorescence quenching anti-FITC antibody (ThermoFisher Scientific) were added in the reaction mix. Mixes were put in a 96-well fluorescence plate, and incubated at 28 °C in a thermostated fluorescence plate reader (Clariostar, BMG Labtech). Samples fluorescence was recorded at 10-min intervals (excitation: 475–490 nm; emission: 515–545 nm). B Initial rates of fluorescence decays in experiments of (A). Plots were fitted with exponential decay and the rate constants were calculated and averaged from two independent experiments. R² values were 0.95, 0.94 and 0.98 for BaxWT, BaxWT+Tom22(1–52) and BaxR⁹E, respectively. The negative control (−) was a mix in which no plasmid was added, and the positive control (CHAPS) included 0.01% CHAPS. C BaxWT was co-synthesized with Tom22(1–52) and BaxR⁹E was synthesized alone in the presence of nanodiscs. Nanodiscs were purified and concentrated on Vivaspin concentrators (cut-off 50 kDa; Sartorius). They were negatively stained with uranyl acetate and observed by TEM (see “Methods”). Selected images showing the presence of a pore are shown. The scale bar is 10 nm. D, E Pixel densities were measured on images along a line crossing the largest diameter of the pores.

TEM of negatively stained nanodiscs showed that, following the co-synthesis of Bax/Tom22(1-52) or the synthesis of BaxR⁹E alone, a structure was detected which resembles a pore (Fig. 6C) with a diameter of about 5 nm (Fig. 6D, E). This size was consistent with both electrophysiology experiments on MOM [46, 47] and reconstituted oligomers [46], and with TEM observations of precipitated/resolubilized oligomers of BaxP¹⁶⁸A reconstituted in nanodiscs by the co-formation method [32]. These data showed that the direct nanodisc insertion of BaxWT co-synthesized with Tom22, as well as that of BaxR⁹E synthesized alone, could generate the formation of a pore suitable for the release of cytochrome c.

Discussion

Tom22-induced conformational changes lead to Bax membrane insertion

Setting up a model combining the cell-free synthesis of Bax to its insertion into nanodiscs led us to reappraise the involvement of the mitochondrial receptor Tom22 in Bax membrane insertion. Tom22 is one of the three receptors of the TOM complex, also including Tom20 and Tom70. Its highly negatively charged N-terminal domain [38] is involved in the binding and unfolding of precursors of mitochondrial proteins ([48], for review). It is also involved in the assembly of the TOM complex [48]. Both Tom22 and Tom20 also exhibit a chaperone-like activity [49]. We had previously reported that the presence of nanodiscs unexpectedly induced the precipitation of Bax [32]. Here, we report that the co-synthesis of Tom22 prevented this precipitation and promoted the insertion of Bax into nanodiscs (Fig. 1). To decipher the mechanisms underlying the effect of Tom22, we first investigated the molecular events leading to nanodisc-induced Bax precipitation.

The interaction of Bax with nanodiscs induced the exposure of the GALLL motif in Hα1 of Bax molecules resulting in their precipitation through hydrophobic interactions: indeed, substitutions to more hydrophilic residues (A²⁴R, L²⁶Q) prevented Bax precipitation (Fig. 2G). Conversely, the deletion of ART, that constitutively exposes Hα1 [14], induced the precipitation of Bax, even in the absence of nanodiscs (Fig. 2B).

The same GALLL motif is involved in the MOM targeting of Bax [14] and in the interaction between Bax and Tom22 [15]. Knocking down Tom22, or inactivating it by proteolysis or by a specific antibody, decreased Bax mitochondrial localization and apoptosis [15]. In the same study, peptide mapping identified ²¹KTGALLLQ²⁸ as the main domain of interaction of Bax with Tom22. However, the involvement of Tom22 in Bax mitochondrial targeting has been debated, and contradictory conclusions have been drawn [15–20]. The present report, based on a minimal model containing only Bax, Tom22 and a lipid bilayer stabilized in nanodiscs, unambiguously demonstrated that Tom22 not only prevented Bax precipitation but further promoted its insertion into nanodiscs (Fig. 1C–E). Once the GALLL motif was exposed, it could interact with Tom22 which triggered Bax membrane insertion. In the absence of Tom22, the hydrophobic interaction between GALLL motifs led to Bax precipitation. Mutations A²⁴R and L²⁶Q prevented Bax precipitation by decreasing the hydrophobicity of the GALLL motif but impaired the interaction with Tom22, while the deletion of ART constitutively exposed the GALLL motif leading to BaxΨ precipitation regardless of the presence of nanodiscs, but to its insertion into nanodiscs when Tom22 was present.

The membrane insertion of Bax requires the extrusion of Hα9 from the hydrophobic groove ([3, 4, 50] for review). It has been proposed that the interaction of Bim at the non-canonical binding site, including Hα1, Hα6 and the α1-α2 loop [8] could trigger long-range conformational changes leading to Hα9 extrusion [9], which was further supported by molecular dynamics simulations [51] and by the observation that mutations in the α1-α2 loop prevented the extrusion of Hα9 [10]. We observed that the interaction of Tom22 with exposed Hα1 led to the insertion of Hα9 (Fig. 4B). Conversely, the deletion of Hα9 or the introduction of the helix-breaking mutation T¹⁷⁴P abolished the protective effect of Tom22 on Bax precipitation (Fig. 4C, E). This showed that, after Hα1 had been exposed, any failure of Hα9 membrane insertion resulted in Bax precipitation, further emphasizing the crucial role of Hα9 extrusion as the key step towards Bax insertion.

Although Bax is not generally considered as a mutational hotspot in cancers, several misense mutations found in tumors might alter its pro-apoptotic function [52, 53]. Furthermore, substitutions that had been primarily generated to study Bax conformational changes targeted residues later found to be mutated or modified in some cancers. Namely, we have reported the possible occurrence of an electrostatic interaction between R⁹ and D¹⁵⁴ stabilizing soluble Bax [41]. Interestingly, BaxR⁹ can be monomethylated [54], which is expected to destabilize the interaction with D¹⁵⁴. A D¹⁵⁴Y substitution has also been found in some tumors (https://www.uniprot.org/uniprotkb/Q07812/variant-viewer; https://www.intogen.org/search?gene=BAX). This led us to investigate the consequences of substitutions of these two interacting residues. The R⁹E mutant did not precipitate in the presence of nanodiscs (Fig. 5A) and was spontaneously inserted (Fig. 5B, C) in a way that depended on Hα9 (Fig. 5D, E). This mutation thus induced the spontaneous insertion of Bax, regardless of the presence of Tom22.

The D¹⁵⁴Y substitution did not prevent the rescue of Bax precipitation by Tom22 (Fig. 5F), but impaired Bax insertion (Fig. 5G). This suggested that the Tom22-triggered conformational change leading to Hα9 insertion involved D¹⁵⁴. Docking simulations between Bax and Tom22 have suggested that Tom22 might interact more strongly with soluble Bax than with Hα9-inserted Bax [55]. In the same study, simulating the breaking of the hydrogen bond between S¹⁸⁴ and D⁹⁸ resulted in the movement of the C-terminus of Bax, starting at D¹⁵⁴. These data were consistent with the hypothesis that a transient interaction between Tom22 and soluble Bax was followed by a movement of the C-terminus of Bax around D¹⁵⁴, leading to Hα9 insertion and then the release of membrane-inserted Bax from the interaction with Tom22.

Tom22-dependent Bax pore formation

Although BaxWT alone might induce some basal permeabilization of liposomes, both the co-synthesis with Tom22 or the synthesis of BaxR⁹E alone increased the rate of permeabilization to dextran-10kDa (Fig. 6A, B). Under these conditions, we could observe nanodiscs containing a pore displaying a ~5 nm diameter (Fig. 6C–E), which was consistent with electrophysiology data on MOM [46, 47, 56] and reconstituted Bax oligomers [46]. This suggested that the stimulation of Bax insertion by Tom22 and the R⁹E mutation was not associated to a random insertion of Bax molecules but rather to the formation of a pore similar to the one found in MOM. The observed pore size was much smaller than the one of large structures observed by high-resolution fluorescence microscopy and atomic force microscopy, reaching 24 to 176 nm in diameter [57–59], which are compatible with the formation of megapores involved in the release of mtDNA during inflammatory responses [60, 61]. Conversely, the smaller 5nm-pore might correspond to early apoptotic steps, when small apoptogenic factors, such as cytochrome c (~3 nm) and smac/diablo (~3.5 nm) are released. It was however somewhat larger than the 3.5 nm size that had been measured in nanodiscs containing Bid-BH3/low pH-activated Bax [30]. On the basis of cryoEM images and molecular dynamics simulations, it was proposed that Bax monomers (or possibly dimers) might form lipidic pores by rearranging phospholipid molecules in nanodiscs [31]. NMR structural studies of the Bax core (Bax Hα2-Hα5) in bicelles are compatible with a pore of which the walls were made of Bax dimers interacting with lipids, which might be further organized in dodecamers to generate a pore large enough to promote the release of cytochrome c [62]. Electrophysiology data showed that Bax pores exist under different forms displaying different conductance substates [63], which might accurately describe the continuum of Bax-induced permeabilization to small apoptogenic factors such as cytochrome c and smac/diablo, then to larger apoptogenic factors AIF and endonuclease G, and up to massive permeability events leading to the release of mitochondrial DNA in inflammatory responses ([64–66] for reviews). However, the conformation of membrane-inserted Bax in “small apoptotic pores” and “large inflammation pores” might not be identical. Although they exhibit a certain degree of flexibility [67], the nature of nanodiscs imposes more physical constraints, both in terms of size and lipid packing, than, for example, liposomes [68]. It has also been reported that Bax is more efficiently inserted in lipid bilayers having a large radius of curvature (large liposomes and large mitochondria) than in lipid bilayers have a smaller radius of curvature (small liposomes and fragmented mitochondria) [69]. However, because nanodiscs are planar lipid bilayers, the lower efficiency of Bax insertion into nanodiscs cannot be explained by the curvature. Bim or cBid are sufficient to insert Bax into liposomes [35, 70], but not into nanodiscs (Fig. 3A, B). It is tempting to speculate that this might reflect different types of pores formed from Bax monomers/dimers having distinct conformations and, owing to the physical constraints they impose, nanodiscs may provide a mean to stabilize small pores vs megapores. It is noteworthy that, following the WEHI-539-induced release of primed Bax from its interaction with Bcl-xL, Tom22 was required for Bax membrane insertion both in nanodiscs and isolated mitochondria, suggesting that the molecular mechanisms involved were similar.

On the respective roles of BH3 direct activators and Tom22 on Bax membrane insertion and pore formation

The induction of apoptosis is a prerequisite for Bax/Tom22 interaction in cellulo [15]. It can therefore be hypothesized that BH3 activators and Tom22 might cooperate during the process of Bax activation. The concerted action of Bim and Tom22 would lead to the extrusion then membrane insertion of Bax (Fig. 7). Once inserted, Bax could actually play the role of a receptor to itself [71]. The interaction of Bax with cBid or Bim at the canonical binding site, that induces the exposure of the BH3-domain and the conformational change of Hα5/Hα6 [72, 73], might therefore prime soluble Bax for its interaction with membrane-inserted Bax, leading to the stepwise formation of oligomers [74] (Fig. 7).

Fig. 7. Representation of Bax interaction with nanodiscs and Tom22.

Hypothetic model of the concerted involvement of BH3-only proteins and Tom22 in Bax insertion in the MOM. The interaction of Bim with Bax at the non-canonical binding site promoted the exposure of Hα1, allowing the interaction of Bax with Tom22. Bax/Tom22 interaction led to the extrusion and insertion of Hα9. The interaction of cBid or Bim with Bax induced the exposure of the BH3-domain and the remodeling of the Hα5/Hα6 [72–74], allowing the recruitment of cBid/Bim-activated Bax by membrane-inserted Bax, leading to the formation of membrane-inserted oligomers.

In our minimal model, BH3 activators were not required, and the interaction of Bax with the lipid bilayer seemed sufficient to expose Hα1. In cancer cells, Bax activation by BH3 proteins is limited by the overexpression of anti-apoptotic proteins, such as Bcl-xL, leading us to investigate if Tom22 was still involved in Bax activation in the presence of Bcl-xL. Following the rupture of Bax/Bcl-xL interaction by WEHI-539, Bax could be translocated from Bcl-xL-containing nanodiscs and inserted into new nanodiscs only when Tom22 was present (Fig. 3C, D). A similar conclusion was drawn from experiments of Bax transfer from the same nanodiscs containing Bax and Bcl-xL to mitochondria treated or not with an anti-Tom22 antibody (Fig. 3E–G). This demonstrated that Tom22 was required for Bax membrane insertion after it was primed by the engagement then release of its interaction with Bcl-xL [44]. This emphasized the crucial role of Tom22 in Bax membrane insertion under conditions reflecting the context of cancer cells. This might be fully relevant in the context of the use of BH3-mimetics as anti-tumor agents. It should be noted, however, that the interaction with Tom22 is probably not the only pathway leading to Bax membrane insertion. We have reported previously that the co-synthesis of Bcl-xL with Bax was able to stimulate the insertion of Bax into nanodiscs [36], an effect that could be related to the stimulation of Bax mitochondrial localization by both Bcl-2 [75] and Bcl-xL [76, 77]. Also, the protein VDAC2 has been shown to stimulate Bax mitochondrial localization and was required for the subsequent Bax-induced permeabilisation [78]. This suggests that there is more than one pathway to target Bax to the MOM, both under active and inactive conformations.

Conclusion

Since the initial report showing that Tom22 was involved in the MOM targeting of Bax [15], no mechanistic explanation had been provided and the actual role of Tom22 had been questioned [19, 20]. The present study connects the interaction of Bax with Tom22 to the movement of Hα9, as the primary event leading to Bax membrane insertion. It also explains why Bax could not be spontaneously inserted in nanodiscs [32]. This study paves the path for future structural investigations on Bax in nanodiscs, in which Bax forms a relatively small pore, consistent with the limited release of small proteins such as cytochrome c. We also propose a model combining the action of Tom22 and BH3-activators Bim and cBid on Bax insertion, and extend our observations to the role of Tom22 in the membrane insertion of Bax after it is released from its inhibition by its anti-apoptotic partner Bcl-xL.

Materials and methods

Plasmids construction and cell-free protein synthesis

Full-length, untagged Bax, Tom22, Tom20 and Bcl-xL were cloned in the Nde1/Xho1 sites of the pIVEX 2.3a plasmid. Kras4b was cloned in the pIVEX 2.4d plasmid in frame with the N-terminal His6 tag. A double TAA stop codon was included to secure translation arrest during in vitro synthesis. Site-directed mutagenesis was done by an optimized Quickchange protocol [79] and mutations were verified by sequencing (Eurofins).

The plasmid pET28b encoding His7-MSP1E3D1 (Addgene) was transformed into Escherichia coli BL21DE3 and the protein was purified as described previously [32, 36]. A strepII-tagged version of the protein has been constructed by site-directed mutagenesis. The pAR1219 encoding T7-RNA polymerase was transformed into E. coli BL21DE3*, and the protein was purified according to [80]. Nanodiscs were formed with a mixture of phospholipids (48% POPC, 32% POPE, 12% DOPS, 8% cardiolipid (w/w)) (Anatrace), and purified by SEC on a Superdex 200 (10/300) column connected to an Äkta purifier chromatography system (see [32] for details). The size and homogeneity of nanodiscs preparations was checked by DLS. Nanodiscs were stored at 4 °C and typically used within 2 months. Other lipid compositions have been tested: POPC alone, POPC/POPG 3/1 (w/w), E. coli phospholipid extract (Sigma), which did not change the precipitation of Bax nor the rescue by Tom22 and Bcl-xL (not shown) [32]. The continuous exchange cell-free synthesis protocol was detailed previously [32, 36, 80]. Syntheses were done in inverted microtubes containing 100 µL of Reaction Mix in the round compartment of the cap and 1700 µL of Feeding Mix, separated by a dialysis membrane (cut-off 6–8 kDa), overnight at 28 °C under gentle agitation (80 rpm).

After the syntheses, the volume of the reaction mixes was carefully weighted and, after a 20,000 × g centrifugation, the pellet was re-suspended in the same volume, so that the same proportions of pellets and supernatants were loaded on SDS-PAGE. After the separation of nanodiscs and soluble proteins on Ni-NTA (Qiagen), the same proportions of unbound material and imidazole-eluted material were analyzed (Fig. S6). It follows that, for each gel, the amount of proteins in pellets (p) vs supernatants (sn), and in unbound fraction (u) vs bound fraction (b) are directly comparable.

SDS-PAGE and western blots

Proteins were solubilized in Laemmli buffer 2× and heated at 65 °C for 20 min. Samples were loaded on SDS-PAGE (Mini Protean, Biorad). Tris-glycine gels were made of 12.5% of acrylamide/bisacrylamide (37.5/1). Gels were run at 15 mA per gel. Protein transfer on PVDF membranes (Hybond, Cytiva) was done with the Transblot system (Biorad) for 1h30 at 350 mA. western blotting was done in PBST or TBST, depending on the antibody (Table S1). Western blots were revealed by chemiluminescence (Luminata Forte, Millipore) and recorded with a digital camera (G-Box, Syngene).

NEM-PEG labeling

NEM-PEG labeling was done according to [10]. The maleimide function reacts covalently with cysteine residues only when they are exposed to solvent, thus increasing the molecular weight of the target protein by ~5 kDa. Cysteine residues embedded in membranes remain intact. Protein samples were incubated with 0.2 mM methoxypolyethyleneglycol maleimide (NEM-PEG, 5 kDa; Sigma-Aldrich) for 1 min on ice. The reaction was stopped by adding 20 mM NEM for 10 min. The increase of the apparent molecular weight is actually closer to ~10 kDa, due to the high hydrophilicity and linear structure of PEG.

Dextran-FITC release from liposomes

Liposomes permeabilization was measured through the quenching of external Dextran-FITC by an anti-FITC antibody. A cell-free synthesis reaction was set up in 100 µL, in the presence of 10 µL liposomes loaded with Dextran-FITC (10 kDa, Sigma) (liposomes preparation is detailed in [32]). 4 µg of anti-FITC antibody (Rabbit Monoclonal 6HC5LC9; ThermoFisher Scientific) was added to quench external FITC fluorescence. The mix was then added to black 96-well plates and incubated at 28 °C in a fluorescence plate reader (Clariostar, BMG Labtech). Fluorescence was measured at 10-min intervals (ex: 475–490 nm, em: 515–545 nm) and the initial rate was measured by applying the formula of a two-phase decay [81].

Bax translocation between nanodiscs

Bax and Bcl-xL were co-synthesized in the presence of nanodiscs formed with His7-tagged MSP and purified on Ni-NTA and dialyzed in ND buffer (200 mM NaCl, 10 mM Hepes, pH 7.4) to eliminate imidazole [36]. In parallel experiments, Tom22 was synthesized in the presence of nanodiscs formed with StrepII-tagged MSP, purified on Strep-Tactin (IBA) and dialyzed in ND buffer to eliminate biotin. The eluate from Ni-NTA containing Bax/Bcl-xL in His7-nanodiscs was mixed with an equal amount of StrepII-nanodiscs, empty or containing Tom22 (1.13 µM of each type of nanodiscs, expressed as MSP concentration). 250 nM WEHI-539 (MedChem) was added to the mixes, that were incubated overnight at 4 °C. Mixes were then loaded on Strep-Tactin and the unbound and bound fractions were collected and analyzed by western blot.

Bax-induced permeabilization of mitochondria

Bax and Bcl-xL were co-synthesized in the presence of nanodiscs formed with His7-tagged MSP and purified on Ni-NTA as above [36].

HEK293FT cells and HCT116 Bax-KO cells (Dr. Vogelstein, Baltimore, USA) were grown in DMEM or McCoy’s 5 A media, respectively (Thermofisher) supplemented with glutamine, fetal calf serum, streptomycin and penicillin, at 37 °C and 5% CO₂. Cells were then scraped in 20 mL PBS and harvested (800 × g, 5 min). The cells pellet was re-suspended in 10 mL MB buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM Hepes, pH 7.4) supplemented with an antiproteases cocktail (Complete, Roche). Cells were homogenized in a Dounce homogenizer (30 passages for HEK-293 cells, 60 passages for HCT116 Bax-KO cells). Cell debris were removed by centrifugation (600 × g, 5 min). The supernatant was centrifuged again (800 × g, 5 min), and mitochondria were collected by centrifugation (8000 × g, 10 min). The pellet was re-suspended in 2 mL MB buffer and centrifuged again (8000 × g, 10 min). The mitochondrial pellet was re-suspended in 0.2 mL MB buffer and used immediately.

Two hundred micrograms of mitochondria were incubated for 1 h in the presence or in the absence of 1 µg anti-Tom22 antibody (Abcam). Nanodiscs containing Bax/Bcl-xL (1 µM MSP) and 1 µM WEHI-539 were sequentially added, and mitochondria were incubated overnight at 4 °C. They were collected by centrifugation (8000 × g, 10 min) and analyzed by SDS-PAGE and western blot.

Dynamic light scattering

Dynamic light scattering (DLS) was measured in a DynaPro Nanostar (Wyatt) on 10 µL samples. Triplicates of 10-acquisition measurements were done and averaged for each point.

Transmission electron microscopy

Transmission Electron Microscopy (TEM) was done as described previously [32]. Nanodiscs were concentrated on Vivaspin concentrators (50 kDa, Sartorius). Negative staining was done according to [82]. Samples were observed on a Tecnai F20 FEG electron microscope (FEI, ThermoFisher Scientific) operated at 200 kV using an Eagle 4k_4k camera (ThermoFisher Scientific). Measurements were manually done with Image J (https://imagej.net/ij/) on dm3 files.

Miscellaneous

Unless indicated otherwise, chemicals were from Clinisciences and Sigma-Aldrich.

Uncropped western blots are shown in original data files. Where indicated, quantification has been done on non-saturated blots and statistical analyzes were done with GraphPad 6.05 tools.

Author contributions

ARE and SM conceptualized the study and wrote the manuscript. ARE designed and performed biochemistry experiments with the help of MP and SM. LD performed TEM experiments. ARE and SM analyzed data and did the figures. ARE, LD, MP and SM discussed data, read and edited the manuscript.

Data availability

Original data are available upon request.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41420-024-02108-x.