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. Author manuscript; available in PMC: 2010 Mar 24.
Published in final edited form as: Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2009 Feb;26(1):130–137.

The Cytosolic Domain of Bcl-2 Forms Small Pores on Model Mitochondrial Outer Membrane After Acidic pH-Induced Membrane Association

Jun Peng 1,2, Suzanne M Lapolla 1, Zhi Zhang 1, Jialing Lin 1
PMCID: PMC2844873  NIHMSID: NIHMS182197  PMID: 19334571

Abstract

The permeability of mitochondrial outer membrane (MOM) is regulated by proteins of the Bcl-2 family via their interactions at the membrane. While pro-apoptotic Bax protein promotes MOM permeabilization (MOMP) releasing cytochrome c after activation by BH3-only protein, anti-apoptotic Bcl-2 protein protects MOM. However both Bax and Bcl-2 can form pores in model membranes. Unlike Bax pore that has been extensively studied and directly linked to MOMP, much less is known about Bcl-2 pore. We thus investigated pore-forming property of recombinant Bcl-2 lacking the C-terminal transmembrane sequence (Bcl-2ΔTM) in liposomal membranes of MOM lipids. We found that: (1) Bcl-2 formed pores at acidic pH that induced association of Bcl-2 with liposome; (2) Bcl-2 pore size was dependent on Bcl-2 concentration, suggesting that oligomerization is involved in Bcl-2 pore formation; (3) Unlike Bax pore that can release large molecules up to 2 mega-Da, Bcl-2 pore was smaller releasing molecules of a few kilo-Da. Therefore, Bcl-2 and Bax may form different size pores in MOM, and while the large pore formed by Bax may release cytochrome c during apoptosis, the small pore formed by Bcl-2 may maintain the normal MOM permeability.

Keywords: Mitochondrial outer membrane (MOM), Permeability, Bcl-2, Bax

INTRODUCTION

Cell death by apoptosis eliminates excess, redundant, abnormal cells in animals and hence is crucial for animal development and tissue homeostasis. Bcl-2 family proteins are key regulators of apoptosis, functioning as either suppressors or promoters. Aberrant expression of these proteins results in the dysregulation of apoptosis that leads to some of the apoptotic diseases, including cancer, autoimmunity and neurodegenerative disorders (1, 2). In mammals, over 25 Bcl-2 family members have been identified, all of which contain at least one of the four conserved sequences called Bcl-2 homology (BH) motifs 1, 2, 3 and 4 (3). According to the function and sequence homology, Bcl-2 family is divided to three subfamilies. The anti-apoptotic Bcl-2 subfamily proteins such as Bcl-2 and Bcl-XL contain all four BH motifs. The pro-apoptotic Bax subfamily members such as Bax and Bak display homology in BH1-3 motifs, whereas the other pro-apoptotic BH3-only subfamily proteins such as Bid and Bad share the sequence homology only in the BH3 motif (4-7).

One possible mechanism by which Bcl-2 family proteins regulate apoptosis is that these proteins homo- or hetero-associate with each other to influence the permeability of mitochondrial outer membrane (MOM) (8). It has been demonstrated that after activated by some BH3-only proteins, either Bax or Bak is sufficient to induce mitochondrial outer membrane permeabilization (MOMP) (9-12), releasing pro-apoptotic proteins such as cytochrome c and Diablo/Smac that trigger apoptosis by activating caspases and nucleases (12-16). Bcl-2-like proteins may bind to active Bax and/or Bak proteins to prevent them from damaging the MOM, if these Bcl-2-like proteins are not bound and inhibited by some other BH3-only proteins (9, 16-18).

Although the molecular mechanism of how Bcl-2 family proteins regulate membrane permeability remains controversial, it is wildly accepted that certain Bcl-2 family proteins may form pores to induce the MOMP, based on the structural studies. The three-dimensional structures of several Bcl-2 family members, including both anti-(Bcl-XL, Bcl-2, Bcl-w) and pro-apoptotic (Bax, Bid) members, are strikingly similar to each other and to the pore-forming domains of diphtheria toxin and bacterial colicins, consisting of two hydrophobic core helices surrounded by five to eight amphipathic helices and their connecting loops (19-25). Consistent with the structural similarity, recombinant Bcl-2 and Bax have been shown to form pores in synthetic lipid bilayers (26-28).

Since Bax can release several pro-apoptotic proteins from mitochondria to promote apoptosis, many studies have focused on Bax pore formation. It has been shown that after activated by some BH3 only proteins, Bax can induce the release of cytochrome c from purified mitochondria, and the release of dextrans up to 2,000 kDa from liposomes (29, 30). The oligomerization is required for Bax to permeabilize the mitochondria, and to form pores in liposomes (30-32). All these mentioned activities of Bax, including Bax oligomerization, Bax pore formation in liposomes and Bax-induced cytochrome c release from mitochondria, can be inhibited by anti-apoptotic Bcl-2 (9, 17, 18, 33). Interestingly Bcl-2 protein itself has also been shown to form pores in liposomal membranes although it is supposed to promote cell survival by maintaining the integrity of membranes (9, 26, 27). However, unlike Bax pores that have been extensively studied, less is known about Bcl-2 pores. Due to the lack of knowledge about Bcl-2 pore formation, it is not clear why both pro- and anti-apoptotic proteins possess pore forming ability but function oppositely, which has become one of the major obstacles for fully understanding the mechanisms of how Bcl-2 family proteins regulate the permeability of MOM and apoptosis.

In this study, we used a unique combination of biochemical, biophysical and molecular methods to investigate the properties of pores formed by the recombinant Bcl-2 proteins lacking C-terminal transmembrane domain (Bcl-2ΔTM). We found: (1) Bcl-2ΔTM forms pores only at acidic pH that induced association of Bcl-2ΔTM with liposome; (2) different from Bax that forms large pores to release pro-apoptotic proteins from mitochondria or to release up to 2000 kDa molecules from liposomes, Bcl-2ΔTM forms much smaller pores that only permeate molecules less than several kDa, close to the size of the molecules that are known to be freely crossing the MOM, which might help account for their different functions during apoptosis; (3) the size of Bcl-2ΔTM pore depends on Bcl-2ΔTM concentration, suggesting that oligomerization may be involved in Bcl-2ΔTM pore formation.

MATERIALS AND METHODS

Materials

All phospholipids were from Avanti Polar Lipids (Alabaster, AL). Fluorescent dye Cascade Blue (CB, MW ~ 0.5 kDa), CB-labeled dextran of 3 kDa or 10 kDa, and rabbit anti-CB polyclonal antibody were obtained from Molecular Probes. Bismaleimidohexane (BMH) was purchased from Pierce.

Preparation of plasmids and proteins

The construction of the plasmid pET22b-hBcl-2Δ22 encoding human Bcl-2 protein with LE (H)6 replacing the C-terminal 22 residues was generated as described (34). The sequence of all plasmids was verified by DNA sequencing. Expression and purification of His6-tagged Bcl-2ΔTM was done basically as described before (34). The purity of the resulted Bcl-2ΔTM protein was examined by SDS-PAGE and Coomassie Brilliant Blue to be > 95%; and the identity of the purified protein was determined by Western blotting using a polyclonal antibody specific for human Bcl-2 (data not shown). The purified proteins were stored in 50mM Tris-HCl (pH 8.5), 140mM NaCl, 2mM EDTA, 5mM DTT and 10% (v/v) glycerol at −80°C.

Preparation of liposomes

Liposomes were made by an extrusion method as described before (35). Briefly, lipid mixtures were made with the lipid composition similar to that of Xenopus MOM: phosphatidylcholine (PC)/phosphatidylethanolamine (PE)/phosphatidylinosital (PI)/phosphatidylserine (PS)/cardiolipin (CL) = 48/28/10/10/4 (mole %). The dried lipid mixture was re-suspended in 0.55 ml of solution containing 3 μM CB dye (0.5 kDa) or CB dye-labeled dextran of 3 or 10 kDa. Free CB dyes were separated from liposomes with entrapped CB dye by gel filtration at room temperature using a 0.7cm × 50cm column with Sephadex G-25 resin for 0.5 kDa CB and CL-2B resin for CB-labeled 3 kDa or 10 kDa dextrans. The concentration of entrapped CB or CB-dextran was determined using a CB fluorescence vs. concentration. From CB fluorescence measurements trapping efficiencies were found to be between 5–10%. In addition, 14C-PC was added into the lipid mixture to determine the lipid concentration in final liposome fractions from gel filtration chromatography.

Monitoring Bcl-2ΔTM pore formation in liposomal membranes by steady-state fluorescence spectroscopy

Steady-state fluorescence emission intensity was measured using the SLM-8100 spectrofluorometer equipped with a 450 W Xenon lamp. The excitation and emission wavelengths were 405 and 433nm for 0.5 kDa CB dye, 385 and 417nm for 3 kDa and 10 kDa CB-dextrans. The bandpass for both excitation and emission light was 4 nm. Both end-point and kinetics measurement were done in a 4×4 mm quartz microcuvette containing the sample that was mixed with a mini-magnetic bar thoroughly. The sample contained liposomes (final total lipids concentration = 100 μM) with entrapped CB dyes and 6 μg/ml anti-CB antibodies in 250 μl of citric-phosphate buffer (CPB). The initial emission intensity (F0) was determined after equilibration of the sample at 25 °C for 5 min. Purified His6-Bcl-2ΔTM protein was then added to concentrations as indicated in Figure 3-5. The sample was incubated at 25 °C for 5 hours and the emission intensity (F) was measured in end-point measurements. Triton X-100 was then added to 0.1% for complete disruption of liposomes and release of all CB dyes. The emission intensity (Ft) was measures again. The quenching efficiency was calculated by ΔFBcl-2 ΔTM /ΔFTriton, where ΔFBcl-2 ΔTM = F0 – F and ΔFTriton = F0 – Ft. All experiments were performed in the CPB buffer with 100 mM NaCl and done with mitochondria-mimic liposomes.

Fig. 3. Bcl-2ΔTM concentration-dependent release of fluorescent molecules of various sizes from liposomes.

Fig. 3

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The release of 0.5 kDa CB dyes (■); 3 kDa CB-labeled dextrans (●); or 10 kDa CB-labeled dextrans (▲) from 100 μM liposome by various concentrations of Bcl-2ΔTM at pH 5.0, was monitored as described in Fig. 2. Data shown are means from three independent experiments with S.D. (error bars).

Monitoring Bcl-2-liposome association by flotation

1 mM liposomes and 4μM His6-Bcl-2ΔTM protein were incubated in 138 μl of CPB containing 10 mM EDTA for 1 hour at 25°C at pH 5.0 or pH 7.4. The resultant sample was then mixed with 162 μl of 2.5 M sucrose (buffered by CPB buffer with the same pH value) to bring the final sucrose concentration to 1.35 M. The resulted sample was added to a centrifuge tube and overlaid with 500 μl of 0.8 M sucrose and then 200 μl of 0.25 M sucrose. The sample was centrifuged in Beckman Optima Ultracentrifuge with TLA 100.2 rotor at 436,000 × g for 6 hrs at 4 °C. Four 250 μl fractions were drawn, starting from the top of the gradient, resulting in fractions S1-S4. The pellet was re-suspended in 250 μl of CPB buffer, resulting in fraction P. The distribution of the liposomes among these fractions was estimated by measuring the radioactivity of the 14C that resulted from the trace of [14C ]-PC incorporated into the liposomes. Proteins from each fraction were precipitated by 25% TCA and analyzed by SDS-PAGE and Coomassie Brilliant Blue.

RESULTS

Determining the pore formation of Bcl-2ΔTM in liposomal membranes

To determine Bcl-2 pore formation in membranes, we used a recombinant His6-tagged Bcl-2ΔTM protein since it is structurally and functionally (in terms of pore-formation) related to the pore-forming domain of diphtheria toxin and colicins. The transmembrane (TM) in full length Bcl-2 functions as a membrane targeting and anchoring domain, probably not involved in pore-formation directly. We used liposomes with lipid composition similar to that of mitochondrial outer membrane (MOM) since the bilayer of the liposomes would mimic that of MOM. The simple liposomal system allows us to test different requirements for Bcl-2 pore-forming reaction more easily.

Bcl-2 pore formation in liposomal membranes was detected by a unique spectroscopic approach. We encapsulated fluorophores into liposomes surrounded by antibodies against the fluorophores, followed by the addition of purified Bcl-2ΔTM proteins. The Bcl-2ΔTM-pore-mediated dye release from the liposomes was monitored by quenching of fluorescence emission of the fluorophores. Since both fluorophores and antibodies do not diffuse across the liposomal membrane spontaneously, the quenching only occurs when the fluorophores are small enough to leak through the pore formed by the Bcl-2ΔTM protein. In addition, by using fluorophores of different sizes within the liposomes, the size of Bcl-2 pore can be determined.

Quenching of CB fluorescence by anti-CB antibodies is effective and independent of pH

To determine the conditions under which quenching can be used to measure the dye release from liposomes, we first titrated CB-labeled 3 kDa dextrans into a solution containing anti-CB antibody, samples without antibody were set as negative controls. Approximately 1 min after the addition of CB-dextran, fluorescence intensity was measured. Fig. 1 shows that 3 μg/ml anti-CB antibodies quenched more than 70% of the CB fluorescence emitted by up to 0.04 μM CB-dextrans. Therefore, in following experiments we added 6 μg/ml anti-CB antibodies to samples containing certain concentration of liposome that can only release 0.03 μM or less CB dyes upon complete lysis. Thus, the quenching will quantitatively monitor any intermediate dye releases such as those caused by Bcl-2. In addition the extents of quenching at pH 5.0 and 7.4 were very similar (Fig.1), suggesting that the quenching can be used to monitor the dye release at both low pH and neutral pH. Similar quenching results were obtained for the 10 kDa CB-labeled dextran or 0.5 kDa CB (data not shown), suggesting that the quenching itself is independent of dye size and thus can be used to monitor the release of different sized dyes at equilibrium that is dependent on pore size.

Fig. 1. pH-independent quenching of CB fluorescence by anti-CB antibodies.

Fig. 1

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The percent quenching of various concentrations of CB-labeled 3-kDa dextran by 3 mg/ml of anti-CB antibody is shown: (■) at pH 7.4; or (○) at pH 5.0. Percent quenching equals to [F(−Ab) –F(+Ab)]/F(−Ab), where F(−Ab) or F(+Ab) is the fluorescence emission intensity of CB in the absence or presence of anti-CB antibody, respectively.

Bcl-2ΔTM forms pores at pH 5.0 or below

The pore-forming domain of diphtheria toxin and colicins forms pores at acidic pHs. The cytosolic domain of Bcl-2 or Bcl-2ΔTM may form pores at similar pH since it is structurally related to these pore-forming domains. We therefore assayed the pore-forming activity of Bcl-2ΔTM at various pHs. We incubated Bcl-2ΔTM and the liposomes loaded with 3 kDa CB-dextran at 25 °C for 5 hours, with a protein to lipid molar ratio of 4:1000. The extent of Bcl-2ΔTM-dependent dye release or Bcl-2ΔTM pore formation was determined by the value of ΔFBcl-2 ΔTM /ΔFTriton, in which ΔFBcl-2 ΔTM is the normalized reduction of fluorescence emission intensity after incubation of the Bcl-2ΔTM proteins with liposomes, and ΔFTriton is the same parameter determined after the liposomes are completely lysed by 0.1% Triton X-100 to release all CB dyes. As shown in Fig. 2, the CB-dextrans were released from the liposomes and quenched by the anti-CB antibodies only at pH 5.0 or below. No such release was observed when pH was 5.5 or above. These data demonstrate that pore formation by Bcl-2ΔTM in the liposome occurs only at acidic pH. Similar results were obtained with the liposomes containing the 0.5 kDa CB dyes (data not shown), suggesting that pH 5.0 or below is a condition required for the formation of all types of pores formed by Bcl-2ΔTM.

Fig. 2. pH-dependent release of fluorescent molecules from liposomes.

Fig. 2

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Release of 3 kDa CB-dextran from 100 μM MOM-mimic liposome by Bcl-2ΔTM at various pHs, was shown as the extent of the fluorescence quenching by the anti-CB antibody located outside of the liposome. The extent of Bcl-2ΔTM-induced dye release was determined by the value of ΔFBcl-2 ΔTM /ΔFTriton after a 5-hr incubation, as described in Materials and Methods. Data shown are means from three independent experiments using 0.4 mM Bcl-2ΔTM protein (grey bar); or an equal volume of the protein buffer (white bar), with standard deviations (S.D., error bars).

Bcl-2ΔTM pore size depends on Bcl-2ΔTM concentration

To determine the effect of Bcl-2ΔTM concentration on pore size, we performed the above fluorescence quenching assays at various Bcl-2ΔTM to lipids ratios using liposomes loaded with different sized CB-labeled molecules. As shown in Fig. 3, Bcl-2ΔTM-mediated CB dye releases from all liposomes are functions of the molar ratio of Bcl-2ΔTM to lipids, though these functions are release of CB-10 kD dextran molecules was observed even at the highest ratio tested. The half maximal release (R50) for 0.5 kD CB or 3 kD CB-labeled dextran occurred at ~1.5 or 3.0 Bcl-2ΔTM per 1,000 lipids, respectively. Therefore, the larger the entrapped molecule is, the higher the concentration of Bcl-2ΔTM is needed for its release, suggesting that the size of Bcl-2 pore is dependent on the Bcl-2ΔTM concentration. Furthermore, there is an upper limit for Bcl-2 pore size, at least with physiologically possible Bcl-2 concentrations.

The observation that the size of Bcl-2 pore is dependent on Bcl-2ΔTM concentration implies that Bcl-2ΔTM association may be involved in pore formation, and that the larger the Bcl-2ΔTM oligomer, the larger the pore size. At a low molar ratio of protein to lipids, only a few Bcl-2ΔTM monomers distribute to each liposome on average. The chance to form smaller oligomeric pores is higher than that to form larger oligomeric pores. Therefore, more small dyes are released (and quenched) than large dyes at the low Bcl-2ΔTM to lipids ratio. At a high Bcl-2ΔTM to lipids ratio, both small dyes and large dyes are released to a similar extent, because under this condition, almost all liposomes will contain enough proteins to form an oligomeric pore that is large enough to release both small and large dyes. In contrast, the above data do not support the model that Bcl-2 pores are formed only by monomeric proteins, since this model predicts that the release of both small and large dyes should be similar no matter at lower or higher protein to lipids ratio because all pores would have a uniform size.

Acidic pH facilitates Bcl-2ΔTM protein association with liposome

Why Bcl-2ΔTM could not form pores in liposomes at a neutral pH? One possibility is that acidic pH may help Bcl-2ΔTM protein associate with the liposomal membrane. Bcl-2 have a slightly net negative charge at neutral pH; and so far Bcl-2 proteins used in all in vitro studies lack the hydrophobic C-terminal anchor, which facilitates the protein purification, but also relieves the protein of its normal means of membrane targeting. Therefore in vitro, it may be necessary to have acidic pH to protonate the negatively-charged residues of Bcl-2ΔTM, making it more likely to associate with liposomal membrane that is negatively charged on the surface.

To test this possibility, we performed flotation to examine the homo-association of Bcl-2ΔTM and the binding of the protein to liposomal membranes. After incubation of Bcl-2ΔTM and liposomes at pH 5.0 or 7.4 for 1 hour, the sample was fractionated by a 3-step sucrose gradient float-up centrifugation. The liposomes together with associated Bcl-2ΔTM proteins would float to the top fraction of gradient, whereas the Bcl-2ΔTM proteins still in solution would remain in the bottom fraction; and the Bcl-2ΔTM protein aggregates would be pelleted. As shown in Fig. 4, 60-62% of total loaded lipids are recovered from the top fraction, demonstrating that this fraction contained most of the total liposomes. The distribution of Bcl-2ΔTM in the gradient was determined by SDS-PAGE analysis of the fractions. At pH 5.0, most of Bcl-2ΔTM proteins were detected in the top fraction with the liposomes (Fig. 4A). In contrast, at pH 7.4, most of Bcl-2ΔTM stayed in the bottom fraction and the pellet fraction and none associated with liposomes in the top fraction (Fig. 4B), suggesting acidic pH facilitates Bcl-2ΔTM protein association with liposome.

Fig.4. Effect of acidic pH on Bcl-2ΔTM protein association with liposomes.

Fig.4

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After incubation of Bcl-2ΔTM and liposomes at pH 5.0 (A) or pH 7.4 (B), the sample was fractionated by a step sucrose gradient float-up centrifugation. The liposome-bound protein migrated into the upper supernatant fractions (S1-2), whereas the soluble protein remained in the bottom fractions (S3-4). The aggregated protein was in the pellet fraction (P). The distribution of liposomes in the gradient was monitored by counting the 14C radioactivity and the results are indicated below each lane as the percentage of total liposomes. Data shown are representative Coomassie stained SDS-PAGE gels of three independent experiments.

DISCUSSION

Bcl-2 family proteins have been known to regulate the permeability of the mitochondrial outer membrane. Although the mechanisms by which these proteins affect membrane permeability remain unknown, two controversial models have been proposed: the Bcl-2/VDAC/MPT model and Bcl-2 pore model. The three-dimensional structures of some Bcl-2 family proteins are strikingly similar to the pore-forming domains of bacterial toxins. Consistent with the structural similarity, anti-apoptotic Bcl-2 and pro-apoptotic Bax have been reported to form channels in synthetic lipid membranes.

Bax can trigger the release of Cytochrome c (17kDa), SMAC/DIABLO (27kDa) and AIF (67kDa) from purified mitochondria, suggesting that Bax pore size is larger than the size of these proteins. Satio et al. found that in reconstituted liposomes Bax could form pores of 22 Å in diameter, which is larger than that of globular Cytochrome c (17 Å). Kuwana et al. showed that tBid and Bax together could form pores that can release dextran molecules of 2,000 kDa. Bcl-2 has been shown to inhibit the membrane damage caused by Bax pore formation, but why Bcl-2 itself can form pores in these membranes remains unclear. Our studies demonstrated that Bcl-2ΔTM pores can release 500 to 3,000 Da molecules but not 10,000 Da ones, and it has been known that MOM contains many kinds of porins, which forms aqueous channels that are permeable to all molecules of 5,000 Da or less, including small proteins. Therefore, unlike Bax pore, Bcl-2ΔTM pore has a much smaller size and only permeates molecules less than several kDa, close to the size of the molecules that are known to be freely crossing MOM, suggesting that Bcl-2 might function as inhibitors of apoptosis by forming pores to maintain the normal membrane permeability.

Acidic pH has been shown to be required for Bcl-2ΔTM and its closest homologue, Bcl-XLΔTM, to form pores in liposomal membranes. Our data suggest that acidic pH promotes the association of Bcl-2ΔTM with membrane. Possibly the acidic pH changes Bcl-2ΔTM’s conformation, exposing the hydrophobic core helices (helix 5 & 6), and therefore facilitating the insertion of the helices into the membranes. Acidic pH also induces some conformational changes in Bcl-XLΔTM such as increasing solvent-exposure of hydrophobic sites while retain the overall secondary structure. In addition to changing the conformation, acidic pH may also promote protonation of some ionizable residues such as glutamate, aspartate and histine, as shown previously for the pore-forming diphtheria toxin and some membrane-inserting peptides (36-39). Notably there are four glutamates, one aspartate and one histine in the helices 5-6 region of Bcl-2. Protonation of these residues will reduce the free energy cost for insertion of the two putative pore-forming helices into the lipid bilayer.

ACKNOWLEDGMENTS

We thank Drs. Adam Zlotnick, Ann Louise Olson, Chibing Tan and Olga Nikolaeva for valuable suggestions, and NIH grant GM062964 to Jialing Lin for partial support.

The abbreviations used are

ANT

adenine nucleotide translocator

CB

Cascade Blue

Bcl-2ΔTM

Bcl-2 lacking the C-terminal transmembrane sequence

BH

Bcl-2 homology

DTT

dithiothreitol

EDTA

ethylene-di-amine tetra-acetic acid

MOM

mitochondrial outer membrane

MW

relative molecular weight

S.D.

standard deviation

SDS-PAGE

sodium lauryl sulfate-polyacrylamide gel electrophoresis

VDAC

voltage dependent anion channel

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