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© The Rockefeller University Press,
0021-9525/2001//237 $5.00
The Journal of Cell Biology, Volume 152, Number 2,
, 2001 237-250
Original Article |
Essential Role of Voltage-Dependent Anion Channel in Various Forms of Apoptosis in Mammalian Cells
tsujimot{at}gene.med.osaka-u.ac.jp
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (
), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.
Key Words: VDAC • apoptosis • Bcl-2 • Bax • cytochrome c
© 2001 The Rockefeller University Press
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Introduction |
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Mitochondria play a crucial role in apoptosis by releasing several apoptogenic molecules, including cytochrome c and Smac/Diablo from the intermembrane space into the cytoplasm in response to a variety of death-promoting stimuli (for reviews see Adams and Cory 1998; Green and Reed 1998; Du et al. 2000; Tsujimoto and Shimizu 2000a,Tsujimoto and Shimizu 2000b; Verhagen et al. 2000). Once in the cytoplasm, cytochrome c binds to Apaf-1, triggering oligomerization of the Apaf-1/cytochrome c complex that leads to recruitment and activation of a major apical caspase, caspase-9. In turn, caspase-9 activates various effector caspases such as caspase-3 (for review see Thornberry and Lazebnik 1998). It has been shown that Bcl-2 family proteins regulate mitochondrial membrane permeability to control cytochrome c release: proapoptotic Bax, Bak, and BH3-only proteins like Bid and Bik induce cytochrome c release, whereas antiapoptotic Bcl-2 and Bcl-xL prevent it (Eskes et al. 1998; Jürgensmeier et al. 1998; Marzo et al. 1998; Narita et al. 1998; Finucane et al. 1999; Pastorino et al.. 1999; Shimizu and Tsujimoto 2000).
Recently, we have shown that Bax/Bak and Bcl-xL, but not Bik and Bid, can bind directly to the voltage-dependent anion channel (VDAC) and modulate its activity (Shimizu et al. 1999, Shimizu et al. 2000a,Shimizu et al. 2000b; Shimizu and Tsujimoto 2000). The VDAC is a mitochondrial outer membrane channel, which usually functions as the pathway for the movement of various substances in and out of the mitochondria (for review see Colombini 1989), and is considered to be a component of the oligoprotein permeability transition (PT) pore complex that plays a role in the PT (for reviews see Bernardi et al. 1994; Zoratti and Szabó 1995). Our biochemical and electrophysical studies have shown that Bax and Bak enhance VDAC activity so that cytochrome c passes through the channel, whereas Bcl-xL closes the VDAC (Shimizu et al. 1999, Shimizu et al. 2000a,Shimizu et al. 2000b; Shimizu and Tsujimoto 2000). We have also shown that nonfunctional mutants of Bax and Bcl-xL lose their effect on VDAC activity (Shimizu et al. 1999, Shimizu et al. 2000a,Shimizu et al. 2000b). Furthermore, Bax/Bak induces apoptotic mitochondrial changes, including cytochrome c release and mitochondrial membrane potential () loss, in mitochondria isolated from wild-type yeast, but not VDAC1-deficient yeast (Shimizu et al. 1999). We have also shown that Bax expression induces cytochrome c release in wild-type yeast cells, but not in VDAC1-deficient yeast cells (Shimizu et al. 2000c). Based on these findings, we have proposed that the VDAC plays an essential role in Bax/Bak-induced apoptotic mitochondrial changes and thus in the process of apoptosis in mammalian cells (Shimizu et al. 1999, Shimizu et al. 2000a,Shimizu et al. 2000b), although no direct evidence has been available. Other models for apoptogenic cytochrome c release have also been proposed. Cytochrome c release might be mediated by physical rupture of outer membrane resulting from mitochondrial swelling (Vander Heiden et al. 1997) or destabilization of membrane induced by Bax as well as tBid (Basanez et al. 1999; Kudla et al. 2000). Alternatively, Bax and Bak form oligomer channels in membrane that are permeable to cytochrome c, and tBid facilitates the insertion into the mitochondrial membrane or oligomerization of Bax and Bak (Eskes et al. 2000; Saito et al. 2000; Wei et al. 2000).
Microinjection of specific antibodies into cells has proven very useful to analyze the biological functions of particular proteins (Imamoto et al.. 1995; Hieda et al. 1999). Therefore, we generated anti-VDAC antibodies that blocked VDAC activity, and showed that Bax-induced, but not Bid-induced, cytochrome c release and apoptosis were significantly inhibited by these antibodies, and that these antibodies also significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. These results provide evidence that the VDAC plays an essential role in apoptotic mitochondrial changes and apoptosis in mammalian cells.
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Materials and Methods |
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Protein Purification
Recombinant human Bax (rBax) was expressed as a His-tagged protein in Escherichia coli strain XL1-blue using the Xpress System (Invitrogen), as described elsewhere (Narita et al. 1998). Irrelevant control proteins were prepared using the empty vector. Recombinant human Bid, truncated Bid (tBid), Bik, and BakC (lacking the COOH-terminal 21 amino acid residues) were expressed as GST fusion proteins in Escherichia coli strain DH5
and were purified on a glutathione-Sepharose column. Then Bid, tBid, Bik, and BakC were released from GST by cleavage with thrombin. The purity of Bax, Bid, tBid, Bik, and BakC was shown to be >80%. All purified proteins were finally dissolved in the same control buffer composed of 20 mM Hepes-K+, pH 7.4, and 1 mM dithiothreitol. Rat liver mitochondrial VDAC was purified as described previously previously (Shimizu et al. 1999), and showed a single band on SDS-polyacrylamide gel.
Generation of Anti-VDAC Antibodies
Two oligopeptides corresponding to parts of the human VDAC1 were synthesized on a Multiple Peptide Synthesizer (model 396; Advanced Chemtech) using diisopropylcarbodiimide/1-hydroxybenzotriazole-activated fluorenylmethoxycarbonyl-protected amino acids. The purity of each peptide was determined to be >90% by matrix-assisted laser desorption ionization time of flight mass spectrometry. These peptides were used to immunize rabbits, as described elsewhere (Imamoto et al. 1992). Anti-VDAC antibodies raised against the two oligopeptides (designated as Ab#20 and Ab#25) were purified from rabbit serum on a column of Sepharose conjugated with purified rat VDAC. Antibodies that bound to VDAC-Sepharose were eluted with 0.1 M glycine-HCl (pH 2.5) and neutralized with NaOH. We used three different preparations of Ab#25 (Ab#25-1, 25-2, and 25-3) that came from different animals, but mainly used Ab#25-1 unless otherwise mentioned.
To remove antiseptic, the antibodies were dialyzed extensively against a buffer composed of 20 mM Hepes-K+ (pH 7.4), and then were concentrated in a Micro Centricon 30 (Amicon Corp.) for use in microinjection and mitochondrial experiments.
Immunoprecipitation and Western Blot Analysis
Bax–VDAC interactions in the presence of anti-VDAC antibodies or control IgG were assessed by coimmunoprecipitation, as described elsewhere (Shimizu et al. 1999) using rat liver mitochondria. The mitochondria were lysed and immunoprecipitated with an anti-Bax antibody (N20) and the amount of coimmunoprecipitated VDAC was estimated by Western blot analysis. Interaction between rBax and VDAC on RBCs was also assessed by coimmunoprecipitation as described (Shimizu et al. 1999).
Reconstitution of VDAC and Bax in Liposomes
Plain liposomes and VDAC liposomes were prepared as described elsewhere (Shimizu et al. 1999). Liposomes in buffer consisting of 30 mM sodium sulfonate and 20 mM Tricine-NaOH (pH 5.3) were preincubated with 0.2 µg/µl antibodies for 3 min at room temperature, and with rBax (0.2 µg/µl) or the equivalent amount of irrelevant protein. VDAC activities were measured by assessing [14C]sucrose uptake, as described previously (Shimizu et al. 1999). In brief, 20 µl of liposomes treated with antibodies was incubated with 5 µl of [14C]sucrose (97%, 200 µCi/ml) in the presence of rBax or irrelevant protein at 25°C, and filtered by centrifugation using a 30K limiting filter (Millipore) to remove free [14C]sucrose. Then the [14C]sucrose incorporated into the liposomes was measured with a gamma-scintillation counter (model 1414; Wallac). Bax liposomes and irrelevant protein liposomes were prepared at pH 7.4 by the same procedure as VDAC liposomes, using the same amount of rBax as VDAC protein or an equivalent amount of irrelevant protein. Bax channel activity was measured by the same radiotracer uptake procedure, except that [3H]glucose (97%, 20 µCi/ml) was used instead of [14C]sucrose.
Preparation of Isolated Mitochondria
Livers from male Donryu rats were homogenized with a glass Teflon Potter homogenizer. Mitochondria were isolated in 0.3 M mannitol, 10 mM potassium Hepes (pH 7.4), 0.2 mM EDTA, and 0.1% fatty acid–free BSA (MT-1 medium), as described previously (Shimizu et al. 1998). Then the mitochondria were washed twice with the same medium without EDTA (MT-2 medium).
Measurement of Mitochondrial Biochemical Parameters
For energization, isolated mitochondria (1 mg protein/ml) were incubated at 25°C in MT-2 medium plus 1 mM potassium phosphate and 4.2 mM succinate, unless otherwise indicated. Mitochondria were incubated with antibodies for 5 min, and then were treated with rBax, rBid, or Ca2+ at the indicated concentrations, after which and cytochrome c release were measured. was assessed by measuring the -dependent uptake of rhodamine 123 using a spectrophotometer (F-4500; Hitachi), as described elsewhere (Shimizu et al. 1998). For detection of cytochrome c release and mitochondrial association of Bax, mitochondria were spun, and the pellet and supernatant were subjected to Western blot analysis using anticytochrome c and anti-Bax antibodies. Mitochondrial respiration was measured using an O2 electrode in the presence of succinate (state IV) or succinate plus ADP (state III). of cells was measured by incubating cells with 1 µM JC-1, followed by examination under a fluorescence microscope (IX70; Olympus).
Microinjection
HeLa cells (a human cervical cancer cell line) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Microinjection experiments were performed using a micromanipulator (Narishige), essentially as described previously (Hieda et al. 1999). Maximum and minimum injected volume were calculated to be 1.7 x 10–14 and 1.7 x 10–15 liters (Matsuoka et al. 1994). Thus, when 12 µg/µl of antibody was injected, the injected amount was estimated to be 0.2–0.02 pg. 1 h after injection of antibodies (free from antiseptic) with or without Cy5-labeled mouse IgG into the cytoplasm of cells, rBax or rtBid with or without green fluorescent protein (GFP) was injected in the cytoplasm of the same cells. The concentrations of proteins used for microinjection are described in the figure legends. In some experiments, cells were stained with Hoechst 33342 (10 µM) or annexin V–Cy3 using an Annexin V–Cy3 apoptosis detection kit (MBL). Cell morphology was examined under a transmission microscope or a fluorescence microscope.
Immunostaining
Cells were washed twice with PBS and fixed with 3.7% formaldehyde in PBS at room temperature for 10 min. After permeabilization with 0.5% Triton X-100 at room temperature for 5 min, cells were incubated with 5% skim milk. Then the primary antibody in 5% skim milk was added for 
12 h at 4°C. After washing twice, cells were treated with the respective secondary antibody conjugated with Alexafluor488 or 568 and were examined under a fluorescence microscope.
Lysis of RBCs
Whole blood was obtained from a healthy donor with consent. RBCs were prepared by washing the whole blood with a large amount of 0.9% NaCl, followed by centrifugation and suspending in 0.9% NaCl. The RBCs were treated with antibodies or BH4 oligopeptides for 5 min, after which rBax, rBak, or Kanagawa hemolysin was added at the indicated amount. Then the RBCs were spun, and released hemoglobin was measured by the absorbance at 543 nm (the isospecific point of reduced and oxidized hemoglobin) using a spectrophotometer (UV-160A; Shimadzu).
FACS® Analysis
Mitochondria and RBCs were incubated with antibodies at the indicated concentrations for 30 min at 4°C, and then washed with MT-1 medium and 0.9% NaCl, respectively. Subsequently, the mitochondria and RBCs were incubated with 20 µg/ml of anti–rabbit IgG-Alexa488 for 1 h at 4°C. After washing twice, samples were applied to a flow cytometer (FACSCaliburTM; Becton Dickinson) for analysis.
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Loss and Cytochrome c Release in Isolated Mitochondria-helix and 12 β-strands that cross the membrane to create a β-barrel (Fig. 1 A). Furthermore, since these residues are well conserved among three isoforms of human and mouse, the antibodies were expected to react with all isoforms of various mammals. Both antibodies raised in rabbits (Ab#20 recognizing amino acids 151–165 and Ab#25 recognizing amino acids 104–120) were affinity purified and were shown to be highly specific for human and rat VDAC (Fig. 1 B). As shown in Fig. 1 C, Ab#20 and Ab#25, but not normal rabbit IgG (NRI), efficiently inhibited VDAC activity, as assessed by [14C]sucrose uptake into VDAC liposomes. Ab#25 and Ab#20, but not NRI, also inhibited the Bax-induced enhancement of VDAC activity (Fig. 1 C). In contrast, both Ab#20 and Ab#25 showed no effect on Bax channel activity, as assessed by [3H]glucose uptake into Bax liposomes (Fig. 1 D; data not shown).
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loss and cytochrome c release. In contrast, these mitochondrial changes were efficiently inhibited by Ab#20 and Ab#25 in a concentration-dependent manner (Fig. 2A and Fig. B, top three panels), indicating that the VDAC is required for Bax-induced cytochrome c release and loss in mammalian mitochondria under our experimental conditions, consistent with our previous findings in yeast mitochondria (Shimizu et al. 1999, Shimizu et al. 2000c). The amount of Ab#20 and Ab#25 used was near the saturation level for VDAC on the mitochondria, as judged from flow cytometric analysis of mitochondria stained with Ab#25 (Fig. 2 C) and also by comparing the amount of each antibody with the amount of VDAC in the mitochondria using PAGE (data not shown).
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As can be seen in Fig. 2 B (forth and fifth panels from the top), Ca2+-induced cytochrome c release, which was previously shown to be mediated by the PT (Narita et al. 1998), was also inhibited by Ab#20 and Ab#25 (25-1 and 25-2 were derived from different animals), indicating that the VDAC is essential for the PT.
It has previously been shown that Bax-induced cytochrome c release is largely dependent on the PT (i.e., blocked by PT inhibitors such as cyclosporine A and bongkrekic acid, and accompanied by loss) (Jürgensmeier et al. 1998; Marzo et al. 1998; Narita et al. 1998) and only partly occurs in a PT-independent manner (Narita et al. 1998; Shimizu and Tsujimoto 2000), although other groups have reported that Bax-induced cytochrome c release is not dependent on the PT, particularly in the absence of Ca2+ (Eskes et al. 1998; Finucane et al. 1999; Kluck et al. 1999). To determine whether inhibition of Bax-induced cytochrome c release by Ab#20 and Ab#25 was specific to PT-associated cytochrome c release, we examined the effect of these antibodies on Bax-induced cytochrome c release in the presence of a Ca2+ chelator that completely inhibited the PT (Zoratti and Szabó 1995; data not shown). As shown in Fig. 2 B (bottom), PT-independent cytochrome c release induced by Bax was also inhibited by both Ab#20 and Ab#25, indicating an essential role of the VDAC in Bax-induced cytochrome c release regardless of PT dependency. Note that a higher concentration of rBax was used to induce cytochrome c release in the presence of EGTA than that without EGTA.
Anti-VDAC Antibodies Inhibit Bax-induced Apoptosis
To determine whether the VDAC had an essential role in Bax-induced apoptosis in mammalian cells, we carried out an experiment in which Ab#20 and Ab#25 were microinjected into the cytoplasm of HeLa cells. Consistent with the predominant localization of VDAC in the mitochondria, Ab#25 was mainly observed in the mitochondria by immunostaining, when injected at a lower concentration (3 µg/µl) (Fig. 3 A), whereas it was observed in the mitochondria with a small amount throughout the cytoplasm when injected at a higher concentration of 12 µg/µl (data not shown), suggesting that a concentration of 3 µg/µl was less than the saturation level for mitochondrial VDAC. Therefore, microinjection experiments were mainly done with Ab#20 and Ab#25 at 12 µg/µl, which was close to the saturation level for mitochondrial VDAC. Injection of these antibodies showed little toxicity in HeLa cells (data not shown). To test the effect of Ab#20 and Ab#25 on mitochondrial respiration, cells were incubated in glucose-free medium for 24 h. Since glycolysis was halted under these conditions, inhibition of mitochondrial respiration (e.g., by the addition of oligomycin, an F1 ATPase inhibitor) led to mitochondrial loss (Fig. 3 B, second panel). However, when cells were microinjected with Ab#20 or Ab#25, remained high during incubation in glucose-free medium (Fig. 3 B, far right panel; data not shown), indicating that these antibodies did not significantly affect mitochondrial respiration, consistent with the results obtained using isolated mitochondria (Fig. 2 D).
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Anti-VDAC Antibodies Inhibit Bax-induced Lysis of RBCs
It has previously been reported that addition of rBax induces lysis of RBCs, and this hemolysis has been considered to be due to formation of the Bax channel on the RBC membrane (Antonsson et al. 1997). Since VDAC is also known to be located on the plasma membrane (Cole et al. 1992; Buettner et al. 2000), the possibility was raised that Bax-mediated RBC lysis was also mediated by the VDAC. The presence of the VDAC on RBC plasma membrane was confirmed by flow cytometry (Fig. 7 A), which also showed that it was oriented with the epitope for Ab#25 facing outside, consistent with previous observations (Cole et al. 1992). Since RBCs do not possess mitochondria, which was confirmed by the absence of cytochrome c (Fig. 7 B), and since they have no ER and Golgi apparatus, the VDAC shown by Western blotting (Fig. 7 B) probably largely represented VDAC on the plasma membrane. The VDAC on RBCs was 1 kD smaller than that in HeLa cells (Fig. 7 B), but reacted with all the anti-VDAC antibodies (31HL, Ab#25 [Fig. 7 B], Ab#20, and an antibody whose epitope was amino acids 177–192 [data not shown]), and, therefore, the VDAC on RBCs probably represents a splicing variant or is subjected to RBC-specific modifications. Several erythrocyte-specific alterations of proteins have been reported, for example, hexokinase and pyruvate kinases (Lacrinique et al. 1992; Murakami and Piomelli 1997). The possibility was not excluded that the VDAC on RBCs represents a protein highly related to VDAC or another member of the VDAC family. The presence of the VDAC on RBC plasma membrane was also confirmed by tryptic digestion of VDAC but not glyceraldehyde 3-phosphate dehydrogenase (GPDH), a cytosolic glycolytic enzyme (Fig. 7 C), and by the presence of VDAC in ghost RBCs (Fig. 7 D). Like mitochondrial VDAC, VDAC in RBCs bound to rBax (Fig. 7 E).
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FL) lacking the ability to close VDAC (Shimizu et al. 2000b), inhibited hemolysis in a dose-dependent manner, although to a lesser extent comparing with Ab#20 and 25. This partial inhibition of Bax-induced hemolysis by the BH4 peptide was partly due to toxicity of the BH4 peptide to induce extensive aggregation that was prominent at higher concentrations (Fig. 7 J). As shown in Fig. 7 K, neither Ab#20 nor Ab#25 inhibited hemolysis induced by Kanagawa hemolysin, which is known to occur via formation of pores by the hemolysin itself. These results indicated that, as in the mitochondria, Bax increased plasma membrane permeability by acting on the VDAC.
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Discussion |
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loss in mitochondria isolated from wild-type yeast cells but not from VDAC1-deficient yeast cells (Shimizu et al. 1999, Shimizu et al. 2000c), led us to propose that cytochrome c is released through the VDAC during apoptosis and the Bcl-2 family of proteins regulate apoptosis by modulating VDAC activity in mammalian cells. Several other models have been proposed to explain how Bax induces cytochrome c release: (a) by rupture of the outer mitochondrial membrane due to swelling (Vander Heiden et al. 1997, Vander Heiden et al. 1999) that could result from Bax–adenine nucleotide translator (ANT) interaction (Marzo et al. 1998) or due to Bax-mediated destabilization of the lipid membrane (Basanez et al. 1999); or (b) via the multimeric channels of Bax (Eskes et al. 2000; Saito et al. 2000). In this study, using neutralizing anti-VDAC antibodies, we obtained direct evidence that the VDAC is essential for Bax-induced apoptotic mitochondrial changes (including cytochrome c release and loss) and for apoptotic death of mammalian cells. We also showed that anti-VDAC antibodies could significantly inhibit etoposide-, paclitaxel-, and staurosporine-induced apoptosis. These results support our model that the VDAC is involved in apoptogenic cytochrome c release and apoptosis that are dependent on members of the Bax subfamily. Although the results in this study confirmed that VDAC is essential for Bax-induced apoptosis, they did not formally confirm that cytochrome c indeed passes through VDAC in mammalian cells during apoptosis. However, our previous demonstration that cytochrome c can pass through the VDAC in the presence of Bax/Bak in the proteoliposome system (Shimizu et al. 1999, Shimizu et al. 2000a) strongly suggests that VDAC is a channel for apoptotic cytochrome c release in mammalian cells. To confirm this notion requires further investigation. Since our neutralizing anti-VDAC antibodies inhibited VDAC activity, leading to the inhibition of apoptotic mitochondrial changes and cell death, our results excluded the possibility that the VDAC needs to be inhibited for the induction of apoptosis, leading to hyperpolarization and subsequent mitochondrial swelling (Vander Heiden et al. 1997, Vander Heiden et al. 1999) that is blocked by the action of Bcl-2 to maintain VDAC function (Vander Heiden et al. 2000). We also showed that the VDAC is essential for Bax-induced rupture of RBCs, although this phenomenon was previously considered to be mediated by large pores formed by Bax alone (Antonsson et al. 1997). Thus, the VDAC is essential for Bax to modulate membrane permeability at two independent sites, the mitochondrial membrane and the plasma membrane, further supporting the idea that the VDAC is the functional target of Bax. Given that none of the Bcl-2 family proteins is secreted, a Bax-mediated increase of plasma membrane permeability does not seem to be physiologically relevant. However, the VDAC on the plasma membrane is involved in cell volume regulation (Thinnes et al. 2000), so there might be secreted protein(s) that modulate this VDAC in a similar fashion to Bax/Bak.
It has previously been suggested that Bax induces cytochrome c release via two different mechanisms in isolated mitochondria: a mechanism that is accompanied by loss and is blocked by PT inhibitors such as cyclosporine A (Jürgensmeier et al. 1998; Marzo et al. 1998; Narita et al. 1998) or a mechanism that is not accompanied by loss and is resistant to PT inhibitors (Eskes et al. 1998; Finucane et al. 1999; Kluck et al. 1999), although it seems likely that the former mechanism is more physiologically relevant because Bax induces cytochrome c release together with loss in mammalian cells (Xiang et al. 1996; Marzo et al. 1998; Pastorino et al. 1998; Shimizu and Tsujimoto 2000; our unpublished observations). In the present study, our neutralizing anti-VDAC antibodies inhibited Bax-induced cytochrome c release that was largely PT inhibitor-sensitive. Importantly, the same antibodies inhibited Bax-induced cytochrome c release in the absence of Ca2+, that was PT inhibitor resistant. Thus, irrespective of PT dependence, Bax-induced cytochrome c release always requires the VDAC (Fig. 8). Accordingly, the likely scenario is that Bax triggers VDAC opening and cytochrome c release, leading to opening of inner membrane channel(s) such as ANTs via a mutual interaction that results in loss and the PT, which might subsequently induce another phase of cytochrome c release.
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We have previously shown that Bax-induced apoptotic mitochondrial changes are largely dependent on mitochondrial respiration (Shimizu and Tsujimoto 2000). Various respiratory substrates and products, including succinate and adenine nucleotides, pass through the VDAC (for review see Colombini 1989). Our finding that the neutralizing anti-VDAC antibodies blocked cytochrome c release, but not respiration, suggests that only a small fraction of VDAC activity is sufficient for respiration although a larger fraction is required for apoptogenic cytochrome c release, or that the antibodies may not close the mitochondrial VDAC completely so that substances involved in respiration still pass through the channel.
We have previously shown that BH3-only proteins such as Bid (also tBid) and Bik do not bind to VDAC (Shimizu and Tsujimoto 2000). We have also shown that Bid/Bik-induced cytochrome c release is substantially different from Bax/Bak-induced cytochrome c release: (a) Bax/Bak-induced, but not Bid/Bik-induced, cytochrome c release is accompanied by loss; and (b) PT inhibitors and respiratory chain inhibitors block Bax/Bak-induced, but not Bid/Bik-induced, cytochrome c release (Shimizu and Tsujimoto 2000). Based on these observations, we have proposed that Bax/Bak target VDAC to induce cytochrome c release, whereas Bid/Bik induces cytochrome c release by a mechanism not involving the VDAC (Shimizu and Tsujimoto 2000; Tsujimoto and Shimizu 2000b). Consistently, we also showed here that VDAC is required for Bax-induced, but not for Bid-induced, cytochrome c release and cell death. These results, together with our observation that Bax/Bak but not Bid/Bik induce cytochrome c release in mitochondria isolated from yeast cells (unpublished observation), suggest that Bid/Bik probably targets a distinct molecule on the mitochondrial outer membrane to induce cytochrome c release (Fig. 8). It has been suggested that Bid (tBid) exerts its activity by heterodimerizing with antiapoptotic members of the Bcl-2 family (Wang et al. 1996), or by facilitating oligomerization of Bax and Bak to form pores permeable for cytochrome c (Eskes et al.. 2000; Wei et al. 2000). Bid (tBid) has also been suggested to have an ability to form a pore by itself (Schendel et al. 1999) or destabilizing lipid membrane (Kudla et al. 2000). The substantial difference between Bax/Bak-induced and Bid/Bik-induced cytochrome c release and apoptosis (Shimizu and Tsujimoto 2000), however, makes the possibility unlikely that Bid functions only through heterodimerizing with Bcl-2/Bcl-xL or facilitating Bax/Bak oligomerization, because these two models suggest no difference between Bid-induced and Bax/Bak-induced cytochrome c release and cell death. The possibility is also unlikely that Bid/Bik forms a channel by itself or destabilizes mitochondrial membrane for cytochrome c release, because, unlike Bax/Bak, Bid is unable to induce cytochrome c release in yeast mitochondria (our unpublished results). Since Bid/Bik-induced cytochrome c release is prevented by Bcl-2/Bcl-xL, these antiapoptotic Bcl-2 family members might form heterodimers with Bid/Bik to inhibit their activity or directly close the Bid/Bik target channel in a similar manner as occurs with the VDAC (Fig. 8).
In conclusion, we obtained evidence that the VDAC is essential for Bax-induced (but not Bid-induced) apoptotic cytochrome c release and apoptotic death and apoptosis induced by etoposide, paclitaxel, and staurosporine in mammalian cells. These results, taken together with our previous observation that Bax opens the VDAC and releases cytochrome c in a proteoliposome system (Shimizu et al. 1999, Shimizu et al. 2000a), suggest that the VDAC is a channel for apoptotic cytochrome c release that is directly regulated by the Bcl-2 family of proteins.
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Acknowledgments |
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This study was supported in part by a grant for Scientific Research on Priority Areas, a grant for Center of Excellence Research, a grant for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, and by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan.
Submitted: 26 June 2000
Revised: 13 November 2000
Accepted: 21 November 2000
Abbreviations used in this paper: ANT, adenine nucleotide translator; BH, Bcl-2 homology;
, mitochondrial membrane potential; GFP, green fluorescent protein; GPGH, glyceraldehyde 3-phosphate dehydrogenase; NRI, normal rabbit IgG; PT, permeability transition; rGFP, recombinant GFP; VDAC, voltage-dependent anion channel.
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