Bid Induces the Oligomerization and Insertion of Bax into the Outer Mitochondrial Membrane

Abstract

In many types of apoptosis, the proapoptotic protein Bax undergoes a change in conformation at the level of the mitochondria.
This event always precedes the release of mitochondrial cytochrome c, which, in the cytosol, activates caspases through binding to Apaf-1. The mechanisms by which Bax triggers cytochrome c release are unknown. Here we show that following binding to the BH3-domain-only proapoptotic protein Bid, Bax oligomerizes
and then integrates in the outer mitochondrial membrane, where it triggers cytochromec release. Bax mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading to apoptosis.

Full text

MOLECULAR AND CELLULAR BIOLOGY,
0270-7306/00/$04.00⫹0
Feb. 2000, p. 929–935 Vol. 20, No. 3
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Bid Induces the Oligomerization and Insertion of Bax
into the Outer Mitochondrial Membrane
ROBERT ESKES,† SOLANGE DESAGHER, BRUNO ANTONSSON,
AND JEAN-CLAUDE MARTINOU*
Serono Pharmaceutical Research Institute, Ares Serono International S.A.,
CH-1228 Plan-les Ouates, Geneva, Switzerland
Received 21 June 1999/Returned for modification 9 August 1999/Accepted 25 October 1999
In many types of apoptosis, the proapoptotic protein Bax undergoes a change in conformation at the level
of the mitochondria. This event always precedes the release of mitochondrial cytochrome c, which, in the
cytosol, activates caspases through binding to Apaf-1. The mechanisms by which Bax triggers cytochrome c
release are unknown. Here we show that following binding to the BH3-domain-only proapoptotic protein Bid,
Bax oligomerizes and then integrates in the outer mitochondrial membrane, where it triggers cytochrome c
release. Bax mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading
to apoptosis.
Bcl-2 family members play a key role in processes underlying
programmed cell death or apoptosis (17, 25, 36). The Bcl-2
family is composed of both antiapoptotic (Bcl-2, Bcl-x
L
, Bcl-w,
Mcl-1, A1, NR-13, BHRF1, LMW5-HL, ORF16, KS-Bcl-2,
E1B-19K, and CED-9) and proapoptotic (Bax, Bak, Bok, Bik,
Blk, Hrk, BNIP3, Bim
L
, Bad, Bid, and EGL-1) molecules (1).
These proteins can form homo- and heterodimers that involve
amino acid sequences known as Bcl-2 homology (BH) do-
mains. Four of these domains (BH1 to BH4) have been iden-
tified (20, 25, 36, 44). The BH3 domain of the proapoptotic
members appears to be required for the interaction between
anti- and proapoptotic molecules (5).
The principal site of action of some of the Bcl-2 family
members appears to be the mitochondrion (9, 19, 21, 37, 41).
Mitochondria play a major role in many types of apoptosis. In
particular, this organelle releases apoptosis-inducing factor
(40) and cytochrome c (3, 9, 19, 21, 37, 45). The latter triggers
caspase 9 activation through Apaf-1–caspase 9 complex forma-
tion (26). Bcl-2 family members play a key role in regulating
cytochrome c release. While Bcl-2 and Bcl-x
L
suppress cyto-
chrome c release (3, 21, 45), Bax stimulates this event both in
vitro in isolated mitochondria and in intact cells following
heterologous expression (3, 9, 19). The mechanisms by which
these proteins perform their function are currently unknown.
The three-dimensional structures of Bcl-x
L
(34) and Bid (6,
31) revealed structural similarities between these proteins and
the channel-forming domains of the bacterial toxins colicins
and diphtheria toxins. Consistent with such structural similar-
ity, some members of this family including Bax, Bcl-2, and
Bcl-x
L
are also able to form ion channels in synthetic lipid
membranes (2, 33, 38, 39). The channel-forming activity of
these proteins has not yet been demonstrated in vivo. How-
ever, it is now clear that, at least during apoptosis, these pro-
teins are associated with intracellular membranes, in particular
with mitochondrial membranes. The events that trigger mem-
brane association of the Bcl-2 family members are still poorly
understood, although it is probable that, like colicins and diph-
theria toxins, Bcl-2 family members may have to undergo a
change in conformation before undertaking membrane inser-
tion.
We have previously shown that at an early stage of apoptosis
in cerebellar granule cells deprived of serum and potassium or
in HeLa cells exposed to staurosporine, Bax undergoes a
change in conformation (8). Similarly, the structure of Bak,
another proapoptotic member of the Bcl-2 family, was re-
ported to undergo conformational change during various types
of apoptosis well before cytochrome c release from mitochon-
dria (8, 12). For both Bax and Bak, this change in conforma-
tion appears to expose the N-terminal domain, which other-
wise is cryptic and nonaccessible to antibodies. Interestingly,
Bid, a BH3-domain-only protein which interacts with Bax, was
able to trigger this conformational change in Bax (8). The goal
of our present experiments was to understand the role of the
switch in Bax conformation and how this key event could be
related to cytochrome c release from mitochondria. Here we
report that following Bid-induced conformational change, Bax
oligomerizes and inserts tightly within the outer mitochondrial
membrane without a requirement for any proteolytic event.
The integration of Bax in the outer mitochondrial membrane is
followed by cytochrome c release, which, in contrast to Bax
membrane integration, is highly dependent on magnesium.
MATERIALS AND METHODS
Cell culture. HeLa cells and the stable HeLa cell line that constitutively
overexpresses Bcl-2 (HeLa–Bcl-2) (10) were cultured in a 1:1 mixture of basal
Iscove medium and Ham’s F-12 medium (Seromed) supplemented with 10%
fetal calf serum and 2 mM
L-glutamine.
Subcellular fractionation. At different times after the induction of apoptosis,
HeLa cells were harvested in isotonic mitochondrial buffer (MB) (210 mM
mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM HEPES [pH 7.5]) supplemented
with complete protease inhibitor cocktail (Boehringer Mannheim). The cells
were broken by six passages through a 25G1 0.5- by 25-mm needle fitted on a
5-ml syringe, and the suspension was centrifuged at 2,000 ⫻ g in an Eppendorf
centrifuge at 4°C. This procedure was repeated twice, and supernatants from
each step were pooled before being subjected to centrifugation at 13,000 ⫻ g at
4°C for 10 min. The supernatant was further centrifuged at 600,000 ⫻ g for 10
min at 4°C to yield the light membrane pellet (not analyzed) and the final soluble
fraction (S100). The heavy membrane material was pooled and resuspended in
* Corresponding author. Mailing address: Serono Pharmaceutical
Research Institute, Ares Serono International S.A., 14 Chemin des
Aulx, CH-1228 Plan-les Ouates, Geneva, Switzerland. Phone: 41-22-
706-9822. Fax: 41-22-794-69-65. E-mail: Jean-Claude.Martinou
@Serono.com.
† Present address: J. W. G.-University Frankfurt am Main, ZIM—
Med Klinik III—Molekulare Ha¨matologie, D-60596 Frankfurt Ger-
many.
929

MB-EGTA (MB with 0.5 mM EGTA instead of EDTA) and centrifuged at
500 ⫻ g for 3 min at 4°C to eliminate residual nuclei. The resulting supernatant
was centrifuged at 10,000 ⫻ g for 10 min at 4°C to further purify the mitochon-
drial fraction. The protein concentration was estimated by the method of Brad-
ford (4) with bovine serum albumin as the standard.
In vitro assay for Bax insertion and cytochrome c release. Mitochondria (100
␮g of proteins) were incubated in the presence or absence of various recombi-
nant proteins in 100 ␮l of MBC buffer (MB with EGTA supplemented with 4
mM MgCl
2
,5mMNa
2
HPO
4
, 5 mM succinate, and 5 ␮M rotenone) for 15 min
at 30°C and then centrifuged for 5 min at 13,000 ⫻ g and 4°C. The supernatants
and the pellets were used for the determination of cytochrome c release. For
alkali extraction, the mitochondrial pellets were resuspended (1 mg of protein/
ml) in freshly prepared 0.1 M Na
2
CO
3
(pH 11.5) and incubated for 20 min on ice.
The membranes were then pelleted by centrifugation (600,000 ⫻ g for 20 min at
4°C). Mitochondrial membrane pellets corresponding to 10 ␮g of proteins (the
alkali-resistant fractions) and the corresponding volume of supernatants (the
alkali-sensitive fractions) were separated by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis on 4 to 20% Tris-Gly gels (NOVEX). Their respective
contents of cytochrome c and Bax were estimated by Western blotting with a
polyclonal anti-cytochrome c antibody (dilution 1:2,500) or a polyclonal antibody
directed against Bax (Upstate Biotechnology). Equal loading of the mitochon-
drial pellet was verified by using an antibody either against cytochrome c oxidase
subunit IV (Cox IV) or against cytochrome c oxidase subunit II (Cox II) (both
from Molecular Probes) or an antibody against the voltage-dependent anion
channel VDAC (Calbiochem). Antigen-antibody complexes were detected by
using horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G and
enhanced chemiluminescence detection reagents.
Cross-linking. The mitochondria (0.5 mg of proteins) were incubated with the
recombinant proteins, then pelleted by centrifugation, and resuspended in MB-
EGTA, and disuccinimidyl subernate (DSS) (in dimethyl sulfoxide DMSO;
Pierce) or bis(sulfosuccinimidyl) subernate (BS
3
) (in 5 mM sodium citrate buffer
[pH 5.0]; Pierce) was added from a 10-fold stock solution to a final concentration
of 2 mM. After incubation for 30 min at room temperature, the cross-linker was
quenched by the addition of 1 M Tris-HCl (pH 7.5) to a final concentration of 20
mM. After quenching, the membranes were dissolved in RIPA buffer and cleared
by centrifugation at 12,000 ⫻ g. The lysate was immunoprecipitated with anti-Bax
2D2 monoclonal antibody (Genzyme) and then analyzed by Western blotting
with the polyclonal anti-Bax antibody.
Digitonin treatment of mitochondria. Following incubation with Bid, mito-
chondria (100 ␮g) were pelleted, dissolved in 100 ␮l of digitonin (1.2 mg/ml), and
incubated for 25 min on ice. The mitoplasts were pelleted by centrifugation and
dissolved in 100 ␮l of RIPA buffer. The fractions were analyzed by Western
blotting as mentioned above.
Production of recombinant proteins. His-tagged Bid, Bid mutants, human
Bcl-x
L
, and human mutant Bcl-x
L
(Bcl-x
L
m: G
138
3A), both Bcl-x
L
protins lack-
ing 24 amino acids at the COOH terminus, were produced as described previ-
ously (8).
RESULTS
Bax integration into the mitochondrial membrane in stau-
rosporine-treated HeLa cells. In many cultured cells, Bax is
found in both the cytosolic and mitochondrial fractions (15, 43;
unpublished observation). To test whether mitochondrial Bax
was integrated in the mitochondrial membranes, we performed
an alkali extraction of proteins from mitochondria isolated
from HeLa cells. Bcl-x
L
, Bak, and Cox II were analyzed in
parallel. In contrast to Bcl-x
L
or Bak, which were resistant to
the alkali extraction, most Bax was lost during this treatment,
indicating that under normal conditions Bax is loosely attached
to mitochondria while most Bcl-x
L
and Bak proteins are in-
serted in the membrane (Fig. 1A and B). When HeLa cells
were treated with staurosporine, Bax became resistant to alkali
extraction in a time-dependent manner. After 12 h of incuba-
tion with staurosporine, almost all the Bax was found to be
inserted in the mitochondrial membranes (Fig. 1A). These
results, in agreement with previous results reported by Goping
et al. (11), demonstrate that in response to a death stimulus,
Bax becomes inserted in the mitochondrial membranes.
The major objective of this study was to determine the
mechanisms that are responsible for Bax insertion in mito-
chondrial membranes. We reported previously that during
staurosporine-triggered apoptosis of HeLa cells, mitochondrial
Bax undergoes a change in conformation, rendering its N ter-
minus accessible to antibodies (8). This event was accompa-
nied by a release of cytochrome c. Moreover, we reported that
Bid, which translocates to mitochondria during apoptosis, was
able to trigger a change in Bax conformation when added
FIG. 1. Bax integration into the mitochondrial membranes during apoptosis
and after Bid treatment. (A) Mitochondria from HeLa cells cultured in the
absence or presence of 1 ␮M staurosporine for increasing times were isolated
and treated with 0.1 M Na
2
CO
3
to produce alkali-sensitive (Att [attached]) and
-resistant (Ins [inserted]) fractions. Both fractions were analyzed by Western
blotting for the presence of Bax. Cox II was used as a gel-loading control. (B)
Mitochondria from HeLa cells were incubated with 100 nM recombinant wild-
type Bid and two Bid mutants (BidmIII-1 and BidmIII-3) for 15 min at 30°C,
recovered by centrifugation, and treated with 0.1 M Na
2
CO
3
as above. Various
proteins were analyzed by Western blotting in alkali-sensitive (Att.) and -resis-
tant fractions (Ins.). (C) Cytochrome c was analyzed by Western blotting in the
mitochondrial suspension following incubation with wild-type and mutant Bid.
FIG. 2. Bid induces the insertion of Bax into the outer mitochondrial mem-
brane. Isolated mitochondria (100 ␮g) from HeLa cells were incubated with 100
nM Bid for 15 min at 30°C and treated with digitonin (1.2 mg/ml) for 25 min at
4°C. The digitonin-sensitive (Out. [outer mitochondrial membrane]) and resis-
tant (In. [inner mitochondrial membrane]) fractions were analyzed by Western
blotting for the presence of VDAC, Cox IV, Bax, and Bcl-x
L
.
930 ESKES ET AL. MOL.CELL.BIOL.

directly to isolated mitochondria. Bid was therefore a likely
candidate as the protein responsible for driving the Bax inte-
gration into the mitochondrial membrane. Figure 1B shows
that addition of 100 nM Bid to mitochondria isolated from
HeLa cells rendered Bax resistant to alkali extraction, indicat-
ing that it had undergone membrane integration. Similar re-
sults were obtained when mitochondria were incubated in the
presence of 100 nM Bid and 100 ␮M z-VAD-fmk, a broad-
spectrum caspase peptide inhibitor, indicating that Bid-in-
duced Bax membrane insertion was independent of caspase
activation (results not shown). The mitochondrial levels of
Bcl-x
L
, Bak, Cox IV, and VDAC, all membrane-integrated
proteins, did not change following Bid treatment (Fig. 1B). Bid
association with the mitochondria remained sensitive to alkali
treatment, suggesting that in contrast to Bax, this protein does
not itself integrate into the mitochondrial membranes (Fig.
1B).
Bid interacts with other Bcl-2 family members via its BH3
domain (42). To determine if binding of Bid to Bax is a pre-
requisite for Bax insertion, two Bid BH3 mutants with selec-
tively lowered affinity for either Bax (BidmIII-3: G
94
3A) (8,
42) or Bcl-2 (BidmIII-1: M
97
D
98
3AA) (8, 42) were tested.
While BidmIII-1 was as effective as wild-type Bid, BidmIII-3
was unable to stimulate Bax insertion (Fig. 1B) and cyto-
chrome c release from mitochondria (Fig. 1C). Together, these
FIG. 3. Bid induces Bax oligomerization. Isolated mitochondria from HeLa
cells were incubated with 1 ␮M Bid for 15 min at 30°C, and the mitochondrial
pellet was treated with two different cross-linkers as described in Materials and
Methods. After cross-linking, the mixture was immunoprecipitated with anti-Bax
monoclonal antibody and analyzed by Western blotting with an anti-Bax poly-
clonal antibody.
FIG. 4. Time course study of Bax oligomerization, Bax membrane insertion,
and cytochrome c release after addition of Bid to isolated mitochondria. Isolated
mitochondria from HeLa cells were incubated with recombinant Bid at 30°C for
increasing times and analyzed for Bax oligomerization (A), Bax membrane
insertion (B), and cytochrome c release (C). Att., attached (alkali sensitive); Ins.,
inserted (alkali resistant); Cyt.c, cytochrome c.
FIG. 5. Bcl-x
L
and Bcl-2 inhibit Bid-induced oligomerization and insertion of
Bax into the outer mitochondrial membrane. (A to C) Mitochondria isolated
from HeLa cells were incubated with 100 nM Bid in the presence of 1 ␮M
recombinant Bcl-x
L
or Bcl-x
L
m at 30°C for 15 min. Mitochondria were used to
study Bax insertion into the outer mitochondrial membrane (A), cytochrome c
release (B), and Bax dimerization (C). (D) Mitochondria from HeLa cells over-
expressing Bcl-2 were isolated and incubated with 100 nM Bid at 30°C for 15 min
and used to analyze Bax insertion into the outer mitochondrial membrane. Att.,
attached (alkali sensitive); Ins., inserted (alkali resistant); Ctl., Cont., control;
Cyt.c, cytochrome c.
VOL. 20, 2000 Bid-INDUCED BAX INSERTION IN MITOCHONDRIA 931

results strongly indicate that Bid induces the insertion of Bax
by interacting directly with Bax.
Bax integration in the outer mitochondrial membrane. We
next tested whether Bax inserts into the outer or the inner
mitochondrial membrane by using digitonin to selectively dis-
solve the outer mitochondrial membrane. VDAC and Cox IV,
which are present in the outer and inner mitochondrial mem-
branes, respectively, were used to set up the conditions for
optimal extraction. In the presence of 1.2 mg of digitonin/mg of
protein, mitochondria from HeLa cells lost most of the VDAC
protein while Cox IV remained attached to the mitochondria,
indicating conditions for selective extraction of proteins from
the outer mitochondrial membrane (Fig. 2). Like VDAC, Bax
was found to be sensitive to digitonin extraction, confirming
that in nonapoptotic cells Bax is attached to the outer mito-
chondrial membrane. A small amount of Bax remained insen-
sitive to digitonin extraction, which could reflect Bax present at
contact sites, although we cannot exclude the possibility that a
minor portion of the protein is also localized in the inner
mitochondrial membrane. Importantly, however, the fraction
of digitonin-resistant Bax was unaltered following incubation
with Bid for 15 min. In contrast, an increase in the amount of
digitonin-sensitive Bax was detected (Fig. 2). The increase in
Bax levels detected in the presence of Bid reflects the fact that
Bax becomes tightly attached to mitochondrial membranes,
while in the absence of Bid, substantial amounts of Bax can be
easily lost from mitochondria during repetitive washes. Bcl-x
L
was also present mainly in the outer mitochondrial membrane,
and its distribution remained unchanged following treatment
with Bid (Fig. 2). We conclude from these experiments that
following its interaction with Bid, Bax integrates into the outer
mitochondrial membrane.
Bax dimerization. It was reported previously that Bax is
present in a monomeric form in the cytoplasm (16) and that
dimerization is a prerequisite for mitochondrial translocation
(13). This prompted us to test whether Bid was able to trigger
Bax dimerization and whether this precedes membrane inte-
gration. Intact mitochondria were treated with either the mem-
brane-permeable DSS or the non-membrane-permeable BS
3
cross-linking agents. In the absence of Bid, Bax was detected as
a monomer. However, in the presence of 100 nM Bid, two
Bax-immunoreactive bands of approximately 40 and 60 kDa
were clearly detectable (Fig. 3). These are likely to correspond
either to Bax homodimers and trimers or to Bax heterodimers.
We also detected an additional Bax-immunoreactive band run-
ning at ⬃40 kDa that could correspond to a Bax monomer
linked to an unknown protein (Fig. 3). Similar results were
obtained with two different anti-Bax antibodies. No immuno-
reactivity of these protein bands was detected with antibodies
directed against Bcl-2, Bcl-x
L
, Bag-1, Bid, or VDAC.
We next examined the temporal relationship between Bax
oligomerization, its membrane insertion, and mitochondrial
cytochrome c release. Figure 4A shows that upon addition of
Bid to isolated mitochondria, Bax dimers and trimers were
observed within 2.5 to 5 min and their levels peaked at 10 min.
Importantly, Bax oligomerization preceded Bax insertion into
the membrane, which appeared evident only between 5 and 10
min after Bid addition (Fig. 4B). Release of cytochrome c from
mitochondria also occurred between 5 and 10 min after Bid
addition, although maximal levels were not observed until after
15 min (Fig. 4C). These results suggest that Bax dimerization
(or oligomerization) precedes its membrane integration and
the efflux of cytochrome c from mitochondria.
Regulation of Bid-induced Bax integration in the outer mi-
tochondrial membrane by Bcl-x
L
and Bcl-2. We have shown
previously that Bcl-2 and Bcl-x
L
are able to inhibit the change
in conformation of Bax in cells exposed to an apoptotic stim-
ulus or following the addition of Bid to isolated mitochondria.
Here we tested whether Bcl-x
L
could prevent Bid-induced Bax
oligomerization and insertion into the membrane. We found
that in the presence of Bcl-x
L
, Bid was unable to trigger Bax
membrane integration, cytochrome c release, or Bax oligomer-
ization (Fig. 5A to C). In contrast, a mutant of Bcl-x
L
(Bcl-
x
Lm
), which fails to bind Bax, did not inhibit these sequential
events. Bid was also unable to trigger Bax integration when
added to mitochondria from HeLa cells overexpressing Bcl-2
(Fig. 5D).
Membrane insertion of Bax without cytochrome c release in
the absence of magnesium ions. We have reported previously
that the ability of Bax to trigger cytochrome c release from
mitochondria was highly dependent on magnesium ions but
independent of the opening of the permeability transition pore
FIG. 6. In contrast to cytochrome c release, Bid-induced insertion of Bax does not require the presence of Mg
2⫹
. (A) Mitochondria from HeLa cells were incubated
with 100 nM Bid for 15 min at 30°C in the presence or absence of 2.5 mM MgCl
2
or MnCl
2
before analysis of Bax insertion into membranes. (B) Mitochondria were
incubated in the presence or absence of 100 nM Bid and various salts including MgCl
2
(2.5 mM), MnCl
2
(2.5 mM), KCl (5 mM), NaCl (5 mM), and LiCl (5 mM). Both
supernatants and mitochondrial pellets were analyzed for cytochrome c release. Ctl., control; Att., attached (alkali sensitive); Ins., inserted (alkali resistant); Cyt. c,
cytochrome c.
932 ESKES ET AL. MOL.CELL.BIOL.

(9). We have now extended these observations by testing
whether magnesium is important for membrane insertion of
Bax. Figure 6A shows that Bax was resistant to alkali extraction
following Bid addition to mitochondria in both the presence
and absence of 2.5 mM Mg
2⫹
, indicating that Bax integration
in the membrane occurs independently of Mg
2⫹
. However,
only mitochondria incubated with Bid in the presence of 2.5
mM Mg
2⫹
were found to release cytochrome c (Fig. 6B).
Similar results were obtained with 2.5 mM MnCl
2
, which can
substitute for MgCl
2
in many cases (Fig. 6). However, other
ions such as sodium, potassium, or lithium, all tested at 5 mM,
were much less effective (Fig. 6B). We conclude that the pres-
ence of magnesium is important for Bax-induced cytochrome c
release only after Bax is inserted in the mitochondria.
DISCUSSION
Bax, a proapoptotic protein that is absolutely required for
apoptosis in many neuronal cell types and in ovarian follicles
(7, 32), exerts at least part of its activity by triggering cyto-
chrome c release from mitochondria (9, 19, 21, 37, 41). The
mechanisms by which Bax stimulates cytochrome c efflux are
still unclear. In previous reports, several events occurring dur-
ing apoptosis at the level of the Bax protein, including trans-
location to mitochondria (15, 43), change in conformation (8),
dimerization (11, 13), and membrane integration (11, 13), have
been described. Despite this, the mechanisms responsible for
Bax insertion into the mitochondrial membrane and the local-
ization of Bax within mitochondria are still unclear. Here we
show that Bid, a BH3-only Bax-interacting protein, is able to
trigger Bax integration in the outer mitochondrial membrane.
We have reported previously that Bid is able to induce a
change in Bax conformation leading to the exposure of its
N-terminal domain. Interestingly, the N-terminal domain of
Bax exerts a repressing activity on the targeting of Bax to
mitochondrial membranes, possibly by interfering with the hy-
drophobic C-terminal membrane-anchoring domain (11). A
Bid-induced change in Bax conformation may therefore rep-
resent the first of a series of events leading to Bax insertion
into membranes. Bax dimerization appears to be another crit-
ical event for Bax integration in membranes, since enforced
dimers of Bax specifically target mitochondria and trigger
FIG. 7. Model for the activation of Bax by Bid during apoptosis. Following an apoptotic stimulus, Bid binds to Bax and triggers a change in the conformation of
Bax. As a result, Bax dimerizes (or oligomerizes) and inserts into the outer mitochondrial membrane, which results in cytochrome c (Cyt. c) release from mitochondria.
VOL. 20, 2000 Bid-INDUCED BAX INSERTION IN MITOCHONDRIA 933

some mitochondrial dysfunction (13). However, in this study,
the Bax-enforced dimers failed to trigger cytochrome c release
from mitochondria, suggesting that Bax dimers may not rep-
resent the correct quaternary structure either for membrane
insertion or for cytochrome c release (13). Consistent with the
data reported by Gross et al. (13), our results suggest that
active Bax homodimers or oligomers form following interac-
tion with Bid. However, we cannot exclude the possibility that
Bax also forms large complexes with other proteins. Both Bcl-2
and Bcl-x
L
were able to prevent Bax oligomerization and in-
sertion, which is consistent with our previous results that both
antiapoptotic proteins counteract the Bid-induced change of
conformation of Bax by binding directly with Bax. Neverthe-
less, it remains possible that Bcl-x
L
or Bcl-2 inhibits Bax-in-
duced cytochrome c release independently of binding to Bax
(22), by forming channels which would counterbalance Bax
action.
The mechanisms by which Bax, following its membrane in-
sertion, triggers cytochrome c release from mitochondria are
still unclear. It has been proposed that Bax may stimulate the
opening of the permeability transition pore (PTP) through
interaction with the adenine nucleotide translocator (30). As a
result of PTP opening, mitochondria would swell, leading to
rupture of the outer mitochondrial membrane and passive
release of cytochrome c (23, 24). There are, however, types of
apoptosis in which a shrinkage rather than a swelling of mito-
chondria has been reported (18, 27–29, 46), and in sympathetic
neurons deprived of nerve growth factor, the release of cyto-
chrome c can be reversed if caspase activation is prevented
(29). Moreover, we have found that cyclosporin A, an inhibitor
of the PTP, was unable to inhibit Bid-induced Bax insertion
into mitochondrial membranes (results not shown) and Bax-
induced cytochrome c release from mitochondria (9). To-
gether, these studies suggest that the release of cytochrome c
from mitochondria may be a well-controlled event, occurring,
under certain circumstances, independently of the opening of
the PTP. One possible mechanism is that Bax forms a channel
large enough to allow the release of cytochrome c as well as
other proteins of a similar size. The integration of Bax within
membranes would be the first of a series of events leading to
channel formation. Nonetheless, we report that Bax insertion
into mitochondria is not sufficient to trigger cytochrome c
release. Indeed, in the absence of Mg
2⫹
, cytochrome c was not
released despite Bax insertion into the mitochondrial mem-
branes.
All the experiments described above were performed with
full-length Bid, which has been reported previously to translo-
cate to mitochondria during staurosporine-induced apoptosis
of HeLa cells (8). In contrast to Bax, we found that Bid does
not integrate in the mitochondrial membrane. This is in con-
trast to what was found after cleavage of Bid by caspase 8
during interleukin-3 deprivation of FL5.12 cells. In these stud-
ies, the cleaved form of Bid (p15 BID) translocated to mito-
chondria and became an integral membrane protein (14). We
have found that Bid cleaved by caspase 8 is at least 10-fold
more efficient in triggering Bax insertion into the mitochon-
drial membrane and cytochrome c efflux from mitochondria
(R. Eskes et al., unpublished data).
Bax integration was shown to occur in the outer mitochon-
drial membrane as assessed by digitonin extraction. We have
not been able to detect any translocation of Bax from the outer
to the inner mitochondrial membrane during the process of
cytochrome c release. This contrasts with the results of others,
who reported Bax redistribution from the outer to the inner
mitochondrial membrane following treatment of mitochondria
with atractyloside (30). Our results suggest strongly that Bax
exerts its apoptotic function by being physically present in the
outer mitochondrial membrane. This localization is compatible
with the ability of Bax to interact with the outer mitochondrial
membrane channel VDAC, as previously reported (35). This
localization would also be consistent with the hypothesis that
Bax itself may form a channel to allow the release of cyto-
chrome c. Nevertheless, a fraction of Bax, as well as Bak and
Bcl-x
L
, was found to be integrated in the outer mitochondrial
membrane of mitochondria isolated from nonapoptotic cells. It
is possible that in the absence of an apoptotic stimulus, both
Bax and Bak are inactivated in the membrane by forming
Bax–Bcl-x
L
or Bak–Bcl-x
L
heterodimers, although we cannot
exclude the possibility that these proteins are also functional
and play a physiological role in nonapoptotic cells.
In summary, our results are consistent with a model of cel-
lular apoptosis in which Bid interacts with Bax to trigger a
change in Bax conformation leading to dimerization (or oli-
gomerization) and integration into the outer mitochondrial
membrane (Fig. 7).
ACKNOWLEDGMENTS
We thank S. Arkinstall and K. Maundrell for critical reading of the
manuscript, C. Herbert for artwork, and T. Wells for encouraging
support.
Part of this work was also supported by grants from the European
Community (Biotech grant BIO4CT96 0774 to J.-C. Martinou).
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