From the Serono Pharmaceutical Research Institute, Serono International S.A., 14 chemin des Aulx, CH-1228 Plan-les Ouates, Geneva, Switzerland
Received for publication, November 30, 2000, and in revised form, December 27, 2000
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ABSTRACT |
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Bax is a Bcl-2 family protein with proapoptotic
activity, which has been shown to trigger cytochrome c
release from mitochondria both in vitro and in
vivo. In control HeLa cells, Bax is present in the cytosol and
weakly associated with mitochondria as a monomer with an apparent
molecular mass of 20,000 Da. After treatment of the HeLa cells
with the apoptosis inducer staurosporine or UV irradiation, Bax
associated with mitochondria is present as two large molecular weight
oligomers/complexes of 96,000 and 260,000 Da, which are integrated into
the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and
insertion into the mitochondrial membrane. The outer mitochondrial
membrane protein voltage-dependent anion channel and
the inner mitochondrial membrane protein adenosine nucleotide
translocator do not coelute with the large molecular weight Bax
oligomers/complexes on gel filtration. Bax oligomerization appears to
be required for its proapoptotic activity, and the Bax oligomer/complex
might constitute the structural entirety of the cytochrome
c-conducting channel in the outer mitochondrial membrane.
Apoptosis is mediated through two major pathways, the death
receptor pathway and the mitochondrial pathway (1). The mitochondrial pathway is controlled and regulated by the Bcl-2 family of proteins (2-4). This protein family can be divided into antiapoptotic (Bcl-2,
Bcl-XL, Bcl-w, Mcl-1, A1) and proapoptotic (Bax, Bak, Bok/Mtd, Bcl-Xs, Bid, Bad, Bik/Nbk, Bim, Blk) members (5-7). Although
the overall amino acid sequence homology between the family members is
relatively low, they contain highly conserved domains, referred to as
Bcl-2 homology domains (BH1 to -4). The activity of these proteins
appears to be regulated, at least partly, by formation of homo- and
heterocomplexes (8-13). The conserved BH domains are involved in these
interactions. In the proapoptotic proteins, Bax and Bak, the BH3 domain
is essential for complex formation as well as for their "killing"
effect (4, 14-17). The hydrophobic C-terminal domain present in some
of the proteins has been implicated in targeting the proteins to
intracellular membranes (11, 18). Thus, many members of the family
including Bcl-2, Bcl-XL, Bak, and Bax are acting at the
level of mitochondria.
It now appears clear that mitochondria have an important function in at
least some apoptotic signaling cascades (3, 19- 20). Following
a death stimulus, many proteins from the mitochondrial intramembrane space, including cytochrome c, adenylate
kinase, Smac/DIABLO, procaspases, and apoptosis-inducing factor, have been reported to be released into the cytosol (21-26). The release of
cytochrome c has been shown to be a fast process, depleting the mitochondria of cytochrome c within a few minutes (27). In the cytosol, cytochrome c forms a complex with Apaf-1,
dATP, and procaspase 9 (28). The complex formation leads to caspase 9 activation followed by downstream activation of other caspases, ultimately leading to cell death. Mounting evidence points to Bax and other proapoptotic family members as the central regulators of
the release of proteins from the mitochondrial intramembrane space.
Overexpression of Bax in cells or the addition of purified recombinant
Bax directly to isolated mitochondria triggers the release of
cytochrome c (29-32). However, the mechanism through which
Bax triggers the permeability of the outer mitochondrial membrane is unclear.
The three-dimensional solution structure of the full-length Bax
protein has recently been solved by NMR (33). The Bax structure shows a
high similarity to the overall conformation of the two other Bcl-2
family proteins for which structural information is available, the
antiapoptotic protein Bcl-XL and the BH3 domain-only protein Bid (34-36). The proteins contain central hydrophobic helices ( We have shown that in contrast to oligomeric Bax, monomeric recombinant
Bax cannot form channels in liposomes nor trigger cytochrome
c release from isolated mitochondria (41). Recently, Bax
tetramers were shown to form a channel large enough to allow the
release of cytochrome c from liposomes (42). Here we show that, in cultured cells exposed to the apoptosis inducer
staurosporine or UV irradiation, Bax forms oligomers, which possibly
form complexes with yet unidentified mitochondrial membrane proteins.
The Bax oligomers are found inserted into the mitochondrial membrane. Moreover, we show that in the presence of Bcl-2, Bax oligomer formation
and insertion into the mitochondrial membrane is inhibited.
The Superdex 200 (16/60) column was from Amersham Pharmacia
Biotech, and 14% polyacrylamide gels and 10% NuPage gels were from Novex (San Diego, CA).
CHAPS1 was from Roche
Molecular Biochemicals, and Triton X-100 was from Fluka. Polyclonal
anti-Bax antibodies were purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY), monoclonal anti-Bax antibodies were from R & D
Systems (Minneapolis, MN), monoclonal anti-Bcl-2 antibodies were from
Genosys (Cambridge, UK), polyclonal anti-Bcl-XL antibodies
were from Transduction Laboratories (Lexington, KY), monoclonal
anti-Bak antibodies were from Oncogene Research Products (Boston, MA),
monoclonal anti-VDAC and polyclonal anti-catalase antibodies were from
Calbiochem, monoclonal anti-HA antibodies were from Babco (Richmond,
CA), monoclonal anti-Myc and polyclonal anti-His antibodies were from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), monoclonal anti-COX
antibodies were from Molecular Probes, Inc. (Eugene, OR), monoclonal
anti-Golgi 58-kDa protein antibodies were from Sigma, and the
monoclonal anti-calnexin and anti-Hsp70 antibodies were from
Affinity Bioreagents (Golden, CO). The polyclonal rabbit anti-ANT
antibody was a kind gift from Prof. Theo Wallimann (43).
Cell Culture--
HEK cells, HeLa cells, and a stable HeLa cell
line constitutively overexpressing Bcl-2 (HeLa-Bcl-2) (44) were
cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 µg/ml streptomycin, and 50 IU/ml penicillin. Apoptosis was induced by culturing the cells in
medium containing 1 µM staurosporine for 16 h;
alternatively, the cells were UV irradiated (280 mJ cm Isolation of Mitochondria--
The cells suspended in MB
supplemented with complete protease inhibitor mixture (Roche Molecular
Biochemicals) were disrupted by passage through a 25G1 0.5 × 25 needle. The sample was passed through the needle five times and
subsequently centrifuged at 2000 × g for 3 min. The
supernatant was saved, and the pellet was resuspended in MB. The
breakage procedure was repeated four times. The combined supernatants
were centrifuged at 2000 × g for 3 min to remove
nuclei and unbroken cells. The supernatant was subsequently centrifuged
at 13,000 × g for 10 min. This supernatant was kept
and centrifuged at 100,000 × g for 30 min to give the cytosolic cell fraction. The pellet fraction, corresponding to the
mitochondrial fraction, was suspended in MB and recentrifuged at
13,000 × g for 10 min. Alternatively, the first
13,000 × g mitochondrial pellet was suspended in 4 ml
of MB, and 2-ml portions were layered on top of a discontinuous sucrose
gradient consisting of 20 ml of 1.2 M sucrose, 10 mM Hepes-NaOH, 1 mM EDTA, pH 7.5, on top of 17 ml of 1.6 M sucrose, 10 mM Hepes-NaOH, 1 mM EDTA, pH 7.5. The sample was centrifuged at 27,000 rpm
in a Beckman SW28 rotor for 2 h at 4 °C. Mitochondria were
recovered at the interface of 1.2 and 1.6 M sucrose and
washed in MB. All manipulations were carried out at 4 °C.
Treatment of Isolated Mitochondria with Recombinant Caspase
8-Cleaved Bid--
Isolated mitochondria (3 mg/ml) suspended in 1 ml
of KCl buffer (15 mM Hepes-NaOH, 125 mM KCl, 4 mM MgCl2, 5 mM
NaH2PO4, 0.5 mM EGTA, 5 mM succinate, pH 7.5) were incubated with 50 nM
recombinant caspase 8-cleaved Bid for 30 min at 30 °C. At the end of
the incubation, the sample was centrifuged at 13,000 × g for 10 min, and the mitochondria were washed with MB buffer.
Preparation of Mitochondrial Extracts--
The washed
mitochondrial fraction was suspended in MB containing 2% CHAPS or 2%
Triton X-100, incubated on ice for 1 h, sonicated, and centrifuged
at 100,000 × g for 30 min. The supernatant corresponds to the solubilized mitochondrial fraction. Protein concentrations were
determined using the Bio-Rad protein assay kit.
Sodium Carbonate Extraction of the Mitochondria--
The
mitochondrial pellet was suspended in 0.1 M
Na2CO3, pH 12, to a final protein concentration
of 3 mg/ml and incubated on ice for 20 min. At the end of the
incubation, the sample was centrifuged at 100,000 × g
for 1 h. The supernatant, which contains nonmembrane-integrated proteins, was saved, and CHAPS was added to a final concentration of
2%. The pellet was suspended in MB containing 2% CHAPS, incubated on
ice for 1 h, sonicated, and centrifuged at 100,000 × g for 30 min. The supernatant, which contains solubilized
integral membrane proteins, was saved.
Gel Filtration Analyses--
Gel filtrations were performed at
4 °C on a Superdex 200 (16/60) column equilibrated in 25 mM Hepes-NaOH, 300 mM NaCl, 0.2 mM
DTT, 2% (w/v) CHAPS, pH 7.5, and run at a flow rate of 1 ml/min. When
Triton extracts were analyzed, CHAPS was replaced by 2% Triton X-100.
The column was calibrated with gel filtration standard proteins from
Amersham Pharmacia Biotech giving the following elution volumes:
thyroglobulin (669,000 Da), 49.9 ml; ferritin (440,000 Da), 55.9 ml;
catalase (232,000 Da), 65.5 ml; BSA (67,000 Da), 77.5 ml; ovalbumin
(43,000 Da), 83.2 ml; chymotrypsinogen A (25,000 Da), 92.9 ml;
ribonuclease A (13,700 Da), 97.4 ml. The void volume of the column was
determined by blue Sepharose that eluted at 43.3 ml. A 500-µl sample
was loaded onto the column, and the eluate was monitored at 280 nm.
After 20 min elution (20 ml), fractions of 2 ml were collected,
and aliquots from the fractions were analyzed by Western blotting. The
samples were separated on 14% SDS-PAGE under reducing conditions and
transferred to polyvinylidene difluoride membranes. The proteins were
detected with the specific antibodies as indicated in the figures, and
the blots were developed with the ECL system from Amersham Pharmacia Biotech.
Transfection of HEK Cells with Tagged Bax--
Bax was tagged
with His, HA, and Myc at the N terminus, and the constructs were cloned
into the pC1 vector (Stratagene). HEK cells were then transfected with
the three different Bax constructs (Fugene6) (Roche Molecular
Biochemicals). The transfection efficiency was between 50 and 60%. 100 µM ZVAD was added 60 min prior to the addition of 1.6 µg of each of the Bax plasmids, and the cells were cultured for
16 h. At the end of the incubation, the cells were harvested, and
mitochondria were isolated by differential centrifugation as described above.
Bax Activation, Cross-linking, and
Immunoprecipitation--
Mitochondria isolated from transfected HEK
cells corresponding to 0.5 mg of mitochondrial protein was incubated
with 1 µM caspase 8-cleaved Bid in KCl buffer (15 mM Hepes-NaOH, 125 mM KCl, 4 mM
MgCl2, 5 mM NaH2PO4,
0.5 mM EGTA, 5 mM succinate, pH 7.5) for 15 min
at 30 °C. At the end of the incubation, the mitochondria were
recovered by centrifugation and resuspended in 400 µl of MB
containing 1 mM EGTA. The cross-linkers
bis-(sulfosuccinimidyl)suberate (Pierce) and disuccinimidyl suberate
(Pierce) were added to final concentrations of 2 mM, and
the sample was incubated for 30 min at room temperature. The reaction
was stopped by the addition of Tris-HCl, pH 8.0, to a final
concentration of 20 mM and subsequent incubation for 15 min
at room temperature. The mitochondria were recovered by centrifugation
and solubilized by incubation in radioimmune precipitation buffer (19 mM Tris-HCl, 140 mM NaCl, 1% Nonidet P-40,
0.1% SDS, 1% sodium deoxycholate) for 30 min at 4 °C. The sample
was centrifuged at 12,000 × g for 10 min, and the
supernatant was treated with 50 µl of protein G-Sepharose for 3 h at 4 °C. Protein G-Sepharose was removed by centrifugation, and 4 µg of anti-His antibody and 50 µl of protein G-Sepharose were added and incubated on rotation for 16 h at 4 °C. Protein G-Sepharose was recovered by centrifugation and washed four times with 400 µl of
radioimmune precipitation buffer. The washed Sepharose was suspended in
sample buffer, and the samples were analyzed on 10% NuPage gels in a
Mops running buffer system (Novex). Proteins were transferred to
nitrocellulose membranes and analyzed by Western blot with four
different antibodies, anti-HA, anti-Myc, anti-Bax, and anti-VDAL.
Cytosolic samples were centrifuged at 100,000 × g for
5 min and then treated with cross-linker and analyzed as above.
Recombinant Proteins--
Full-length Bax with a His tag at the
N terminus was expressed in Escherichia coli and purified as
previously described (45). Oligomerization was induced by disrupting
the bacteria in buffer containing 1% Triton X-100. During the
purification, Triton was exchanged for 1% octyl glucoside. The
purified protein was stored in 25 mM Hepes-NaOH, 0.2 mM DTT, 1% octyl glucoside, 30% glycerol, pH 7.5, at
Full-length Bid with a His tag at the N terminus was expressed in
E. coli and purified as previously described (46). The purified Bid was stored in 25 mM Tris-HCl, 0.1 mM DTT, 30% glycerol, pH 7.5, at Bax in Extracts from Control and Apoptotic HeLa
Cells--
Cultured HeLa cells have been used to study the quaternary
structure of Bax in apoptotic cells. Apoptosis was induced by culturing the cells in the presence of 1 µM staurosporine for
16 h or by UV irradiation. Consistent with published results, in
untreated HeLa cells, Bax was found both in the cytosol and associated
with the mitochondria (Fig. 1). In
apoptotic cells, Bax almost completely disappeared from the cytosol.
Although a small increase in Bax associated with the mitochondria was
seen in the apoptotic cells, this does not fully account for the
decreased cytosolic Bax concentration (Fig. 1), suggesting that
cytosolic Bax that is not translocated to the mitochondria is
eliminated in apoptotic cells, possibly through proteolysis (47).
Bax Quaternary Structure in Triton X-100 Mitochondrial Extracts
from Control and Apoptotic HeLa Cells--
To study the quaternary
structure of Bax associated with mitochondria from control and
apoptotic HeLa cells, mitochondrial proteins were extracted with 2%
Triton X-100 as described under "Materials and Methods." The
soluble mitochondrial extracts were analyzed on gel filtration, and the
fractions from the column were analyzed by Western blot with an
anti-Bax antibody. Gel filtration separates molecules according to the
Stokes radius and gives an estimate of their molecular weights and thus
of their quaternary structure. Bax extracted from either control or
apoptotic HeLa cell mitochondria eluted as a single peak at fractions
20-24, corresponding to a molecular mass of 260,000 Da (Fig.
2). Monomeric Bax was found neither in
control nor apoptotic mitochondrial extract. Furthermore, analysis of
other mitochondrial membrane proteins that have been suggested to
interact with Bax, including Bak, Bcl-XL, and VDAC, all
coeluted with Bax as high molecular weight complexes in fractions
20-24. Again, no difference was observed between mitochondrial
extracts from control and apoptotic cells (Fig. 2). However, the
mitochondrial membrane protein Cox IV eluted with a main peak in
fractions 28-30 and a minor peak in fractions 22-24. The void volume
of the column was at fraction 12.
Triton X-100 has been reported to induce dimerization of Bax and other
Bcl-2 family members (48). In a previous study, we reported that Triton
X-100 was able to trigger oligomerization of soluble monomeric Bax
(41). Therefore, Triton might have induced the oligomerization of Bax
and possibly other proteins extracted from the mitochondrial membrane,
masking any differences in protein quaternary structure between control
and apoptotic cells. Previous studies on the ability of various
detergents to trigger Bax oligomerization showed that in contrast to
Triton X-100 and octyl glucoside, CHAPS had no effect on Bax quaternary structure (41). Therefore, we examined whether CHAPS could extract Bax
from mitochondria. As shown in Fig. 3,
2% CHAPS was as efficient as 2% Triton X-100 in extracting Bax from
the mitochondrial membrane of both control and apoptotic cells. This is
an important control to ensure that we were not extracting and
analyzing only a subpopulation of Bax from the mitochondria.
Bax Quaternary Structure in CHAPS Mitochondrial Extracts from
Control and Apoptotic HeLa Cells--
Mitochondria were isolated by
differential centrifugation from control and apoptotic HeLa cells,
proteins were extracted with 2% CHAPS as described under "Materials
and Methods," and the soluble mitochondrial extracts were analyzed on
gel filtration. In the mitochondrial extract from control cells, Bax
eluted in fractions 36-40. This corresponds to a molecular mass of
20,000 Da, which is close to the theoretical calculated molecular mass
of monomeric Bax, 22,000 Da (Fig.
4A). In contrast, in the
mitochondrial extract from cells where apoptosis had been induced by
staurosporine treatment, Bax eluted mainly as large oligomers in
fractions 20-28. Two peaks with estimated molecular masses of 260,000 and 96,000 Da were consistently detected (n = 9) (Fig.
4A). The amount of monomeric Bax eluting in fractions 36-38
varied between preparations and could depend on the percentage of cells
that had entered apoptosis at the time of harvest. Identical results
were obtained with a monoclonal anti-Bax antibody, confirming that the
protein detected was indeed Bax (results not shown).
Western blot analysis with marker antibodies for various organelles
showed that the mitochondria isolated by differential centrifugation
was contaminated mainly by ER (Fig. 5).
To ensure that the Bax oligomers were associated with the mitochondria, the mitochondrial fraction was further purified on a sucrose gradient; this purification step improved the purity of the preparation. The
sucrose gradient-purified mitochondria confirmed the result; oligomeric
Bax (fractions 20-28) was found in the mitochondrial extract from
apoptotic cells, whereas monomeric Bax (fractions 36-40) was present
in both apoptotic and control mitochondrial extracts (Fig.
4B).
In addition, inducing apoptosis by exposing HeLa cells to UV
irradiation also induced oligomerization of Bax associated with the
mitochondria. Similar to mitochondrial extract from
staurosporine-treated cells, two peaks of Bax oligomers at fractions
20-22 (Mr 260,000) and fractions 26-28
(Mr 96,000) were detected on gel
filtration (Fig. 6). Furthermore, we
treated isolated HeLa mitochondria with 50 nM caspase
8-cleaved Bid for 30 min. The extract from the Bid-treated mitochondria
contained the same Bax oligomers as mitochondria from
staurosporine- or UV-treated HeLa cells (Fig. 6). Thus, inducing apoptosis by three different stimuli all resulted in Bax
oligomerization.
Composition of the Bax Oligomers--
In addition to its
interactions with Bcl-2 family members, Bax has also been reported to
interact with other mitochondrial membrane proteins. Interactions with
the outer mitochondrial membrane protein VDAC and the inner membrane
protein ANT, both part of the permeability transition pore, have been
reported (49, 50). To investigate whether the Bax oligomers contained
these proteins, the fractions from the gel filtration column were
analyzed for Bak, Bcl-XL, VDAC, and ANT (Fig.
4A).
Two major peaks of Bak were detected, eluting in fractions 24-26 and
30-32. A low amount was also present over a wide molecular weight
range (fractions 14-32), which suggests various forms of oligomers
and/or complexes. However, no difference in the distribution between
control and apoptotic cells was detected. Thus, since the Bax oligomers
are not present in the mitochondrial membrane of nonapoptotic cells,
interactions between Bak and the Bax oligomers are unlikely.
Bcl-XL was similarly detected over a large range (fractions
16-32) with a major peak in fractions 26-32. As for Bak, no
difference between control and apoptotic cells was detected.
VDAC eluted after the Bax oligomers (fractions 20-28) as a sharp peak
at fractions 30-32 (48,000 Da) in the mitochondrial extract from both
control and apoptotic cells. ANT eluted before the Bax oligomers at
fractions 14-16 (650,000 Da). No difference was detected between the
control and apoptotic mitochondrial extracts. These results show that
neither VDAC nor ANT is part of the Bax oligomers extracted from
mitochondria of apoptotic cells.
The Bax Oligomers--
The elution profile of oligomeric
recombinant Bax from the gel filtration column showed two peaks eluting
at fractions 22-24 (Mr 160,000) and 27-29
(Mr 80,000) (Fig. 6). This indicates that recombinant Bax forms two distinct oligomers containing different numbers of monomeric units. It appears that the larger oligomer (160,000 Da) could be a dimer of the smaller oligomer (80,000 Da). In
the mitochondrial extract from apoptotic HeLa cells, we also detected
two Bax peaks, the first peak eluting at fractions 20-22
(Mr 260,000) and the second peak at fractions
26-28 (Mr 96,000) (Figs. 4 and 6). The Bax
oligomers from apoptotic mitochondrial extracts eluted at higher
molecular weights compared with the recombinant protein. This
difference in apparent molecular weight might indicate that in the
mitochondrial membrane the Bax oligomers form stable complexes with as
yet unidentified proteins.
To further investigate the composition of the Bax oligomers, we
overexpressed Bax fused to three different epitope tags (HA, Myc, and
His) in HEK cells. Since overexpression of Bax induces apoptosis, the
transfected cells were cultured in the presence of the caspase
inhibitor ZVAD to prevent transfected cells from dying and thus
selecting for nontransfected cells. We verified by Western blot that
the tagged Bax was localized, like endogenous Bax, both in the cytosol
and the mitochondria (Fig.
7A). Mitochondria were then
isolated from transfected and untransfected HEK cells, membrane
proteins were extracted with 2% CHAPS, and the extracts were analyzed
on gel filtration. To the cytosol from the transfected cells, 2% CHAPS
was added, and the samples were analyzed on gel filtration. In the
mitochondrial extract from untransfected cells and in the cytosol from
transfected cells, only Bax monomers were detected (Fig.
8). However, in the mitochondrial extract
from transfected cells (apoptotic cells), Bax oligomers similar to those in staurosporine or UV-irradiated HeLa cells were detected (Fig.
8). The majority of Bax in the oligomers was endogenous untagged Bax;
thus, with the anti-Bax antibodies we did not detect the upper band
corresponding to HA- and Myc-tagged Bax. To ensure that the tagged
proteins were part of the oligomers, we further analyzed the fractions
of the mitochondria extract from the transfected cells for the presence
of tagged Bax. Although the concentration of the tagged proteins was
low, we detected all three tags in the fractions containing Bax
oligomers, fractions 18-30 (Fig. 8). To further investigate the
composition of the Bax oligomers, we performed cross-linking
experiments on isolated mitochondria from the Bax-transfected cells as
described under "Materials and Methods." Bax was subsequently
immunoprecipitated with anti-His antibodies, and the precipitated
sample was analyzed on Western blot with anti-Bax, anti-HA, and
anti-Myc antibodies. As seen in Fig. 7B, Bax oligomers
precipitated with anti-His antibodies were reactive to Bax, HA, and Myc
antibodies. On the contrary, no immunoreactivity was detected with
anti-VDAC antibodies. These results show that the Bax oligomers from
mitochondria of apoptotic cells are composed of multiple Bax
monomers and are not only Bax monomers in complex with other proteins.
Bax oligomers were not detected in the cytosol (Fig. 7B). It
has been shown that in unactivated monomeric Bax the N terminus is not
exposed and not immunoreactive (46); this presumably explains why we
did not precipitate the monomeric N-terminal His-tagged Bax in the
cytosol. The molecular weights of the Bax-reactive bands in the
mitochondrial extract were estimated, and the corresponding number of
Bax molecules in the complexes were calculated (Fig. 7C).
Bands corresponding to Bax mono-, di-, tri-, and tetramers were
identified. Although higher molecular weight bands can be detected, the
molecular weights cannot be estimated in this region of the SDS-PAGE
separation. One band (Fig. 7C, band d)
between a Bax trimer and tetramer could correspond to a complex with an
unidentified protein of ~15 kDa.
Bax Localization in the Mitochondria--
We have recently shown
that Bid treatment of isolated HeLa mitochondria results in the
insertion of Bax into the outer mitochondrial membrane (51). Here we
investigated whether the change in Bax quaternary structure was
required for integration into the mitochondrial membrane during
apoptosis. We treated mitochondria from control and apoptotic cells
with 0.1 M Na2CO3, pH 12, which
solubilizes proteins attached to the membrane, whereas proteins
integrated into the lipid membrane remain associated with the membrane
fraction. After this treatment, the soluble (attached proteins) and the membrane fraction (integrated proteins) were analyzed by Western blot.
We confirmed the findings by Eskes et al. (51) that
in control cells Bax was only attached to the mitochondrial membrane, whereas in the apoptotic cells most of the protein was inserted into
the membrane (Fig. 9A). Bak
and Bcl-XL were integrated into the membrane in both
control and apoptotic cells. As expected, the membrane protein VDAC was
found to be integrated into the membrane in both samples. As shown in
Fig. 4, mitochondria from apoptotic cells contained both monomeric and
oligomeric Bax. We then investigated the nature of the quaternary
structure of membrane-integrated Bax. As shown in Fig. 9B,
membrane-integrated Bax was present as the oligomer. A low amount of
monomeric Bax was detected in the sodium carbonate-washed mitochondrial
pellet. However, this was also detected in the control cells and is
presumably a contamination of attached protein that was not completely
removed.
Bax Oligomerization Does Not Appear in Bcl-2-overexpressing
Cells--
A HeLa cell line overexpressing Bcl-2 (HeLa-Bcl-2) is
resistant to staurosporine-induced apoptosis (44). We treated these cells with staurosporine for 16 h and analyzed Bax associated with
the mitochondria of treated and control cells. Both control and
staurosporine-treated cells contained only Bax monomers. No Bax
oligomers were detected in mitochondria isolated from
staurosporine-treated cells (Fig. 10).
Bcl-2 eluted in fractions 26-30 in both control and
staurosporine-treated cells. No difference in the elution profiles of
Bak, Bcl-XL, and VDAC was detected between control and
apoptotic cells. To determine whether Bax had been inserted into the
mitochondrial membrane in the staurosporine-treated cells, we treated
the isolated mitochondria with sodium carbonate. As seen in Fig.
9C, the monomeric Bax was only found attached to the
mitochondrial membrane; no Bax was inserted in the membrane of this
staurosporine-treated cells. Bcl-2 was found to be inserted into the
mitochondrial membranes from both control and staurosporine-treated cells (Fig. 9C).
The release of cytochrome c from mitochondria has been
shown to play a crucial role in many apoptotic signaling cascades
through the activation of the downstream effector caspases (28).
Several studies point to Bax as a trigger of cytochrome c
release (29-31). Based on the ability of this protein to form channels
in synthetic lipid membranes, several models have been proposed that
would allow Bax to form a cytochrome c-conducting channel.
Bax oligomerization and insertion in the outer mitochondrial membrane
resulting in a "Bax-only" channel or hybrid channels formed by Bax
binding to VDAC or ANT represent alternative possible mechanisms (52, 53). Thus, at the molecular level, it remains unclear how Bax triggers
cytochrome c release.
Here, using gel filtration analysis and cross-linking, we show the
formation of high molecular weight Bax oligomers in mitochondrial membranes of apoptotic cells. Previous cross-linking experiments of Bax
in mitochondrial membranes, followed by immunoprecipitation and Western
blot analysis, have revealed the existence of several Bax-immunoreactive bands, suggesting the presence of Bax oligomers (51,
54). The use of Bax fused to various tags allowed us, for the first
time, to demonstrate the formation of Bax oligomers during apoptosis. A
direct relationship between formation of large Bax oligomers and
apoptosis was further demonstrated by gel filtration analysis. Analysis
of membrane proteins requires solubilization with detergents. The
choice of detergent is critical, since the protein quaternary structure
and complex interactions may be affected by the detergent (55, 56). Hsu
and Youle (48) have reported that cytosolic Bax from thymic cells
displayed different conformational states that depended on the
detergent to which they were exposed. That certain detergents induce
significant conformational changes in Bax was shown in a recent
publication by Suzuki et al. (33). In the study by Suzuki
et al. (33), results from NMR experiments support the
formation of large Bax oligomers. In agreement with these data, we
found that Triton X-100, but not CHAPS, was able to trigger
oligomerization of pure recombinant monomeric Bax (rBax) (41).
Importantly, unlike Bax, Bcl-2 and Bcl-XL monomers did not
oligomerize in the presence of various detergents, including Triton
X-100 (41, 57). Indeed, as expected, when we extracted mitochondrial
membrane proteins from control and apoptotic cells with Triton X-100,
Bax consistently eluted on gel filtration chromatography as a high
molecular weight oligomer. In fact, several proteins analyzed,
including Bak, Bcl-XL, and a major component of the permeability transition pore, VDAC, that has been reported to interact
with Bax, coeluted with Bax in a discrete peak corresponding to a
molecular mass of 260,000 Da. However, the mitochondrial membrane
protein Cox IV did not coelute with the 260,000-Da complexes, indicating that the extraction procedure did not induce a general protein aggregation or only a fragmentation of the mitochondrial membrane.
The fact that CHAPS does not trigger oligomerization of recombinant and
cytosolic monomeric Bax indicated that CHAPS was a more appropriate
detergent to study Bax oligomerization in mitochondria during
apoptosis. Using this detergent, we found that while mitochondrial Bax
from control cells eluted as a monomer on gel filtration, the protein
from mitochondria of apoptotic cells consistently eluted as large
molecular weight oligomers, which were distributed in two major peaks
corresponding to masses of 260,000 and 96,000 Da. This Bax elution
profile was obtained in mitochondrial extracts from HeLa cells
undergoing apoptosis induced by either staurosporine or UV irradiation.
Moreover, we found that the addition of caspase 8-cut Bid, a Bax
activator, to mitochondria isolated from HeLa cells was able to trigger
the formation of similar large molecular weight Bax oligomers. These
data suggest that oligomerization of Bax and its ability to form large
oligomers may be a phenomenon common to several apoptotic pathways
involving Bax. The Bax oligomers were always tightly inserted in the
mitochondrial membranes, and they were never detected in the cytosol,
suggesting that they may form at or within the outer mitochondrial
membrane. However, we cannot rule out the possibility that these large
Bax oligomers may form outside of mitochondria and, once formed,
rapidly insert into the outer mitochondrial membrane, preventing their
cytosolic detection. This latter hypothesis would be consistent with
earlier data showing that Bax translocate from the cytosol to
mitochondria during apoptosis (58-60) and that enforced dimerization
of Bax is accompanied by its insertion in the mitochondrial membrane (54). However, in the latter study, the Bax-enforced dimers failed to
trigger cytochrome c release from mitochondria, suggesting that Bax dimers are not the active quaternary structure. A recent study
shows that Bax is able to trigger the release of cytochrome c from liposome. The quaternary structure of the cytochrome
c-conducting Bax channels was estimated to be a tetramer
(42).
Interestingly, we found that in Bcl-2-overexpressing cells that are
resistant to staurosporine-induced apoptosis (44), Bax oligomerization
did not occur. This result is consistent with our previous data showing
that Bcl-2 prevents the conformational change of Bax, which precedes
its activation and membrane insertion (46, 51). In the
Bcl-2-overexpressing cells, Bcl-2 was found both in the cytosol and
inserted in the mitochondrial membrane. It is unclear which of these
two Bcl-2 pools prevents the conformational change that Bax undergoes
during apoptosis.
Recently, we have shown that soluble monomers of rBax isolated from
E. coli in the absence of detergent fail to display channel activity in liposomes and are unable to trigger cytochrome c
release from liver mitochondria (41). However, when rBax is extracted from E. coli membranes with CHAPS, it is recovered as
oligomers. Importantly, these oligomers display channel activity,
indicating that CHAPS extraction is compatible with the preservation of
Bax activity (data not shown). Strikingly, when we analyzed pure rBax extracted from E. coli membranes with CHAPS or Triton X-100
by gel filtration, two oligomer populations of 160,000 and 80,000 Da
were detected. The significantly higher molecular mass of the Bax
oligomers from mitochondrial membranes compared with the rBax oligomers
suggests either a difference in the number of Bax subunits composing
the oligomers or an association with other mitochondrial proteins.
In addition, we used gel filtration to examine the mitochondrial
membrane extracts for the presence of other Bcl-2 family members (Bak,
Bcl-XL, Bcl-2) as well as for VDAC and ANT. We observed that neither VDAC nor ANT copurified with Bax. Moreover, VDAC and ANT
were eluted in different fractions. This result is consistent with
previous data that showed that when ANT was extracted with CHAPS it was
recovered bound to cyclophilin D alone, whereas in Triton X-100
extracts, the complex contained an equimolar amount of VDAC (61, 62).
Since there is the possibility that, in the presence of CHAPS,
interactions between Bax, ANT, and VDAC do not withstand the
extraction procedure, we cannot exclude the possibility that these
proteins interact within membranes. However, in contrast to previous
results (49, 50), we have never been able to demonstrate such
interactions by cross-linking experiments (51) or by
immunoprecipitation (Fig. 7B and data not shown). These data
suggest that even within membranes, interactions between Bax and ANT or
Bax and VDAC may be weak or transient or, alternatively, that only
minor amounts of these proteins interact with each other at any one time.
It is important to note that, in contrast to Bax, the quaternary
structures of Bak, Bcl-XL, Bcl-2, VDAC, and ANT do not
change significantly during apoptosis. All proteins appear to be part of large molecular weight complexes. Interactions between Bcl-2 and
VDAC or Bcl-XL and Bak have previously been reported (49, 63, 64). The copurification of Bcl-2 with VDAC and of
Bcl-XL with Bak is consistent with these data. However,
copurification does not necessarily indicate direct interactions
between the proteins.
In conclusion, we have demonstrated the formation of two high molecular
weight Bax oligomers in mitochondrial membranes of cells undergoing
apoptosis. The formation of these Bax oligomers involves a series of
events, including a change in Bax conformation triggered by Bid or
other "BH3-only" proteins (46) and insertion into the outer
mitochondrial membrane (51). The exact protein composition of these
oligomers now remains to be determined, although we already know that
the core structure is composed of several Bax subunits. Moreover,
it remains to be determined whether these large Bax oligomers
constitute the cytochrome c-conducting channel in mitochondria.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5 and
6) surrounded by amphipathic helices.
Of the three proteins, the Bax structure is the only one that contains
the C-terminal hydrophobic domain. This domain forms a helix (
9)
that protects the BH3-containing hydrophobic cleft on the protein. The
structures of the Bcl-2 proteins are reminiscent of diphtheria toxin
and the colicins A and E1. These toxins are pore-forming proteins that
function as membrane channels that allow passage of ions or small
polypeptides. Bax and other Bcl-2 family proteins have been shown to
possess channel-forming activity in artificial membranes (37-40).
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2) and subsequently cultured for 16 h.
Control cells were cultured in the absence of staurosporine or without
UV irradiation. At the end of the treatment, the cells were harvested
in phosphate-buffered saline containing 1 mM EDTA,
recovered by centrifugation at 750 × g for 10 min,
washed, and suspended in mitochondrial buffer (MB) (210 mM
manitol, 70 mM sucrose, 1 mM EDTA, 10 mM Hepes-NaOH, pH 7.5).
80 °C.
80 °C. Caspase
8-cleaved Bid was generated by mixing 200 µl of Bid (12.4 mg/ml) with
200 µl of caspase cleavage buffer (50 mM Hepes-NaOH, 10 mM DTT, 100 mM NaCl, 10% sucrose, pH 7.5) and
1 µl of caspase 8 (3 mg/ml), and the sample was incubated for 2 h at 20 °C. Over 95% of Bid was cleaved as estimated by SDS-PAGE.
Cleaved Bid was separated from caspase 8 by binding to
Ni2+-nitrilotriacetic acid-agarose; step-eluted by 100 mM imidazole; dialyzed into 25 mM Tris-HCl, 0.1 mM DTT, 30% glycerol, pH 7.5; and stored at
80 °C.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Bax in cytosol and mitochondria from control
and apoptotic HeLa cells. The cells were broken in MB
buffer, and the cytosol and mitochondrial fractions were isolated as
described. The cytosolic (Cyt) and the mitochondrial
(Mit) samples (1 µg of total protein) were analyzed on
SDS-PAGE and Western blot with anti-Bax antibodies.
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Fig. 2.
Analysis of Triton X-100 mitochondrial
extract from control and apoptotic HeLa cells by gel filtration on
Superdex 200. Mitochondria were isolated and treated with 2%
Triton X-100. Solubilized proteins were recovered by centrifugation at
100,000 × g for 30 min. 500 µl of the soluble
fraction containing 2 mg of protein was loaded on a Superdex 200 column
(16/60) equilibrated in 25 mM Hepes-NaOH, 300 mM NaCl, 0.2 mM DTT, 2% (w/v) Triton X-100, pH
7.5, and eluted at a flow rate of 1 ml/min. Fractions of 2 ml were
collected, and every second fraction was analyzed by SDS-PAGE and
Western blot with antibodies against, Bax, Bak, Bcl-XL,
VDAC, and Cox IV.
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Fig. 3.
Extraction of Bax from mitochondrial
membranes. Mitochondria isolated from control and apoptotic cells
were treated with either 2% CHAPS or 2% Triton X-100. The samples
were centrifuged at 100,000 × g for 30 min, and the
soluble and pellet fractions (1 µg of total protein) were analyzed on
SDS-PAGE and Western blot with anti-Bax antibodies. Mit.,
total mitochondria; Mit. Sup., solubilized fraction;
Mit. pellet, nonsolubilized fraction. C, CHAPS;
T, Triton X-100.
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Fig. 4.
Analysis of CHAPS mitochondrial extract from
control and apoptotic HeLa cells by gel filtration on Superdex
200. A, mitochondria were isolated by differential
centrifugation and treated with 2% CHAPS. Solubilized proteins were
recovered by centrifugation at 100,000 × g for 30 min.
500 µl of the soluble fraction containing 2 mg of protein was loaded
on a Superdex 200 column (16/60) equilibrated in 25 mM
Hepes-NaOH, 300 mM NaCl, 0.2 mM DTT, 2% (w/v)
CHAPS, pH 7.5, and eluted at a flow rate of 1 ml/min. Fractions of 2 ml
were collected, and every second fraction was analyzed by SDS-PAGE and
Western blot with antibodies against Bax, Bak, Bcl-XL,
VDAC, and ANT. B, mitochondria were isolated on a sucrose
gradient and subsequently treated as described for A. Every
second fraction from the gel filtration column was analyzed by Western
blot with antibodies against Bax.
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Fig. 5.
Purity of the mitochondria preparations.
Samples of crude HeLa cell extract (C), mitochondria
isolated by differential centrifugation (D), and
mitochondria isolated by sucrose gradient (G) from control
and apoptotic (staurosporine treated) cells corresponding to 1 µg of
total protein were analyzed by SDS-PAGE and Western blot with
antibodies against Bax, Hsp70 (mitochondria marker), calnexin (ER
marker), Golgi 58-kDa protein (Golgi marker), and catalase (peroxisome
marker).
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Fig. 6.
The Bax oligomers. Oligomeric
recombinant full-length Bax (F.L. Bax) and CHAPS-solubilized
mitochondrial extracts from staurosporine-treated (Bax HeLa
Staurosporine) and UV-irradiated HeLa cells and isolated HeLa
mitochondria treated with 50 nM caspase 8-cut Bid were
analyzed on a Superdex 200 column (16/60) equilibrated in 25 mM Hepes-NaOH, 300 mM NaCl, 0.2 mM
DTT, 2% (w/v) CHAPS, pH 7.5, and eluted at a flow rate of 1 ml/min.
Fractions of 2 ml were collected and analyzed by SDS-PAGE and Western
blot with antibodies against Bax. The top part of
the figure shows the scanned profiles of the recombinant
full-length Bax and Bax HeLa staurosporine Western blots.
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Fig. 7.
Composition of the Bax oligomers. HEK
cells were cotransfected with Bax tagged with HA, Myc, and His at the N
terminus. A, samples of the cytosol and mitochondria
fraction corresponding to 10 µg of total protein were analyzed on
Western blot with antibodies against, Bax, HA, Myc, and His.
B, cytosol and mitochondria were isolated and treated with
the cross-linkers bis-(sulfosuccinimidyl)suberate and
disuccinimidyl suberate. After cross-linking, mitochondrial proteins
were solubilized, and Bax was immunoprecipitated with anti-His
antibodies from the solubilized mitochondrial and the cytosolic
fractions. The immunoprecipitated samples were analyzed by reducing
SDS-PAGE and Western blot with anti-Bax, anti-HA, anti-Myc, and
anti-VDAC antibodies. C, The molecular weights of the
Bax-immunoreactive bands (a-e) were estimated, and the
corresponding number of Bax molecules were calculated.
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Fig. 8.
Analysis of CHAPS mitochondrial extract from
HEK cells by gel filtration on Superdex 200. Mitochondria from
untransfected HEK cells and HEK cells transfected with HA-, Myc-, and
His-tagged Bax were isolated and treated with 2% CHAPS. Solubilized
proteins were recovered by centrifugation at 100,000 × g for 30 min. 500 µl of the soluble fraction containing 2 mg of protein was loaded on a Superdex 200 column (16/60) equilibrated
in 25 mM Hepes-NaOH, 300 mM NaCl, 0.2 mM DTT, 2% (w/v) CHAPS, pH 7.5, and eluted at a flow rate
of 1 ml/min. Fractions of 2 ml were collected, and every second
fraction was analyzed by SDS-PAGE and Western blot with antibodies
against Bax. The fractions from the transfected mitochondrial extract
were in addition analyzed by antibodies against HA, Myc, and His. Two
Bax-reactive bands were identified; the upper band corresponds to HA
and Myc tagged Bax, and the lower band corresponds to endogenous and
His-tagged Bax. The cytosol fraction from transfected cells was treated
with 2% CHAPS and analyzed as the mitochondrial extracts with anti-Bax
antibodies.
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Fig. 9.
Localization of Bax at the mitochondrial
membrane. A, mitochondria from control and apoptotic
HeLa cells were treated with 0.1 M
Na2CO3, pH 12, on ice for 20 min. At the end of
the incubation, the sample was centrifuged at 100,000 × g for 1 h. The supernatant, which contained
mitochondrial attached proteins (Att), was saved, and CHAPS
was added to a final concentration of 2%. The pellet was suspended in
MB containing 2% CHAPS, incubated on ice for 1 h, sonicated, and
centrifuged at 100,000 × g for 30 min. The second
supernatant contained the solubilized inserted membrane proteins
(Ins). The two solubilized samples were analyzed by SDS-PAGE
and Western blot with antibodies against, Bax, Bak, Bcl-XL,
and VDAC. B, 500 µl of the solubilized samples were loaded
on a Superdex 200 column (16/60) equilibrated in 25 mM
Hepes-NaOH, 300 mM NaCl, 0.2 mM DTT, 2% (w/v)
CHAPS, pH 7.5, and eluted at a flow rate of 1 ml/min. For each sample,
the fractions corresponding to monomeric and oligomeric Bax were pooled
and were analyzed by SDS-PAGE and Western blot with antibodies against
Bax. Ins, inserted; Att, attached; M,
monomer; O, oligomer. C, mitochondria from
control and apoptotic HeLa cells overexpressing Bcl-2 were treated with
0.1 M Na2CO3, pH 12, on ice for 20 min. At the end of the incubation, the samples were centrifuged at
100,000 × g for 1 h. The supernatant, which
contained mitochondrial attached proteins (Att), was saved, and CHAPS
was added to a final concentration of 2%. The pellet was suspended in
MB containing 2% CHAPS, incubated on ice for 1 h, sonicated, and
centrifuged at 100,000 × g for 30 min. The second
supernatant contained the solubilized inserted membrane proteins
(Ins). The two solubilized samples were analyzed by SDS-PAGE
and Western blot with antibodies against Bax and Bcl-2.
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Fig. 10.
Analysis of CHAPS mitochondrial extract from
control and staurosporine-treated Bcl-2-overexpressing HeLa cells by
gel filtration on Superdex 200. Mitochondria were isolated and
treated with 2% CHAPS. Solubilized proteins were recovered by
centrifugation at 100,000 × g for 30 min. 500 µl of
the soluble fraction containing 2 mg of protein was loaded on a
Superdex 200 column (16/60) equilibrated in 25 mM
Hepes-NaOH, 300 mM NaCl, 0.2 mM DTT, 2% (w/v)
CHAPS, pH 7.5, and eluted at a flow rate of 1 ml/min. Fractions of 2 ml
were collected, and every second fraction was analyzed by SDS-PAGE and
Western blot with antibodies against Bax, Bcl-2, Bak,
Bcl-XL, and VDAC.
DISCUSSION
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ABSTRACT
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DISCUSSION
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Timothy Wells for encouragement and support, Drs. Amanda Proudfoot and Kinsey Maundrell for critical reading of the manuscript, Alena Hochmann for expert technical assistance, and Christopher Herbert for art work.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 41 22 706 9802;
Fax: 41 22 794 6965; E-mail: bruno.antonsson@serono.com.
§ Present address: Dept. Biologie Cellulaire, University of Geneva, 30 qual E. Ansermet, 1211 Geneva 4, Switzerland.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M010810200
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ABBREVIATIONS |
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The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; VDAC, voltage-dependent anion channel; ANT, adenosine nucleotide translocator; MB, mitochondrial buffer; HA, hemagglutinin; Mops, 4-morpholinepropanesulfonic acid; rBax, recombinant monomeric Bax.
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REFERENCES |
---|
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---|
1. | Hengartner, M. O. (2000) Nature 407, 770-776[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Yang, E.,
and Korsmeyer, S. J.
(1996)
Blood
88,
386-401 |
3. |
Green, D. R.,
and Reed, J. C.
(1998)
Science
281,
1309-1312 |
4. | Kelekar, A., and Thompson, C. B. (1998) Trends Cell Biol. 8, 324-330[CrossRef][Medline] [Order article via Infotrieve] |
5. | Kroemer, G. (1997) Nat. Med. 3, 614-620[Medline] [Order article via Infotrieve] |
6. | Jacobson, M. D. (1997) Curr. Biol. 7, R277-R281[Medline] [Order article via Infotrieve] |
7. | Reed, J. C. (1997) Nature 387, 773-776[CrossRef][Medline] [Order article via Infotrieve] |
8. | Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 74, 609-619[Medline] [Order article via Infotrieve] |
9. | Knudson, C. M., and Korsmeyer, S. J. (1997) Nat. Genet. 16, 358-363[Medline] [Order article via Infotrieve] |
10. | Yin, X.-M., Oltvai, Z. N., and Korsmeyer, S. J. (1994) Nature 369, 321-323[CrossRef][Medline] [Order article via Infotrieve] |
11. | Borner, C., Martinou, I., Mattmann, C., Irmler, M., Schaerer, E., Martinou, J.-C., and Tschopp, J. (1994) J. Cell Biol. 126, 1059-1068[Abstract] |
12. | Sedlak, T. W., Oltvai, Z. N., Yang, E., Wang, K., Boise, L. H., Thompson, C. B., and Korsmeyer, S. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7834-7838[Abstract] |
13. |
Hsu, Y.-T.,
and Youle, R. J.
(1997)
J. Biol. Chem.
272,
13829-13834 |
14. | Chittenden, T., Flemington, C., Houghton, A. B., Ebb, R. G., Gallo, G. J., Elangovan, B., Chinnadurai, G., and Lutz, R. J. (1995) EMBO J. 14, 733-736 |
15. |
Hunter, J. J.,
and Parslow, T. G.
(1996)
J. Biol. Chem.
271,
8521-8524 |
16. | Wang, K., Yin, X. M., Chao, D. T., Milliman, C. L., and Korsmeyer, S. J. (1996) Genes Dev. 10, 2859-2869[Abstract] |
17. |
Zha, J.,
Harada, H.,
Osipov, K.,
Jockel, J.,
Waksman, G.,
and Korsmeyer, S. J.
(1997)
J. Biol. Chem.
272,
24101-24104 |
18. | Krajewski, S., Tanaka, S., Takayama, S., Schibler, M. J., Fenton, W., and Reed, J. C. (1993) Cancer Res. 53, 4701-4714[Abstract] |
19. | Newmeyer, D. D., Farschon, D. M., and Reed, J. C. (1994) Cell 79, 353-364[Medline] [Order article via Infotrieve] |
20. | Kroemer, G. (1997) Cell Death Diff. 4, 443-456[CrossRef] |
21. |
Bossy-Wetzel, E.,
Newmeyer, D. D.,
and Green, D. R.
(1998)
EMBO J.
17,
37-49 |
22. | Single, B., Leist, M., and Nicotera, P. (1998) Cell Death Diff. 5, 1001-1003[CrossRef][Medline] [Order article via Infotrieve] |
23. | Kohler, C., Gahm, A., Noma, T., Nakazawa, A., Orrenius, S., and Zhivotovsky, B. (1999) FEBS letter 447, 10-12[CrossRef][Medline] [Order article via Infotrieve] |
24. | Lorenzo, H. K., Susin, S. A., Penninger, J., and Kroemer, G. (1999) Cell Death Diff. 6, 516-524[CrossRef][Medline] [Order article via Infotrieve] |
25. | Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33-42[Medline] [Order article via Infotrieve] |
26. | Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43-53[Medline] [Order article via Infotrieve] |
27. | Goldstein, J. C., Waterhouse, N. J., Juin, P., Evan, G. I., and Green, D. R. (2000) Nat. Cell Biol. 2, 156-162[CrossRef][Medline] [Order article via Infotrieve] |
28. | Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489[Medline] [Order article via Infotrieve] |
29. | Rosse, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B., and Borner, C. (1998) Nature 391, 496-499[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Eskes, R.,
Antonsson, B.,
Osen-Sand, A.,
Montessuit, S.,
Richter, C.,
Sadoul, R.,
Mazzei, G.,
Nichols, A.,
and Martinou, J.-C.
(1998)
J. Cell Biol.
143,
217-224 |
31. |
Jurgensmeier, J. M.,
Xie, Z.,
Deveraux, Q.,
Ellerby, L.,
Bredesen, D.,
and Reed, J. C.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
4997-5002 |
32. |
Finucane, D. M.,
Bossy-Wetzel, E.,
Waterhouse, N. J.,
Cotter, T. G.,
and Green, D. R.
(1999)
J. Biol. Chem.
274,
2225-2233 |
33. | Suzuki, M., Youle, R. J., and Tjandra, N. (2000) Cell 103, 645-654[Medline] [Order article via Infotrieve] |
34. | Muchmore, S. W., Sattler, M., Liang, H., Meadows, R. P., Harlan, J. E., Yoon, H. S., Nettesheim, D., Chang, B. S., Thompson, C. B., Wong, S.-L., Ng, S.-C., and Fesik, S. W. (1996) Nature 381, 335-341[CrossRef][Medline] [Order article via Infotrieve] |
35. | Chou, J. J., Li, H., Salvesen, G. S., Yuan, J., and Wagner, G. (1999) Cell 96, 615-624[Medline] [Order article via Infotrieve] |
36. | McDonnell, J. M., Fushman, D., Milliman, C. L., Korsmeyer, S. J., and Cowburn, D. (1999) Cell 96, 625-634[Medline] [Order article via Infotrieve] |
37. | Minn, A. J., Velez, P., Schendoe, S. L., Liang, H., Muchmore, S. W., Fesik, S. W., Fill, M., and Thompson, C. B. (1997) Nature 385, 353-357[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Schendel, S. L.,
Xie, Z.,
Montal, M. O.,
Matsuyama, S.,
Montal, M.,
and Reed, J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5113-5118 |
39. |
Antonsson, B.,
Conti, F.,
Ciavatta, A. M.,
Montessuit, S.,
Lewis, S.,
Martinou, I.,
Bernasconi, L.,
Bernard, A.,
Mermod, J.-C.,
Mazzei, G.,
Maundrell, K.,
Gambale, F.,
Sadoul, R.,
and Martinou, J.-C.
(1997)
Science
277,
370-372 |
40. |
Schlesinger, P. H.,
Gross, A.,
Yin, X.-M.,
Yamamoto, K.,
Saito, M.,
Waksman, G.,
and Korsmeyer, S. L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11357-11362 |
41. | Antonsson, B., Montessuit, S., Lauper, S., Eskes, R., and Martinou, J.-C. (2000) Biochem. J. 345, 271-278[CrossRef][Medline] [Order article via Infotrieve] |
42. | Saito, M., Korsmeyer, S. J., and Schlesinger, P. H. (2000) Nat. Cell Biol. 2, 553-555[CrossRef][Medline] [Order article via Infotrieve] |
43. | Rojo, M., and Wallimann, T. (1994) Biochim. Biophys. Acta 1187, 360-367[Medline] [Order article via Infotrieve] |
44. | Estoppey, S., Rodriguez, I., Sadoul, R., and Martinou, J.-C. (1997) Cell Death Differ. 4, 34-38[CrossRef] |
45. | Montessuit, S., Mazzei, G., Magnenat, E., and Antonsson, B. (1999) Protein Expression Purif. 15, 202-206[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Desagher, S.,
Osen-Sand, A,
Nichols, A.,
Eskes, R.,
Montessuit, S.,
Lauper, S.,
Maundrell, K.,
Antonsson, B.,
and Martinou, J-C.
(1999)
J. Cell Biol.
144,
891-901 |
47. | Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Hsu, Y.-T.,
and Youle, R. J.
(1998)
J. Biol. Chem.
273,
10777-10783 |
49. | Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483-487[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Marzo, I.,
Brenner, C.,
Zamzami, N.,
Jurgensmeier, J. M.,
Susin, S. A.,
Vieira, H. L. A.,
Prevost, M.-C.,
Xie, Z.,
Matsuyama, S.,
Reed, J. C.,
and Kroemer, G.
(1998)
Science
281,
2027-2031 |
51. |
Eskes, R.,
Desagher, S.,
Antonsson, B.,
and Martinou, J.-C.
(2000)
Mol. Cell. Biol.
20,
929-935 |
52. | Vander Heiden, M. G., and Thompson, C. B. (1999) Nat. Cell Biol. 1, E209-E215[CrossRef][Medline] [Order article via Infotrieve] |
53. | Martinou, J.-C., Desagher, S., and Antonsson, B. (2000) Nat. Cell Biol. 2, E41-E43[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Gross, A.,
Jockel, J.,
Wie, M. C.,
and Korsmeyer, S. L.
(1998)
EMBO J.
17,
3878-3885 |
55. | Helenius, A., and Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79[Medline] [Order article via Infotrieve] |
56. | Newby, A. C., Chrambach, A., and Bailyes, E. M. (1982) Techniques in Lipid and Membrane Biochemistry , pp. 1-22, Elsevier/North-Holland, Amsterdam |
57. |
Conus, S.,
Kaufmann, T.,
Fellay, I.,
Otter, I.,
Rosse, T.,
and Borner, C.
(2000)
EMBO J.
19,
1534-1544 |
58. |
Hsu, Y.-T.,
Wolter, K. G.,
and Youle, R. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3668-3672 |
59. |
Wolter, K. G.,
Hsu, Y. T.,
Smith, C. L.,
Nechushtan, A.,
Xi, X. G.,
and Youle, R. J.
(1997)
J. Cell Biol.
139,
1281-1292 |
60. |
Goping, I. S.,
Gross, A.,
Lavoie, J. N.,
Nguyen, M.,
Jemmerson, R.,
Roth, K.,
Korsmeyer, S. J.,
and Shore, G. C.
(1998)
J. Cell Biol.
143,
207-215 |
61. | Crompton, M., Virji, S., and Ward, J. M. (1998) Eur. J. Biochem. 258, 729-735[Abstract] |
62. | Woodfield, K., Ruck, A., Brdiczka, D., and Halestrap, A. P. (1998) Biochem. J. 336, 287-290[Medline] [Order article via Infotrieve] |
63. |
Griffiths, G. J.,
Dubrez, L.,
Morgan, C. P.,
Jones, N. A.,
Whitehouse, J.,
Corfe, B. M.,
Dive, C.,
and Hickman, J. A.
(1999)
J. Cell Biol.
144,
903-914 |
64. |
Sattler, M.,
Liang, H.,
Nettesheim, D.,
Meadows, R. P.,
Harlan, J. E.,
Eberstadt, M.,
Yoon, H. S.,
Shuker, S. B.,
Chang, B. S.,
Minn, A. J.,
Thompson, C. B.,
and Fesik, S. W.
(1997)
Science
275,
983-986 |