From the Department of Pathology, University of Texas Health Science Center, San Antonio, Texas 78229
Received for publication, January 24, 2001, and in revised form, February 14, 2001
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ABSTRACT |
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ATP depletion results in Bax translocation from
cytosol to mitochondria and release of cytochrome c from
mitochondria into cytosol in cultured kidney cells. Overexpression of
Bcl-2 prevents cytochrome c release, without ameliorating
ATP depletion or Bax translocation, with little or no association
between Bcl-2 and Bax as demonstrated by immunoprecipitation (Saikumar,
P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M.,
and Venkatachalam, M. A. (1998) Oncogene 17, 3401-3415). Now we show that translocated Bax forms homo-oligomeric
structures, stabilized as chemical adducts by bifunctional
cross-linkers in ATP-depleted wild type cells, but remains monomeric in
Bcl-2-overexpressing cells. The protective effects of Bcl-2 did not
require Bcl-2/Bax association, at least to a degree of proximity or
affinity that was stable to conditions of immunoprecipitation or adduct
formation by eight cross-linkers of diverse spacer lengths and chemical
reactivities. On the other hand, nonionic detergents readily induced
homodimers and heterodimers of Bax and Bcl-2. Moreover, associations
between translocated Bax and the voltage-dependent anion
channel protein or the adenine nucleotide translocator protein could
not be demonstrated by immunoprecipitation of Bax, or by using
bifunctional cross-linkers. Our data suggest that the in
vivo actions of Bax are at least in part dependent on the
formation of homo-oligomers without requiring associations with other
molecules and that Bcl-2 cytoprotection involves mechanisms that
prevent Bax oligomerization.
Mitochondria are central to the apoptosis activation pathway in
many physiological and pathological conditions. Members of the Bcl-2
family of proteins are known to affect mitochondrial function and
regulate the release of apoptosis-activating factors (2-5).
Anti-apoptotic members of Bcl-2 family (e.g. Bcl-2 and Bcl-xL) act primarily to preserve mitochondrial integrity
by suppressing the release of cytochrome c (5). In contrast,
pro-apoptotic members (Bax, Bid, etc.) induce the release of cytochrome
c and cause mitochondrial dysfunction (1, 6-8). The
pro-apoptotic protein, Bax, which normally resides in the cytosol,
translocates to mitochondria when triggered by certain stimuli (6, 9). Translocated Bax has been shown to induce cytochrome c
release both in vivo (1, 6, 10) and in vitro (11)
and this is followed by caspase activation (10, 12). The mitochondrial permeability transition, an event that results in disruption of the
mitochondrial potential gradient, has been reported to induce cytochrome c release and apoptosis (13). However, our
observations and several other reports suggest that the effects of Bax
are targeted at the outer mitochondrial membrane and that the
mitochondrial inner membrane remains intact even after Bax-induced
release of cytochrome c (6, 10, 14-16).
How cytochrome c leaves mitochondria during apoptosis after
relocation of Bax to the mitochondrial outer membrane still remains an
unanswered puzzle. Potential mechanisms involve mitochondrial swelling
caused by opening the permeability transition pore in the inner
membrane (17) or by mitochondrial hyperpolarization followed by
swelling and membrane rupture (18). However, it has been reported that
the pro-apoptotic proteins Bid and Bax can release cytochrome
c from isolated mitochondria in the absence of detectable
mitochondrial swelling (19). Although it was believed earlier that Bax
induces the release of cytochrome c by inhibiting Bcl-2
function through binding of the Bcl-2 homology domains
BH1, BH2, and BH3, there is
evidence to suggest that Bax and Bcl-2 function independently in
regulating apoptosis (20, 21). Formation of ion channels in synthetic
lipid bilayer by members of the Bcl-2 family (22) has suggested that
pro-apoptotic members may rearrange in the outer mitochondrial
membranes to allow the efflux of cytochrome c by forming
large channels. Even though both Bcl-2 and Bax are capable of forming
ion channels in artificial membranes, it is unclear how these proteins
can form similar channels and still exert opposing actions. The data
would suggest that Bax function can be inhibited by
Bcl-2/ Bcl-xL, but does not require direct Bax/Bcl-2 or
Bax/Bcl-xL interaction for regulating Bax function (23,
24). For example, enforced dimerization of Bax, as a chimeric protein
with FK506 binding protein, resulted in its translocation to the
mitochondria and induced cell death even in the presence of
Bcl-xL (25). Likewise, Bax mutant proteins that fail to
bind to Bcl-2 are capable of inducing apoptosis (20). In addition, Youle's group (26) have shown that nonionic detergents induce Bax homo- and heterodimerization with Bcl-2 or Bcl-xL and
suggested that such simple dimers alone are not sufficient to
regulate apoptosis. Bax has recently been reported to interact directly
with VDAC1 on the outer
membrane to release cytochrome c or with ANT on the inner
membrane to initiate the permeability transition, indirectly leading to
cytochrome c release (27, 28). More recently, it has been
reported that Bax may cause instability in artificial lipid membranes,
suggesting another mechanism by which Bax may permeabilize the outer
mitochondrial membrane (29). A caveat in this model is that
Bcl-xL, which is known to block cytochrome c
release from intact mitochondria, did not prevent the
membrane-destabilizing effects of Bax.
We have been investigating the roles played by Bax and Bcl-2 in the
regulation of cytochrome c release from mitochondria in a
model of apoptotic cell death induced by cellular ATP depletion. We
have shown previously that severe ATP depletion of cultured rat kidney
proximal tubule cells induced by hypoxia or chemical inhibitors of
mitochondrial respiration triggers the translocation of cytosolic Bax
to mitochondria and cytochrome c release into the cytosol
(1). In this model, mitochondrial insertion of Bax does not compromise
the integrity of inner mitochondrial membranes (14). Thus, the outer
mitochondrial membrane would appear to be a reasonable site for the
permeabilizing actions of Bax, at least in the context of hypoxia.
Using this model, we now report that, following insertion into the
mitochondrial outer membrane, Bax oligomerizes to form a multimeric
structure that could explain the release of cytochrome c. We
also show that the protective actions of Bcl-2 may stem from its
ability to block Bax oligomerization in the mitochondrial outer
membrane without forming physical complexes with the Bax protein.
Materials
Materials purchased from vendors were as follows. Ham's
F-12/Dulbecco's modified Eagle's medium were from Life Technologies, Inc. Monoclonal antibodies to rat Bax (1D1) were kindly provided by
Dr. Richard J. Youle (National Institutes of Health, Bethesda, MD) and
polyclonal antibody to rat adenine nucleotide translocator was provided
by Dr. H. H. Schmid (Hormel Institute, Austin, MN). Anti-cytochrome c monoclonal antibody (clone 7H8.2C12) was
from PharMingen (San Diego, CA); anti-Bcl-2 polyclonal antibody ( ATP Depletion by CCCP
Cells were cultured in serum-supplemented Ham's
F-12/Dulbecco's modified Eagle's medium with 17.5 mM
glucose as described (30) and plated at 105
cells/cm2 in 60- or 100-mm collagen-coated dishes. After
overnight growth, cells were washed with phosphate-buffered saline and
subjected to ATP depletion by incubation in glucose-free Krebs-Ringer
bicarbonate buffer (in mM: 115 NaCl, 1 KH2PO4, 4 KCl, 1 MgSO4, 1.25 CaCl2, and 25 NaHCO3; pre-gassed with 95%
N2, air, and 5% CO2) containing 15 µM CCCP at 37 °C under normoxic conditions. Glycine
was included at 5 mM in the buffer to simulate glycine
contents of tissues in vivo (31, 32), thus preventing early
necrotic injury during incubation (33).
Preparation of Subcellular Fractions
Protocol I--
Cytosolic and membrane fractions were prepared
by selective plasma membrane permeabilization with digitonin (34),
followed by membrane solubilization. Briefly, control and experimental cells in dishes were treated with 0.05% digitonin in isotonic buffer A
(10 mM HEPES, 150 mM NaCl, 1.5 mM
MgCl2, 1 mM EGTA, pH 7.4; ~ 107cells/ml) containing protease inhibitors (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride,
0.8 µM aprotinin, 50 µM bestatin, 15 µM E-64, 20 µM leupeptin, 10 µM pepstatin A), for 1-2 min at room temperature. Cell
permeabilization by digitonin was standardized by measuring 100%
release of lactate dehydrogenase and was also monitored visually under
an inverted microscope. The permeabilized cells were shifted to
4 °C, scraped with a rubber policeman, and collected into centrifuge
tubes. The supernatants (Dig/Cytosol) were routinely collected after
centrifugation at 15,000 × g for 10 min. Following
centrifugation, the pellet was further extracted with ice-cold
detergent (1% Nonidet P-40 or Triton X-100 or CHAPS) in buffer A
containing protease inhibitors for 60 min at 4 °C to release
membrane- and organelle-bound proteins including mitochondrial cytochrome c. Both detergent-soluble (membrane) and
insoluble fractions were collected by low speed (15,000 × g) or high speed (500,000 × g)
centrifugation. The protein patterns of soluble membrane fractions,
after low or high speed centrifugation, both by SDS-PAGE and Western
blotting were indistinguishable. Therefore, solubilized membrane
fractions were routinely collected by centrifugation at 15,000 × g for 10 min. The relative protein levels of Dig/Cytosol, membrane- and detergent-insoluble fractions are 58 ± 4%, 15 ± 0.5%, 27 ± 4% for control, and 48 ± 6%, 14 ± 0.5%, 38 ± 6% for ATP-depleted cells (4 h CCCP), respectively
(data from four independent experiments).
Protocol II--
Subcellular fractionation of cells was also
achieved by Dounce homogenization in isotonic Buffer B (250 mM sucrose, 10 mM HEPES, 10 mM KCl,
1.5 mM MgCl2, 1 mM EGTA, pH 7.4)
and differential centrifugation yielding nuclear (500 × g pellet), mitochondrial (15,000 × g
pellet), microsomal (500,000 × g pellet), and cytosol (500,000 × g supernatant) fractions. Unbroken cells
constituted <1%, as monitored by light microscopy with trypan blue.
Unlike control cells, ATP-depleted cells contained large numbers of
altered mitochondria that sedimented with nuclei. In order to make
valid comparisons, nuclear and mitochondrial fractions were collected together as 15,000 × g pellet. Protein concentration
was estimated with bicinchoninic acid (BCA) reagent (Pierce) following
supplier's protocol using bovine serum albumin as standard.
Protein Cross-linking
All cross-linkers were dissolved in Me2SO just
before using. Cross-linkers were added at 1 mM (0.1 mM for SANPAH) concentration to intact cells (~2 × 106 cells equivalent to 1 mg of total protein), cells
permeabilized with digitonin or detergent extracts of membrane. Optimal
cross-linking conditions for cytosol and membrane extracts were
determined after testing different cross-linker to protein ratios. 1 mM concentration of all cross-linkers except for SANPAH
(0.1 mM) was found to be optimal for a range of protein
concentrations (100-500 µg) in the extracts. After the addition of
cross-linkers, cells or extracts were incubated on a head-to-head
rocker for 30 min at room temperature. Amine targeting cross-linkers
(NHS esters and imido esters) were quenched by adding 0.1 volume of 2 M Tris-HCl (pH 7.4) and incubated with rocking for another
30 min at room temperature. Sulfhydryl targeting cross-linker DPDPB was
removed from cells by washing the pellets or from extracts by protein
precipitation with 3% trichloroacetic acid or 80% acetone. In case of
SANPAH, all the incubations were carried at 4 °C with 20 min of
incubation to react NHS esters and 10 min of exposure to UV light (360 nm) to generate the nonspecifically reactive nitrenes. After
cross-linking, cytosol and membrane fractions from intact cells were
collected as described above. Cleavage of -S-S-bridge-containing
cross-linkers was achieved by incubating extracts with 50 mM DTT for 30 min at 37 °C.
Immunoanalysis
Proteins were resolved by non-reducing or reducing (50 mM DTT) SDS-PAGE in Xcell II mini cell on 10% or 4-12%
(gradient) NuPAGE gels (Invitrogen, CA) using MES or MOPS running
buffer as recommended by the manufacturer. After electrophoresis,
proteins from the gel were electroblotted onto 0.2-µm PVDF membranes
following manufacturer's directions. Western blotting using
appropriate primary antibodies and peroxidase-conjugated suitable
secondary antibodies was performed to analyze proteins.
Chemiluminescent substrates (Pierce) were used to detect
antigen-antibody complexes on the PVDF membrane. Immunoprecipitation
was carried out as follows. Solubilized extracts in lysis buffer (1%
Nonidet P-40 in buffer A with protease inhibitors) were pre-cleared by
mixing with 50 µl of protein G-Sepharose beads (1:1 diluted
suspension) for 1 h at 4 °C, and beads were removed by
centrifugation. The resultant supernatants were incubated with primary
antibodies (2 µg of antibody) at 4 °C for 2 h or overnight. Immunoprecipitates were collected by incubating with protein
G-Sepharose for 1 h, followed by centrifugation for 2 min at
4 °C. The pellets were washed with lysis buffer three times,
followed by three washes with same buffer containing 500 mM
NaCl to increase stringency and reduce nonspecific binding of proteins
to immunoprecipitates. In some cases, high salt washes were omitted
deliberately to preserve proteins in immunoprecipitates that might have
associated nonspecifically or with weak affinity. After final wash, the
protein G-Sepharose beads with immunoprecipitates were suspended in
SDS/sample buffer and analyzed by SDS-PAGE and Western blotting as
described above.
Protein Analysis by Isoelectric Focusing
For IEF, a 6% acrylamide (30% acrylamide, 1.8%
N,N-methylene bisacrylamide) slab gel was prepared with
7.5% Ampholine (1:1 (v/v) mix of ampholytes pH 3.5-9.5 and pH
5.0-8.0; Amersham Pharmacia Biotech) with 1% CHAPS in a gel cassette.
Cytosol and membrane extracts with or without cross-linking were
applied to IEF gels as follows. Protein samples (10-30 µg) were
diluted with an equal volume of 2× sample buffer (40 mM
arginine, 40 mM lysine, 30% glycerol) and were applied on
top of the gel. The gel was focused for 1 h at 100 V, 3 h at
200 V, and 0.5 h at 500 V using anode (7 mM phosphoric
acid) and cathode (20 mM arginine, 20 mM
lysine) buffers at room temperature. Proteins in IEF gels were
electroblotted onto a 0.2-µm PVDF membrane for 1 h in 0.7%(v/v)
acetic acid, pH 3.0, and were detected by appropriate antibodies. The
pH gradient on the IEF gel was determined either by surface electrode
or by pH measurement of deionized water eluate of focused gel slices (0.5-cm width).
Relative Distribution of Bax in Normal and ATP-depleted
Cells--
We have shown previously that cultured rat kidney proximal
tubule cells express high concentrations of Bax, which is localized in
the cytosol (1). ATP depletion by either hypoxia or treatment with CCCP
in the absence of glucose causes Bax translocation to mitochondria and
cytochrome c release into cytosol (1). Provision of growth
medium after hypoxia or chemically induced ATP depletion allows
resynthesis of ATP by glycolysis and causes apoptotic death in cells
with cytosolic cytochrome c (1). As shown in Fig. 1A, Bax is predominantly
cytosolic in control cells (>99% soluble) and migrates to membranes
(mitochondria) in ATP-depleted cells (>90% membrane-bound; Fig.
1A, lanes 2 and 3). The
relative percentages of soluble and membrane-bound Bax in control and
ATP-depleted RPTC were determined by densitometric analysis of
chemiluminescence signals on films after Western blotting. From several
experiments, we found that, after 4 h of ATP depletion, the
average amount of Bax translocated to membranes varies from 80% to
100% of total. The zwitterionic detergent CHAPS extracted
membrane-inserted Bax almost as efficiently (>95%) as nonionic
detergent Nonidet P-40 (Fig. 1A, see lanes
3 and 4 and lanes 6 and
7). Digitonin-released cytosol collected after
centrifugation at 15,000 × g for 10 min (Fig.
1A, lane 2) or 500,000 × g for 15 min (Fig. 1A, lane
5) contained the same amount of Bax, although total protein
was marginally reduced by 12 ± 3% (four independent
determinations) after 500,000 × g centrifugation.
These results further confirmed the soluble nature of Bax protein in
normal cells and validated the use of digitonin to obtain cytosol from
whole cells.
Double immunostaining, using an anti-Bax antibody and an antibody to
cytochrome oxidase, a mitochondrial marker, revealed that the
localization of Bax coincided exactly with that of cytochrome oxidase
during ATP depletion, showing that Bax translocates exclusively to the
mitochondria (1). In accordance with our earlier results, Bax and
cytochrome oxidase were shown in the current study to be present in
different fractions in normal cells but co-localized to the same
fractions in ATP-depleted cells (Fig. 1B, lanes
2 and 3). In order to assess Bax translocation by
conventional cell fractionation, Dounce homogenization followed by
differential centrifugation was carried out. Control cells provided
clean fractions of nuclei, mitochondria, microsomes, and cytosol.
Integrity and specificity of mitochondrial fractions were confirmed by
the presence cytochrome c (data not shown). Interestingly,
although mitochondria appeared filamentous in normal cells, they were
round and aggregated around the nucleus in ATP-depleted cells (1),
suggesting that ATP depletion has changed the shape and densities of
mitochondria. The modified shape and densities of mitochondria posed a
challenge in obtaining clean mitochondrial fractions in ATP-depleted
cells. Therefore, mitochondria and nuclei were collected together to assess Bax translocation. Together with our previous immunocytochemical observations showing that nuclei of ATP-depleted cells are devoid of
Bax and that the protein is visualized exclusively in mitochondria (1),
these fractionation studies provided good evidence that Bax
translocates to mitochondria, but not nuclei or the microsomal membranes during ATP depletion (Fig. 1B, lanes
4-6). Based on these results, we routinely used the crude
membrane fractions that remained after the removal of
digitonin-released cytosol to assess Bax localization. Separate studies
showed that membrane bound Bax was efficiently extracted with
detergents, and remained in the supernatants to the same extent
regardless of whether the extracts were centrifuged at 15,000 × g or 500,000 × g. Thus assay of detergent
extracts of the crude membrane fraction represented a valid method to
assess Bax in mitochondrial membranes. Double immunostaining using
anti-Bax and anti-cytochrome c antibodies revealed that, in
100% of the cells with Bax in mitochondria, a diffuse cytosolic
cytochrome c staining was observed, whereas cells with Bax
in cytosol displayed a filamentous mitochondrial staining (1). Data
presented in Fig. 1C confirm these published observations
and show that, after Bax translocation, cytochrome c is
released from mitochondria into the cytosol.
Mitochondrially Localized Bax Forms Oligomers in the
Membrane--
Although the Bax molecule has mitochondrial targeting
signals in its sequence, it remains unclear what factors keep it in cytosol in normal cells. The molecular modifications that Bax might
undergo before or during translocation to mitochondria are also
unknown. In vitro studies have shown that treatment of
liposomes with Bax can permeabilize lipid membranes to allow transit of cytochrome c and dextrans (35). Calculation of the sizes of Bax-induced membrane pores has suggested that homo-oligomers of at
least four Bax molecules are required to account for the results (35).
These observations and other considerations suggested to us that
translocated Bax in mitochondrial membranes might exist in the form of
oligomers. To preserve the possible oligomeric state of Bax in the
cytosol or mitochondrial membranes, we employed a battery of eight
different bifunctional protein cross-linkers. Cross-linking was usually
performed prior to solubilization of the membranes, since detergent
treatment, by itself, can artificially induce dimerization of Bax with
other Bax molecules and with Bcl-2 (26). These studies showed that
Nonidet P-40 and Triton X-100, but not CHAPS, induced spurious
homodimers and heterodimers (26). As a control to confirm these prior
observations, and to test the efficacy of cross-linkers to stabilize
putative oligomers, we also solubilized membranes in Nonidet P-40,
Triton X-100, and CHAPS prior to cross-linking in some experiments.
In our initial studies, control and CCCP-treated cells were subjected
to chemical cross-linking with a membrane-permeable linker (DSP) at
different time points. Cytosol was collected and analyzed for Bax by
SDS-PAGE and immunoblotting under non-reducing conditions. As shown in
Fig. 2A, after prolonged
incubation with CCCP, Bax disappeared from the cytosol. However, we did
not detect any slow-moving Bax containing adducts in the cytosol at any
of these time points. A photoactivatable cross-linker SANPAH, one of
whose reactive groups can interact with any atom in the vicinity during
cross-linking, also failed to demonstrate Bax adducts in the cytosol
(data not shown), supporting the monomeric nature of cytosolic Bax.
Analysis of DSP cross-linked membrane fractions of ATP-depleted cells
showed that progressively greater amounts of Bax are present in
membranes with increasing durations of ATP depletion (Fig.
2B). In contrast to cytosolic Bax (Fig. 2A), the membrane-inserted Bax formed slow-moving adducts (Fig. 2B,
lanes 3-6). When the cross-linker was cleaved
under reducing conditions with 50 mM DTT, only the 21-kDa
species of Bax was present in these samples (Fig. 2C),
suggesting that the slow-moving adducts indeed represent Bax-containing
complexes. Interestingly, increasing the length of the cross-linkers
from 6.4 to 16.1 Å permitted the demonstration of higher order
oligomers possibly containing six or more molecules of Bax in membranes
(Fig. 2D). The molecular sizes of these adducts obtained
from various experiments were estimated to be multiples of ~21 kDa
(Fig. 2E), suggesting the formation of Bax homo-oligomers.
In order to rule out the possibility that pretreatment of cells with
digitonin might induce Bax oligomer formation, membranes were prepared
with or without digitonin treatment. As shown in Fig. 2F,
digitonin is not responsible for Bax adduct formation in mitochondrial
membranes of ATP-depleted cells.
Isoelectric Focusing Supports Homo-oligomerization of Bax in the
Membrane--
Although data presented in Fig. 2E suggest
that Bax monomers make up membrane-bound oligomers, it does not rule
out the possibility that Bax may form oligomers with other proteins of
similar molecular size. To investigate this possibility, SDS-PAGE
analysis was complemented with isoelectric focusing to distinguish
homo-oligomers from hetero-oligomers. This approach assumes that
Bax-interacting proteins must have isoelectric points different from
Bax. The pro-apoptotic Bax is an acidic protein with a theoretically
estimated pI of 4.69, and this value correlated well with the measured
value of ~4.75 for untreated Bax by isoelectric focusing (Fig.
3B). We employed two types of
cross-linkers to analyze oligomeric Bax. The sulfhydryl reactive agent
DPDPB, which does not alter the net charge on reacting proteins, and
the amine-reactive agent EGS, which lowers the pI of cross-linked
proteins by reacting with basic (amine) groups, were used. CHAPS
extracted membrane oligomeric Bax treated without or with DPDPB
(SDS-PAGE; Fig. 3A, lanes 1 and
2) focused at a pI of ~4.75 (Fig. 3B,
lanes 1 and 2), a value similar to
that of monomeric Bax from cytosol (Fig. 3B, lane
7). Since nonionic detergents but not CHAPS were shown to
induce homo- and hetero-oligomerization of Bax (26), cytosolic Bax was
exposed to either Triton X-100 or CHAPS prior to cross-linking with
DPDPB. In either case, the cross-linked protein focused at the same pI
as untreated monomeric cytosolic Bax (Fig. 3B,
lanes 5-7). Results using the amine-reactive cross-linker EGS were similar, except that Bax migrated as a more acidic species at a pI of ~4.4 due to loss of free basic amine groups(s) in the protein. All other amine-reactive cross-linkers also
reduced the pI of Bax (data not shown). Membrane-bound oligomeric Bax,
stabilized by EGS and extracted with CHAPS (Fig. 3A,
lane 3), and cytosolic Bax, artificially
dimerized in the presence of Triton X-100 and stabilized by EGS (see
Fig. 5A, lane 1), co-migrated during
isoelectric focusing (Fig. 3B, lanes 3 and 4).
Bcl-2 Expression Does Not Prevent Translocation of Bax to
Mitochondria during ATP Depletion--
We have shown earlier by
immunocytochemistry and Western blotting that Bcl-2 does not prevent
Bax translocation to mitochondria following hypoxia or chemically
induced ATP depletion in cultured proximal tubule cells, but is able to
block the release of cytochrome c (1). Bax translocation is
also a critical event in neuronal apoptosis and is not prevented by
overexpression of Bcl-2 during nerve growth factor deprivation (36).
However, Bcl-2 inhibited the release of cytochrome c,
caspase activation, and cell death in these neurons (36). These results
contradict other published results where Bax migration into
mitochondria was blocked by Bcl-2 overexpression (25, 37). The relative
levels of total Bax in RPTC and Bcl-21 cells are not
significantly different. With extended durations of ATP depletion, Bax
translocation in Bcl-21 cells reaches similar levels as in
RPTC (Fig. 4A, compare
lanes 1-5 and 6-10), as we have
reported previously (1). The studies presented here had greater than
70% of total Bax translocated to membranes.
Bcl-2 Prevents Oligomerization of Translocated Bax in the Membrane
without Forming Hetero-oligomers with Bax--
We have reported before
that translocated Bax molecules in membrane fractions of ATP-depleted
Bcl-21 cells have little or no association with Bcl-2 as
shown by cross-immunoprecipitation studies (1). When Bax antibodies
were used, the immunoprecipitates did not contain Bcl-2; conversely,
when Bcl-2 antibodies were used, only trace amounts of Bax were
occasionally found in the precipitates. Even these trace amounts of Bax
that were sometimes present in Bcl-2 immunoprecipitates are likely to
represent low affinity nonspecific adsorption, or Bax/Bcl-2
heterodimers induced artificially by nonionic detergents (26, 38). We
studied the issue further by using eight protein cross-linkers of
different spacer lengths and chemical reactivities to detect
associations of Bax and Bcl-2. Membrane-translocated Bax failed to form
oligomers with either Bcl-2 or other Bax molecules in cells
overexpressing Bcl-2 (Fig. 4, B and C). Lack of
Bax oligomerization was evident with four different chemical
cross-linkers, as shown in Fig. 4B, the photo cross-linker
SANPAH (Fig. 4C), and three other cross-linkers (data not
shown). On the other hand, in RPTC, which do not overexpress Bcl-2,
translocated Bax readily forms large molecular weight adducts even when
<15% of total Bax is present in the membrane (Fig. 2B, lane 3). Moreover, when these membranes of
ATP-depleted RPTC are cross-linked with the photo-activable
cross-linker SANPAH, oligomeric Bax adducts are demonstrated readily in
contrast to the behavior of the same protein in ATP-depleted
Bcl-21 cell membranes (Fig. 4C,
inset). The failure of SANPAH to cross-link Bax to other Bax molecules or to Bcl-2 is not likely to be related to the availability of specific reactive groups nearby in the putative partner. One of the
reactive groups in SANPAH is amine-specific, but the other forms a
highly reactive nitrene that forms adducts nonspecifically with any
atom in the vicinity. Thus, SANPAH has the ability to cross-link amines
in one partner to a wide spectrum of structures in the other partner
within the reach of the spacer arm. On the other hand, steric factors
related to cross-linker spacer lengths and distances between reactive
groups in the partners also need to be considered. A partial answer to
this question is provided by the results using SANPAH, as discussed
above. Additionally, we deliberately solubilized ATP-depleted
Bcl-21 cell membranes in Nonidet P-40, Triton X-100, or
CHAPS prior to cross-linking. Under these conditions as reported
previously by Youle's group (26), Bax/Bax, Bax/Bcl-2, and Bcl-2/Bcl-2
dimers are induced artificially by Nonidet P-40 and Triton X-100 but
not by CHAPS. As the results in Fig. 5
show, Bax/Bax, Bax/Bcl-2, and Bcl-2/Bcl-2 adducts were readily
demonstrable when cross-linking was done after Nonidet P-40 or Triton
X-100 solubilization, but not CHAPS treatment. Finally, the spacer
lengths of the eight cross-linkers that we used vary between 6.4 and
19.9 Å, distances that should cover a wide range of separation of
reactive groups in the partners. Together with the immunoprecipitation
results, these observations provide important data that argue strongly
for the validity of not only the positive observations with respect
oligomeric Bax adducts, but also their prevention by Bcl-2, and the
lack of demonstrable Bax/Bcl-2 associations when cross-linking was
performed before detergent solubilization.
Detergent-induced Bax Homodimerization Is Inhibited by
Bcl-2--
It has been reported previously that detergents induce Bax
homodimerization and Bax/Bcl-2 heterodimerization (26, 38). We have
extended our studies to test whether Bax oligomerizes in the presence
of detergents. Whole cells with cytosolic Bax (normal RPTC) were
extracted with the detergents Triton X-100, Nonidet P-40, and CHAPS and
then subjected to chemical cross-linking with DSP. Bax analysis by
immunoblotting showed that cytosolic Bax formed homodimers in the
presence of detergents Triton X-100 and Nonidet P-40 (Fig.
5A). However, the zwitterionic detergent CHAPS failed to
induce Bax homodimerization (Fig. 5A, lanes
2 and 5). These results agree completely with
previous reports that showed differential effects of detergents on Bax
dimerization (26, 38).
Since Bcl-2 prevented Bax oligomerization in the mitochondrial
membrane, we tested whether Bcl-2 also interferes with
detergent-induced Bax homodimerization. Normal Bcl-21 cells
were solubilized with different detergents to allow membrane Bcl-2 and
cytosolic Bax to interact with each other, cross-linked, and then
analyzed by SDS-PAGE and Western blotting (Fig. 5B,
lanes 1-6 (anti-Bax) and lane
7 (anti-Bcl-2)). In cell extracts from Bcl-2-overexpressing cells (Fig. 5B), Bax homodimerization was partially
inhibited (Fig. 5, compare lanes 1 and
3 in panels A and B).
Although Bcl-2 was able to form heterodimers with Bax in these
detergents, the total amount of Bax/Bcl-2 heterodimers could not
account for the entire decrease in Bax/Bax homodimer formation.
Speculatively, it seems reasonable to consider that Bcl-2 may reduce
Bax homodimerization by competing with Bax for space in detergent
micelles. Although the results show that Bcl-2 has the ability to form
heterodimers with Bax, they also show that the amounts of heterodimers
that form are relatively sparse, considering the concentrations of the
partners in the detergent, suggesting that they cannot form tight
complexes. Further studies are clearly necessary to address the
questions regarding Bax and Bcl-2 interactions among themselves and
each other in detergents as well as in membranes. The Bax homodimers
seen in Triton X-100 extracts without cross-linker (Fig. 5,
lane 4 in panels A and
B) are probably due to presence of oxidizing contaminants
even in the membrane grade detergent. These dimers disappeared with
pretreatment of extracts with 50 mM DTT for 30 min at
37 °C (but not boiling for 10 min). Similarly, the trace amounts of
dimers seen after non-reducing SDS-PAGE of oligomerized membrane Bax
not subjected to cross-linking (Figs. 2D and 3A,
lane 1), which are probably due to incomplete
dissociation and/or partial oxidation, also disappeared under reducing
conditions (Fig. 2F, lanes 1 and
3).
In order to investigate the physical state of Bcl-2 in mitochondrial
membranes, Bcl-21 cells were treated with DSP followed by
membrane extraction with Triton X-100, Nonidet P-40, or CHAPS. Bcl-2
migrated as a monomeric protein on SDS-PAGE with (data not shown) or
without cross-linking (Fig. 5C, lane
1). However, when the membrane extracts in above detergents
were subjected to cross-linking, small amounts of Bcl-2/Bcl-2
homodimers were detected in Triton X-100 or Nonidet P-40 extracts (Fig.
5C, lanes 2 and 4) but not in CHAPS extract (Fig. 5C, lane 3).
Together, these results show that detergents such as Nonidet P-40 and
Triton X-100 can induce the formation of homo- and heterodimers of Bax
and Bcl-2.
Dimer-forming Detergents Reduce Higher Order Bax Oligomers to
Dimers--
The data presented above indicate that the structural
conformation of Bcl-2 and Bax in natural membranes is different from that in detergents. An important difference between detergents and
membranes is that detergents predominantly form micellar structures, whereas membranes are organized as lipid bilayers with asymmetric distribution of proteins and lipids. We therefore tested whether detergent solubilization of membrane-inserted Bax would change its
oligomeric properties. Stabilization of complexes by chemical cross-linking was carried out before or after detergent solubilization of membranes of ATP-depleted RPTC and Bcl-2-overexpressing cells. The
data presented in Fig. 6A show
that translocated Bax in natural membranes exists in oligomeric form.
The higher orders of oligomers were reduced mainly to dimers when
cross-linking was performed after solubilization with either Nonidet
P-40 or Triton X-100 (Fig. 6A, compare lanes
1 and 2 or lanes 3 and
4). Lane 5 containing Triton
X-100-solubilized membranes without cross-linker shows Bax dimers; as
discussed earlier, dimer formation in this case is also attributable to
incomplete dissociation and oxidants present in Triton X-100 (Fig. 5).
As shown before, Bax oligomerization was dramatically reduced in
membranes from ATP-depleted Bcl-2-overexpressing cells (Fig.
6B, lanes 1 and 3). It is
worth noting again that the Bcl-2 protein did not form either
homo-oligomers or hetero-oligomers with Bax in such cells (Fig.
6B, lanes 1 and 3).
However, if membranes had been solubilized with Nonidet P-40 and Triton
X-100 before cross-linking, Bax/Bcl-2 heterodimers formed readily (Fig.
6B, lanes 2 and 4). The
identity of Bax/Bcl-2 heterodimers was confirmed both by molecular
weight calculation and Western blotting with anti-Bcl-2 antibodies
(data not shown). In contrast, membranes solubilized in CHAPS before
cross-linking still contained higher order Bax oligomers in RPTC (Fig.
6C, lane 1) but in ATP-depleted Bcl-21 cells, no Bcl-2 containing adducts were detected
(Fig. 6C, lane 2). Controls with
cross-linking followed by CHAPS extraction also showed similar results
(Fig. 3A, lane 3). Overall, our
results suggest that Bax oligomerization in the mitochondrial outer
membrane is prevented under natural conditions (i.e. without
prior exposure to detergents) by Bcl-2 without requirement for stable
associations with Bax.
VDAC or ANT Form Homodimers but Not Heterodimers with
Bax--
Recently it has been reported that Bax may interact with an
outer membrane protein, the voltage-dependent anion channel
protein (also known as porin), to induce cytochrome c
release (27). Another study reported that Bax might interact with an
inner membrane protein, the adenine nucleotide translocator (28). This
interaction has also been suggested to be an antecedent factor
responsible for cytochrome c release. Therefore we searched
for Bax and Bcl-2 interactions with VDAC or ANT in our ATP depletion
model. Although our cross-linking and isoelectrofocusing studies have
indicated no association of Bax with proteins other than itself,
further studies were carried out to identify Bax-associated proteins. Membrane extracts were immunoprecipitated with antibodies to Bax or
Bcl-2, and the resulting precipitates were analyzed for the presence of
VDAC or ANT by immunoblotting. As shown in Fig.
7A, antibodies to Bax and
Bcl-2 immunoprecipitated only Bax and Bcl-2, respectively, and did not
bring down even traces of VDAC protein (Fig. 7A,
lanes 2 and 3 and lanes
4 and 5). Our attempts to immunoprecipitate VDAC
from nonionic detergent membrane extracts with four commercially available antibodies failed, though these antibodies could recognize VDAC in immunoblots (Fig. 7A, lane 1).
However, when the membrane proteins were solubilized with SDS and
renatured by diluting with Nonidet P-40-containing buffer, these same
antibodies successfully immunoprecipitated VDAC (data not shown),
suggesting that partial renaturation of VDAC exposes otherwise buried
antibody-binding epitopes. Available antibodies against VDAC were
therefore not usable for co-immunoprecipitation studies to identify
association partners, at least in our model system. Conceivably, steric
hindrance related to the complexity of the outer membrane protein
microenvironment may also have been responsible for failed
immunoprecipitation of VDAC with these antibodies. Immunoblotting of
Bax and Bcl-2 immunoprecipitates for ANT also gave negative results
(data not shown), suggesting that neither ANT nor VDAC is associated
with translocated Bax in mitochondria. Immunoprecipitation under less stringent (150 mM NaCl washes only) as well as stringent
(0.5 M NaCl washes included) conditions failed to reveal
the presence of VDAC or ANT in the immunoprecipitates (data not shown).
Therefore, chemical cross-linking approach was used to stabilize any
weak interactions between proteins and identify Bax-associated
proteins. Our results, presented in Fig. 7B, show that Bax
and VDAC each can form homo-oligomers; in the same fractions, Bax/VDAC
associations were not observed as there were no adducts of intermediate
size (~54 kDa). Such associations between Bax and VDAC or ANT should have produced heterodimers in the size range of 52-56 kDa. We did not
detect complexes in that size range in Bax containing mitochondrial
membranes from ATP-depleted cells. Detection of VDAC homo-oligomers
probably supports their role as channel-forming proteins. Additionally,
Bcl-2/VDAC associations could not be demonstrated in
Bcl-2-overexpressing control cells (Fig. 7B,
lanes 8 and 9). Similarly, ANT protein
also did not form associations with Bax or Bcl-2 (Fig. 7C).
In control cells, DTBP did not cross-link ANT molecules, whereas DPDPB,
an agent with longer linker length, did (Fig. 7C,
lanes 1 and 2 and lanes
5 and 6). On the other hand, in
ATP-depleted cells, both DTBP and DPDPB reacted with ANT and revealed dimers (Fig. 7C, lanes 3,
4, 7, and 8). This result suggests that ANT molecules, during ATP depletion, either come closer or undergo
conformational change or both to allow cross-linking by DTBP. Our
failure to identify intermediate forms of ANT/Bax or ANT/Bcl-2
complexes with four other cross-linkers of different spacer arm lengths
and reactive specificities, including the nonspecific highly reactive
photo cross-linker SANPAH (data not shown), together with unequivocal
negative immunoprecipitation results, argues against the necessity for
such interactions for the function of Bax or Bcl-2 proteins.
Isoelectric Focusing Analysis to Identify Homo-oligomerization
Versus Hetero-oligomerization of Bax--
The calculated values of
isoelectric points (pI) of rat Bax (4.69) and human Bcl-2 (7.32) are
close to experimental values of 4.76 (rat Bax) and 7.39 (human Bcl-2,
see Fig. 8A). The calculated pI values of the proteins that have been reported to interact with Bax,
e.g. VDAC (9.04), ANT (10.54), Bcl-2 (7.32) and proteins that are known to be released by Bax from the mitochondria,
e.g. cytochrome c (10.39), adenylate kinase
(9.86), and apoptosis-inducing factor (9.63) suggest that all of these
proteins are basic in nature. In contrast, Bax is an acidic protein (pI
4.75) both in RPTC (see Fig. 3) and Bcl-2-overexpressing cells (Fig.
8A, lanes 1-3) before or after
translocation to membrane. Bcl-2, in normal cells, has two fast moving,
minor isoforms with pI of 6.61 and 6.85 (Fig. 8). These two minor forms
may represent phosphorylation variants of Bcl-2 since phosphorylation
of Bcl-2 at both tyrosine and serine/threonine residues has been
reported (39, 40). Disappearance of these two isoforms with DPDPB
cross-linking (Fig. 8A, lane 5)
probably suggests that these isoforms may have been cross-linked to
non-soluble cell structures (41). Amine-reactive cross-linkers like EGS
have changed Bcl-2 into acidic species (Fig. 8A,
lane 6; pI 6.16-6.85). Based on the formation of
small amounts of Bax/Bcl-2 dimers in the presence of Nonidet P-40
detergent (Fig. 5B), we considered that such a complex would
have an intermediate pI that is an average of Bax and Bcl-2 pI values.
As shown in Fig. 8B, we were able to detect such a complex
with a pI value of ~5.8 on an IEF gel in DPDPB-cross-linked
detergent-solubilized membranes. However, in ATP-depleted
Bcl-21 cell membranes, we failed to find Bax/Bcl-2
complexes not only in SDS-PAGE gels (Fig. 4, B and
C) but also in IEF gels (Fig. 8A), suggesting
that such complexes could only form under artificial conditions. If
VDAC or ANT were to form such complexes with Bax, we should have been able to detect Bax protein at a pI ~6.8 or ~ 7.5, respectively. However, our results (Fig. 8A,
lanes 2 and 3) clearly indicate that
no such complexes are formed in cross-linked membranes, suggesting that
Bax does not interact with either VDAC or ANT in the mitochondria.
Based on our results, we propose a model (Fig.
9) to explain how Bax may permeabilize
the mitochondrial outer membrane to release cytochrome c and
other intermembrane space proteins. The model assumes a channel formed
by Bax that can permeate intermembrane proteins across the
mitochondrial outer membrane.
Regulation of apoptosis by the Bcl-2 family of proteins
occurs primarily at the mitochondrial outer membrane and involves mitochondrial permeabilization or its prevention. The studies presented
in this paper are attempts to understand the mechanisms by which Bax
induces cytochrome c efflux from mitochondria of ATP-depleted cells. Although the signaling mechanisms responsible for
Bax translocation during ATP depletion remain unclear, the experiments
reported here have revealed important insights into the physical states
and associations of Bax molecules after they have been inserted into
mitochondrial membranes. Using chemical cross-linkers, we were able to
detect higher order homo-oligomers of Bax in membrane fractions of
ATP-depleted cells. The presence of Bax oligomers from tetramers to
decamers (Figs. 2, 3, 4, and 6) in the membrane fraction suggests that
Bax may be able to form large structures with potential to allow the
passage of proteins at least of the size of cytochrome c
(~12 kDa). Recently, it has been demonstrated that at least 4 molecules of Bax can form a pore of size 22 Å that is capable of
transporting cytochrome c, a molecule with a Stokes diameter
of 17 Å (35). Similar oligomers were identified with recombinant Bax
protein in the presence of octyl glucoside (42). Isoelectric focusing
analysis of these cross-linked oligomers suggests that Bax multimers
contain homogeneous populations of Bax molecules (Figs. 3 and 8).
Overall, with respect to the current studies, our results using
cross-linkers clearly favor homo-oligomerization of Bax in the
mitochondrial outer membrane.
In contrast, cytosolic Bax failed to form oligomers (Fig.
2A). These findings suggest that Bax containing adducts in
cytosol move instantaneously into mitochondria soon after they have
formed, or that the unique conformation of cytosolic Bax does not allow reactivity with cross-linkers. The latter possibility was ruled out by
the fact that amine-reactive cross-linkers were able to alter the pI of
cytosolic Bax.2 Cross-linking
at different time points after ATP depletion failed to reveal Bax
adducts in the cytosol, and this may rule out the first possibility. On
the other hand, nonionic detergents such as Nonidet P-40 and Triton
X-100 were able to induce dimerization of cytosolic Bax with itself or
Bcl-2, as demonstrated by cross-linking, suggesting that detergents
were altering the conformation of cytosolic Bax (Fig. 5). However,
CHAPS, a zwitterionic detergent, did not induce Bax or Bcl-2
oligomerization, which suggests that this detergent has actions
different from those of nonionic detergents with respect to its ability
to modify Bax or Bcl-2 molecules. These results confirm earlier work
from Youle's group (26, 38) and suggest that Bax monomers move to
mitochondrial membranes in response to unknown stimuli and form
homo-oligomers in situ. Our results also suggest that,
although there may be similarities between lipid bilayers and detergent
micelles in their effects on Bax with respect to their shared ability
to induce and maintain oligomers, there are also key differences. When
cross-linking of membrane proteins was carried out after membrane
solubilization in Nonidet P-40 or Triton X-100, multimers of Bax were
either absent or present in trace amounts, the vast majority of Bax
being present as dimers and monomers (Fig. 6A). This
suggests that only planar membrane bilayers allow Bax multimerization
to higher order structures, and that nonionic detergents might have
dissociated these pre-existing Bax multimers. In contrast, CHAPS, which
does not induce oligomerization of cytosolic Bax, also failed to
dissociate higher order Bax oligomers in mitochondrial membranes (Fig.
6C).
In contrast to results obtained by others, we have shown that Bcl-2
overexpression does not abolish Bax translocation to mitochondria (Fig.
4A). The results presented in this paper demonstrate that Bcl-2 cannot block Bax association with mitochondria in the ATP depletion model. However, Bcl-2 can completely prevent Bax
oligomerization in the mitochondrial outer membrane (Figs. 4
(B and C) and 6B). Thus, at least in
our experimental model, Bcl-2 may inhibit cytochrome c
release and protect cells by preventing oligomerization of Bax rather
than by blocking Bax insertion into mitochondrial membrane. Moreover,
we also found that Bax translocation to mitochondria in rat proximal
tubule cells is independent of apoptotic stimuli. UV (80 J/m2) exposure also induces Bax translocation in both RPTC
and Bcl-21 cells but oligomerization is seen only in RPTC
but not in Bcl-21 cells.2 This suggests that
prevention of Bax translocation is not a necessary prerequisite for the
protective actions of Bcl-2 in mitochondrial membranes.
Our results suggest that prevention of Bax homo-oligomerization by
Bcl-2 does not involve direct interactions of Bcl-2 with Bax, at least
to an extent of close proximity and affinity that could have been
revealed by co-immunoprecipitation, or demonstrable by eight different
cross-linkers with varying spacer arms (6.4-19.9 Å) and reactivities
(imido and NHS esters for amines, pyridyldithio for sulfhydryls, and
photo-activable nitrophenylazide for non-selective linking to atoms in
the vicinity). All of these cross-linkers were able to form Bcl-2
adducts, visualized as Bcl-2 homodimers and Bcl-2/Bax heterodimers,
only when pretreated with nonionic detergents prior to cross-linking;
but failed to detect such adducts when membranes were cross-linked
prior to detergent extraction. Together with immunoprecipitation
studies, our results suggest strongly that mechanisms other than direct
Bcl-2/Bax interactions must be involved in Bcl-2 protection against Bax
cytotoxicity. How then, could Bcl-2 prevent Bax-induced leakage of
cytochrome c? The inability of membrane-bound Bax to form
oligomers in mitochondria containing Bcl-2 could conceivably be
explained by the abundance of Bcl-2 and possible saturation of
membranes with the protein in Bcl-2-overexpressing cells (Fig. 9). Our
failure to observe Bax oligomers in membranes with translocated Bax
from Bcl-2-overexpressing cells suggests that partially inserted Bax
may not have undergone conformational changes required to form
oligomers/channels, perhaps by steric constraints imposed by abundant
Bcl-2 molecules occupying the same microenvironment(s). This
explanation demands that Bax and Bcl-2 share some critical lipid
domains, and possibly cooperating proteins in mitochondrial membranes.
Conceivably, these membrane domains and co-operating proteins are
required for full Bax insertion and function, made possible by exposure
of previously "masked" sequences. If the "shared" domains are
saturated with Bcl-2, Bax molecules are either denied access totally,
or provided access for limited insertion by steric restraints that
prevent assumption of configurations necessary for oligomer/channel
formation. This hypothesis is consistent not only with our present
findings, which show Bax insertion without oligomerization, but also
with the findings of other laboratories, which have reported reduction and even prevention of Bax translocation by Bcl-2 (37, 43).
Previous reports have suggested that Bax heterodimerization with
mitochondrial outer membrane porin (VDAC) or inner membrane protein ANT
may be involved in the release of cytochrome c or other
inter-membrane space proteins. However, we failed to detect associations of Bax with either ANT or VDAC by immunoprecipitation of
extracts from cells with mitochondrially translocated Bax. High salt
washes during immunoprecipitation were deliberately avoided to
encourage low affinity or nonspecific protein interactions in some
experiments; nevertheless, we failed to co-immunoprecipitate ANT or
VDAC with Bax. Moreover, eight different cross-linkers of diverse
spacer lengths and chemical reactivities also failed to reveal adducts
between Bax and VDAC or ANT. On the other hand, these agents readily
demonstrated homo-oligomers of ANT and VDAC. Additionally, the pI of
monomeric Bax in the cytosol and oligomeric translocated Bax in
membranes, with or without stabilization by cross-linkers, were
identical. This supports the idea that Bax oligomers were homogeneous
in composition and contained Bax exclusively. It is particularly
instructive to compare overall the behavior of oligomeric Bax,
membrane-bound Bax extracted with CHAPS (which does not induce
oligomerization by itself), monomeric Bax in the cytosol exposed to
CHAPS with or without cross-linker, cross-linked cytosolic Bax after
dimerization by Triton X-100, and untreated natural Bax. The results
show that neither the detergent nor the cross-linker DPDPB had altered
the net charge of the protein species. Under these conditions, the
identity of pI among all the species argues forcefully for the
formation of Bax homo-oligomers in membrane fractions of ATP-depleted
cells with translocated protein.
In summary, our results are consistent with a model (Fig. 9) of
Bax-dependent mitochondrial permeabilization in which
formation of Bax oligomers in the outer mitochondrial membrane triggers the release of cytochrome c. The anti-apoptotic protein,
Bcl-2, when overexpressed, may either interfere with Bax insertion into membranes (37, 43), or prevent Bax oligomerization in the membrane
following ATP depletion or UV irradiation by mechanisms related to
steric exclusion from critical microdomains in mitochondrial membranes.
This model also considers the possibility of oligomeric Bax somehow
inducing pore formation by other outer membrane proteins to
permeabilize cytochrome c. In the light of recent data, the latter possibility is considered unlikely, suggesting that Bax oligomers by themselves in liposomes are capable of transporting cytochrome c across the lipid bilayer (35). According to our model, Bcl-2 prevents such oligomerization by physically interfering with complete insertion of Bax into the membrane, a requirement for
oligomerization, without forming stable heteromeric complexes. Therefore, partially inserted but loosely bound Bax, in Bcl-2-loaded mitochondria, may quickly equilibrate with cytosolic Bax in other models of apoptosis where Bax association with mitochondria in Bcl-2-overexpressing cells is seemingly reduced. Future studies on
purified, stably cross-linked multimers of Bax and systems that permit
regulated expression of Bcl-2 should help to clarify these issues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C 21) and anti-Bax polyclonal antibody (P-19) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-porin 31 HL (
-VDAC) monoclonal antibodies Ab-1, Ab-2, Ab-3, and Ab-4 were from Calbiochem (San Diego, CA). Horseradish peroxidase-conjugated and preadsorbed secondary antibodies to mouse and rabbit were obtained from Jackson Immunoresearch Laboratories (Westgrove, PA). Rat kidney proximal tubule
cells (RPTC; SKPT-0193 clone 2), and human Bcl-2-overexpressing RPTC
(Bcl-21) were described before (1). Membrane grade Triton X-100 and digitonin were obtained from Roche Molecular
Biochemicals. Other detergents and chemical cross-linkers
disuccinimidyl tartarate, disuccinimidyl glutarate, DSP, DTSSP, DTBP,
EGS, DPDPB, and SANPAH were purchased from Pierce. All other
reagents were of the highest grade available.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Bax redistribution during ATP depletion.
RPTC were exposed to 15 µM CCCP in glucose-free medium
for 4 h. Cell fractions were analyzed for Bax, cytochrome oxidase
(COX), and cytochrome c after Western blotting
with chemiluminescence detection. If more than one antigen was probed,
blots were exposed sequentially to antibodies of indicated antigens.
A, relative distribution of Bax from normal and CCCP-treated
cells after differential detergent fractionation. Cytosol from
digitonin-permeabilized cells was obtained by collecting supernatant
(Dig/Cyto) after centrifugation at 15,000 × g for 10 min (lane 2) or 500,000 × g for 15 min (lane 5). The
cytosol-depleted cell pellet was then extracted with either Nonidet
P-40 or CHAPS and collected as detergent-soluble (NP/Mem or
CH/Mem) and insoluble matrix fractions (NP/Insol
or CH/Insol) after centrifugation as described under
"Experimental Procedures." Insoluble matrix fraction was dissolved
in SDS/sample buffer, and all the fractions were analyzed by loading
proportional amounts of each fraction on reducing SDS-PAGE gels in
relation to 100 µg of total cellular protein. The average protein
distribution in cytosol, membrane, and insoluble fractions is 58:15:27
for normal and 48:14:38 for 4 h ATP-depleted cells. B,
relative distribution of Bax and cytochrome oxidase in different cell
fractions of control and CCCP-treated RPTC. Dig/Cyto
(lane 2) and NP/Mem (lane
3) fractions obtained by protocol I are compared with
cytosol (lane 4), combined nuclei + mitochondria
(lane 5) and microsomal (lane
6) fractions acquired by protocol II in relation to total
cell lysate (lane 1). C, Bax and
cytochrome c distribution in cell fractions of control
(lanes 1-3) and CCCP-treated (lanes
4-6) RPTC.
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Fig. 2.
Membrane translocation and oligomerization of
Bax in membrane fractions. Following incubation of RPTC with CCCP
for 0, 1, 2, 2.5, 3, or 4 h, cells were treated with cleavable
membrane-permeable cross-linker DSP (1 mM). Cytosol and
membrane fractions were obtained as described under "Experimental
Procedures," and proportional amounts corresponding to total protein
were analyzed for Bax by Western blotting under non-reducing conditions
unless indicated. A, prolonged exposure to CCCP resulted in
the disappearance of Bax from cytosol and did not show any slow moving
adducts at all time points (lanes 1-6).
B, progressively larger amounts of Bax accumulated in the
membrane fraction (Nonidet P-40 extract) as slow moving adducts
(lanes 1-6). C, slow moving Bax
adducts in DSP cross-linked cells are derived from Bax monomers. Slow
moving Bax adducts under non-reducing conditions (lanes
2-5) are converted to Bax monomers if disulfide bonds in
the cross-linked preparations are cleaved by incubation with 50 mM DTT (lanes 7-10). D,
cross-linkers with longer spacer arms demonstrated the presence of
increased amounts of higher order Bax oligomers. Membrane extracts of
ATP-depleted cells (4 h CCCP) after incubating with cross-linkers of
different spacer lengths as indicated. E, correlation of
molecular weights of Bax adducts with expected number of Bax molecules
in the corresponding adduct. The molecular weights of adducts
containing Bax were calculated by plotting their migrations against
migrations of molecular weight standards in semi-logarithmic plots from
eight different experiments. Inset shows two representative
lanes of Bax ladders obtained by reducing SDS-PAGE and Western blotting
of EGS cross-linked membranes from ATP-depleted cells. F,
digitonin pre-treatment does not induce Bax adduct formation in the
membranes. Membranes from digitonin-permeabilized cells
(Dig+) or heavy membrane fraction containing mitochondria
and nuclei (Dig ) were treated with (CL+) or
without (CL
) cross-linker (SANPAH) before extraction with
Nonidet P-40. Samples (20 µg) were analyzed for Bax under reducing
conditions. Both preparations showed presence of similar Bax adducts
(lanes 2 and 4). Number of Bax
monomers in all slow moving adducts is indicated in appropriate
panels.
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Fig. 3.
Analysis of Bax by SDS-PAGE
(A) and isoelectric focusing (B) to
identify the nature of Bax oligomers. ATP-depleted cells were
first treated with or without indicated cross-linkers, and membrane
proteins were then extracted with CHAPS. Cytosol from control cells was
first exposed to Triton X-100 or CHAPS followed by cross-linking and
immunoblotting for Bax after SDS-PAGE (A) or isoelectric
focusing (B) under non-reducing conditions. CH,
CHAPS; TX, Triton X-100; CL1, DPDPB;
CL2, EGS; , no cross-linker. The sulfhydryl-reacting
cross-linker, DPDPB, does not change the pI of Bax, whereas EGS altered
the pI of modified Bax.
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Fig. 4.
Effect of Bcl-2 overexpression on Bax
translocation and oligomerization. A, time course of
Bax translocation in Bcl-21 cells and RPTC analyzed by
Western blotting after reducing SDS-PAGE. B, cross-linkers
of diverse spacer lengths and reactivities failed to reveal slow
moving Bax adducts in membranes from Bcl-21 cells analyzed
on non-reducing gels. Results with four cross-linkers are shown after
4 h of CCCP treatment. C, nonspecifically reactive
photoactivatable cross-linker SANPAH also failed to reveal Bax
oligomers in membranes of Bcl-2-overexpressing cells. At various time
points of CCCP incubation, Bcl-21 cells were permeabilized
with digitonin and treated with photo-cross-linker SANPAH before
membrane extraction with Nonidet P-40. Unlike Bcl-21 cells,
CCCP-treated (4 h) RPTC readily revealed slow moving Bax adducts after
cross-linking with SANPAH (see inset).
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Fig. 5.
Detergents induce homo- and heterodimers of
Bax and Bcl-2 that can be cross-linked. Control RPTC
(A) or control Bcl-21 cells (B) were
first solubilized with indicated detergents Triton X-100
(TX), CHAPS (CH), or Nonidet P-40 (NP)
without ( CL) or with (+CL) chemical
cross-linking with DSP. Samples were analyzed for Bax and Bcl-2 after
separation on non-reducing SDS-PAGE. A, the nonionic
detergents Triton X-100 (lane 1) and Nonidet P-40
(lane 3) induced dimerization of Bax, whereas
CHAPS did not (lane 2). B, analysis of
Bax (lanes 1-6) and Bcl-2 (lane
7) shows that in Bcl-2-overexpressing cells, Triton X-100
(lane 1) and Nonidet P-40 (lanes
3 and 7) induced Bax homodimers and Bax/Bcl-2
heterodimers as demonstrated by cross-linking. Bax dimerization is
reduced by Bcl-2. CHAPS failed to induce homodimers or heterodimers.
C, nonionic detergents, Triton X-100 or Nonidet P-40
(lanes 2 and 4), induced weak
homodimerization of Bcl-2 in membrane fractions after extraction, but
CHAPS does not (lane 3). Note that homodimers of
Bax or Bcl-2 were frequently observed following extraction with Triton
X-100, but not with Nonidet P-40 or CHAPS even without the use of
chemical cross-linkers. These dimers were readily disrupted by
treatment with 50 mM DTT for 30 min at 37 °C.
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Fig. 6.
Pre-exposure of Bax oligomers to nonionic
detergents disintegrates higher order oligomers to dimers.
Membranes from CCCP-treated (4 h) RPTC and Bcl-21 cells
were either first subjected to cross-linking followed by extraction
with nonionic detergents (CL+NP or CL+TX) or were
solubilized first with different detergents and then subjected to
cross-linking (NP+CL, TX+CL, or
CH+CL). All samples (cross-linked with DSP) were analyzed
under non-reducing conditions. A, higher order oligomers
diminished markedly when membranes were exposed to nonionic detergents
before being subjected to cross-linking (lanes 2 and 4) compared with cross-linking followed by extraction
(lanes 1 and 3). Lane
5 represents Triton X-100 extract (membrane fraction) of
CCCP-treated cells, without cross-linking, containing translocated Bax.
Dimer represents artificial disulfide linkage not disrupted by SDS-PAGE
under non-reducing conditions. B, in Bcl-2-overexpressing
cell membranes, membrane-translocated Bax did not form higher order
homo-oligomers or heterodimers with Bcl-2, as demonstrated by
cross-linking followed by solubilization (lanes 1 and 3). Moreover, only trace amounts of Bax dimers could be
seen, in contrast to the abundance of dimers and higher order oligomers
that form in membranes in wild type (RPTC) cells (A).
However, when membranes were exposed to the nonionic detergents Nonidet
P-40 (lane 2) or Triton X-100 (lane
4) before cross-linking, Bax/Bcl-2 heterodimers appeared.
The identity of the faint bands seen spanning below the 31-kDa region
is not known. C, CHAPS solubilization prior to cross-linking
still preserved Bax oligomers, which formed after translocation to the
membrane (lane 1). Results with cross-linking
followed by CHAPS solubilization are identical (see Fig. 3A,
lane 3). In Bcl-21 cell membranes
containing translocated Bax, Bcl-2 did not form any slow moving adducts
(lane 2). CH, CHAPS; NP,
Nonidet P-40; TX, Triton X-100.
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Fig. 7.
Mitochondrial VDAC and ANT proteins failed to
co-immunoprecipitate with Bax and did not form cross-linked adducts
with Bax but formed homo-oligomeric adducts. A,
immunoprecipitation of detergent extracts from ATP-depleted RPTC with
anti-Bax (mouse monoclonal, 1D1) and ATP-depleted Bcl-21
cells with anti-Bcl-2 (rabbit polyclonal C 21) antibodies were
carried out as described under "Experimental Procedures." The
immunoprecipitates were analyzed under non-reducing or reducing (data
not shown) SDS-PAGE, followed by immunoblotting with anti-porin 31HL
antibody, and subsequently probed for either Bax or Bcl-2. Membrane
fraction from RPTC was included as a control for VDAC signal
(lane 1). Both
-Bax (lanes
2 and 3) and
-Bcl-2 (lanes
4 and 5) antibodies failed to co-precipitate VDAC
(shown by absence of signal). On the other hand, probing these blots
with Bax or Bcl-2 antibodies showed signal for the presence of Bax
(lanes 2 and 3) and Bcl-2
(lanes 4 and 5), respectively, in
these immunoprecipitates. Intact IgG present in the precipitates is not
shown. Analyzing samples under reducing conditions (data not shown)
revealed extra bands of IgG light (25 kDa) and heavy (50 kDa) chains
with no bands in the 33-kDa region, indicating lack of VDAC in Bax
immunoprecipitates. Similar results were obtained with ANT (data not
shown). B, chemical cross-linking with DSP or DTSSP was
carried out after releasing cytosol with digitonin. Digitonin
permeabilization allowed water-soluble DTSSP to react with
intracellular membranes. Membrane fractions from RPTC after ATP
depletion and control Bcl-21 cells were analyzed for Bax
and VDAC. VDAC was visualized as homo-oligomeric adducts in both
ATP-depleted RPTC (lanes 2 and 3) as
well as in control Bcl-21 cells (lanes
8 and 9). Lack of association of VDAC with Bax in
ATP-depleted RPTC or control Bcl-21 cells was suggested by
the absence of adducts with molecular mass of ~54 kDa (Bax/VDAC
dimer) or ~59 kDa (Bcl-2/VDAC dimer). Bax oligomers in ATP-depleted
RPTC were present (
-Bax), with no evidence of
Bax-VDAC heterodimer formation (absence of ~54 kDa; lanes
5 and 6). C, the inner mitochondrial
membrane protein ANT was visualized predominantly as monomers and
homodimers, with smaller amounts of higher order complexes when treated
with DPDPB in both control and ATP-depleted membranes from
Bcl-21 cells (lanes 1 and
3) or RPTC (lanes 5 and 7).
In contrast, DTBP, a cross-linker with a shorter spacer, failed to
stabilize ANT dimers and its complexes in control cells
(lanes 2 and 6), but stabilized them
in membranes from ATP-depleted cells (lanes 4 and
8). Complexes in the size range expected for Bax/ANT (~55
kDa) or Bcl-2/ANT (~60 kDa) heterodimers were not visualized. When
the membranes were sequentially probed for Bax, homo-oligomers of Bax
were visualized in ATP-depleted cells; again, complexes in the size
range of Bax/ANT oligomers (55 kDa) were not found (data not shown).
Similarly, sequential probing for Bcl-2 also failed to reveal Bcl-2/ANT
complexes (data not shown).
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Fig. 8.
Isoelectric focusing analysis of membrane
fractions to identify hetero-oligomers of Bax. Isoelectric
focusing methodology was described under "Experimental Procedures."
A, CHAPS extract of membranes from ATP-depleted and control
Bcl-21 cells were subjected to IEF with or without
cross-linking, as described under "Experimental Procedures"
( CL, no cross-linker; CL, 1, DPDPB;
CL 2, EGS). The sulfhydryl-reacting cross-linker, DPDPB,
does not change the pI of Bax or Bcl-2 (lanes 2 and 5), whereas the amine-reactive cross-linker, EGS,
changes the pI of these proteins (lanes 3 and
6). Bcl-2 has two minor isoforms with pI lower than the
predominant form indicated by arrowheads (lane
4). After DPDPB cross-linking, these two isoforms were not seen,
suggesting possible cross-linking of these forms to non-soluble cell
structures with free-sulfhydryl groups. In either case, complexes with
pI greater or lesser than the pI of Bax or Bcl-2 were not detected with
or without cross-linking, suggesting lack of interaction with other
proteins including VDAC and ANT. B, control
Bcl-21 cells were solubilized in Nonidet P-40 followed by
cross-linking with DPDPB. Detergent-induced Bax/Bcl-2 heterodimers (see
inset for sample analyzed by SDS-PAGE and immunoblotting)
were detected by immunoblotting after IEF. Both
-Bax and
-Bcl-2
antibodies were utilized sequentially to identify Bax and Bcl-2 in the
immunoblot. The pI of the Bax/Bcl-2 complex is the average of Bax and
Bcl-2 pI values. CL 1, DPDPB; CL 2, EGS;
CL, no cross-linker.
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Fig. 9.
Hypothetical model for Bax-induced
mitochondrial outer membrane permeabilization and Bcl-2
protection. Translocated Bax undergoes conformational changes in
mitochondrial outer membranes to form oligomers. Based on our
results and published work, we propose that higher order oligomers ( 4
Bax molecules) form channels that permit the transit of
apoptosis-activating factors such as cytochrome c. Although
not conclusively demonstrated, our results also suggest that Bax is not
associated with other mitochondrial channel-forming proteins (VDAC and
ANT) and that such interactions may not be required for the
permeabilizing actions of Bax. Alternatively, it is possible but
unlikely that oligomeric Bax do not form pores, but somehow induce pore
formation by rearranging other mitochondrial outer membrane proteins
(data not shown). In Bcl-2-overexpressing cells, oligomerization of Bax
is prevented by Bcl-2 even after being inserted into the membrane. We
suggest that steric hindrance imposed by abundant and possibly
saturating concentrations of Bcl-2 in membrane microdomains may prevent
inserted Bax from undergoing conformational changes required for
oligomerization. This model can also explain, in other examples of
apoptosis where ATP levels are not completely compromised, the apparent
prevention of Bax insertion by saturating concentrations of Bcl-2 in
membrane (25, 37) by assuming that partially inserted Bax may return to
cytosol due to failed conformational changes required for Bax anchoring
in the membrane. OM, outer membrane; IM, inner
membrane; IMS, intermembrane space.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We greatly acknowledge the generous gift of Bax monoclonal antibody 1D1 from Dr. Richard J. Youle and ANT polyclonal antibody from Dr. H. H. Schmid.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK54472 (to P. S.) and DK37139 (to M. A. V.), by a Morrison Trust grant (to P. S.), and by a Texas Advanced Research Program grant (to Z. D.).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: Dept. of Pathology,
University of Texas Health Science Center, 7703 Floyd Curl Dr., San
Antonio, TX 78229. Tel.: 210-567-6597; Fax: 210-567-2367; E-mail: saikumar@uthscsa.edu.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100655200
2 V. Mikhailov, M. Mikhailova, D. J. Pulkrabek, Z. Dong, M. A. Venkatachalam, and P. Saikumar, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: VDAC, voltage-dependent anion channel protein; RPTC, rat proximal tubule cell; Bcl-21, human Bcl-2-overexpressing rat proximal tubule cell; ANT, adenine nucleotide translocator protein; DSP, dithiobis(succinimidyl propionate); DTSSP, 3,3'-dithiobis(sulfosuccinimidyl propionate); DTBP, dimethyl 3,3'-dithiobispropionimidate; EGS, ethylene glycol bis(succinimidyl succinate); DPDPB, 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane; SANPAH, N-succinimidyl 6-[4'-azido-2'-nitrophenylamino]hexanoate; CCCP, carbonyl cyanide-m-chlorophenylhydrazone; DTT, dithiothreitol; IEF, isoelectric focusing; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; NHS, N-hydroxysuccinimide.
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