From the Department of Pathology, The University of Texas Health
Science Center, San Antonio, Texas 78229 and the Center
for Advanced Biotechnology and Medicine, § Howard Hughes
Medical Institute, Department of Molecular Biology and Biochemistry,
Rutgers University, Piscataway, New Jersey 08854
Received for publication, April 9, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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ATP depletion induced by hypoxia or
mitochondrial inhibitors results in Bax translocation from cytosol to
mitochondria and release of cytochrome c from mitochondria
into cytosol in cultured rat proximal tubule cells. Translocated
Bax undergoes further conformational changes to oligomerize into high
molecular weight complexes (Mikhailov, V., Mikhailova, M., Pulkrabek,
D. J., Dong, Z., Venkatachalam, M. A., and Saikumar, P. (2001) J. Biol. Chem. 276, 18361-18374). Here we
report that following Bax translocation in ATP-depleted rat proximal
tubule cells, Bak, a proapoptotic molecule that normally resides in
mitochondria, also reorganizes to form homo-oligomers. Oligomerization
of both Bax and Bak occurred independently of Bid cleavage and/or
translocation. Western blots of chemically cross-linked membrane
extracts showed nonoverlapping "ladders" of Bax and Bak complexes
in multiples of ~21 and ~23 kDa, respectively, consistent with
molecular homogeneity within each ladder. This indicated that Bax and
Bak complexes were homo-oligomeric. Nevertheless, each oligomer could
be co-immunoprecipitated with the other, suggesting a degree of
affinity between Bax and Bak that permitted co-precipitation but not
cross-linking. Furthermore, dissociation of cross-linked complexes by
SDS and renaturation prior to immunoprecipitation did not prevent
reassociation of the two oligomeric species. Notably, expression of
Bcl-2 prevented not only the oligomerization of Bax and Bak, but also
the association between these two proteins in energy-deprived cells.
Using Bax-deficient HCT116 and BMK cells, we show that there is
stringent Bax requirement for Bak homo-oligomerization and for
cytochrome c release during energy deprivation. Using
Bak-deficient BMK cells we further show that Bak deficiency is
associated with delayed kinetics of Bax translocation but does not
affect either the oligomerization of translocated Bax or the leakage of
cytochrome c. These results suggest a degree of functional
cooperation between Bax and Bak in this form of cell injury, but also
demonstrate an absolute requirement of Bax for mitochondrial permeabilization.
Members of the Bcl-2 family of proteins are key regulators of
programmed cell death or apoptosis (1, 2). These proteins are known to
affect mitochondrial function and regulate the release of
apoptosis-activating factors from mitochondria (1-3). Anti-apoptotic members of the Bcl-2 family (Bcl-2, Bcl-XL, Ced-9, Bcl-w, and Mcl-1)
act primarily to preserve mitochondrial integrity by suppressing the
release of cytochrome c. In contrast, pro-apoptotic members (Bax, Bak, Bik, Blk, Bok, Hrk, BNIP3, Bad, Bid, Bim, and EGL-1) mainly
induce the release of stimulators of apoptosis and cause mitochondrial dysfunction.
Apoptosis is regulated by the subcellular localization and
translocation of Bcl-2 family members. Anti-apoptotic members such as
Bcl-2 and Bcl-XL and the pro-apoptotic member Bak reside predominantly in mitochondria (4, 5). In contrast, pro-apoptotic members Bax, Bid,
and Bad that reside in the cytosol are translocated to the outer
mitochondrial membrane in response to stress or apoptotic stimuli and
release intermembrane space proteins (4, 6-8). It was shown through
gene knockout studies that pro-apoptotic Bax and Bak have redundant
function and are required for the induction of apoptosis in response to
a variety of death signals (9, 10). Distinct mechanisms are involved in
translocation of BH3 only proapoptotic proteins Bad and Bid from
cytosol to mitochondria. Phosphorylation and 14-3-3 binding regulate
Bad translocation (11), whereas Bid translocation is regulated by
proteolytic cleavage as part of the Fas and tumor necrosis
factor- Although translocation of Bax to the mitochondrial outer membrane is
required to release cytochrome c during apoptosis induced by
various death stimuli, the mechanism of Bax-mediated membrane permeabilization is still being debated. Recent studies have shown that
Bax forms homo-oligomeric complexes in mitochondrial membranes (16-19). These findings and other experiments on artificial lipid membranes (17) have suggested that Bax undergoes conformational changes
before and after insertion, followed by oligomerization and pore
formation. However, the possibility that Bax could interact with other
proteins in mitochondrial locations continues to be explored.
Oligomerization and/or activation of Bak and Bax were also induced by
Bid, another pro-apoptotic Bcl-2 family member, during death receptor
signaling (15, 19-22). Recent studies have indicated that
non-Bid-mediated mechanisms are also involved in the oligomerization of
Bax and Bak in viral-mediated apoptosis (23).
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 (24). This was attributed to pore formation by Bax homo-oligomerization (16). Bax oligomerization as well as mitochondrial outer membrane permeabilization was prevented by Bcl-2 without forming
physical complexes with Bax (16). Here we report that Bak, another
proapoptotic member of the Bcl-2 family, exists as part of a large
protein complex of unknown composition in mitochondria of normal cells,
but undergoes rearrangement to form homo-oligomeric complexes following
Bax insertion into mitochondria of hypoxic/ATP-depleted cells. Our data
suggest functional dependence between Bax and Bak and strong
association between Bax and Bak complexes in mitochondria. In addition
they also demonstrate a stringent Bax dependence to affect
mitochondrial permeabilization. Accordingly, selective Bak deficiency
delayed the kinetics of Bax translocation, but did not prevent Bax
oligomerization or cytochrome c release. On the other hand,
selective Bax deficiency prevented both Bak oligomerization and
cytochrome c release.
Materials--
DMEM,1
Ham's F-12/DMEM, McCoy's 5A medium, and minimum essential medium were
from Invitrogen. Antibodies were from suppliers as indicated: mouse
monoclonal antibodies, anti-rat Bax (1D1; Neomarkers, Fremont, CA),
anti-mouse Bax (5B7, Sigma), anti-Bcl-2 and anti-cytochrome
c (BD Biosciences); rabbit polyclonal antibodies, anti-Bax
(N-20; Santa Cruz Biotechnologies, Santa Cruz, CA) and anti-Bak
(Upstate Biotechnology, Lake Placid, NY); rat monoclonal, anti-human
Bid (Zymed Laboratories Inc.). Horseradish
peroxidase-conjugated or fluorescent label-conjugated secondary
antibodies to mouse or rabbit were obtained from Jackson ImmunoResearch
Labs (West Grove, PA). Rabbit polyclonal anti-mouse Bid that also
recognized rat Bid was kindly provided by Dr. Stan Krajewski, The
Burnham Institute, La Jolla, CA. RPTC and its clone
(Bcl-21), which is a overexpressing human Bcl-2 protein,
were described before (16). HeLa cells were obtained from American Type
Culture Collection (Manassas, VA). Bax(+/ ATP Depletion, Subcellular Fractionation, and Chemical
Crosslinking--
RPTC were cultured in serum-supplemented Ham's
F-12/DMEM as described before (24), HCT116 Bax( Assessment of Mitochondrial Membrane Potential--
To examine
changes in the mitochondrial membrane potential, experimental cells
were loaded with rhodamine 123 (50 µM), a
membrane-permeant mitochondria-specific tracer dye, for 10-15 min at
37 °C in a CO2 incubator. Cells were washed with
phosphate-buffered saline and observed under a fluorescent microscope.
Size Exclusion Chromatography--
Size exclusion chromatography
was performed using the AKTA purifier 10 (Amersham Biosciences) at room
temperature with a Superose 6 (10/30) gel filtration column
equilibrated in column buffer (buffer A supplemented with 2% (w/v)
CHAPS and 1 mM DTT) at a flow rate of 0.4 ml/min. For gel
filtration experiments, whole cells were extracted with column buffer
containing protease inhibitors. Extracts were centrifuged at
500,000 × g for 15 min before loading onto the
Superose 6 column. After loading the column with 200-µl samples,
fractions of 0.5 ml were collected and concentrated with trichloroacetic acid/acetone precipitation before analysis by Western
blotting. Gel filtration protein standards were used to calibrate the
column. Protein standards were thyroglobulin (669 kDa), 11 ml; ferritin
(440 kDa), 12.6 ml; catalase (232 kDa), 14.3 ml; bovine serum albumin
(67 kDa), 15.1 ml; ovalbumin (43 kDa), 15.6 ml; chymotrypsinogen A (25 kDa), 17.5 ml; and cytochrome c (12.4 kDa), 19.1 ml. The
void volume of the column was 7.5 ml.
Immunoanalysis--
Western blotting was done as described
before (16, 24). Briefly, proteins were resolved by SDS-PAGE in Xcell
II mini cells using 10 or 4-12% (gradient) NuPAGE gels (Invitrogen)
with MES running buffer in the presence (EGS cross-linking) or absence (dithiobis(succinimidyl propionate) cross-linking) of DTT. Proteins in
the gels were transferred to polyvinylidene difluoride membranes (0.2 µm) by electroblotting. For Western blot analysis, appropriate primary antibodies and peroxidase-conjugated secondary antibodies were
used. Chemiluminescent substrates (Pierce) were used to detect antigen-antibody complexes on the polyvinylidene difluoride membrane. Molecular weights of cross-linked Bax and Bak complexes were calculated by plotting their migrations against migrations of molecular weight standards in semilogarithmic plots.
Immunoprecipitation was carried out as described earlier (16).
Solubilized extracts (100-500 µg) in lysis buffer were precleared and the resultant supernatants were incubated with primary antibody (2 µg) at 4 °C for 2 h. 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. The immunoprecipitates dissolved in SDS/sample
buffer were analyzed by Western blotting as described above.
Immunocytochemistry was performed as described before (24). Cells
plated on coverslips were fixed with a modified Zamboni's fixative
followed by exposure to primary antibodies, anti-Bax (mouse monoclonal)
and anti-Bak (rabbit polyclonal) or anti-cytochrome c
(monoclonal, clone 2G8.B6 kindly provided by Dr. R. Jemmerson of
University of Minnesota Medical school, MN) followed by
Alexa-conjugated anti-rabbit and CY3-conjugated anti-mouse antibodies.
Pre-embedding EM immunocytochemistry in normal and apoptotic cells
after ATP depletion and repletion was performed as previously described
(28). Briefly, cells were fixed with 4% formaldehyde in 0.1 M phosphate buffer at pH 7.4 for 45 min, washed,
saponin-permeabilized, and incubated with anti-Bax antibody (1D1)
followed by Nanogold-labeled Fab' anti-mouse secondary antibody
(Nanoprobes, Yaphank, NY). Silver enhancement (HQ kit; Nanoprobes) was
done following the manufacturer's recommendations. Samples were
treated with 0.2% OsO4, washed, stained with uranyl
acetate, dehydrated in ethanol, and embedded in Epoxy resin.
Bax Translocation, and Reorganization/Oligomerization of Bax and
Bak during ATP Depletion--
ATP depletion by hypoxia or treatment
with CCCP, a mitochondrial uncoupler, was shown to cause Bax
translocation to mitochondria and release of cytochrome c
into cytosol in cultured rat kidney proximal tubule cells (16, 24). The
BMK cells deficient in both Bax and Bak showed resistance to tumor
necrosis factor-
To identify Bax-Bak interactions, cells were fractionated into
cytosolic and membrane fractions (16) followed by chemical cross-linking with dithiobis(succinimidyl propionate) or EGS. Western
blotting of cross-linked proteins showed that progressively greater
amounts of Bax translocated to mitochondria during increasing durations
of ATP depletion and oligomerized into dimers and higher oligomers in
multiples of ~21 kDa, the monomer (16) (Fig. 1C, lanes 1-5). Concurrently, mitochondrial Bak, normally
present as monomers as well as very large protein complexes, rearranged to form Bak oligomeric "ladders" (Fig. 1, C and
D). As in the case of Bax (16), the calculated molecular
weights of Bak complexes in these ladders correspond to
multiples of ~23 kDa, the monomer (Fig. 1D, lane
4). This suggests that these newly formed complexes of Bak are
homo-oligomers. The mobility and molecular sizes of Bak oligomers were
quite distinct from those of Bax oligomers (Fig. 1, C and
D). The large Bak complexes greater than 250 kDa that were
observed in normal cells at the top of the gel (Fig. 1D,
lane 3) were diminished in amount during ATP depletion with concomitant appearance of Bak oligomers (Fig. 1D, lane
4). It has been reported that Bak oligomerization is associated
with t-Bid translocation to mitochondria after Bid cleavage by
caspase-8 (17). Analysis of Bid protein in RPTC during ATP depletion
(Fig. 1E) indicated no significant change of Bid protein
levels in the cytosol. The small amount of Bid detected in membrane
fractions of control cells was unchanged during ATP depletion.
Furthermore, we did not detect cleaved products of Bid such as t-Bid
(~15 kDa) in the membrane fraction, suggesting that Bid is not
involved in Bax translocation and oligomerization.
Bax translocation was observed also in HeLa cancer cells that had been
subjected to hypoxia or ATP depletion. The data presented in Fig.
2A show Bax translocation and
release of cytochrome c from mitochondria into cytosol
during hypoxia in HeLa cells. As in the case of RPTC, hypoxia did not
lead to Bid cleavage (Fig. 2A) or affect its localization.
Chemical cross-linking of membrane proteins in HeLa cells showed
oligomerization of translocated Bax (Fig. 2B, lane
4') and dimerization of Bak (Fig. 2B, lane 4). Bak reorganization was mainly seen as Bak dimer in HeLa cells; this appears to be because of overshadowing of Bak oligomers by very
large amounts of Bak containing protein complexes of heterogeneous molecular size in control as well as in hypoxic cells (Fig.
2B, lanes 2 and 4).
Formation of Oligomeric Complexes of Bax and Bak--
We used size
exclusion chromatography to characterize Bax and Bak protein complexes
in RPTC. Total cell lysates in 2% CHAPS lysis buffer from normal and
ATP-depleted RPTC were fractionated on a Superose 6 gel filtration
column and eluted fractions were analyzed by Western blotting. Results
presented in Fig. 3A indicated that Bax is monomeric in normal cells with a peak elution at about 25 kDa. In contrast, Bax from ATP-depleted cells eluted in fractions with
apparent molecular weights between 25,000 and 1340,000 with a
peak at about 440,000 consistent with the formation of large Bax
complexes (Fig. 3A). On the other hand, Bak eluted in a
broad range between 43 to 5000 kDa even in normal cells, with a peak at
about 232 kDa (Fig. 3B). The distribution of Bak as part of a large complex in mitochondria of normal cells was evident also after
protein cross-linking; Bak adducts of >250 kDa size were observed
(Fig. 1C). At present, the identity of protein(s) associated with Bak in mitochondria is unknown. ATP depletion induced a small but
significant alteration in Bak elution profile with a peak at about 440 kDa (Fig. 3B). The overlapping elution profiles of Bax and
Bak suggest possible association between these proteins in the
mitochondria of ATP-depleted cells.
Immunoelectron microscopy confirmed the translocation and localization
of Bax in mitochondrial membranes in the form of complexes. Bax was
seen exclusively in the cytosol of normal cells but clustered around
mitochondria in apoptotic cells after ATP depletion and repletion
induced by reincubation in growth medium (Fig. 3C,
panels 1 and 2, respectively). Localization of
Bax clusters largely on the surfaces of mitochondria is consistent with
our proposal that translocated and oligomerized Bax forms channels
resident in mitochondrial outer membranes (16). We attempted
immunoelectron microscopy of Bak also; although suitable for Western
blotting, the antibodies currently available caused unacceptable
background signals in immuno-EM images (not shown).
Oligomerization of Bax and Bak Is Inhibited by Bcl-2--
We have
shown earlier that Bcl-2 overexpression prevents Bax oligomerization
and cytochrome c release in RPTC after hypoxia or ATP
depletion (16). We have now tested the effect of Bcl-2 on Bak
oligomerization using chemical cross-linking and gel filtration. As
shown in Fig. 4A, Bcl-2
overexpression prevented the rearrangement of both translocated Bax and
mitochondrial Bak during ATP depletion induced by CCCP. By gel
filtration, alterations in the elution profile of Bax in ATP-depleted
Bcl-21 cells were only modest (Fig. 4B). On the
other hand, the Bak elution profile was compressed following CCCP
treatment of Bcl-21 cells (Fig. 4C, bottom
panel) as in the wild type RPTC (Fig. 3B, bottom
panel). The compression of elution profiles corresponded to
reduction in the amounts of normally present large Bak adducts retained
at the top of the gel after chemical cross-linking (Fig. 4A,
lanes 6 and 8). Nevertheless, smaller Bak
oligomers did not form in ATP-depleted Bcl-2 overexpressing cells
relative to controls (Fig. 4A, lane 8). These
results suggest that Bcl-2 inhibits not only the oligomerization of Bax
following translocation of the protein, but also the rearrangement of
mitochondrial Bak to form small homo-oligomers.
Bax Oligomers Are Associated with Bak Oligomers--
Whereas gel
filtration studies have helped to identify the formation of large
protein complexes, they failed to characterize the organization of
these complexes. Therefore, chemical cross-linking along with
immunoprecipitation was undertaken to clarify how Bax and Bak complexes
are organized during ATP depletion. For immunoprecipitation, CHAPS
extracts were used because nonionic detergents such as Nonidet P-40 and
Triton X-100 induce conformational change and oligomerization of Bax
(16, 29). The anti-Bax antibody (1D1) recognizes a buried epitope of
Bax in normal cells and precipitates little or no Bax (Fig.
5A, lane 7 and
9, bottom panel) (24). However, Bax is
precipitated by the anti-Bax antibody in ATP-depleted RPTC and
Bcl-21 cells (Fig. 5A, bottom panel,
compare lanes 8 and 10 with lanes 7 and 9) because of Bax conformational changes that result in
membrane translocation. A modest but significant increase of Bak
precipitation was seen with anti-Bak antibodies also, following ATP
depletion (Fig. 5A, upper panel, compare
lanes 1 and 3 with 2 and
4). Although chemical cross-linking yielded distinct
nonoverlapping ladders of Bak and Bax (Fig. 1, B and
C), Western blotting of anti-Bax immunoprecipitates of
ATP-depleted wild type RPTC revealed the presence of Bak in the
precipitate (Fig. 5A, top panel, lane 8). Similarly, anti-Bak immunoprecipitates from ATP-depleted cells contained Bax (Fig. 5A, bottom panel, lane
2). Therefore, co-immunoprecipitation results indicated possible
association between Bak and Bax oligomers in the mitochondria of
ATP-depleted wild type RPTC (Fig. 5A, lanes 2 and
8). As expected, overexpression of Bcl-2 inhibited
co-immunoprecipitation of Bax and Bak from ATP-depleted cells (Fig.
5A, lanes 4 and 10). Furthermore,
Bcl-2 was not co-precipitated with either Bax antibodies or Bak
antibodies (not shown) suggesting lack of physically stable association
between pro-apoptotic Bak or Bax with anti-apoptotic Bcl-2. To
detect Bak-Bax oligomers with increased sensitivity, chemically
cross-linked extracts were also subjected to co-immunoprecipitation. Interestingly, distinct ladders of Bax dimers, trimers, and higher order oligomers were co-precipitated with anti-Bak (Fig. 5B,
lane 6), and Bak dimers, trimers, and tetramers were
co-precipitated with anti-Bax in ATP-depleted RPTC (Fig. 5B, lane
2) confirming the association between Bax and Bak oligomers.
Dissociation and Reassociation of Bax and Bak Oligomers--
The
presence of nonoverlapping ladders of Bax and Bak raised the question
of how Bax homo-oligomers would interact with Bak homo-oligomers. To
address this question, in some experiments, mitochondrial membranes
from ATP-depleted cells were extracted with SDS buffer (0.35%) with or
without prior chemical cross-linking (labeled +SDS in Fig.
6). The SDS extracts were heated to
70 °C for 10 min to dissociate noncovalently interacting molecules. Proteins were renatured by 10-fold dilution of SDS in the presence of
2% CHAPS at room temperature. After SDS dilution, proteins were
immunoprecipitated with either anti-Bak or anti-Bax antibodies. In
another group of experiments, immunoprecipitation was performed without
prior SDS treatment (labeled
The most important inference from these observations on the effect of
SDS exposure prior to immunoprecipitation of cross-linked Bax and Bak
is that regardless of prior SDS treatment, co-precipitated ladders of
Bax and Bak are nonoverlapping with respect to the molecular sizes of
the respective oligomers (Fig. 6B, lanes 1, 2 and 3, 4). The results pose a
paradox with respect to how Bax and Bak oligomers are formed and how
they associate with each other. It seems apparent that the observations
can only be explained by the formation of largely homogeneous Bax and
Bak homo-oligomers in ATP-depleted mitochondrial membranes. The
propensity of these oligomers to co-precipitate may indicate a degree
of affinity between Bax and Bak oligomers that cannot be preserved by
cross-linking, but one which may be compatible with a role for Bax
translocation and homo-oligomerization in subsequently inducing the
recruitment and formation of Bak oligomers. Thus, oligomerization of
translocated Bax may be a pre-requisite to interact with Bak and cause
Bak reorganization.
Functional Interdependence of Bax and Bak--
To investigate the
role of Bax in Bak reorganization and cytochrome c release,
we have subjected Bax(+/
To determine whether Bax can independently oligomerize and release
cytochrome c in the absence of Bak, we used transformed baby
mouse kidney cells (25) derived from wild type (Bax(+/ The release of cytochrome c from mitochondria is a
crucial step in apoptotic signaling through the activation of caspases (30-32). Several studies point to a major role for Bax in cytochrome c release based on its ability to form channels in
artificial lipid membranes (33) and large oligomeric complexes in the
mitochondrial outer membrane (16-18). Although the formation of
transmembrane channels by Bax oligomers is a likely explanation, the
question of how Bax triggers cytochrome c release after its
translocation to mitochondria continues to be debated. Here, we
demonstrate that following Bax redistribution from the cytosol to
mitochondria, formation of Bax oligomers in the mitochondrial outer
membrane is also accompanied by reorganization of resident Bak
molecules to homo-oligomerize. Analysis of the molecular sizes of Bax
and Bak oligomers by cross-linking and SDS-PAGE indicated that both Bax
and Bak oligomers are largely if not exclusively homogeneous. Our
results show also that these homo-oligomeric complexes of both Bak and
Bax co-precipitated with each other during immunoprecipitation.
Unlike Bax, Bak was found to be constitutively present in the
mitochondria of RPTC (Fig. 1A, panel 1), in the
form of large complexes by gel filtration (Fig. 3A) and
chemical cross-linking (Fig. 1C). After hypoxia or ATP
depletion, both Bax and Bak were co-localized in mitochondria (Fig.
1A, panel 2). Following translocation of Bax to
mitochondria, Bak reorganized to form smaller oligomeric complexes that
are absent in normal cells (Fig. 1, C and D). The molecular weights of Bax and Bak oligomers indicated that these complexes are homogeneous and are therefore homo-oligomers (Fig. 1D). By analogy to mechanisms that lead to pore formation by
bacterial toxins, conformational changes may occur in Bax that lead to
oligomerization following membrane insertion (34, 35). The observation
that both Bax and Bak exhibit conformational changes during hypoxia or
ATP depletion raises the question whether these two proteins are
causally linked to the release of apoptogenic cytochrome c and Smac proteins from mitochondria. Based on previous reports that Bak
oligomerization is primarily mediated by Bid, we explored the role of
Bid in hypoxia or ATP depletion-induced apoptosis. Previous studies
have shown that death stimuli by Fas ligand or tumor necrosis
factor- Protein complex formation in mitochondrial membranes was also analyzed
by size sieving chromatography. We chose CHAPS to solubilize membrane
proteins because of its smaller aggregation number and its inability to
induce conformational change in the Bcl-2 family of proteins (16, 29).
Although co-purification of proteins in fractions separated by gel
filtration does not necessarily mean interactions between the proteins,
the converse should be true. That is, proteins with tight interactions
between them should co-purify. In normal cells, both Bax and Bak have
distinct elution profiles after gel filtration. The broader elution
profile (43-5000 kDa) of Bak in normal cells (Fig. 3) suggests
possible association of Bak with a protein or protein complex in the
mitochondrial outer membrane. In contrast, the elution profile of Bax,
which is cytosolic in normal cells, is sharper and indicative of its monomeric nature (Fig. 3). However, the elution profiles of both Bak
(~43-1340 kDa) and Bax (~25-1340 kDa) were found to overlap significantly in ATP-depleted cells (Fig. 3). Our immunoelectron microscopy data on translocated Bax in mitochondria of ATP-depleted cells are in agreement with results reported by Youle's group (37) in
terms of the clustering of Bax on mitochondrial surfaces. However, we
did not detect large clusters of Bax molecules outside the mitochondria
in cells affected by ATP depletion-induced apoptosis (Fig.
3C, panel 2). The immuno-EM findings are also
consistent with our previous studies (16, 24) and current results
showing Bax translocation, oligomerization, and cytochrome c release.
By several types of analysis including cross-linking, gel filtration,
and immunoprecipitation, our results have further clarified the
inhibitory effect of Bcl-2 expression on the deleterious effects of
pro-apoptotic Bax and Bak. Bcl-2 prevented the relatively close association of Bax and Bak molecules with themselves to form chemically cross-linkable homo-oligomers, as well as the possibly looser association between these two different species of homo-oligomers to
form larger coimmunoprecipitable complexes that could not be stabilized
by cross-linking. More Bax and Bak were precipitated by their
respective antibodies from ATP-depleted cells than from normal cells
(Fig. 5). This can be attributed to increased antibody recognition of
these antigens caused by unfolding of the COOH terminus of Bax and
translocation of the protein (13, 29), and dissociation of Bak from
large Bak containing complexes of otherwise unknown composition, native
to mitochondrial membranes (Fig. 4). Relevant to the formulation of
strategies to address still unresolved questions regarding how Bcl-2
protects cells, expression of this protein prevented neither the ATP
depletion-induced mitochondrial translocation of Bax (16, 24), nor the
associated dissolution of large Bak containing complexes (Figs. 4 and
5). However, Bcl-2 did prevent or markedly inhibited Bax and Bak
homo-oligomerization and cross-immunoprecipitation of Bax and Bak that
could otherwise be demonstrated easily in the absence of Bcl-2
expression (Figs. 4 and 5). The molecular interactions that underlie
these remarkable results will need close attention in future studies.
Co-immunoprecipitation of Bax oligomers with Bak antibodies and Bak
oligomers with Bax antibodies (Fig. 5) and the nonoverlapping nature of
Bax and Bak ladders after co-precipitation (Fig. 5B) together suggest that these different homo-oligomeric species interact
in the membrane. Interestingly, dissociation and reassociation studies
have thrown light on the nature of interaction between Bax and Bak.
Regardless of treatment with SDS prior to renaturation and
cross-immunoprecipitation of cross-linked membranes, Bax as well as Bak
containing oligomers (ladders) were largely, if not exclusively,
homogeneous, i.e. they were homo-oligomeric. This suggests
that molecular interactions between individual monomers in either Bax
or Bak ladders were close, to the extent that they could be stabilized
by cross-linking. The failure to cross-link proteins does not
necessarily mean they are not bound to each other. Steric factors
related to the chemical nature of the cross-linkers and/or protein
interactions are involved in cross-linking of proteins, and negative
results need to be interpreted with caution. This was addressed by
using a nonspecific heterobifunctional cross-linker SANPAH
(N-succinimidyl-6-[4'-azido-2'-nitrophenylamino]
hexanoate), which cross-links proteins through an amine reactive and a
photoactivable nitrene that reacts nonspecifically with any atom within
the reach of the spacer arm, and we obtained predominantly
homo-oligomers (16). On the other hand, interactions between Bax and
Bak must have been relatively loose, because they permitted
co-immunoprecipitation, but could not be stabilized by cross-linking to
any significant extent (for the exception see Fig. 6B,
lane 6). If Bax-Bak interactions had been tight to the
degree that both molecules contributed to the formation of
hetero-oligomeric complexes (Bax-Bak pore), Bax and Bak ladders
resolved by SDS-PAGE should have shown overlapping patterns. Obviously,
this was not the case.
Our results show that trimers and higher order cross-linked oligomers
of Bax reassociate with Bak during CHAPS renaturation after SDS
denaturation to a much greater extent than do Bax-Bax dimers or Bax
monomers (Fig. 6B, lane 6). The appearance of
Bax-Bak heterodimers only as a minor component of these reassociated
complexes (Fig. 6B, lane 6) further reinforces
the concept that pores in the membrane are mainly constituted of
homo-oligomeric Bax and Bak. Considered in their entirety, our results
suggest that interaction between Bax and Bak takes place after Bax has
undergone conformational change to oligomerize. Therefore, Bax
oligomerization probably precedes Bak reorganization and oligomerization.
Gene knockout studies with Bax, Bak, and Bax plus Bak have
indicated that cells with single gene knockout are capable of releasing cytochrome c, but those with double knockout are not (10,
25). Indeed, mice deficient of both Bax and Bak died perinatally and suffered multiple developmental defects that were more severe than in
animals deficient for only Bax or Bak (9). This finding was attributed
to suppression of developmentally regulated apoptosis caused by the
double knockout. It has been reported that Bak knockout mice are
developmentally normal and reproduce normally (9), whereas Bax knockout
mice are reproductively defective and display some phenotypic
abnormalities (38, 39). With respect to our findings, one possibility
is that Bax and Bak are capable of forming pores independent of each
other. On the other hand, Bak may require Bax or a similar molecule to
form pores optimally. The latter argument is based on the observations
that some colon cancer cells do not exhibit redundancy with respect to
the apoptogenic roles of Bak and Bax (26, 40). A third explanation may
involve formation by both Bak and Bax of pores larger than those formed
by either Bax or Bak alone. Because heteromeric Bax-Bak complexes were
not seen after chemical cross-linking, our studies do not favor a hetero-oligomeric pore, at least in the hypoxic model of apoptosis in
kidney epithelial, HeLa, and HCT116 cells. Research to date suggests
that Bak reorganization to form homo-oligomers requires another Bcl-2
family protein such as t-Bid (21) or Bax (current study). In
unpublished work,2 we have
investigated the permeabilization of isolated mitochondria from
Bax(
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
signaling pathways (7, 8). Regulation of Bax
translocation is distinct from that of Bad and Bid and involves a
conformational change that exposes the amino and carboxyl termini
leading to mitochondrial translocation (12-15). Nevertheless, the
exact mechanisms that cause conformational changes in Bax are still unknown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and Bax(
/
) HCT116
colon cancer cell lines were kindly provided by Dr. Bert Vogelstein,
Johns Hopkins University School of Medicine, Baltimore, MD. The wild
type (Bax(+/
)/Bak(+/+)), Bax-deficient (Bax(
/
)/Bak(+/+)),
Bak-deficient (Bak(
/
)/Bax(+/
), and both Bax and Bak-deficient
(Bax(
/
)/Bak(
/
)) BMK cells were described before (25).
Detergents and chemical cross-linkers were purchased from Pierce. Gel
filtration column (Superose 6) and standards were obtained from
Amersham Biosciences (Piscataway, NJ). All other reagents were of the
highest grade available.
/
) and Bax(+/
)
cells (26), BMK cells, and HeLa cells were cultured in McCoy's 5A
medium, DMEM, and minimum essential medium supplemented with 10% fetal bovine serum, respectively. Cells were plated at 1-2 × 105 cells/cm2 in 60- or 100-mm dishes. After
overnight growth, cells in glucose-free Krebs-Ringer bicarbonate buffer
(in mM: 115 NaCl, 1 KH2PO4, 4 KCl,
1 MgSO4, 1.25 CaCl2, and 25 NaHCO3)
for RPTC, HeLa, and HCT116 or glucose- and serum-free DMEM for BMK were
subjected to ATP depletion induced by a mitochondrial inhibitor
(uncoupler CCCP) or hypoxia (incubated in an anaerobic chamber) for the
indicated times. Necrotic injury in ATP-depleted cells was prevented by inclusion of 5 mM glycine in the buffer to simulate glycine
contents of tissues in vivo (27). Cytosolic and membrane
fractionation and chemical cross-linking were done as described before
(16). Briefly, cells were permeabilized at room temperature with
0.015-0.02% digitonin for 1-2 min in isotonic buffer A (10 mM HEPES, 150 mM NaCl, 1.5 mM
MgCl2, 1 mM EGTA, pH 7.4) containing protease
inhibitors (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride
hydrochloride, 0.8 mM aprotinin, 50 mM
bestatin, 15 mM E-64, 20 mM leupeptin, 10 mM pepstatin A). The permeabilized cells were shifted to
4 °C, scraped, and collected into centrifuge tubes. The supernatants (digitonin/cytosol) were collected after centrifugation at 15,000 × g for 10 min at 4 °C. The pellet was further extracted
with ice-cold lysis buffer (2% CHAPS in buffer A containing protease inhibitors) for 60 min at 4 °C to obtain membrane fraction. Cells permeabilized with digitonin or membranes extracted with CHAPS were
incubated with bifunctional cross-linkers (1 mM
dithiobis(succinimidyl propionate) or EGS with linker lengths of 12 and
16 Å, respectively) on a head-to-head rocker for 30 min at room
temperature. After quenching the unreacted cross-linkers with 1/10
volume of 2 M Tris-HCl (pH 7.4), cells or extracts were
incubated for another 30 min at room temperature with rocking. After
cross-linking, membranes were extracted with 2% CHAPS in buffer A. In
some experiments, membrane extracts were prepared in 0.35% SDS plus 1 mM DTT in buffer A instead of CHAPS. Mitochondria from
Bax(
/
) and Bax(+/
) cell homogenates were obtained by differential
centrifugation (16).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced cell death (25). However, cells deficient
in Bax or Bak alone were susceptible to tumor necrosis
factor-
-induced cell death suggesting that Bak can substitute for
Bax in forming mitochondrial pores to release cytochrome c.
This prompted us to further investigate the role of Bax and Bak in
outer membrane permeability of mitochondria in energy-deficient cells.
Double immunolabeling of RPTC with anti-Bak (green) and
anti-Bax (red) revealed that Bak is constitutively present
in mitochondria whereas Bax is predominantly localized in the cytosol
of normal cells (Fig. 1A,
panel 1). ATP depletion by CCCP resulted in co-localization
of both Bak and Bax in the mitochondria (Fig. 1A,
panel 2). We have shown earlier that following Bax
translocation, cytochrome c is released into cytosol (16, 24). To find out whether the development of permeability transitions can explain cytochrome c release after prolonged exposure to
CCCP, cells depleted of ATP by CCCP were allowed to recover in complete growth medium without CCCP, but in the presence of z-VAD, a caspase inhibitor that blocks downstream apoptotic events. During recovery, cells were loaded with rhodamine 123, whose accumulation in
mitochondria is dependent on the presence of the potential gradient
across the mitochondrial inner membrane. The results presented in Fig. 1B clearly show that both control and recovering cells were
able to a mount potential showing of perinuclear mitochondrial
distribution of rhodamine fluorescence (Fig. 1B,
panels 1 and 3). On the other hand, in
CCCP-treated cells, rhodamine 123 remained in the cytosol indicating
loss of mitochondrial potential (Fig. 1B, panel
2). Experiments done in parallel showed that cells treated
similarly with CCCP had leaked cytochrome c into cytosol
without or with an additional period of recovery (not shown). The
ability of mitochondria to accumulate rhodamine 123 despite having
leaked cytochrome c precluded the possibility that
permeability transitions had occurred, because the development of
transitions is inconsistent with the ability of membranes to maintain a
barrier to the free diffusion of protons.
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Fig. 1.
Bax translocation and oligomerization of
mitochondrial Bax and Bak in ATP-depleted cells.
A, immunolocalization of Bak and Bax in normal and
ATP-depleted RPTC. Bak and Bax were localized with double
immunolabeling. Control and ATP-depleted cells were fixed and labeled
for both Bax and Bak as described under "Experimental Procedures."
Panel 1 shows simultaneous visualization of Bak and Bax in
normal cells. Inset shows identical cells observed in
different channels with Bak (green) in mitochondria and Bax
(red) in the cytosol. Panel 2 shows Bak and Bax
distribution in ATP-depleted cells after 3 h of CCCP treatment.
Inset shows Bak (green) and Bax (red)
now co-localized to mitochondria. B, RPTC were exposed to
CCCP for 4 h (CCCP). Another group of CCCP-treated
cells were allowed to recover in full growth medium without CCCP in the
presence of z-VAD, a caspase inhibitor to block downstream events of
apoptosis (Recovery). Both groups of cells as well as
control cells (Control) were loaded with rhodamine 123 for
10-15 min and photographed using fluorescence microscopy.
C, following incubation of RPTC with 15 µM CCCP for 0, 1, 2, 3, or 4 h, cells were treated
with the cleavable membrane-permeable cross-linker
dithiobis(succinimidyl propionate) (1 mM). Membrane
fractions were obtained as described under "Experimental
Procedures," and analyzed for Bax (lanes 1-5) or Bak
(lanes 6-10) by Western blotting under nonreducing
conditions. Prolonged exposure to CCCP resulted in progressive
accumulation of Bax in the membrane fraction as slow moving complexes
(lanes 3-5). Similarly, slow moving Bak complexes appeared
after ATP depletion in parallel with Bax accumulation in mitochondria
(lanes 6-10). Closed arrowheads indicate Bax
oligomers and open arrowheads indicate Bak oligomers.
D, membrane extracts of ATP-depleted cells (4 h CCCP)
after incubation with the noncleavable cross-linker EGS were analyzed
by Western blotting under reducing conditions. The molecular weights of
Bax and Bak complexes were calculated by plotting their migrations
against migrations of molecular weight standards in semilogarithmic
plots from several experiments. Two representative lanes of Bak ladders
and Bax ladders are shown and they show homo-oligomers of Bak and Bax,
respectively. E, time course analysis of Bid
distribution by Western blotting in the cytosol and membranes of
ATP-depleted cells.
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Fig. 2.
Hypoxia-induced Bax translocation and Bak
oligomerization in HeLa cells. A, HeLa cells were
incubated for 0, 1, 2, 3, 4, and 5 h of hypoxia in an anaerobic
chamber. Cytosol and membrane fractions were obtained as described
under "Experimental Procedures," and analyzed for Bax, Bid, and
cytochrome c (Cyt.c) by Western blotting
(lanes 1-6). Hypoxic exposure resulted in the accumulation
of Bax in the membrane fraction and cytochrome c in the
cytosol. B, following hypoxic incubation, HeLa
membranes were treated with the chemical cross-linker EGS (1 mM) and analyzed for Bak (lanes 1-4) or Bax
(lanes 1'-4') by Western blotting.
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Fig. 3.
Co-elution of Bak and Bax oligomers in
energy-deprived cells. CHAPS-solubilized membrane extracts from
normal and ATP-depleted (4 h CCCP) RPTC were analyzed on a Superose 6 gel filtration column (10/30) equilibrated in Buffer A containing 2%
(w/v) CHAPS and 1 mM DTT and eluted at a flow rate of 0.4 ml/min. Fractions of 0.5 ml were collected and alternative fractions in
the separation range were analyzed by SDS-PAGE and immunoblotting with
Bax or Bak antibodies as described under "Experimental Procedures."
A, the scanned elution profile of Bax in normal
(RPTC/Con) and ATP-depleted cells
(RPTC/CCCP). B, the scanned
elution profile of Bak from normal
(RPTC/Con) and ATP-depleted cells
(RPTC/CCCP). C, EM
immunocytochemistry of Bax in normal and apoptotic RPTC. ATP depletion
followed by recovery in complete growth medium induced apoptosis
because of resynthesis of glycolytic ATP. Arrowheads
represent cytosolic Bax in panel 1. M represents
mitochondria in panel 2.
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Fig. 4.
Bcl-2 overexpression blocks Bax and Bak
oligomerization. A, cross-linking of Bcl-2
overexpressing RPTC (Bcl-21) failed to reveal slow
moving Bax adducts (lane 4) or Bak adducts (lane
8) after ATP depletion (4 h CCCP). In contrast, RPTC show Bax
(lane 2) and Bak (lane 5) oligomers after ATP
depletion. B, the scanned elution profile of Bax in
normal (Bcl-21/Con) and
ATP-depleted cells (Bcl-21/CCCP).
C, the scanned elution profile of Bak from normal
(Bcl-21/Con) and
ATP-depleted cells
(Bcl-21/CCCP).
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Fig. 5.
Co-immunoprecipitation of Bak and Bax
oligomers. A, immunoprecipitation of 2% CHAPS extracts
of whole cells (~300 µg) from control and ATP-depleted RPTC and
Bcl-21 cells with anti-Bax (mouse monoclonal, 1D1) and
anti-Bak (rabbit polyclonal) antibodies were carried out as described
under "Experimental Procedures." The immunoprecipitates were
analyzed under reducing SDS-PAGE, followed by immunoblotting with
anti-Bak and anti-Bax antibodies. These blots were subsequently
re-probed for either Bax or Bak. Both Bak (lanes 1 and
3) and Bax (lanes 7 and 9) antibodies
failed to co-precipitate Bax and Bak, respectively, from control cells.
On the other hand, ATP-depleted (4 h CCCP) cells showed signals for the
presence of Bax (lane 2, bottom) and Bak
(lanes 8, top), respectively, in Bak and Bax
immunoprecipitates. Bax in cytosol is poorly precipitated by anti-Bax
antibody in CHAPS extracts from normal cells (lanes 7 and
9, bottom panel). Only Bax translocated to mitochondria is
recognized well by this antibody in CHAPS extracts from ATP-depleted
RPTC and Bcl-21 cells (lanes 8 and 10, bottom panel). A moderate increase in Bak recognition by
polyclonal anti-Bak antibody is also noted (compare lanes 1 and 3 with lanes 2 and 4, top panel).
There is little or no cross-precipitation of Bax or Bak with each other
in ATP-depleted Bcl-21 cells (Lanes 4 and
10). B, digitonin insoluble, 2% CHAPS extracted
membrane fractions (~100 µg) after chemical cross-linking with EGS
from both ATP-depleted and control cells were subjected to
immunoprecipitation as described under "Experimental Procedures"
with monoclonal anti-Bax (lanes 1-4) and polyclonal
anti-Bak antibodies (lanes 5-8). Immunoblotting of Bax
immunoprecipitates with anti-Bak (lanes 1-4) and Bak
immunoprecipitates with anti-Bax antibodies (lanes 5-8) was
performed after SDS-PAGE under reducing conditions. Distinct ladders of
Bak in Bax immunoprecipitates and Bax in Bak immunoprecipitates were
observed in lysates from ATP-depleted RPTC but not in control or
Bcl-21 cells with or without ATP depletion.
SDS in Fig. 6). Western blotting analysis of Bak (or Bax) immunoprecipitates of noncross-linked mitochondrial membranes revealed that little or no Bax (or Bak) is
co-precipitated if they had been exposed to SDS prior to treatment with
the precipitating antibody (shown for Bak immunoprecipitate in Fig.
6A). Thus, in the absence of chemical cross-linking, all complexes of Bax and Bak were separated into monomers during SDS treatment. Failure to co-precipitate Bax with Bak from dissociated complexes suggests that Bak and Bax monomers did not reassociate with
each other after exposure to SDS. On the other hand,
immunoprecipitation of Bak from chemically cross-linked mitochondrial
proteins after SDS denaturation and dilution in CHAPS yielded Bax
oligomers containing three or more molecules (Fig. 6B,
lanes 5 and 6). These immunoprecipitates contained little or no Bax monomers and dimers (Fig. 6B,
lane 6) relative to conditions where they had not been
previously exposed to denaturation with SDS (Fig. 6B,
lane 5). Of interest, there were modest amounts of Bax-Bak
heterodimers in the precipitates that resisted dissociation by SDS.
However, higher order Bax-Bak oligomers could not be identified.
Anti-Bax antibodies precipitated all forms of Bak including monomers
and dimers, with or without SDS denaturation and dilution in CHAPS
(Fig. 6B, lanes 3 and 4). In the
absence of interaction between monomeric forms of Bax and Bak, this
result suggests that all forms of Bak interact with oligomeric Bax.
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Fig. 6.
Dissociation and reassociation of Bax and
Bak. A, membrane fractions without cross-linking
( CL) from ATP-depleted RPTC (5 × 106
cells) were extracted with either CHAPS (
SDS) or 0.35%
SDS (+SDS) plus 1 mM DTT in buffer A. The
samples extracted with SDS were heated at 70 °C for 10 min to remove
noncovalent interactions. The samples were renatured by diluting with
2% CHAPS buffer to a final SDS concentration of 0.035%. Diluted
samples were immunoprecipitated with anti-Bak antibodies and
precipitates were analyzed for Bax and Bak by Western blotting. SDS
treatment increased Bak precipitation by anti-Bak antibodies, but it
inhibited Bax co-precipitation. B, membranes were treated
with EGS cross-linker (+CL) before extracting with CHAPS or
SDS buffer. The SDS extracts were renatured in CHAPS as described above
and subjected to immunoprecipitation with anti-Bak or Bax antibodies.
The immunoprecipitates were separated on SDS-PAGE and analyzed by
Western blotting. Bax was immunoprecipitated with 1D1 monoclonal
antibody and probed with polyclonal anti-Bak antibody (lanes
3 and 4) followed by polyclonal anti-Bax (N-20)
antibody (lanes 1 and 2) after stripping Bak
antibody of the membrane. Bak was immunoprecipitated with polyclonal
antibody and probed with monoclonal anti-Bax antibody (lanes
5 and 6). Mainly Bax trimers and higher oligomers are
co-precipitated with Bak. All forms of Bak co-precipitated with Bax
probably by associating with Bax oligomers. Small amounts of Bax-Bak
dimers were seen when signal from Bax dimers is suppressed (lane
6).
) and Bax(
/
) colon cancer cells to ATP
depletion by CCCP. Bax was totally absent in Bax(
/
) cells. Unlike
RPTC or HeLa cells, Bax(+/
) cells contained ~30-40% of total Bax
in mitochondria, and the remainder was present in the cytosol of normal
cells (Fig. 7A, lanes
1 and 2). When mitochondria isolated from Bax(+/
)
cells were incubated for 30 min at 30 °C, the majority of
mitochondrially associated Bax was released into the medium (Fig.
7A, lane 4). This observation indicates a loose
association of Bax with mitochondria in normal Bax(+/
) cells.
Moreover, mitochondrially associated Bax in normal cells did not occur
in oligomeric form as shown by chemical cross-linking (Fig.
7B, lane 10). Upon incubation of Bax(+/
) cells
with CCCP, formation of Bax oligomers was evident following chemical
cross-linking, with concomitant release of cytochrome c
(Fig. 7, B, lanes 11 and 12, C, lanes 5 and 6). Bak was abundant in
both Bax(+/
) and Bax(
/
) cells (Fig. 7B, lanes
1-6). As in the case of HeLa cells (Fig. 2B),
CCCP-induced Bak oligomers were seen largely in the form of dimers
after cross-linking in Bax(+/
) cells (Fig. 7B, lanes
5 and 6). With ATP depletion, Bak dimers increased
significantly in Bax(+/
) cells, but not in Bax(
/
) cells (Fig.
7B, lanes 1-6). These results are most
consistent with a role for Bax in the reorganization/oligomerization of
Bak that takes place during ATP depletion. Regardless of these considerations, the results also showed that there is an absolute requirement for Bax expression to release cytochrome c in
significant amounts during ATP depletion (Fig. 7C, compare
lanes 2, 3 and lanes 5, 6).
The small amount of cytochrome c release seen in Bax(
/
)
cells can be attributed to the fragility of energy-deprived cells
subjected to plasma membrane permeabilization methods. A potential role
of permeability transitions in the release of cytochrome c
during ATP depletion by CCCP or hypoxia was ruled out by the ability of
affected mitochondria to mount potential and accumulate potentiometric
dyes after removal of CCCP (Fig. 1C) or reoxygenation (24)
in complete growth medium. Moreover, our results showing little or no
release of cytochrome c in Bax(
/
) cells after prolonged treatment with CCCP (Fig. 7C) also rule out a role for the
permeability transition in this phenomenon.
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Fig. 7.
Requirement of Bax for Bak oligomerization
and cytochrome c release in Bax( /
), Bax(+/
)
colon cancer cells. A, Western blot analysis of
cytosol (Cyto, lane 1) and mitochondria
(Mito, lane 2) obtained by differential
centrifugation of Bax(+/
) cell homogenates (16), for Bax and Bak.
Proteins were loaded in proportional amounts (Cyto:Mito,
3:1) on SDS-PAGE. Isolated mitochondria from normal Bax(+/
) cells
were incubated in isotonic buffer (10 mM HEPES, 10 mM KH2PO4, 125 mM KCl,
4 mM MgCl2, 1 mM succinate, and 1 mM ADP, pH 7.4) at 0 °C (lane 3) or 30 °C
(lane 4) for 30 min. Mitochondrial pellet and supernatant
(Supt.) collected by centrifugation were analyzed for Bax by
Western blotting. Bax release into supernatant indicates a loose
association of Bax with mitochondria in normal cells. B,
Bax(
/
) and Bax(+/
) HCT116 cells were treated with 2.5 µM CCCP for 7.5 h at 37 °C in Krebs-Ringer
buffer. Following incubation of HCT116 cells with CCCP, membrane
fractions were obtained and cross-linked with EGS as described under
"Experimental Procedures," and analyzed for Bak (lanes
1-6) or Bax (lanes 7-12) by Western blotting under
reducing conditions. A typical blot from four independent experiments
is shown. Prolonged exposure to CCCP resulted in the formation of
oligomeric Bax in the membrane fraction as slow moving complexes
(lanes 11 and 12). Similarly, slow moving Bak
dimers appeared after ATP depletion in parallel with Bax oligomers
(lanes 5 and 6). Note that there is a significant
increase in Bak dimers only in ATP-depleted Bax(+/
) cells but not
Bax(
/
) cells. The identities of slow moving bands intermediate in
size between monomer and dimer in control cells (lanes 1 and
4) and corresponding to trimer in both ATP-depleted
Bax(
/
) and Bax(+/
) cells (lanes 2, 3,
5, and 6) are not known. C,
cytosolic digitonin extracts of Bax(
/
) and Bax(+/
) colon cancer
cells after ATP depletion (7.5 h CCCP) were analyzed for released
cytochrome c (Cyt.c in cytosol) by Western
blotting.
)/Bak(+/+)), Bax (Bax(
/
)), Bak (Bak(
/
))-deficient and Bax/Bak double
deficient (Bax(
/
)/Bak(
/
)) mice. We observed progressive
translocation of Bax to mitochondria with increasing durations of ATP
depletion in wild type BMK cells (Fig.
8A, lanes 1-5).
However, Bak(
/
) cells showed delayed kinetics of Bax translocation
(Fig. 8A, lanes 6-10). After 3 h of ATP
depletion in wild type cells, the vast majority of cytosolic Bax had
translocated to mitochondria and >90% of cells had released
cytochrome c into the cytosol (Fig. 8B,
panel 1). On the other hand, in Bak knockout cells, Bax
translocation was significantly delayed (Fig. 8A,
lanes 6-10). Corresponding to this, less than 20% of cells
showed cytochrome c release (Fig. 8B, panel
3). In contrast, both Bax knockout and Bax/Bak double knockout
cells showed resistance to cytochrome c release (Fig. 8B, panels 2 and 4). Chemical
cross-linking of membranes revealed that translocated Bax is
oligomerized into dimers and higher order oligomers in energy-deprived
wild type BMK cells (Fig. 8C, lane 2). Similarly
treated Bak(
/
) cells also showed Bax oligomerization (Fig.
8C, lane 6). These results clearly suggest that
Bax does not require Bak to oligomerize after translocation to
mitochondria. However, Bak deficiency seems to affect the kinetics of
Bax translocation probably because of molecular changes in mitochondria
that could have occurred because of Bak deficiency. Similarly,
mitochondrial Bak, normally present as monomers as well as larger
protein complexes bigger than dimers, rearranged to form Bak
homo-oligomeric ladders in wild type BMK cells during energy
deprivation (Fig. 8D, compare lanes 1 and
2). As in the case of Bax, the calculated molecular weights
of rearranged Bak complexes in these ladders correspond to dimers,
trimers, and higher order homo-oligomers. In contrast, Bax knockout
cells failed to show Bak rearrangement to form homo-oligomers even
after prolonged incubation under ATP-depleted conditions (Fig.
8D, lanes 5 and 6). These results
clearly suggest that Bax is required to induce Bak reorganization and
cytochrome c release during hypoxia or ATP
depletion.
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Fig. 8.
Bak is not required for Bax oligomerization
in BMK cells. A, time course of Bax translocation
in wild type (Bax(+/ )/Bak(+/+)) and Bak-deficient
(Bax(+/
)/Bak(
/
)) cells during ATP depletion (1 µM
CCCP). B, immunocytochemistry of cytochrome
c in control and ATP-depleted cells (CCCP, 3 h)
observed by confocal fluorescent microscopy. Wild type cells show
~90% of cells release cytochrome c into the cytosol
(panel 1) whereas, only ~20% of cells leaked cytochrome
into their cytosol in Bak-deficient cells (panel 3) during
ATP depletion. During the same length of exposure to ATP depletion,
both Bax-deficient and Bax/Bak-deficient cells failed to release
cytochrome c. Recovery of these cells in complete growth
medium showed apoptosis only in wild type and Bak knockout cells (not
shown) further confirming the absolute requirement of Bax for energy
deprivation-induced cytochrome c release. C,
oligomerization of Bax in wild type (lanes 1 and
2) and Bak-deficient cells (lanes 5 and
6) shown by immunoblotting of EGS cross-linked membrane
proteins after ATP depletion (4 h CCCP). Bax-deficient cells do not
show any signal (lanes 3 and 4).
D, chemical cross-linking and immunoblotting show
anti-Bak reactive slow moving Bak adducts in control wild type BMK
cells. Note that these adducts disappear and new Bak complexes that
resemble multimeric Bak molecules are formed during ATP depletion (4 h
CCCP) in wild type cells (compare lanes 1 and 2).
Bax-deficient cells (Bax(
/
)/Bak(+/+)) failed to show Bak
reorganization during the same length of exposure to ATP depletion (4 h
CCCP; compare lanes 5 and 6).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activate caspase-8 to cleave Bid, a BH3 only protein, to a
truncated form (t-Bid) that is targeted to mitochondria, inducing Bak
to oligomerize (21). However, in both RPTC and HeLa cells, Bid did not
undergo either proteolytic cleavage or mitochondrial translocation
during ATP depletion by CCCP or hypoxia. Moreover, failure to prevent
Bax translocation and cytochrome c release from mitochondria
of ATP-depleted cells by z-VAD (24, 36), a broad spectrum caspase
inhibitor, is also indicative of the noninvolvement of t-Bid.
Therefore, it seems likely that Bax triggers a conformational change in
Bak to homo-oligomerize following mitochondrial insertion.
/
) and Bax(+/
) cells exposed to recombinant t-Bid in
vitro. The results showed that release of cytochrome c
and Smac by t-Bid from isolated mitochondria has an absolute
requirement of Bax. Bak alone was not effective.2 In the
current work with cells selectively deficient in Bax or Bak, we
demonstrate existence of complex relationships between Bax and Bak. In
the absence of Bax, Bak alone not only failed to undergo reorganization
but also failed to induce the release of cytochrome c upon
ATP depletion (Figs. 8, B and D and
7C). On the other hand, homo-oligomerization of Bax or
cytochrome c release does not require Bak (Fig.
8C). However, optimal Bax translocation does seem to need
the presence of Bak (Fig. 8A). Whether this should be
attributed to Bak alone or a Bak associated common target for Bax and
Bak needs to be determined.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Stan Krajewski, the Burnham
Institute, for Bid antibodies and Dr. Bert Vogelstein, Johns Hopkins
Medical School, for kindly providing Bax(+/) and Bax(
/
) colon
cancer cells.
![]() |
FOOTNOTES |
---|
* This work was supported by NIDDK National Institutes of Health Grants DK-54472 (to P. S.) and DK 37139 (to M. A. V.).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, The 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, November 25, 2002, DOI 10.1074/jbc.M203392200
2 V. Mikhailov and P. Saikumar, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; Bcl-21, human Bcl-2 overexpressing rat proximal tubule cells; RPTC, rat proximal tubule cells; BMK, baby mouse kidney; EGS, ethyleneglycolbis(succinimidyl succinate); CCCP, carbonyl cyanide-m-chlorophenylhydrazone; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; t-Bid, truncated Bid.
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REFERENCES |
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