From the INSERM UMR 419, 9 quai Moncousu F-44035
Nantes Cedex 01, France, ¶ CNRS UMR 5095, 1 rue Camille Saint
Saëns, F-33007 Bordeaux Cedex, France
Received for publication, September 3, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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The translocation of Bax Apoptosis is finely regulated by intracellular events,
which at its onset, appear to be under the control of members of the BCL-2 family (1). The main site of action of these proteins appears to
be the mitochondrion, particularly through the control of the release
of apoptogenic factors from this organelle (2, 3). Members of the BCL-2
family can be anti-apoptotic or proapoptotic and totally or
partially mitochondrial-bound or cytosolic (4). In most cells, one of
the crucial and most regulated steps in the implementation of apoptosis
is the control of the translocation of Bax Bcl-2 is anchored into MOM bilayer by a short hydrophobic domain close
to the C terminus (CT) with its N terminus (NT) exposed toward the
cytosol (7-9). Bcl-2 belongs to a class of membrane proteins called
tail-anchored (TA) proteins, which are associated with different
intracellular membranes, usually MOM and the endoplasmic reticulum
(10). Based on the homology with Bcl-2, it has been proposed that Bax
became inserted to mitochondria by its hydrophobic Goping et al. (17) have described the presence of a sequence
enclosed in the first 20 amino acid residues regulating Bax Because of the importance of the translocation of Bax to the
mitochondria during apoptosis, we have re-addressed the question of the
localization of mitochondrial-targeting sequences in Bax Reagents--
Unless specified, all of the reagents used in this
study were from Sigma. Monoclonal Bax antibody (clone 4F11) was from
Immunotech (Marseille, France). The mitochondrion-selective
probe MitoTracker Green-FM was obtained from Molecular Probes
(Interchim, Marseille), and fluorogenic peptide Ac-DEVD-AMC was from
Bachem (Voisins, France).
Plasmids and Cell Transfections--
NT and CT mutants of Bax
were obtained by site-directed mutagenesis using the PCR-based Gateway
method and were subcloned into a pDEST 12.2 plasmid according to the
manufacturer's instructions (Invitrogen). Red fluorescent proteins
(RFP) constructions were subcloned into pDsRed-1N1 and pDsRed-C1
plasmids according to the manufacturer's instructions
(Clontech, Ozyme, St. Quentin Yvelines, France).
Transformer Site-directed mutagenesis kit from Clontech was used for internal mutations. Primers
used for mutagenesis are listed in Table
I. To obtain in vitro
translated proteins, RFP constructs were subcloned into pGEM T-easy
vector (Promega, Charbonnieres, France) after digestion by
SacI/NotI for pDsRed-1N1 and by
ApaI/NdeI for pDsRed-C1. A Bax-deficient cell
line derived from a human Glioblastoma Multiforme
(BdGBM)2 was
transfected with the empty vectors or Bax mutants cloned. Plasmid DNA
(5 µg) were introduced into 106 BdGBM cells by
electroporation (GenePulser, Bio-Rad) using 200 V/cm and 250 microfarads. The transfected cells were selected in a medium containing
neomycin (250 µg/ml) for 48 h. After selection, the bulk of
transfected cells was used for the in vitro experiments to
avoid clonal bias as described previously (16). The expression of RFP
in stably transfected cells was determined by cytometry on a FACScan II
(BD Biosciences) by measuring the natural fluorescence of the protein
in the cells after exclusion of dead cells on the basis of their
forward and side scatter characteristics.
Acellular Assay of Bax Insertion--
Mitochondria were prepared
from normal rat liver, and cell-free association of Bax with the
mitochondria was performed as described previously (16).
[35S]Met (Amersham Biosciences) labeled
proteins were synthesized from cDNAs using the TNT-coupled
transcription/translation system from Promega. Post-translational
insertion of labeled proteins into mitochondrial membranes and alkaline
treatment of mitochondrial-bound proteins were performed with a
standard import buffer as described previously (16). Radiolabeled
proteins bound to the mitochondria were recovered in the pellet after
centrifugation of the incubation mixture for 10 min at 4 °C and at
8000 × g. [35S]Met-Bax associated with
isolated mitochondria were analyzed on a SDS-PAGE and scanned with a
PhosphorImager (Amersham Biosciences). Insertion of Bcl-2 or of
a innermembrane protein was routinely performed in parallel to control
the quality of mitochondria. The amount of proteins present in the gel
slab was subsequently quantified with IPLab gel program (Signal
Analytics, Vienna, VA).
Quantification of Apoptosis--
Transfected BdGBM cells (2 × 106 cells) were cultured in the presence or in the
absence of doxorubicin (doxo, 20 ng/ml) for 24 h before
quantification of apoptosis by measuring the LDH and DEVDase activities
(16, 19). The LDH activity was measured using a Cytotox96®
assay from Promega as instructed by the manufacturer. After the induction of apoptosis, the cells were washed several times with phosphate-buffered saline plated in 96-well plates and the caspase-3 fluorogenic substrate (Ac-DEVD-AMC) was added in the presence of 0.01%
Triton X-100.
Confocal Analysis--
The cells were incubated for 30 min with
5 µg/ml MitoTracker Green-FM at 37 °C and then fixed with 1:1
methanol/acetone for 15 min at Cell Fractionation--
Transfected BdGBM (2 × 106) cells were collected and centrifuged at 800 × g for 10 min at 4 °C. The cell pellets were washed with
ice-cold phosphate-buffered saline and then resuspended in CEB (v/v)
(250 mM sucrose, 50 mM HEPES, pH 7.4, 50 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 10 µM cytochalasin B, 1 mM EGTA, and 1 mM phenylmethylsulfonyl
fluoride) as described previously (19). Cells were allowed to swell for
30 min on ice. The cells were then homogenized with 30 strokes in a
2-ml glass Dounce homogenizer. The homogenates were centrifuged at
800 × g for 10 min at 4 °C, and the resulting
supernatants were centrifuged for an additional 15 min at 13,000 × g at 4 °C to obtain the mitochondrial pellets (P13).
These supernatants were then centrifuged at 100,000 × g for 30 min in a Airfuge system (Beckman), and the
resulting cytosolic fraction was further referred to as the S100. The
presence of Bax constructs was detected in mitochondrial pellets and
S100 using standard immunoblot methods.
Yeast Experiments--
Human Bax variants were cloned at the
pme1 site of the pYES3/CT plasmid (Invitrogen) under the control
of the GAL1 promoter. Cells were grown aerobically in a
synthetic medium supplemented with lactate as a carbon source (0.17%
yeast nitrogen base, 0.1% potassium phosphate, 0.5% ammonium sulfate,
0.2% Drop-Mix, and 2% lactate, pH 5.2). The induction of Bax variants
was achieved by adding 0.5% galactose, and cells were harvested after
different times (see "Results"). Mitochondria were isolated as
described previously (20). Cytochrome c release was measured
by spectrophotometry of mitochondrial and cytosolic fractions (21). Bax
variants and RFP construct association with mitochondrial membrane was determined by alkaline extraction as follows. Mitochondria (1 mg/ml)
were suspended in 10 mM KOH-Hepes buffer, pH 7.2, containing 0.6 M mannitol added plus or minus 0.1 M sodium carbonate, and 0.1 or 1% Triton X-100. After a
10-min incubation on ice, membranes were recovered by a 15-min
centrifugation at 100,000 × g, proteins were separated
by SDS-PAGE, and immunoblots were performed as described by Oliver
et al. (19).
A Sequence Located in the First 39 Amino Acids of Bax Regulates Its
Targeting to the Mitochondria--
We have previously shown that the
deletion of Bax CT does not affect the association of Bax
We next examined the intracellular localization of these constructs
after transient transfection in BdGBM.2 As shown in Fig.
2A, a similar amount of Bax
constructs was produced by the expression of the various transgenes. We
performed cell fractionation to assess the subcellular localization of
the constructs analyzed above. As shown in Fig. 2B, the
results obtained were consistent with the acellular assay as Bax
As reported previously (16), the mere expression of Bax in the BdGBM
cells is not sufficient to trigger cell death (cf cell morphology in Fig. 2C); thus, we quantified the
death-inducing capacity of the different constructs after treatment
with 20 ng/ml doxo for 24 h. Cell death was assessed using both
the release of the cytosolic enzyme lactate dehydrogenase (LDH) into
culture and the direct measure of caspase activity in cell extracts
(see "Materials and Methods"). As shown in Fig.
3, both LDH release and DEVDase
activities were enhanced in cells transfected with Bax The
We have generated chimeric proteins by fusion of sequences derived from
Bax Competition between Bax
Because acellular analysis suggested that the NT-RFP constructs could
exhibit a dominant negative function toward Bax, we analyzed stably
transfected cells in RFP constructs, the sensitivity to apoptosis upon
transient expression of Bax
Altogether, these results suggest a competition between Bax H Mutations That Affect Bax
We next analyzed by cell fractionation and confocal microscopy the
subcellular localization of these constructs in BdGBM cells. The
mutations that affected Bax
We have analyzed the influence of the expression of these mutants on
the apoptotic response induced by doxo (cf "Materials and
Methods"). As shown in Fig. 8, the A24R
substitution had little or no influence on Bax Effect of the Mutations of Bax Single membrane-spanning proteins of the MOM can be inserted by
their N terminus (e.g. TOM70) or by their C terminus
(e.g. Bcl-2) (25). Because of sequence homology, the protein
Bax has been thought to belong to the class of TA proteins (1). The two
cardinal features of TA proteins are as follows: (i) a distribution between several intracellular compartments such as mitochondria, endoplasmic reticulum, and the Golgi apparatus and (ii) the fact that
the targeting information of the protein is encoded in the CT (10). Bax
fails to comply with these two points as it is usually predominantly
cytosolic and/or mitochondrial in healthy cells and because its CT
cannot support the targeting of a reporter protein (this study and 11, 14). Indeed, we have observed that the translocation of Bax upon the
induction of apoptosis to mitochondria appears to occur independently
of its CT (16, 19). In addition, contrary to what was found for Bcl-2
(7-9), the nature of the sequence located in the Bax CT could
influence its proapoptotic activity without affecting is intracellular
localization (11, 19). Thus, it has been proposed that the Bax CT could
be involved in protein-protein interactions that could regulate its
proapoptotic function (26). Goping et al. (17) have shown
that the Bax NT contained a sequence involved in the retention of the
whole protein in the cytosol. The deletion of this sequence called ART induced a massive translocation of Bax from the cytosol to the mitochondria (16, 17). It has been proposed that the cytosolic conformation of Bax was secured by the interaction between both extremities or by the CT with the BH3 domain and that its translocation to mitochondria involved a conformational change leading to the exposure of the Bax NT (11, 16-17). Interactions among Bax molecules or with other proapoptogenic proteins such as Bid appear to be crucial
to induce a change in the conformation of Bax and/or its interaction
with mitochondria (5-6). However, these changes seem to be necessary
but not sufficient to induce apoptogenic outer membrane permeability
(27).
We have recently described a new variant of Bax The amino acids 20-37 corresponds to the H The fact that the Bax NT-RFP constructs, which contain the
H Interestingly, the H We propose that the apoptotic-induced conformational changes in Bax
frees the H, a pro-apoptotic
member of the BCL-2 family from the cytosol to mitochondria, is a
central event of the apoptotic program. We report here that the
N-terminal (NT) end of Bax
, which contains its first
helix
(H
1), is a functional mitochondrial-addressing signal both in
mammals and in yeast. Similar results were obtained with a newly
described variant of Bax called Bax
, which lacks the first 20 amino
acids of Bax
and is constitutively associated with mitochondria.
Deletion of H
1 impairs the binding of Bax
to mitochondria,
whereas a fusion of the N terminus of Bax
, which contains H
1
with a cytosolic protein, results in the binding of the chimeric
proteins to mitochondria both in a cell-free assay and in
vitro. More importantly, the mitochondria-bound chimeric proteins
inhibit the interaction of Bax
with mitochondria as well as
Bax-apoptogenic properties. The mutations of the H
1, which inhibit
Bax
and Bax
translocation to mitochondria, also block the
subsequent activation of the execution phase of apoptosis. Conversely,
a deletion of the C terminus does not appear to influence Bax
and Bax
mitochondrial addressing. Taken together, our results
suggest that Bax is targeted to mitochondria by its NT and thus through
a pathway that is unique for a member of the BCL-2 family.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
from the cytosol to the
mitochondria (4). Anti-apoptotic proteins such as Bcl-2 or Bcl-XL act
as inhibitors of Bax function, whereas proapoptotic BH3 only members
can either hinder this inhibition (e. g. Bad) or directly
activate Bax
(e. g. Bid), which in both cases would
promote the association of Bax
with the mitochondrial outer
membrane (MOM)1 (5, 6).
9 helix located
at its C-terminal end (for example, see Ref. 11). Several studies have
shown that the Bax
CT is not required for its interaction with
mitochondria, whereas others have found it mandatory for Bax
function (12-16). We have observed that a chimeric Bcl-XL construct in
which the natural Bcl-XL CT was substituted by that of Bax
was no
longer able to associate with mitochondria in the cell-free system,
whereas the reverse substitution (i.e. Bcl-XL CT fused to
Bax
without CT) led to a massive binding to the organelle (14).
Nechustan et al. (11) have shown that unless the serine 184 was properly mutated or deleted, the wild type Bax CT was not a
membrane-addressing signal. From these experiments, one could conclude
that the membrane-targeting information of Bax is not encoded in its
the wild type CT. In addition, because Bax
is usually located
preferentially in the cytosol and/or in the mitochondria of healthy
cells, it does not appear to behave like a classical TA protein
(10).
insertion into mitochondria called the ART sequence, an acronym for
"apoptosis-regulating targeting" sequence. Recently, we have determined by site-directed mutagenesis that a proline (Pro-13) located
within the ART sequence was crucial for the maintenance of the
cytosolic Bax
conformation and that its substitution for a valine
increased its apoptogenicity (16). We have also discovered that human
gliomas expressed a natural variant of Bax
named Bax
, which
lacks the ART sequence (18). Bax
is highly apoptogenic and is a
deletion of its CT, quite remarkably enhanced its association with
mitochondria (16, 18). These results suggest that the CT plays a role
in the control of the cytosolic conformation of Bax and not in its
addressing to the mitochondria (16).
and Bax
. We report here that such a sequence is located in the NT of Bax
and that inactivating mutations within this sequence affect Bax
-
and Bax
-intracellular localization and/or proapoptotic function.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Sequences of the oligonucleotides used for the obtention of Bax or
Bax
fusion proteins and/or mutants
20 °C. After saturation with 3%
bovine serum albumin in phosphate-buffered saline, the cells were
incubated with anti-Bax (1 µg) for 1 h at room temperature.
After extensive washing, a second anti-mouse IgG coupled to rhodamine
was added overnight at 4 °C. Images were collected on a Leica TCS NT
microscope with a 63 × 1.3 NA Fluotar objective (Leica,
Rueil, France). Quantification of the overlay of labeling was
done using MetaMorph 4.6 program (Universal Imaging Corp.). The
correlation factor (CF) is a measure of the strength of the
relationship between two variables, x (labeling specific for
the mitochondria) and y (labeling of constructs of interest), and was calculated as instructed by the manufacturer (www.universal-imaging.com). The RFP was used as a standard for a
cytosolic localization. In average, calculations were performed after
analysis of 50 different cells in three independent experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
with
mitochondria but enhances that of a variant, which lacks the first 20 amino acids of the Bax
sequence (16). Deletion of the first 20 amino acids of Bax
structurally corresponds to a variant called Bax
, which is encoded by a transcript distinct from that of Bax
(18). To further explore the association of Bax
with mitochondria, we prepared a series of NT deletion constructs (Fig.
1A) and analyzed in a
cell-free system the binding to mitochondria of the corresponding in vitro translated proteins (Fig. 1B). As
already shown by several reports (14, 17), the association of Bax
with mitochondria in the cell-free system is extremely low but can be
enhanced by the deletion of the first 19 amino acids. Conversely, a
construct lacking the first 37 amino acids of Bax
(Bax-(
1-37)) does not bind to mitochondria (Fig.
1B).
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Fig. 1.
Existence of a mitochondrial
addressing/targeting signal in the NT of Bax. A,
schematic representation of Bax mRNA. The localization on Bax
mRNA of the Bcl-2 homology domains (BH1, BH2, and BH3) as well
as amphipathic
helices, the ART, and the C-terminal helix
9 (CT)
domains were deduced from data published by Suzuki et al.
(23). Constructs with NT and CT deletions of Bax
and Bax
are
also schematically represented. B, cell-free association of
NT- and CT-truncated Bax with purified rat liver mitochondria. Rat
liver mitochondria were prepared as described under "Materials and
Methods." 4 fmol of [35S]Met-Bax constructs were added
to 50 µg of mitochondria and incubated for 1 h at 37 °C
before mitochondria isolation and SDS-PAGE/fluorographic analysis of
[35S]Met-Bax. 1 fmol of in vitro translated
constructs (i25%) were run in parallel for visualization of
efficiency of the binding to mitochondria. The calculations of the
amount of 35S-labeled protein bound to mitochondria for
each construct (histograms) were obtained from at least three different
experiments.
was found essentially in the mitochondrial pellet (mito) and
Bax
-(
1-37) only in the cytosolic fraction (S100). Bax
and
Bax-(
169-192) were found in both compartments, however, with a
predominant cytosolic localization (Fig. 2B). Confocal
microscopy was used as another technique to confirm the mitochondrial
localization of these different constructs. As illustrated in Fig.
2C, the product of the Bax
transgene was essentially
cytosolic, whereas that of Bax
was predominantly mitochondrial-bound as reported previously (18). We quantified the
correlation between labeling of mitochondria with that of Bax
constructs using a MetaMorph program and compared it with that of the
cytosolic protein RFP (see "Materials and Methods"). As observed in
the cell-free assay and in the subcellular fractionation, the deletion
of the first 18 amino acids of Bax
(i.e. Bax
-(
1-37)) abrogates the binding of the proapoptotic protein to
mitochondria in vitro, which exhibit a correlation factor of
a cytosolic protein (i.e. CF = 0.6). On the other hand,
Bax
was mitochondrial (CF = 1), and Bax
and
Bax-(
169-192) with a CF of 0.79 and 0.78, respectively, were
localized in both compartments (Fig. 2D). Thus, based on the
acellular assay, the confocal analysis, and the cell fractionation results, we conclude that that the first 18 amino acids
of Bax
contained a sequence involved in Bax targeting to
mitochondria.
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Fig. 2.
Effect of NT and CT deletions on Bax
intracellular localization and apoptogenic activity. A,
expression of Bax constructs (Bax , Bax
, Bax-(
1-19),
Bax-(
1-37), and Bax-(
163-192)) in the BdGBM visualized by
immunoblot. mock, cells transfected with pDEST12.2.
B, subcellular localization of Bax constructs by cell
fractionation. BdGBM cells expressing Bax constructs trans-genes were
fractionated as described under "Materials and Methods." The
cytosolic fraction (S100) was analyzed by Western blot for
the presence of Bax using actin as a control. Mitochondrial pellet
(Mito) was analyzed also by Western with anti-Bax antibodies
using the subunit IV of the cytochrome c oxidase
(COX) as a mitochondrial marker. The Western blots shown are
representative of three different experiments. C,
subcellular localization of Bax constructs by confocal microscopy.
BdGBM cells were transiently transfected with Bax constructs as
described under "Materials and Methods." Mitochondria were labeled
with the MitoTracker Green-FM (mito). Pictures are
representative of three different experiments with a magnification of
×60. D, correlation factor between a mitochondrial probe
and Bax constructs localizations. The correlation plot function
graphically represents the intensities of each probe on a pixel by
pixel basis. The correlation factor is calculated giving a measure of
both positive and negative colocalizations for each of the Bax
constructs and the cytosolic protein RFP. Means ± S.D. were
calculated from the analysis of 50 different cells for each
construct.
and Bax
although with different amplitudes as described previously (18). On the
other hand, in contrast with another report (22), little or no
induction of cell death was observed with the Bax-(
1-37) construct
(Fig. 3). It is noteworthy that a deletion of the putative
trans-membrane domain of Bax (CT = amino acids 169-193) had no
effects on both the subcellular localization and apoptogenic activity
Bax
(Figs. 2 and 3).
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Fig. 3.
Quantification of apoptosis triggered by Bax
trans-genes expression in BdGBM cells. Cells transfected with the
different Bax constructs were treated with doxo for 24 h before
measuring the DEVDase activities in the cells and the LDH activity in
the culture media (see "Materials and Methods"). Data shown are
calculated from four different experiments.
1 Helix of Bax Alone Is Sufficient to Target a Cytosolic
Protein to the Mitochondria--
According to NMR studies (23), the
first 18 amino acids of Bax
(i.e. amino acids 20-37 of
Bax
) contains an amphipathic
helix (H
1). Numerous studies
have shown that such
helices are present in membrane-targeting
sequence (24).
NT to the N terminus of the cytosolic RFP (see "Materials and
Methods") and analyzed the binding of these constructs in the
cell-free system using in vitro translated chimeric
proteins. We also constructed chimeric proteins in which the wild type
9 helix and a mutated form of this sequence (i.e.
deletion of the serine 184, Bax-(
S184)) were fused to the CT of the
RFP. These constructs were similar to those described by Nechustan
et al. (11) with the exception that the RFP was used instead
of the green fluorescent protein. We confirmed using the cell-free
system their original observation because Bax
CT did not lead to
the association of the RFP with the mitochondria, whereas the
Bax-(
S184) mutant exhibited higher affinity for mitochondria (Fig.
4A). As also shown in Fig.
4A, the first 37 amino acids of Bax
triggered the
association of the RFP with the mitochondria in contrast to that
observed with the RFP alone, which remained cytosolic. On the other
hand, H
1 alone (Bax-(20-37)) and, to a lesser degree, the sequence
encompassing the first 19 amino acids of Bax
were promoting an
efficient association of the RFP with mitochondria when compared with
the chimera formed from the fusion of Bax CT to the CT of RFP
(i.e. Bax-(169-192)). However, it should be noted that the
Bax-(1-37)-RFP construct exhibited an interesting behavior as its
association with mitochondria was an intermediate between that of
Bax-(20-37)-RFP and that of Bax-(1-19)-RFP (Fig. 4A). These result suggest that the H
1 (Bax-(20-37)) is more efficient to
target Bax to mitochondria than the full-length sequence (Bax-(1-37)) and the ART sequence (Bax-(1-19)), independently of the presence of
other Bax domains. Interestingly, none of these constructs were
resistant to alkali treatment, which suggests that the NT of Bax is a
targeting but not a membrane-anchoring signal. These results were
confirmed by cell fractionation (Fig. 4B) and confocal microscopy/MetaMorph analysis (Fig. 4, C and D).
The Bax-(1-19)-RFP showed a more cytosolic pattern (CF = 0.82)
than the Bax-(20-37)-RFP, which is mainly associated with mitochondria
(CF = 1) (Fig. 4, B-D). Quite interestingly, the
Bax-(1-37)-RFP fusion protein exhibited a mixed cytosolic and
mitochondrial localization (CF = 0.85) reminiscent of Bax
subcellular localization in these cells (Fig. 4, B-D). The
wild type RFP-Bax CT and the Bax-(
S184) mutant (see above) behaved
as described by Nechustan et al. (11) as the wild type CT
construct was mostly cytosolic (CF = 0.7) and the mutated
construct was mainly mitochondria-bound (CF = 0.95) (Fig. 4,
B-D).
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Fig. 4.
A sequence located within the amino acids
20-37 of Bax contains a mitochondrial
addressing/targeting sequence. A, mitochondrial binding
of chimeric proteins formed by the fusion of NT or CT of Bax to the
RFP. Cell-free binding of in vitro translated chimeric
proteins to purified rat liver mitochondria was performed as described
under "Materials and Methods." Input, 25% of added
in vitro translated proteins; mit, mitochondria
bound; AT, alkaline treated. B, subcellular
localization of Bax constructs by cell fractionation. BdGBM cells
expressing Bax constructs trans-genes were fractionated, and the
cytosolic fraction (i.e. S100) and the mitochondrial pellet
(Mito) were analyzed as described in Fig. 2B.
Western blots shown are representative of three different experiments.
C, confocal microscopic analysis of Bax-RFP construct
subcellular localizations. The above chimeric proteins were transiently
expressed in BdGBM using RFP fluorescence and MitoTracker Green-FM. In
agreement with cell free experiments, the Bax-(1-19)-RFP and the
RFP-Bax-(169-192) exhibited a diffuse cytosolic distribution, whereas
Bax-(20-37)-RFP and the RFP Bax-(
S184) were predominantly
associated to mitochondria. The construct Bax-(1-37)-RFP showed both
cytosolic and mitochondrial localizations. Each field was visualized
independently at the appropriate wavelength for MitoTracker and the
RFP. Magnification = ×60. D, correlation factor
between a mitochondrial probe and Bax constructs localizations. The
correlation function was calculated as in Fig. 2D.
Means ± S.D. were calculated from the analysis of 50 different
cells for each constructs.
and Bax NT-RFP Constructs for Binding
to Mitochondria--
We next analyzed, using the cell free assay, the
binding of Bax
to mitochondria in the presence of increasing
concentrations of the different Bax NT-RFP constructs. As shown in Fig.
5A, the addition of
Bax-(1-19)-RFP did not interfere with the binding of Bax
to
mitochondria in the cell-free assay. Conversely, both Bax-(1-37)-RFP
and Bax-(20-37)-RFP constructs inhibited this association although
with an efficiency that appears to be related with the relative
affinity of these constructs for the organelle in the cell-free system
(Fig. 5A). The specificity of the interference of the
association of Bax with the Bax NT-RFP constructs was assessed by the
coincubation of the Bax CT constructs: the wild type RFP-Bax CT and the
Bax-(
S184) mutant. As illustrated in Fig. 5B, neither construct competed for Bax
binding, although RFP-Bax-(
S184) association to mitochondria was roughly similar to that observed for
Bax-(1-37)-RFP.
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Fig. 5.
Bax-(1-37)-RFP and Bax-(20-37)-RFP inhibit
Bax association to mitochondria in the
cell-free assay. 4 fmol of in vitro translated Bax
were added to purified rat liver mitochondria in the presence of
increasing concentrations of Bax NT-RFP (A) or RFP-Bax CT
(B) constructs. 35S-Labeled proteins bound to
mitochondria were analyzed by PAGE and autoradiography. One
fluorography of the three independent experiments performed for each
construct is shown (top) as well as the quantification of
the binding of Bax and the Bax-RFP constructs (bottom)
(mean ± S.D.).
or Bax
. We used cells expressing
the same amounts of constructs as quantified by fluorescence of the RFP
(Fig. 6A). These cells
transfected with either Bax
or Bax
were treated with doxo, and
cell death was quantified both by assessing the activity of LDH in the
culture medium and the cellular DEVDase activity. Compared with
control, the coexpression of Bax-(1-37)-RFP and Bax-(20-37)-RFP
constructs inhibited Bax
and Bax
sensitization to doxo-induced
apoptosis, whereas Bax-(1-19)-RFP constructs had only a slight effect
(Fig. 6, B and C). It should be noted that the
expression of RFP-Bax CT constructs including that of the mitochondria
bound RFP-Bax-(
S184) did not affect Bax function in these
experiments.
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Fig. 6.
Bax-(1-37)-RFP and Bax-(20-37)-RFP
inhibit Bax and Bax
sensitization to doxo-induced apoptosis in
vitro. A, the expression of the wild type
RFP and the Bax-RFP constructs in stably transfected BdGBM was
determined by flow cytometry as described under "Materials and
Methods." Clones used in the experiments were chosen because they
expressed similar amounts of RFP. Staining of the parental mocked
transfected cells is indicated by the thin line in
A. These cells were stably transfected with the RFP
constructs, were transiently transfected by Bax
(B) or
Bax
(C), and treated with doxo as described under
"Materials and Methods." Apoptotic amplitude was monitored by
measuring the cellular DEVDase activity and the LDH released into the
culture medium.
1-RFP
constructs and Bax
or Bax
. This competition is likely to occur
by the inhibition of the insertion of Bax
or Bax
into
mitochondria, and this was confirmed by confocal microscopy (data not shown).
Binding to Mitochondria and Its
Proapoptotic Activity Also Affect Bax
--
As part of a
mutagenesis screening of Bax NT, we substituted Ala-24 for Arg (A24R)
and Leu-26 for Gly (L26G, Bax
numbering) in both Bax
and Bax
(Fig. 7A).
The L26G substitution provoked a break in the H
1, and the A24R
reinforced the basic character of the region surrounding the H
1, two
cardinal points of mitochondrial presequences (24). As shown in Fig.
7B, in both cases, the acellular association of in
vitro translated Bax
and Bax
mutants with mitochondria was
significantly decreased, although only slightly for the A24R
substitution (Fig. 7B). Interestingly, the effect of the
H
1 disruption (L26G) was more important in Bax
than in Bax
,
and the additional change in the H
1 (double substitution L26G/L27V)
did not significantly enhance that of the single L26G mutation (Fig.
7B). In both cases, a disruption of the H
1 had a greater
effect on the binding of Bax than a change in the overall positive
charge of the molecule. The latter result suggests that the amphipathic
nature of the H
1 is more important for mitochondrial association
than its basic character.
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Fig. 7.
Mutations in the H 1
affects Bax association with mitochondria in a cell-free and in
vitro assay. A, schematic representation of
mutants of the H
1 present in Bax
and Bax
. B,
cell-free association of Bax
and Bax
and mutants of the H
1
with purified rat liver mitochondria. The binding of Bax
and
mutants was performed as described under "Materials and Methods."
The A24R (Bax
numbering) substitution has a slight but significant
influence on Bax
binding to mitochondria. On the other hand, the
substitution of L26G with or without the additional substitution of
L27V impaired the binding of both Bax
and Bax
. Histograms show
the amount of protein (fmol) present in the mitochondrial pellet. The
data shown and means ± S.D. were obtained from at least four
independent experiments. C, subcellular localization of Bax
constructs by cell fractionation. BdGBM cells expressing Bax H
1
mutants were fractionated, and the cytosolic fraction (S100)
and the mitochondrial pellet (mito) were analyzed as shown
in Fig. 2B. Western blots shown are representative of three
different experiments. D, confocal microscopy analysis of
H
1 mutants subcellular localization. The above mutated Bax
or
Bax
proteins were transiently expressed in BdGBM cells and analyzed
as in described under "Materials and Methods." Magnification = ×60. E, correlation factor between a mitochondrial probe
and Bax
or Bax
construct localizations. The correlation
function was calculated as in Fig. 2D. Means ± S.D.
were calculated from the analysis of 50 different cells for each
construct.
or Bax
binding to mitochondria in
the cell-free assay also affected its intracellular localization, and
this was particularly striking for Bax
in which the L26G substitution almost abolished its constitutive binding to mitochondria (Fig. 7, C-E).
or Bax
proapoptotic activities. Conversely, in both cases, the L26G mutants
nullified the proapoptotic activities of the Bax variants (Fig. 8), a
result consistent with the inhibition of its addressing to
mitochondria.
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Fig. 8.
The mutants of
H 1, which affected the binding of Bax in the
cell-free system inhibited both Bax
(A) and Bax
(B) apoptogenic properties. Quantification of
apoptosis triggered by Bax mutants expression in BdGBM is shown. Cells
transfected by the different Bax H
1 mutants were treated with doxo
for 24 h before measuring the DEVDase activities in the cells and
the LDH activity in the culture media ("Materials and Methods").
The data shown and ± S.D. were obtained from at least four
independent experiments.
NT in Yeast--
It has been
reported that Bax-induced growth arrest in yeast was preceded by its
binding to mitochondria and, as in mammals, by an increase in the
release of cytochrome c into the cytosol (21). We have
investigated the effect of a deletion of Bax NT and of mutations in the
H
1 on both in vivo binding of Bax to yeast mitochondria
and cytochrome c release-promoting activity. The deletion of
the first 19 amino acids of Bax
enhanced both the binding of the
protein to mitochondria (data not shown) and cytochrome c
release (Fig. 9). As observed in human
mitochondria, the A24R substitution affected both the binding of
Bax-(
1-19) to mitochondria (data not shown) and its cytochrome
c release activity (Fig. 9). However, contrary to what was
observed in human cells, the L26G mutation alone did not affect
Bax-(
1-19) activity and the additional mutation of the adjacent Leu
(L27V) was required to decrease its cytochrome c release
activity (Fig. 9). These results suggest that the interaction of the
H
1 with mitochondria is similar but not identical in yeast and in
mammalian cells.
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Fig. 9.
Cytochrome c release induced
by mutants in the H 1 in S. cerevisiae. Mitochondria were isolated from Bax
H
1 mutant-transfected yeast, and cytochrome c content was
measured by spectrophotometry (see "Materials and Methods").
Results are expressed as the means ± S.D. of at least three
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, Bax
, which is
constitutively associated with mitochondria (18). A deletion of the CT
of Bax
enhanced both its mitochondrial association and its
apoptogenic properties, suggesting again that the CT of Bax was mainly
involved in the control of a cytosolic conformation (16). Thus, this
study investigates the presence of a mitochondrial targeting signal of
Bax in the NT. We constructed a series of NT-truncated variant of Bax
and Bax
and found that a sequence encompassing amino acids
20-37 (Bax
numbering) was important for the association of Bax
with mitochondria both in a cell-free assay and in vitro
(Figs. 1 and 2). The deletion of this sequence in Bax
inhibits both
its constitutive mitochondrial localization and its proapoptotic
activity (Figs. 1 and 2). Our results are in opposition with that of
Wood and Newcomb (22), which show that the deletion of this particular
sequence enhanced its proapoptotic function. However, in the latter
case, the transfection was performed in Bax-expressing cell lines, and
thus, it is conceivable that the Bax-(
1-37) construct could by
acting as a BH3-only protein trigger apoptosis not by itself but
through the activation of endogenous Bax, a feature impossible in
Bax-deficient cell line.
1 of Bax
and Bax
(23) and, as such, is a probable candidate for an import signal of Bax
and Bax
to mitochondria. To test this hypothesis, we have
constructed several chimeric proteins in which different sequences of
the NT with or without the H
1 were fused to the N-terminal of the
RFP. These constructs behaved both in a cell-free system and in
vitro like the NT truncated Bax constructs described above. The
Bax-(1-37)-RFP and the Bax-(1-19)-RFP constructs were partly
cytosolic, whereas the Bax-(20-37)-RFP was found mainly to be
associated with the mitochondria (Fig. 4). Conversely, the RFP-Bax-(169-192) fusion protein, which contained the entire
9 helix, was unable to address the RFP to mitochondria unless Ser 184 was
deleted (Fig. 4). The insertion of the mitochondrial-bound Bax NT-RFP
constructs was found to be alkaline-sensitive (Fig. 4), suggesting that
this segment was not a transmembrane domain but rather a targeting
sequence. This result is consistent with the fact that a trans-membrane
domain usually consists of 20-25 non-polar amino acids (24) and thus
is significantly longer than H
1. Because Bax localization was not
affected by the deletion of the CT, the membrane-anchoring domains of
Bax have to be localized elsewhere. Preliminary results suggest that in
Bax
, the
5 and
6 helices, which are the putative pore-forming
domains of Bax, are involved in this process as already shown by
Nouraini et al. (28). Interestingly, several outer membrane
proteins such as TOM22 have been shown to possess physically distinct
import and membrane anchor sequences that regulate their interaction
with mitochondria (29).
1, can compete for Bax insertion in the cell-free assay (Fig. 5)
and act as dominant negative in vitro (Fig. 6), strongly
suggesting that this targeting signal involves common mitochondrial or
cytosolic receptor(s), remains to be determined. Preliminary results
suggest that protease-sensitive component(s) located in the outer
membrane are involved in the docking of Bax
to mitochondria.
Experiments are currently being carried out to quantify and to identify
these components. The importance of the NT-H
1 in Bax targeting and function was further emphasized by the observation that mutations that
affected Bax binding in the cell-free system (Fig. 7) also abolished
its subcellular localization and the subsequent induction of the
demolition phase of apoptosis (Fig. 8). Similar features were found in
Saccharomyces cerevisiae, although additional mutations were
required to abolish the addressing property of the H
1 (Fig. 9).
These results strengthen the idea that the H
1 fulfills a basic role
in Bax interaction with mitochondria.
1 exhibit some structural homologies with the
BH4 domain of Bcl-2 and Bcl-XL, although it has been clearly demonstrated that Bax lacked such a "functional BH4-like" domain (30). Indeed, the mutations reported in this work, which affect the
binding of Bax to mitochondria (A24R and L27V), were primarily intended
to introduce amino acids present in Bcl-2 and Bcl-XL sequences to
reinforce the BH4-like aspect of the H
1 (30). We show here that
these mutations impair Bax proapoptotic function by interfering with
its addressing to mitochondria and not by introducing a BH4-like
anti-apoptotic function. Nechustan et al. (11) have shown
that structural changes in Bax, which accompany the induction of
apoptosis, result in the exposition of the H
1, and three-dimensional
analyses of the detergent-induced changes in the conformation of Bax
indicate that the NT (i.e. until the H
2) is still
flexible and solvent-exposed in Bax oligomers (23). Thus, the H
1 is
probably still capable of addressing Bax oligomers formed during the
onset of apoptosis to mitochondria.
1 from its interaction with the rest of the molecule,
thus enabling the addressing of Bax to mitochondria through this helix.
This association is necessary but not sufficient to trigger
mitochondria outer membrane permeability, which can only be achieved
through an "apoptogenic" Bax insertion within MOM. The precise
mechanism of Bax insertion and especially the role of other proteins of
MOM and/or the involvement of other domains of Bax in this insertion
remain to be established.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Philippe Juin (INSERM UMR 419) and Naoufal Zamzami (CNRS UMR 1599) for fruitful discussions, Dr. Caroline Colombeix (IFR 26) for fruitful advice on MetaMorph program, and Gwenola Bougras for expert assistance with FACS analysis.
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FOOTNOTES |
---|
* This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche sur le Cancer, and the Ligue Nationale contre le Cancer.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.
§ Supported by a fellowship from the Ligue Départementale contre le Cancer Doubs/Montbeliard.
To whom correspondence should be addressed. Fax:
33-24-008-4082; E-mail: fval@nantes.inserm.fr.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M208955200
2 P.-F. Cartron, M. Juin, P. H. Oliver, K. Meflah, and F. M. Vallette, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MOM, mitochondria outer membrane;
ART, apoptosis regulation of targeting;
BdGBM, Bax-deficient Glioblastoma Multiforme;
CT, C-terminal of human
Bax;
doxo, doxorubicin;
LDH, lactate dehydrogenase;
H1, helix
1;
NT, N-terminal of human Bax;
RFP, red fluorescent protein;
CF, correlation factor;
TA, tailor-anchored.
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