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INTRODUCTION |
Rel/NF-
B transcription factors are ubiquitously expressed and
respond to more than 150 stimuli to regulate an equally wide array of
genes (1). While NF-
B was well known to participate in the control
of immune (2) and inflammatory responses (3), it recently appeared as
an important player in regulating the balance between cell survival and
apoptosis (4, 5). NF-
B is maintained inactive by inhibitory subunits
of the I
B family such as I
B-
. I
B-
interacts with NF-
B
via its ankyrin motifs, masking nuclear localization signals on NF-
B
subunits. The release of transcriptionally competent NF-
B dimers is
achieved after phosphorylation-induced degradation or dissociation of
I
B-
molecules. At least two kinases (IKK
and IKK
) that are
specific for the conserved tandem serines in I
B-
molecules have
been identified. They show an extensive homology and form homo- as well
as heterodimers within the cell. IKKs are localized in the signalsome,
a multiprotein complex (700-900 kDa). This complex also contains
NEMO/IKK
, (6) a structural component that is crucial for the correct
assembly and functionality of the signalsome (for a review, see Refs. 7 and 8). An alternative mechanism that links tyrosine kinases to NF-
B
activation has been described that uses phosphorylation of tyrosine 42 of I
B-
to dissociate I
B-
·NF-
B complexes (9).
Phosphorylation of I
B-
on serines 32 and 36 allows its specific
recognition by the E3RS ubiquitin protein ligase, which then transfers
ubiquitin to lysines 20 and 21. The polyubiquitinated I
B-
molecule is thereafter recognized and degraded in situ by the proteasome 26 S complex (7). The N-terminal domain of I
B-
thus appears to integrate NF-
B activation signals. We used this regulatory domain as a bait in a yeast two-hybrid screening of a Jurkat
cDNA library to search for protein interactors and therefore potential regulators of NF-
B activation. In this report, we describe and characterize the interaction between I
B-
and ANT, the
mitochondrial ATP/ADP translocator (10). ANT is a central component of
the mitochondria permeability transition pore (11). The opening of this
pore, during induction of apoptosis allows the release of molecules,
such as caspases or apoptosis-inducing factor, which are important to
amplify the cell suicide response. The possible functional importance
of the I
B-
/ANT interaction in the context of regulation of
apoptosis is discussed.
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MATERIALS AND METHODS |
Biological Reagents and Cell Culture--
Anti-I
B-
antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Rabbit antiserum anti-I
B-
, anti-Myc epitope, and
anti-F1
ATPase monoclonal antibody were kindly provided
by Jean Imbert (U119, Marseille, France), Jean-François Tanti
(INSERM, EPI E99-11, Nice, France), and Joël Lunardi
(Commissariat à l'Energie Atomique, Grenoble, France), respectively.
Human embryonic kidney 293 cells were maintained in Dulbecco's
modified Eagle's medium containing 2 mM glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, 1 mM
pyruvate, and 10% fetal calf serum (Life Technologies, Inc.). Cells
were transfected by the calcium phosphate method as previously
described (12).
HTLV-I1-infected MT4 cells
(13) and cells of the human leukemic T-cell line Jurkat were maintained
in RPMI 1640 medium containing 50 units/ml penicillin, 50 µg/ml
streptomycin, 1 mM pyruvate, and 5% fetal calf serum.
Yeast Two-hybrid and cDNA Cloning--
I
B-
cDNA
encoding amino acids 2-72 was subcloned into the GAL4 DNA-binding
vector pAS2.1 (CLONTECH, Palo Alto, CA). The resulting plasmid, pAS-I
B2-72, was used as the bait in a two-hybrid screening of a human Jurkat T cell cDNA library
(CLONTECH) in the S. cerevisiae HF7c
strain according to the Matchmaker Two-Hybrid System II Protocol
(CLONTECH). Positive yeast clones were selected for
prototrophy for histidine and expression of
-galactosidase. Yeast
DNA was recovered. Sequencing of positive clones have been performed
using the T7 Amersham Pharmacia Biotech sequencing kit. Full-length ANT
cDNA was amplified by PCR from the human Jurkat T cell cDNA library.
Expression Vectors--
cDNAs encoding full-length ANT or its
C-terminal domain were subcloned downstream and in frame with
glutathione S-transferase (GST) open reading frame in the
pGex-4T-2 plasmid.
Full-length ANT cDNA was also subcloned in pcDNA-Myc plasmid
(gift of Dr. J-F. Tanti, INSERM E99-11).
Expression and Purification of Recombinant Proteins from
Escherichia coli Pull-down Assays--
Plasmids coding for the GST
fused proteins were transformed in E. coli strain DH5
and
induced with 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside for 2 h at
30 °C. GST-fused proteins were purified by incubating precleared
bacterial lysates (phosphate-buffered saline (PBS)-1% Nonidet P-40
sonication) with glutathione-Sepharose beads (Amersham Pharmacia
Biotech). The absorbed proteins were incubated with recombinant
hexahistidine-I
B-
purified for 1 h at 4 °C in binding buffer (25 mM Hepes, pH 7.4, 50 mM NaCl, 2.5 mM CaCl2, 1 mM MgCl2, 0.5% Nonidet P-40, 1% bovine serum albumin) supplemented with Complete protease inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany). After extensive washes in the same buffer without
bovine serum albumin, I
B-
was detected by immunoblotting with
monoclonal anti-I
B-
antibodies.
Immunoprecipitation and Immunoblotting--
Transfected 293 cells were incubated for 30 min at 4 °C in lysis buffer (20 mM Hepes, pH 7.4, 50 mM NaCl, 0.5% Nonidet
P40, 1 mM CaCl2, 1 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture). Lysates were cleared at 10,000 × g for 10 min at 4 °C, and supernatants were incubated
with antisera against I
B-
or Myc epitope that were preabsorbed on
Protein A-Sepharose and Protein G-Sepharose, respectively. The
immunocomplexes were washed three times in lysis buffer. Proteins were
resolved by SDS-PAGE and transferred to Immobilon membranes (Millipore, Bedford, MA) by standard procedures. Immunoblot analysis was performed with the indicated antibodies. Bound antibodies were revealed with
horseradish peroxidase-coupled immunoglobulins (Dako, Trappes, France)
using the ECL Western blotting detection system (Amersham Pharmacia
Biotech) according to the manufacturer's instructions.
Mitochondrial Purification--
Subcellular fractionation was
performed according to Ref. 14. 108 Jurkat cells were
washed with PBS and resuspended in buffer A (250 mM
sucrose, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and
protease inhibitor mixture). Cells were Dounce homogenized (2-cm
diameter, 100 strokes, 4 °C). Unbroken cells and nuclei were
pelleted by centrifugation at 2500 rpm for 5 min. Supernatants were
further centrifuged at 10,000 × g for 30 min. The
resultant supernatants and pellets were designated as cytosolic and
mitochondrial fractions, respectively.
Mitochondria were highly purified according to Ref. 15. Briefly, after
Dounce homogenization, cell extracts were laid over the top of a
Percoll/metrizamide discontinuous gradient. After centrifugation at
20,000 rpm for 15 min, mitochondria were collected at the 17/35%
interface. They were then diluted in buffer A, centrifuged for 30 min
at 10,000 × g, and recovered in the pellet.
Cross-contamination with cytosol was assessed using a specific enzyme
marker: lactate dehydrogenase. The level of cross-contamination was
calculated as the percentage of the total cellular enzyme activity in
the different fractions.
Proteinase K Treatment--
Mitochondria or whole cell extracts
were incubated in buffer A without protease inhibitors in the presence
of 5-15 ng/ml proteinase K (Sigma) for 10 min on ice. Protease
inhibitors were added to stop the reaction, and proteins were analyzed
by Western blotting, as described below.
Immunoelectron Microscopy Study--
For immunoelectron
microscopy, cell pellets from control or Fas-stimulated cells (6 h, 100 ng/ml) were fixed in 3.7% paraformaldehyde and embedded at low
temperature into LR White resin (Hard LR White, London, UK). Ultrathin
sections were laid to 300-mesh nickel grids, washed with PBS, and then
incubated for 60 min at room temperature with I
B-
antibody
(1:200) or with anti-p65 antibody (1:500). After washing with PBS, the
grids were incubated for 60 min with 10-nm colloidal gold-conjugated
rabbit anti-mouse secondary antibody (British BioCell International,
Cardiff, UK). The grids were washed with PBS and then with distilled
water and stained with uranyl acetate. Sections were examined with a
JEOL 1200 EXII electron microscope.
Electrophoretic Mobility Shift Assays--
The cytosolic and
mitochondrial fractions from indicated cells were prepared as described
above. Mitochondria were lysed in totex buffer (20 mM
Hepes, pH 7.9, 350 mM NaCl, 20% glycerol, 1% Nonidet
P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). 30 µg of
protein from total mitochondria extract or 50 µg of protein from
digitonin supernatant were subjected to an electrophoretic mobility
shift assay analysis. I
B-
and NF-
B complexes were dissociated
with a 0.6% deoxycholate treatment during 10 min at room temperature.
The NF-
B probe used was a synthetic double-stranded oligonucleotide
containing the NF-
B binding site of the interleukin-2 gene promoter
(5'-GATCCAAGGGACTTTCCATG-3'). The end-labeled probe was incubated with
extract samples for 20 min at 37 °C. Complexes were separated by
electrophoresis on a 5% nondenaturing polyacrylamide gel in 0.5× TBE.
The dried gels were subjected to autoradiography (X-Omat; Eastman Kodak
Co.).
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RESULTS |
Isolation of ANT as a Potential I
B-
-binding
Protein--
Activation of the transcription factor NF-
B is
regulated by phosphorylation of the N-terminal domain of the inhibitory
subunit I
B-
. We used this N-terminal regulatory domain (I
B-
amino acids 2-72) as the bait in a yeast two-hybrid-based approach to identify new I
B-
interactors and therefore potential NF-
B
regulators. By the screening of a commercial human Jurkat cell library,
~6 × 105 double yeast transformants were obtained,
among which 232 grew on selective medium and only 88 turned blue when
tested in a filter lift
-galactosidase assay. Fifty-six clones were
further characterized by sequencing.
Primary nucleotide sequencing revealed one cDNA clone encoding a
protein not previously described in GenBankTM and two
clones corresponding to two isoforms of the adenine nucleotide translocator: ANT1-(171-297) and ANT2-(152-298).
The interaction between ANT and I
B-
was further characterized in
the yeast two-hybrid system. Full-length I
B-
could not be used in
the two-hybrid system because of a constitutive activity of reporter
genes (not shown; see Ref. 16). As shown in Table I, the DB-I
B-
-(2-72) fused protein
cannot act as activator of transcription when coexpressed in yeast with
GAL4 AD nor in combination with AD-T Ag (large T antigen) fusion
protein (lanes 1 and 2). The AD-ANT
fused protein cannot act as activator of transcription when coexpressed
in yeast with GAL4 DB nor in combination with the DB-p53 fusion protein
(lanes 7 and 8). On the contrary, co-expression of I
B-
-(2-72) with the portion of ANT1 or ANT2 isolated during the screening (lanes 4 and
5) or with the full-length ANT1 cDNA (lane
6) induced expression of the two reporter genes, thus
demonstrating a direct and specific interaction between the two
partners. We performed in vitro binding assay to confirm, in
an independent experimental setting, the physical interaction between
ANT and I
B-
proteins. The C-terminal part of ANT isolated during
the screening as well as the full-length ANT protein sequences were
fused to GST and used in pull-down experiments with recombinant I
B-
. The size of the recombinant proteins was checked by SDS-PAGE and Coomassie staining (Fig.
1A, upper
panel). The GST protein or a nonrelevant fusion
protein could not interact with I
B-
(Fig. 1A,
lower panel, lanes 2-5).
By contrast, the C-terminal domain of ANT1 (lanes
6 and 7) as well as the full-length protein (lanes 8 and 9) interact specifically
and in a dose-dependent manner with I
B-
.
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Table I
Specific testing of ANT/I B- interactions in the yeast
two-hybrid system
Yeast HF7c cells were cotransfected with expression vectors encoding
the indicated GAL4 DNA-binding domain (DB)-fused and GAL4 transcription
activation domain (AD)-fused proteins. Colonies were tested for their
ability to grow on medium lacking histidine, tryptophan, and leucine,
and -galactosidase activity was monitored by a standard filter
assay. Growth on selective medium and -galactosidase activity are
scored as a range from no growth and no -galactosidase activity ( )
to activity generated by the positive control (+).
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Fig. 1.
In vitro and in vivo
binding of ANT to
I B- .
A, in vitro interaction.
Upper panel, GST fusion proteins were produced in
E. coli and purified on glutathione-Sepharose beads.
Recombinant proteins were analyzed by SDS-PAGE and Coomassie Blue
staining. Lower panel, different GST-fused
proteins still bound to glutathione-Sepharose beads were incubated with
purified recombinant poly-His-I B- . The presence of I B- in
pull-down complexes was visualized by Western blot analysis using
I B- antibodies. B, in vivo interaction. 293 cells were transiently transfected with expression vectors encoding
wild type full-length I B- and Myc-tagged ANT. Cell lysates were
prepared 30 h after transfection and incubated with
anti-I B- , anti-Myc antibodies, or rabbit nonimmune serum.
Co-precipitated proteins were detected by immunoblot analysis using
anti-I B- or anti-Myc antibodies.
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These in vitro results confirm the specificity of the ANT
and I
B-
interaction.
Co-precipitation of ANT and I
B-
in Transfected Intact
Cells--
293 cells were transiently transfected with two expression
vectors encoding Myc-tagged ANT and I
B-
. Cellular levels of
I
B-
and ANT were visualized by direct Western blot analysis of
total lysates with antibodies to I
B-
(Fig. 1B,
lane 2 versus lane 1) or to Myc epitope (lane 10 versus lane 9). Cell soluble extracts were then subjected to immunoprecipitation with anti-I
B-
(lanes 3, 4, 11, and
12) or anti-Myc (lanes 5,
6, 13, and 14) rabbit polyclonal
antibodies or with a nonimmune serum (lanes 7,
8, 15, and 16). The precipitates were
fractionated by SDS-PAGE and blotted with polyclonal I
B-
(lanes 1-8) or anti-Myc (lanes
9-16) monoclonal antibodies. Anti-I
B-
precipitates
contained an anti-Myc reactive band (lane 12)
that comigrates with MycANT (lane 10).
Quantification of the bands lead to an estimate of 14% of total ANT
associated with I
B-
. On the contrary, I
B-
could not be
revealed after anti-Myc immunoprecipitation (lane
4). No anti-Myc or anti-I
B-
reactive proteins were
precipitated by a nonimmune serum (lanes 8 and
16). These results demonstrate that a specific interaction between ANT and I
B-
could occur in intact cells.
Localization of I
B-
and p65 NF-
B in Purified Mitochondria
from Jurkat Cells--
Since ANT is a mitochondrial protein, we looked
for the presence of I
B-
in this organelle. Mitochondria
preparation after subcellular fractionation exhibited minor
contamination by cytosol, as revealed by assays for specific
mitochondrial enzymes: lactate dehydrogenase (2.6%; data not shown).
I
B-
was analyzed in total cell extracts (Fig.
2A, upper
panel, lane 1) or in cytosolic
(lane 2) or mitochondrial (lane
3) extracts. Equal amounts (250 µg) of proteins from
cytosol or mitochondria were loaded. Although the majority of I
B-
was localized in the cytosolic fraction (lane 2),
the protein could also be observed in the mitochondria (lane
3). The localization of p65 NF-
B was investigated. As
shown in the lower panel, p65 was also present in
the cytoplasm (lane 2) as well as in the
mitochondria (lane 3).

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Fig. 2.
I B- and p65
localization in subcellular fractions and purified mitochondria from
Jurkat cells. Subcellular fractions and gradient-purified
mitochondria were prepared as described under "Materials and
Methods." A, visualization of I B- and p65 NF- B in
mitochondrial fraction. Subcellular fractions (total (T),
cytosol (C), and mitochondria (M),
lanes 1-3) or gradient fractions
(lanes 4 and 5) from resting Jurkat
cells were subjected to Western blot analysis using anti-I B- ,
anti-p65, and anti-cytochrome c antibodies. B,
immunoelectron microscopy analysis of I B- in purified
mitochondria. Mitochondria were fixed in 3.7% paraformaldehyde and
embedded into resin. The grids were incubated with anti-I B-
antibodies, and binding was revealed with gold-conjugated rabbit
anti-mouse secondary antibody before examination under an electron
microscope. C, mitochondrial I B- and p65 NF- B, but
not Bcl2, are resistant to proteolysis by proteinase K. Extracts from
mitochondrial fractions (lanes 1-3) or whole
cell extracts (lanes 4-6) were incubated with
the indicated doses of proteinase K for 10 min at 4 °C. The proteins
were then visualized by Western blotting with specific antibodies.
D, release of I B- and p65 NF- B from mitochondria
after digitonin treatment. Highly purified mitochondria were treated
with increasing concentrations of digitonin. After centrifugation, the
mitoplasm was recovered from the pellet, whereas the supernatant
contained released proteins. These fractions were subjected to a
Western blot analysis using the indicated antibodies.
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In order to exclude a possible contamination of the mitochondrial
fraction by cytosolic proteins or other cellular compartments such as
the Golgi apparatus or the ER, mitochondria were further enriched and
purified on a Percoll/metrizamide gradient. Lactate dehydrogenase
activity was below 1% in the highly purified mitochondrial fraction
(not shown). Western blot analysis with specific antibodies confirmed
the presence of I
B-
and p65 in mitochondria (lane 5, upper and middle
panels). Using densitometry scanning of the autoradiography,
we calculated that approximately 7% of cellular total I
B-
is
located in mitochondria. Cytochrome c was mainly detected in
the mitochondrial fraction (lane 5,
lower panel) and only faintly in the cytosol
(lane 4, lower panel).
Immunoelectron microscopy analysis using anti-I
B-
antibodies as
primary antibodies was then performed on purified mitochondria (Fig.
2B). No signal could be detected when gold-conjugated
secondary antibody was used alone or in combination with a nonrelevant
primary antibody (see controls on Fig. 4, A and
B). I
B-
staining was associated with the membrane or
the internal crests, but not with the matrix.
We next assessed the sensitivity of I
B-
, p65, and Bcl2 in
mitochondrial fractions to proteinase K (Fig. 2D,
lanes 1-3, left panels).
While Bcl2, which is associated with the external mitochondrial membrane, was completely degraded (lower panel,
lanes 2 and 3), I
B-
and p65
remained unaffected (upper and middle
panels, lanes 2 and 3),
suggesting that I
B-
and p65 are inaccessible to proteinase K. The
two proteins are not intrinsically resistant to proteinase K, since in
whole cell extracts (right panels), I
B-
and
p65 were degraded by proteinase K to the same extent as Bcl2
(lanes 5 and 6). This suggests that
I
B-
and p65 are located inside the mitochondria.
I
B-
and p65 Are Localized in the Mitochondrial Intermembrane
Space--
To determine the sublocalization of I
B-
and
p65 in the organelle, gradient-purified mitochondria were treated with
increasing concentrations of digitonin in order to solubilize the outer
membrane (17). After centrifugation, pellets and supernatants were
subjected to a Western blot analysis. As shown in Fig. 2D, increasing
concentrations of digitonin (0.1-0.4%) induced a disappearance of
I
B-
and p65 from the mitochondria (lanes 3,
5, and 7 versus lane
1) concomitant to their appearance in the supernatant
fractions (lanes 4, 6, and
8 compared with lane 2). As a control,
digitonin was shown to extract intermembranal cytochrome c.
By contrast, the release of the internal membrane-located
F1 ATPase appeared to require a higher digitonin
concentration (0.4%; lane 8).Therefore,
I
B-
and p65 NF-
B appeared to be located in the mitochondrial
intermembrane space, since they can be released after lysis of the
outer membrane. Surprisingly, we observed several times that
I
B-
·NF-
B complexes were more easily and more rapidly
solubilized by digitonin than cytochrome c. This unexpected
result could suggest that cytochrome c may stick to some
mitochondrial proteins or membrane lipids while I
B-
·NF-
B are
more soluble proteins. Indeed, when F1
ATPase was
extracted by 0.4% digitonin, a significant amount of cytochrome
c still remained associated with mitochondria. Nevertheless, this difference in behavior toward digitonin treatment does not necessarily mean that under physiological apoptotic conditions I
B-
·NF-
B would be more easily released from mitochondria
than cytochrome c. Considering the respective size of these
proteins, it is likely that they use different ways to leave the
mitochondrial intermembrane space.
Mitochondrial I
B-
·NF-
B Complexes--
I
B-
immunocomplexes from cytosolic or mitochondrial fractions were analyzed
by Western blot for the presence of p65. As shown in Fig.
3A, no p65 was found in
control precipitates (lanes 1 and 2),
while p65 associated with I
B-
in cytosolic (lane
3) as well as mitochondrial extracts (lane
4).

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Fig. 3.
A, interaction of I B- with p65
NF- B in mitochondrial fractions. Protein lysates from cytoplasm or
isolated mitochondria were incubated with anti-I B- or rabbit
preimmune serum antibodies. Co-precipitated proteins were detected by
immunoblot analysis using anti-p65 antibodies. B,
mitochondria contain associated I B- ·p65 complexes. Cytoplasmic
extracts (30 µg) or digitonin extracts from mitochondria (50 µg)
were subjected to an electrophoretic mobility shift assay analysis with
a B-specific-radiolabeled probe. 0.6% deoxycholate (Doc)
was used to dissociate I B- ·NF- B complexes. The competition
was performed with a 50-fold excess of unlabeled probe
(cold). Nonspecific binding was indicated
(ns).
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These data indicate the presence of specific I
B-
·p65 NF-
B
complexes in the mitochondria.
To further demonstrate the I
B-
·NF-
B association, cytosolic
or digitonin-treated mitochondrial extracts were treated or not with
deoxycholate in order to dissociate NF-
B from I
B-
. Free
NF-
B was then visualized by electrophoretic mobility shift assay
(Fig. 3B). Deoxycholate (Doc) treatment of
cytosol (lane 2) or mitochondria preparation
(lane 5) released NF-
B that was competent for
specifically binding a 32P-labeled
B probe, as verified
by competition assays with unlabeled probe (lanes
3 and 6). Taken together, these results confirm
the existence of I
B-
·NF-
B complexes in the mitochondria.
Immunoelectron Microscopy Analysis of I
B-
and p65 Subcellular
Localization--
Immunoelectron microscopy analysis was then
performed on intact Jurkat cells, using anti-I
B-
or anti-p65
antibodies as primary antibodies. No signal could be detected when
gold-conjugated secondary antibody was used in combination with a
nonrelevant anti-cytokeratin antibody (Fig.
4A) or alone (Fig.
4B).

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Fig. 4.
Immunoelectron microscopy analysis of
I B- subcellular
localization in Jurkat cells. Cell pellets were fixed in 3.7%
paraformaldehyde and embedded into resin. The grids were incubated with
specific antibodies as indicated, whose binding was revealed with
gold-conjugated rabbit anti-mouse secondary antibody before examination
under an electron microscope. A, cytokeratin staining;
B, secondary antibody alone; C and D,
anti-I B- staining; E and F, anti-p65
staining. Higher magnification was used to demonstrate mitochondrial
staining (right panels).
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Analysis of anti-I
B-
antibody-labeled cells revealed numerous
beads distributed mainly in the cytosol with few in the nucleus (Fig.
4C). Beads were also found associated with mitochondria. No
labeling could be detected within the endoplasmic reticulum or the
plasma membrane, ruling out a nonspecific binding of the antibody to
lipid membranes. A greater magnification of mitochondria showed that
I
B-
staining was localized mainly at the membrane level or in the
internal crests (Fig. 4D).
Analysis of anti-p65 antibody labeled cells revealed a similar
distribution of the molecule both in intact cells (Fig. 4E) and mitochondria (Fig. 4F).
Taken together, these results further confirm a mitochondrial
localization of I
B-
as well as of p65 NF-
B.
I
B-
·NF-
B Complexes Are Released from Mitochondria after
Fas-induced Apoptosis--
The release of pro- or antiapoptotic
molecules from the mitochondrial intermembrane space during apoptosis
in several cellular systems is well documented (11). We analyzed the
fate of I
B-
, p65 NF-
B, and cytochrome c after
induction of apoptosis upon engagement of the Fas surface molecule with
the CH11 antibody. After cell treatment with increasing doses of CH11
antibody, mitochondria were isolated and analyzed for the presence of
I
B-
. As shown in Fig.
5A, engagement of the Fas
receptor resulted in a dose-dependent release of I
B-
(lanes 4-6, upper panel)
as well as of p65 (lanes 4-6, middle
panel). The specificity of these observations was confirmed
by the parallel release of cytochrome c (lanes
4-6, lower panel) from mitochondria.
In contrast, little to no degradation of these molecules could be
visualized in the cytosol after CH11 treatment (lanes
1-3).

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Fig. 5.
Release of mitochondrial
I B- after Fas
engagement of the Fas death receptor. A, cells were
treated for 18 h with increasing concentrations of anti-Fas
antibody (CH11), and mitochondria fractions were prepared as described
under "Materials and Methods." Protein extracts were analyzed by
Western blot with the indicated antibodies. B, cells were
treated or not with CH11 antibody as above, followed by electron
microscropy analysis after I B- or p65 staining.
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Electron microscopy analysis of CH11-treated Jurkat cells revealed cell
shrinkage, perinuclear chromatin condensation, and alterations of the
mitochondrial structures, including swelling, disappearance of
cristae membranes, and destructuralizing of the surrounding
membranes (data not shown). In these cells, I
B-
staining was
highly reduced in the mitochondria (Fig. 5B, compare a with b). Similarly, CH11 treatment resulted in
the disappearance of p65 reactivity (compare c with
d).
These data suggest that I
B-
and p65 complexes located in the
intermembrane space of the mitochondria can be released in the cytosol
following stimulation of Jurkat cells with apoptotic signals.
Mitochondrial I
B-
Is Not Degraded in MT4 Cells--
We
then performed experiments in order to determine if the mitochondrial
I
B-
·NF-
B complexes were sensitive to activation signals. We
used HTLV-I-infected MT4 cells that display a constitutive activation
of NF-
B. The HTLV-I Tax protein is known to activate NF-
B by
binding to the NEMO/IKK
component of the signalsome, resulting in a
constitutive activation of the I
B-
kinases (18). MT4 cells
express low amounts of I
B-
(Fig. 6,
lane 1) as visualized by Western blot analysis.
Cell treatment (1 h, 50 µM) with the proteasome inhibitor
ALLN, shown to prevent degradation of I
B-
, resulted in an
increase in the cellular levels of I
B-
(lane 2). By contrast, the amount of I
B-
in the mitochondria
was not affected by the ALLN treatment (compare lane
4 with lane 3). The nitrocellulose
filter was then hybridized with a phosphospecific I
B-
antibody. Despite constitutive stimulation of IKK kinases, no
phosphorylated I
B-
could be detected in cytosolic extracts (lane 6), suggesting that phosphorylated
I
B-
is immediately degraded. Indeed, ALLN treatment allowed the
visualization of phospho-I
B-
(lane 7). By
contrast, no signal could be detected in the mitochondrial fraction
(lane 8), even in the presence of ALLN
(lane 9), suggesting that despite continuous
firing of I
B kinases, mitochondrial I
B-
is protected from
phosphorylation.

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Fig. 6.
Mitochondrial complexes are sequestered from
NF- B activation signals. MT4,
HTLV-I-infected cells were pretreated for 1 h with the proteasome
inhibitor ALLN (50 µM) when indicated. Cytosolic and
mitochondrial extracts were resolved by SDS-PAGE and analyzed by
Western blotting with I B- -specific antibodies (upper
panel) or phospho-I B- -specific antibodies
(lower panel).
|
|
 |
DISCUSSION |
In this report, we show that I
B-
·NF-
B complexes are
present in the mitochondrial intermembrane space via interaction of the
N-terminal domain of I
B-
with the internal membrane protein ANT
(adenine nucleotide
translocator).
The interaction was originally detected in a yeast two-hybrid screening
and checked by in vitro pull-down experiments with GST-ANT
and recombinant I
B-
. A coprecipitation of the two molecules was
observed in transfected 293 cells, demonstrating that the two proteins
could interact in intact cells. Finally, immunoelectron microscopy
allowed the unambiguous demonstration of I
B-
·NF-
B complexes
in the mitochondrial intermembrane space. NF-
B and I
B-
were
unaffected by a proteinase K treatment of intact mitochondria and could
be extracted by digitonin lysis of the outer membrane.
The ANT protein is localized in the inner mitochondrial membrane and
exchanges cytosolic ADP for mitochondrial ATP (10). ANT interacts with
several proteins of the outer membrane (peripheral benzodiazepine
receptor, porin/VDAC, Bax) as well as the matrix (cyclophilin) to form
the permeability transition pore (PTP) or megachannel (19). The PTP
appears as an important regulator of the apoptotic process. Opening of
the pore leads to loss of the mitochondrial transmembrane potential,

m, that can ultimately culminate in matrix swelling
and outer membrane rupture, allowing the release of apoptogenic
proteins such as cytochrome c, apoptosis-inducing factor,
and procaspases (11, 20-22). The exit of cytochrome c is
controlled by proteins of the Bcl-2 family that are anchored on the
mitochondrial outer membrane. Antiapoptotic members of the family
(Bcl-2, Bcl-xl) prevent cytochrome c release in contrast to
the proapoptotic members Bax and Bak (23). Bax has been shown to
interact with ANT to induce PTP opening and cytochrome c
release (24).
Several pharmacological compounds interfere with PTP. For instance,
bongkrekic acid and atractyloside are, respectively, blocker and
inducer of apoptosis by binding two different conformational stages of
ANT (20, 25). Cyclosporin A also blocks mitochondrial apoptotic signals
by interacting with cyclophilin (26), while engagement of the
benzodiazepine receptor facilitates apoptosis (27).
The observed interaction of I
B-
with ANT suggests that
mitochondrial I
B-
·NF-
B complexes could be involved in the
regulation of the apoptotic cascade. Apart from its well known
functions in coordination of immune and inflammatory responses, NF-
B
appears to promote cell survival (4, 5). For instance, inactivation of
the p65 NF-
B (relA) gene resulted in embryonic
death due to massive liver apoptosis (28). This property could be
demonstrated in various cell lines by expression of a dominant negative
form of the inhibitory subunit I
B-
. Replacement of serines 32 and 36 by alanines prevents phosphorylation/degradation of I
B-
and thus activation of NF-
B. Cells expressing the I
B-
superrepressor clearly become more susceptible to apoptosis induced by
tumor necrosis factor, chemotherapeutic drugs, or
-rays
(29-31).
In order to determine if the mitochondrial pool of I
B-
·NF-
B
complexes could be reached by activation signals, we used
HTLV-1-infected MT4 cells that display a constitutive activation of
NF-
B, due to an interaction of the viral Tax protein with IKK
to
activate the IKK complex. Inhibition of the degradation pathway by ALLN increased cytosolic I
B-
levels and allowed detection of the serine phosphorylated form. In sharp contrast, mitochondrial I
B-
was not affected by ALLN treatment. These results show that
mitochondrial I
B-
·NF-
B complexes are sequestered from
NF-
B-activating signals.
Induction of apoptosis after engagement of the Fas death receptor leads
to the exit of I
B-
·NF-
B from mitochondria. While cytochrome
c can leave mitochondria through open VDAC (diameter 2.4-3
nm) (32), the liberation of procaspases and I
B-
·NF-
B complexes requires rupture of the outer membrane. We believe that this
release in the cytosol could increase the number of NF-
B complexes
susceptible for activation.
Apoptosis should be considered as a multistep and integrative process.
Before producing an appropriate outcome response, cells have to
integrate pro- and anti-apoptotic influences. Moreover, cross-talks
between apoptotic and survival pathways have already been demonstrated.
NF-
B could be one of the important players at this cross-road.
NF-
B is activated by the Akt kinase survival pathway (33, 34). The
second face of the cross-talks is represented by the caspase-mediated
inactivation of survival pathways. Transcription factors appear as key
targets. SRF, which regulates growth factor-induced survival genes, is
cleaved and inactivated during apoptosis (35). So is p65 NF-
B, whose
cleavage produces a protein lacking its transactivation domain that has
a dominant negative effect on NF-
B-induced survival genes (36).
I
B-
could also be cleaved by caspase 3, to an N-terminal
truncated and stable inhibitor of NF-
B activation (37).
Apoptotic pathways have characteristics of a self-amplifying process.
Procaspases are released from the mitochondria (38), and activated
caspases are able to induce the exit of cytochrome c to
exacerbate the response (39). In contrast, NF-
B promotes, in many
cells, survival effects via transcription of genes such as those for
cIAP1 and cIAP2 that code for caspases inhibitors (40, 41). It is thus
possible that the release of I
B-
·NF-
B from the mitochondria
is an additional control level. Reception of a survival signal at this
point would limit or delay apoptosis, whereas continuous caspase
activation will lead to NF-
B inactivation. Alternatively,
I
B-
·NF-
B could exert a transcriptionally independent regulatory function on apoptosis. This has been demonstrated for p53,
which translocates to mitochondria during p53-mediated apoptosis. Translocation precedes mitochondrial dysfunctional changes and appears
independent of p53 transcriptional activity (42). It is thus
conceivable that I
B-
·NF-
B could regulate opening of the PTP
through its association with ANT.
The results presented here suggest that a reservoir of
I
B-
·NF-
B complexes in the intermembrane mitochondrial space
could be involved in modulating apoptosis responses.