From the Abramson Family Cancer Research Institute
and the Departments of § Cancer Biology and ** Biochemistry
and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania
19104 and the
Department of Biology, University of Maryland,
College Park, Maryland 20742
Received for publication, February 20, 2001, and in revised form, March 15, 2001
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ABSTRACT |
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The diffusion of metabolites across the
outer mitochondrial membrane is essential for coupled cellular
respiration. The outer membrane of mitochondria isolated from growth
factor-deprived cells is impaired in its ability to exchange metabolic
anions. When added to mitochondria, recombinant
Bcl-xL restores metabolite exchange across the outer
membrane without inducing the loss of cytochrome c from the
intermembrane space. Restoration of outer membrane permeability to
anionic metabolites does not occur directly through Bcl-xL
ion channels. Instead, recombinant Bcl-xL maintains the
outer mitochondrial membrane channel, VDAC, in an open
configuration. Consistent with these findings, when ADP-induced
oxidative phosphorylation is limited by exogenous The primary pathway for metabolite diffusion across the
outer mitochondrial membrane
(OMM)1 is through the
voltage-dependent anion channel (VDAC), a large conductance
channel that in its open configuration is permeable to molecules of up
to ~5000 daltons (1, 2). However, a conserved property of the primary
VDAC isoform from all organisms tested is its ability to adopt multiple
conductance states (3, 4). Treatment of isolated mitochondria with
agents that favor VDAC closure, such as OMM permeability has also been reported to play an important role in
apoptosis. Failure of the OMM to sequester cytochrome c in
the intermembrane space can induce apoptosis (11). The pro- and anti-
apoptotic members of the Bcl-2 family of proteins have been shown to
exert their activity by regulating mitochondrial functions (12, 13).
Interestingly, some members of the Bcl-2 family (such as Bcl-2 and
Bcl-xL) are permanent residents of the OMM whereas many
pro-apoptotic members of the family (such as Bax, Bad, and Bid) can be
translocated from the cytosol to the OMM following an apoptotic signal.
The anti-apoptotic protein Bcl-xL exhibits both structural
and functional similarity to prokaryotic pore-forming proteins. Both,
Bcl-2 and Bcl-xL inhibit cytochrome c release by
regulating OMM integrity or permeability (12, 13). It has been proposed
that VDAC may have a role in regulating cytochrome c release
by forming together with Bax, a high conductivity channel that can
release cytochrome c from the intermembrane space to the
cytosol (14).
Recently we have found that early in apoptosis, prior to the loss of
OMM integrity and the release of cytochrome c, the OMM becomes impermeable to small metabolites (15). This impermeability might lead to further disruptions of mitochondrial homeostasis and
ultimately to the loss of OMM integrity. Following growth factor
withdrawal, both the loss of OMM permeability and cell death are
inhibited in Bcl-xL-expressing cells (15). However, the
mechanism by which Bcl-xL maintains OMM permeability
remains controversial. In this study, we describe a direct biochemical activity of Bcl-xL protein in regulating the conductivity
of VDAC. We demonstrate that Bcl-xL can increase the
probability of VDAC to be in an open configuration under conditions
that favor VDAC closure. Therefore, it is suggested that in response to
an apoptotic signal that would result in VDAC closure,
Bcl-xL promotes the open configuration of VDAC and
maintains metabolite exchange between the mitochondria and the cytosol.
Cell Culture--
FL5.12 cells were maintained at 37 °C and
5% CO2 in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 50 µM 2-mercaptoethanol, and 300 pg/ml
recombinant mouse interleukin-3 (IL-3) (PharMingen). For growth factor
withdrawal, cells were washed three times in serum-free medium and
resuspended in full medium with (control) or without IL-3.
Recombinant Bcl-xL Protein
Production and Analysis--
Full-length human
Bcl-xL was cloned into the pET-16b bacterial
expression vector (Novagen), and recombinant protein was purified from
BL21(DE3)pLysS Escherichia coli on a Ni2+
column using a standard commercial kit (Novagen). Following elution of
the protein with increasing concentrations of imidazole (100 mM to 1 M) the different fractions were
analyzed by SDS-polyacrylamide gel electrophoresis followed by
Coomassie Blue staining. The fractions that contained the recombinant
protein (>95% purity) were combined and dialyzed overnight in 20 mM Hepes buffer, pH 7.4. The recombinant protein was
concentrated to 2 mg/ml using a centrifugal filter (Millipore), divided
into aliquots, and kept frozen ( Preparation of VDAC from Rat Liver--
Mitochondria were
isolated from rat liver by differential centrifugation as previously
described (16). Following removal of soluble protein by hypotonic shock
and centrifugation, the mitochondrial membranes were stored in 1 mM KCl, 1 mM Tris-Cl, pH 7.5, 15% (v/v)
Me2SO as 1-ml aliquots at Mitochondria Isolation and Analysis--
Mitochondria were
isolated from FL5.12 cells and phosphocreatine levels were measured by
HPLC as described previously (15). Where indicated, mitochondria were
incubated at room temperature for 30 min in the presence or absence of
recombinant Bcl-xL prior to the measurement of
phosphocreatine or Western blot analysis.
Mouse liver mitochondria were isolated by tight dounce homogenization
and differential centrifugation in 10 mM Hepes buffer, pH
7.4, containing 200 mM mannitol, 70 mM sucrose,
1 mM EGTA, 1 mg/ml bovine serum albumin. Where indicated,
recombinant Bcl-xL was incubated with mitochondria at the
specified concentration for 10 min at room temperature. Immediately
prior to use in respiration studies, mitochondria were diluted into 20 mM Hepes buffer, pH 7.4 containing 250 mM
sucrose, 10 mM KCl, 5 mM succinate, 3 mM KH2PO4, 1.5 mM
MgCl2, 1 mM EGTA, 1 mg/ml bovine serum albumin. Electrophysiological Analysis--
Membranes were made from
monolayers of diphytanoylphosphatidylcholine by the method of Montal
and Mueller (19) and as modified (20). Recordings were made under
voltage-clamp conditions using calomel electrodes with saturated KCl
bridges. Current was filtered at 90 Hz using a Butterworth filter.
Recombinant Bcl-xL Protein Restores Outer
Membrane Permeability to Phosphocreatine in Mitochondria of Survival
Factor-deprived Cells--
Mitochondria isolated from growth
factor-deprived cells have lost the ability to exchange organic
metabolites such as ATP, ADP, and phosphocreatine with the cytosol
(15). It was previously suggested that this is because of a decrease in
OMM permeability to small anionic metabolites. In
IL-3-dependent FL5.12 cells, changes in OMM permeability
following IL-3 withdrawal can be detected by measuring phosphocreatine
levels in the intermembrane space of mitochondria (15) (Fig.
1A). To determine whether
Bcl-xL can restore outer membrane anion permeability when
added directly to mitochondria impaired in phosphocreatine exchange,
recombinant Bcl-xL was generated. Recombinant
Bcl-xL efficiently incorporated into mitochondria isolated
from growth factor-deprived cells (Fig. 1B). Addition of
recombinant Bcl-xL resulted in an 80% (±15%) decrease in
the amount of phosphocreatine present in the intermembrane space (Fig.
1C). This indicates that Bcl-xL can facilitate
the equilibration of phosphocreatine across the OMM. Bcl-xL
did not appear to induce phosphocreatine release by nonspecifically
disrupting the integrity of the OMM as the release of cytochrome
c from these same mitochondria was not observed (Fig.
1D). As a positive control for cytochrome c
release, mitochondria were subjected to hypotonic shock. Hypotonic
shock results in matrix swelling and the nonspecific disruption of the
OMM. Under these conditions both cytochrome c and
phosphocreatine were released from isolated mitochondria. Hexokinase,
another protein reported to interact with the OMM (21), failed to
induce either phosphocreatine or cytochrome c release (data
not shown). Together, these data demonstrate that Bcl-xL
can act at the mitochondria to restore outer membrane permeability to
anions without disrupting the ability of the membrane to retain intermembrane space proteins.
Bcl-xL Is Not a Phosphocreatine
Channel--
Bcl-xL forms ion channels in vitro
(22, 23), suggesting that a channel activity of Bcl-xL may
be directly responsible for the equilibration of phosphocreatine across
the outer membrane. To test this possibility, the ability of
Bcl-xL channels to pass phosphocreatine was examined by
reconstitution into planar phospholipid membranes. Recombinant
Bcl-xL was inserted into an uncharged phospholipid membrane
in the presence of a 2-fold chemical gradient of
Na Bcl-xL Regulates VDAC Properties--
The
major outer membrane channel, VDAC, is permeable to anionic metabolites
in the open state, but exhibits a large reduction in permeability to
anions such as phosphocreatine when the channel adopts closed
configurations (15, 25). Thus, VDAC must be closed for phosphocreatine
to be retained in isolated mitochondria. In vitro, the
presence of a membrane potential closes VDAC and has been shown to
favor Bcl-xL channel formation (22). To determine whether
Bcl-xL can alter OMM permeability via an effect on VDAC when residing in the same membrane, the ability of recombinant Bcl-xL to alter VDAC channel properties was examined.
Purified rat liver VDAC reconstituted into a planar phospholipid
membrane exhibits decreased channel conductance (closed states) when a voltage is applied across the membrane. In results from a typical experiment, average conductance through VDAC channels is illustrated before and after Bcl-xL addition (Fig.
3A). Bcl-xL caused
an increase in conductance (channel opening) at potentials between
To further explore the effect of Bcl-xL on VDAC channel
properties, single-channel experiments that determine the conductance of each state and transitions between states were performed. Sample traces from one such experiment show the conductance (slope of trace) and transitions between states (vertical connecting
lines) as voltage was varied between ± 50 mV (Fig.
3B). The probabilistic nature of single-molecule behavior
does not allow one to observe differences merely by examining one
trace. From analysis of many such traces, the probability of observing
the channel in its highest conducting state (the open probability) was
determined (Fig. 3C). VDAC channels have two gating
processes working at opposite potentials (26). Bcl-xL
increased the open probability primarily for the gating process that
occurs at positive potentials, indicating an asymmetric effect. Because
this effect was observed when Bcl-xL was added to both
sides of the membrane, the asymmetry is likely a property of VDAC. A
second-order effect on VDAC conductance was also observed. The
single-channel conductance of VDAC in both the open and closed states
was reduced by about 5% when Bcl-xL was added. This may
reflect an interaction between the proteins or may be attributed to a
physical interference with VDAC conductance caused by the close
proximity of the proteins in the membrane.
Recombinant Bcl-xL Maintains VDAC Open in
Isolated Mitochondria under Conditions That Promote VDAC
Closure--
The single-channel experiments indicate that
Bcl-xL increases the conductance of VDAC by increasing the
probability of the channel to be open. This also explains the ability
of Bcl-xL to increase the permeability of the OMM. To
explore this property further, the ability of Bcl-xL to
maintain outer membrane permeability to relevant anions such as ADP was
tested under conditions that are known to close VDAC.
The delay in the transition between state III (ADP present) and state
IV (ADP converted to ATP) was used to calculate the change in outer
membrane permeability by using the method of Lee et al. (9,
27). The respiration data were fitted to a theoretical expression that
accounts for the diffusion through VDAC in the outer membrane and the
saturable transport across the inner membrane by the ANT, using
Equation 1,
Recombinant Bcl-xL protein favors the open state of
VDAC in a planar phospholipid membrane. This results in an increase in the probability of VDAC opening when a potential ( Whereas VDAC appears to close following growth factor withdrawal (15),
the signal that induces VDAC closure is not known. It is possible that
the translocation of pro-apoptotic proteins such as Bax to mitochondria
might directly or indirectly result in VDAC closure. Alternatively,
VDAC closure may result as a consequence of changes in cellular
metabolism. For instance, an early cellular response to growth factor
withdrawal is a decrease in both the mitochondrial membrane potential
and the rate of electron transport (30). This decrease in mitochondrial
function may result in NADH accumulation because oxidative
phosphorylation is required to efficiently regenerate NAD from NADH.
Alternatively, changes in metabolite flux across the OMM may be
responsible for inducing a potential across the outer membrane
(31).
There are several possible ways by which Bcl-xL could
maintain the open configuration of VDAC. Bcl-xL may alter
the gating properties of VDAC by physically interacting with it. Based
upon co-immunoprecipitation studies of detergent-solubilized membranes, it has been reported that Bcl-xL can interact with VDAC
(28). However, it remains unclear whether this reflects an actual
physical association of the proteins within the membrane, or the innate properties of hydrophobic proteins following membrane dissolution. Alternatively, Bcl-xL may insert into the membrane and
alter the local electrical or lipid environment of the membrane and
inhibit VDAC closure indirectly. If the action is indirect, it must be exerted in close proximity to VDAC. In any event, the action of Bcl-xL is to favor the open configuration of VDAC. Factors
favoring the closed state, such as high electric fields, can overcome
the effects of Bcl-xL.
Recombinant Bcl-xL can regulate metabolic anion exchange
across the OMM. This demonstrates for the first time a direct function for an anti-apoptotic Bcl-2 protein in an organelle to which it is
targeted. The regulation of VDAC gating properties may at least partially explain the ability of Bcl-xL to promote cell
survival. By facilitating the continued exchange of metabolites across
the OMM during periods of cellular stress, Bcl-xL protects
against a disruption in mitochondrial physiology that results in the
release of cytochrome c from mitochondria (30). Furthermore,
the demonstration that metabolite flux through VDAC can be regulated by
Bcl-xL may explain how Bcl-2 proteins impact on both the
redox state and pH of the cell (32, 33). Maintaining ADP-coupled
oxidative phosphorylation should limit lactate production, reduce the
amount of glucose consumed by glycolysis, and preserve greater
substrate availability for the production of reducing equivalents
through the pentose phosphate shunt. Changes in both pH and redox state have been associated with apoptosis. Thus, the ability of Bcl-2 proteins to regulate transport across mitochondrial membranes could
account for their ability to affect a wide variety of apoptotic pathways.
-NADH, recombinant
Bcl-xL can sustain outer mitochondrial membrane
permeability to ADP.
-NADH limits respiration by promoting the
closed configuration of VDAC. Together these results demonstrate
that following an apoptotic signal, Bcl-xL can maintain
metabolite exchange across the outer mitochondrial membrane by
inhibiting VDAC closure.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NADH, limits metabolite
flux across the outer membrane and inhibits mitochondrial function
(5-9). This ability of VDAC to adopt a closed configuration and
inhibit ADP-coupled respiration has been suggested to contribute to the
Crabtree effect (9), a paradoxical response in which treatment of
respiring cells with glucose leads to inhibition of oxidative
phosphorylation (10).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C) until used. For Western
blot analysis, the 13.6 anti-Bcl-xL polyclonal antibody and
the 7H8-2C12 anti-cytochrome c antibody (PharMingen) were used.
85 °C. VDAC protein was
purified from single aliquots of the mitochondrial membranes using a
hydroxyapatite/celite column as previously reported (17) and as
modified (18). The VDAC protein was dissolved in 3% Triton X-100, 50 mM KCl, 10 mM Tris-Cl, 1 mM EDTA,
pH 7.0, 15% Me2SO, and stored at
20 °C (short term)
or
85 °C (long term).
-NADH and ADP were added at concentrations of 100 µM.
Oxygen consumption was measured in a functionally airtight
water-jacketed chamber at room temperature using a polarographic oxygen electrode.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Bcl-xL can
increase OMM permeability to phosphocreatine without disrupting
membrane integrity. A, mitochondria were isolated from
FL5.12 cells cultured in the presence (+IL-3) or absence
( IL-3) of IL-3 for 12 h, and the amount of
phosphocreatine was determined by HPLC. The phosphocreatine peak was
confirmed by co-injection of purified phosphocreatine. The average
phosphocreatine peak area, normalized to mitochondrial protein, is
shown for each treatment. The data presented are the mean ± 1 S.E. from four independent experiments. The increase in phosphocreatine
observed upon growth factor withdrawal was statistically significant by
Student's t test (p < 0.05). B,
mitochondria were isolated from growth factor-deprived cells and
incubated in the absence (Control) or presence
(+Bcl-xL) of recombinant Bcl-xL (3 µg/ml) or subjected to hypotonic shock as indicated. The amount of
Bcl-xL present after 30 min of treatment was determined by
Western blot analysis as shown. C, the amount of
phosphocreatine present in mitochondria isolated from growth
factor-deprived cells incubated for 30 min in the absence
(Control) or presence (+Bcl-xL) of
recombinant Bcl-xL (3 µg/ml) was determined by HPLC. The
average phosphocreatine peak area, normalized to mitochondrial protein,
is shown for each condition. The data presented are the mean ± S.E. from four independent experiments. The decrease in phosphocreatine
observed upon Bcl-xL addition was statistically significant
by Student's t test (p < 0.05).
D, mitochondria were isolated from growth factor-deprived
cells and incubated for 30 min in the absence (Control) or
presence (+Bcl-xL) of recombinant
Bcl-xL (3 µg/ml) or were subjected to hypotonic shock as
indicated. Following treatment, the mitochondria were pelleted by
centrifugation, and the amount of cytochrome c present in
the supernatant was determined by Western blot analysis.
2. All
permeant ions tend to flow down their concentration gradient carrying
charge across the membrane. The more permeant ions will carry a greater
charge across the membrane resulting in a current. The voltage that
brings this current to zero (the reversal potential) is related to the
relative permeability of the ions. The current through the membrane,
recorded as a function of voltage, is illustrated before and after
addition of Bcl-xL (Fig. 2).
The intercept, indicated by the arrow, shows the voltage at
which no current flowed through the Bcl-xL channels. For
comparison, the reversal potential would have been 0 mV in the absence
of selectivity, and +9 mV if only phosphocreatine were able to
permeate. The reversal potential measured for Bcl-xL
channels (
14.1 ± 1.5 mV (mean ± 1 S.D.)) is consistent
with a channel that is unable to pass phosphocreatine (Fig. 2).
Furthermore, this reversal potential was indistinguishable from that
recorded under identical conditions for gramicidin channels (
14.1 ± 0.5 mV) (data not shown). Because it is well
established that gramicidin channels are ideally selective for cations
over anions and that they are impermeable to molecules larger than water (24), the channels formed by both gramicidin and
Bcl-xL must be impermeable to phosphocreatine. Thus,
Bcl-xL ion channels do not directly increase the
permeability of the outer membrane to metabolic anions such as
phosphocreatine. Bcl-xL must influence the properties of
the outer membrane indirectly, possibly by promoting the ability of
other channels to pass complex anions.
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Fig. 2.
Bcl-xL
channels are not permeable to phosphocreatine. Triangular voltage
waves (10 mHz, ±25 mV) were applied to a planar phospholipid membrane
separating asymmetrical 200 mM/100 mM solutions
of Na 2,
and current was recorded before and after addition of recombinant
Bcl-xL (1.4 µg/ml) as indicated. The reversal potential
measured after Bcl-xL addition demonstrates that the
resultant channels are permeable to sodium but not to phosphocreatine.
The arrow denotes the reversal potential expected for a
channel with ideal selectivity for Na+. The data presented
are representative of three independent experiments.
25
mV and +25 mV. This increase correlates with the amount of
Bcl-xL added in a dose-dependent manner. The
increase in membrane conductance reflects an increase in VDAC channel
conductance rather than an additive effect of independent
Bcl-xL and VDAC channels because the conductance of
Bcl-xL channels is much lower than that of VDAC channels.
The failure of Bcl-xL addition to increase conductance through VDAC at potentials greater than +25 mV or less than
25 mV
define a limit past which Bcl-xL does not influence VDAC
closure.
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Fig. 3.
Bcl-xL
promotes the maintenance of VDAC in an open configuration.
A, the current through isolated rat liver VDAC incorporated
into a planar phospholipid membrane was recorded during the application
of successive triangular waves (4 mHz, ±50 mV) before and after the
addition of recombinant Bcl-xL at the indicated amounts.
The average conductance through VDAC in response to triangular voltage
waves is shown before and after Bcl-xL addition. The data
presented are the average conductances measured from 14 and 16 waves
respectively and are representative of six independent experiments.
B, triangular voltage waves were applied to a planar
phospholipid membrane containing a single VDAC channel. Representative
current records of the VDAC channel are shown before
(Control) and after (+Bcl-xL)
addition of recombinant Bcl-xL (15 µg/ml). C,
multiple current records were analyzed to determine the likelihood that
a single VDAC channel was in the highest conducting state (open
configuration) as a function of voltage. This open probability is
graphed in the absence (Control) and presence
(+Bcl-xL) of recombinant Bcl-xL as
indicated.
-NADH favors
VDAC closure in planar phospholipid membranes (8) and decreases the
permeability of the outer membrane to ADP in isolated mitochondria (9).
Addition of recombinant Bcl-xL to mitochondria isolated
from mouse liver resulted in a dose-dependent incorporation
of Bcl-xL onto the mitochondria (Fig.
4A). Examining
ADP-dependent respiration in the presence and absence of
recombinant Bcl-xL tested whether Bcl-xL can
affect the permeability of the OMM. Consistent with the high outer
membrane permeability expected when VDAC is open, the addition of
recombinant Bcl-xL had no measurable effect on ADP-dependent respiration in the absence of
-NADH (data
not shown). However, recombinant Bcl-xL prevented
-NADH
addition from prolonging the time it takes the mitochondria to complete
the respiratory burst induced by a defined amount of ADP (Fig.
4B). ADP-dependent respiration requires both the
diffusion of ADP across the outer membrane and the facilitated
transport of ADP across the inner membrane by the adenine nucleotide
transporter (ANT). A steady-state level of ADP is maintained in the
intermembrane space resulting in equal fluxes across both membranes. At
physiological levels of ADP, the diffusion-based flux through the outer
membrane can become rate-limiting. This is manifested as a delay in the
time that the change in PO2, which corresponds to the
conversion of a given bolus of ADP to ATP, is achieved.
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Fig. 4.
Bcl-xL
incorporates into mitochondria and promotes increased OMM permeability
under conditions that favor VDAC closure. A,
mitochondria were isolated from mouse liver and incubated with
recombinant Bcl-xL for 10 min at the indicated
concentration. Following treatment, the mitochondria were washed, and
the amount of Bcl-xL that incorporated into the
mitochondria was determined by Western blot analysis as shown. The
first lane (*) contains recombinant Bcl-xL in the absence
of mitochondria. B, mitochondria isolated from mouse liver
were incubated for 10 min with -NADH (100 µM) in the
presence or absence of recombinant Bcl-xL prior to
measuring the change in oxygen tension over time. Oxygen consumption
curves recorded for mitochondria in the absence (Control)
and presence (+Bcl-xL) of recombinant
Bcl-xL are shown. In both cases, oxidative phosphorylation
(state III respiration) was stimulated by the addition of ADP (100 µM) where indicated. After mitochondria convert all the
ADP added to ATP, the rate of oxygen consumption returns to that
observed prior to ADP addition (state IV). In all cases, mitochondria
exhibited a respiratory control ratio greater than 5. C, the
rate of ADP-stimulated oxygen consumption in the presence of
-NADH
(100 µM) was converted to the change in medium [ADP] as
a function of time, and these data were fitted to an equation to
determine the difference in [ADP] across the OMM as the ADP in the
medium was converted to ATP. The difference in ADP concentration across
the OMM over time is shown for mitochondria in the absence
(Control) or presence (+Bcl-xL) of
the indicated amount of recombinant Bcl-xL after addition
of 100 µM ADP to the mitochondria. D, the rate
of ADP-stimulated oxygen consumption in the presence of
-NADH (100 µM) was converted to the change in medium [ADP] as a
function of time, and these data were fitted to an equation to
determine the permeability of the OMM in the presence of different
amounts of recombinant Bcl-xL. The percent increase in OMM
permeability is shown as a function of Bcl-xL
concentration. The error bars illustrate the range of values
containing the 95% confidence interval for the increase in OMM
permeability at each concentration of Bcl-xL.
where PA is the OMM permeability × area and
Vmax and Km represent
kinetic properties of ANT. The fitted parameters were the permeability
of the outer membrane and the kinetic constants for the ANT. Whereas
the ANT kinetic parameters varied little, significant differences in
outer membrane permeability were observed in response to
(Eq. 1)
-NADH and
were prevented by the presence of recombinant Bcl-xL. Bcl-xL prevented
-NADH from retarding the completion of
an ADP-induced respiratory burst in a dose-dependent
fashion, resulting in reductions in the calculated ADP concentration
gradient across the OMM (Fig. 4C). These parameters were
used to calculate the changes in OMM permeability in the presence of
-NADH, in response to increased amounts of recombinant
Bcl-xL (Fig. 4D). The results suggest that Bcl-xL promotes adenine nucleotide flux across the OMM
under conditions that would normally favor VDAC closure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
25 mV to +25 mV) is
applied. Furthermore, mitochondria that have incorporated recombinant
Bcl-xL do not demonstrate a
-NADH-induced delay in completing a defined change in PO2 in response to a given
dose of ADP. Thus, Bcl-xL can maintain the permeability of
the outer membrane to ADP in the presence of a VDAC closing stimulus.
Previously, it was suggested that Bcl-xL might modulate
VDAC activity by keeping it in a closed configuration and preventing
cytochrome c release (28). However, overexpression of
Bcl-2/Bcl-xL does not result in a reduction in respiration
rate as would be expected if either Bcl-2 or Bcl-xL were to
close VDAC, and it seems unlikely that under normal conditions
Bcl-xL closes VDAC. It is possible though that under some
conditions Bcl-2/Bcl-xL stabilize VDAC and prevent formation of a cytochrome c-releasing pore (13, 29). The
data presented in this study support the hypothesis that
Bcl-xL functions to maintain VDAC in an open configuration
under conditions that favor VDAC closure. This open configuration of
VDAC does not enable the passage of cytochrome c, but
maintains free metabolite exchange across the OMM, supporting oxidative phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank Robert Mizani for assistance with data analysis and members of the Thompson laboratory and James Lear for thoughtful discussions and critiques of the manuscript. We also thank Jeff Rathmell for assistance with mice and William DeGrado for providing access to equipment.
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FOOTNOTES |
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* This work was supported by the University of Chicago Medical Scientist Training Program and Cancer Research Fund Women's Board (to M. G. V.) and by a European Molecular Biology Organization fellowship (to E. G.).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.
¶ These authors contributed equally to this work.
To whom correspondence should be addressed: Abramson Family
Cancer Research Inst., 450 BRBII 421 Curie Blvd., Philadelphia, PA
19104. E-mail: drt@mail.med.upenn.edu.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M101590200
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ABBREVIATIONS |
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The abbreviations used are: OMM, outer mitochondrial membrane; VDAC, voltage-dependent anion channel; IL-3, interleukin-3; ANT, adenine nucleotide transporter; HPLC, high performance liquid chromatography.
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REFERENCES |
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