From the Medical Research Council Dunn Human
Nutrition Unit, Hills Road, Cambridge CB2 2XY and the
§ Department of Biochemistry, University of Cambridge,
Tennis Court Road, Cambridge CB2 1GA, United Kingdom
Received for publication, December 21, 2000, and in revised form, February 20, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We assessed the ability of human
uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative
phosphorylation when expressed in yeast at physiological and
supraphysiological levels. We used three different inducible UCP2
expression constructs to achieve mitochondrial UCP2 expression levels
in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein.
Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no
increase in proton conductance, even in the presence of various
putative effectors, including palmitate and
all-trans-retinoic acid. Only when UCP2 expression in yeast
mitochondria was increased to 4 µg/mg, more than an order of
magnitude greater than the highest known physiological concentration,
was proton conductance increased. This increased proton conductance was
not abolished by GDP. At this high level of UCP2 expression, an
inhibition of substrate oxidation was observed, which cannot be readily
explained by an uncoupling activity of UCP2. Quantitatively, even the
uncoupling seen at 4 µg/mg was insufficient to account for the basal
proton conductance of mammalian mitochondria. These observations
suggest that uncoupling of yeast mitochondria by UCP2 is an
overexpression artifact leading to compromised mitochondrial integrity.
Uncoupling protein 1 (UCP1)1 uncouples brown
adipose tissue mitochondria, causing physiologically important,
hormonally regulated, thermogenic proton cycling across the inner
membrane. The functions of the UCP1 homologues, UCP2 and UCP3 (1-4),
are currently uncertain (5-12). They have been demonstrated to
uncouple mitochondrial oxidative phosphorylation in a number of
experimental models, including proteoliposomes (13), yeast heterologous
expression systems (1, 2, 14-16), and transgenic mice (17). It is clear that, under some experimental conditions, heterologous or transgenic expression of these proteins can cause an increase in the
proton conductance of the inner membrane (16, 18). However, it is less
obvious whether these experimental observations of uncoupling are due
to a native protein activity of the UCP1 homologues, or represent a
more general disruption of mitochondrial function. None of the effects
observed in genetically manipulated model systems has been repeated in
natural systems where changes in the levels of UCP2 and/or UCP3 occur
as a response to some environmental or physiological condition
(19-21).
We have demonstrated that expression of UCP1 in yeast mitochondria can
cause a nonspecific uncoupling that is not due to protein activity
per se (22, 23). This uncoupling artifact is present only at
higher levels of UCP1 expression. At these levels, UCP1 expression in
yeast also interferes with mitochondrial substrate oxidation.
Similarly, Heidkaemper et al. (24) have concluded that both
UCP1 and UCP3 can be expressed in an incompetent form that interferes
with ATP production. They suggest that most of the UCP3 expressed in
yeast mitochondria is nonfunctional. There is considerable evidence
that, under some expression regimes, a substantial proportion of the
UCP1 expressed in yeast mitochondria is in fact not functional (23). In
experiments with mammalian models, Cadenas et al. (18)
showed that transgenic mice overexpressing UCP3 in skeletal muscle
mitochondria (17) have lower state 3 rates of succinate oxidation.
These observations suggest that UCP expression has compromised
mitochondrial function in ways not related to uncoupling. This raises
the question of whether the observed uncoupling following UCP2 or UCP3
expression might also be an artifact of expression and not represent a
significant native activity of the protein. This is especially of
concern because the amount of UCP2/UCP3 expressed in yeast mitochondria has not been quantified.
Recently, information has become available regarding the levels of UCP2
that are found in mammalian mitochondria. Here we use this information,
and three different yeast heterologous expression systems that yield
different amounts of UCP2, to assess the effects of physiological, and
supraphysiological, levels of UCP2 expression in yeast mitochondria. We
relate the different levels of UCP2 expression to measured proton
conductance and attempt to distinguish between native UCP2 activity and
expression artifact.
Expression of UCP2 in Escherichia coli--
Human UCP2 was
expressed in E. coli, where it accumulated as inclusion
bodies that were subsequently harvested and used as a semipure source
of UCP2 with which to calibrate UCP2 expression levels in yeast
mitochondria. A PCR product for hUCP2 was made from a human mRNA
library provided by Dr. Jan Digby (Department of Clinical Biochemistry,
University of Cambridge, Cambridge, United Kingdom), and its sequence
was verified. It was ligated into XbaI and EcoRI
restriction sites of the pET expression vector pMW172 (25). Competent
C41 strain E. coli were transfected with either pET-UCP2 or
the empty pET vector. Cultures were incubated in TB media with 100 µg/ml ampicillin at 37 °C at 250 rpm until the
A600 reached 0.5-0.6. Expression of UCP2 was
induced by addition of 1 mM
isopropyl-
Cells were lysed in B-PER reagent (Pierce) for 10-15 min at room
temperature, centrifuged at 27,200 × g for 15 min and
resuspended in B-PER containing 200 µg/ml lysozyme for 5-10 min to
lyse any remaining cells. Inclusion bodies were harvested by
centrifugation at 27,200 × g for 15 min. The pellet
was washed three times by resuspension in buffer containing 150 mM potassium phosphate, 25 mM EDTA, 1 mM dithiothreitol, 1 mM ATP, pH 7.8 (13), and centrifugation at 27,200 × g. The final pellet was
solubilized in 1.5% n-lauryl sarcosine for 45 min at room
temperature. Insoluble material was removed by centrifugation at
27,200 × g for 15 min. The supernatant (solubilized
UCP2 inclusion bodies) was stored at Purity of Solubilized UCP2 Inclusion Bodies--
Solubilized
UCP2 inclusion bodies were electrophoresed on 19-cm 12%
SDS-polyacrylamide gels for 2 h at 370 V (Fig. 1a).
UCP2 content was assessed with three different stains over a range of
protein loadings. For Coomassie Brilliant Blue R250 staining, protein
loaded per lane was 2-20 µg (Fig. 1a). For staining with silver (Bio-Rad) and SYPRO Orange (Bio-Rad), 0.1-1.0 µg of protein was loaded. Gels were dried overnight and then scanned using a Scanmaker 12 USL (Microtek) scanner. Band intensities were quantified using NIH Image 1.60 (available via FTP). The UCP2 content of inclusion bodies was quantified by comparing the UCP2 signal either to
the signal obtained with bovine serum albumin (fraction V, assumed 90%
pure) or to the total protein signal obtained within the lane. The mean
estimate of the purity of the preparation used to calibrate UCP2
expression in yeast was 55 ± 7%.
Western Blots--
Mitochondrial samples and UCP2 inclusion
bodies were loaded onto a 12% SDS-polyacrylamide gel and run at 160 V
for 1 h in a Tris-glycine running buffer (28.8 g of glycine,
6 g of Tris in 1 liter) containing 0.1% SDS. Protein was
transferred to a polyvinylidene difluoride membrane, using a Bio-Rad
Trans-Blot® SD semidry electrophoretic transfer cell at 10 V for 35 min in running buffer lacking SDS and containing 20%
methanol. The membrane was blocked in a phosphate-buffered saline
solution containing 0.1% Tween 20 and 5% (w/v) MarvelTM nonfat dry
milk powder for 1 h at room temperature.
Membranes were incubated overnight at 4 °C with either of two
primary antibodies. One antibody (N-19; Santa Cruz Biotechnology) was
raised to a 19-amino acid epitope at the N terminus of human UCP2. It
was used at a 1/1000 dilution in blocking buffer). Following several
washing steps, these membranes were incubated with an alkaline
phosphatase-conjugated anti-goat secondary antibody (Sigma), diluted
1/6000 in blocking buffer, for 45 min at room temperature. A second
antibody (M-14; Calbiochem), raised to an epitope representing amino
acids 144-157 of the mouse UCP2 sequence (which is 100% conserved
between mouse and human UCP2), was also used. Membranes were incubated
with a 1/2000 dilution of this primary antibody. Following several
washing steps, they were exposed to an alkaline phosphatase-conjugated
anti-rabbit secondary antibody (New England Biolabs) diluted 1/4000 in
blocking buffer. For both antibodies, membranes were washed twice in
blocking buffer and twice in a buffer containing 10 mM
Tris-HCl, 10 mM NaCl, 1 mM MgCl2,
pH 9.5, then developed with a Phototope®-Star Western blot
detection kit (New England Biolabs) and exposed for up to 1 h to
Kodak X-Omat AR scientific imaging film. Films were scanned and
analyzed as outlined above. UCP2 contents of yeast mitochondria were
interpolated from a UCP2 inclusion body calibration series loaded on
the same gel.
Expression of UCP2 in Saccharomyces cerevisiae--
Cells of the
S. cerevisiae diploid, W303 (a/
UCP2low (pBF242) was made by ligating the UCP2 PCR product into
KpnI and BamHI restriction sites of the pYES2
vector (Invitrogen), so that a 144-base untranslated region was present
between the transcription start site and the initiation ATG.
UCP2mid (pRUCP2) was made by ligating the UCP2 PCR product into
KpnI and SacI restriction sites of the pYES2
vector, so that a Kozak (27) sequence (ACCATGG) was present at the
initiation ATG.
UCP2high (pBF346) was made by blunt-end ligation of the UCP2 PCR
product into the pKV49 vector, so that a Kozak sequence (ATAATGG) was
present at the initiation ATG.
Precultures of yeast transformed with the pYES2 constructs were grown
overnight in selective lactate (SL) media (28) (2% L-lactic acid, 0.67% yeast nitrogen base, 0.1% casamino
acids, 0.12% (NH4)2SO4, 0.1%
KH2PO4, 0.1% glucose, 20 mg/liter tryptophan, and 40 mg/liter adenine) to an A600 of ~2.0,
then transferred to a selective galactose medium (2%
D-galactose, 0.67% yeast nitrogen base, 0.1% casamino
acids, 40 mg/liter adenine, 20 mg/liter tryptophan) at 1/100 dilution
for overnight (about 16 h) growth.
Precultures of yeast transformed with pKV49 (pBF346 and pKV49-empty
vector) were grown similarly in modified SL media, with a leucine
dropout amino acid mixture (25× stock consists of, in g/liter, 0.5 adenine, 0.5 uracil, and 0.5 His, 0.75 Tyr, 0.75 Lys, 0.5 Arg, 0.5 Met,
0.75 Ile, 0.125 Phe, 0.5 Pro, 0.375 Val, 0.5 Thr, 0.875 Ser, 0.25 Glu,
0.25 Asp, 0.5 Gly, 0.5 Asn, 0.5 Ala, 0.5 Cys) in place of casamino
acids. Precultures were grown to an A600 of
~2.0 and then transferred to an identical SL medium at 1/40 dilution
for overnight growth. When cultures had reached an
A600 of 0.5-0.8, 1% D-galactose
was added to induce expression of UCP2. Cells were harvested after
4 h (or more; see "Results").
Isolation of Yeast Mitochondria--
Mitochondria were isolated
following (29) from yeast cultures with A600 of
between 1.0 and 1.5. Yeast cells were harvested by centrifugation at
2500 × g for 5 min at room temperature, resuspended in
Milli-Q grade water, recentrifuged, then resuspended in buffer containing 100 mM Tris-HCl and 20 mM
dithiothreitol, pH 9.3, and incubated for 10 min at 30 °C. The cells
were recentrifuged, washed twice in buffer containing 100 mM Tris-HCl and 500 mM KCl, pH 7.0, and
resuspended in 5 ml of isotonic spheroplasting buffer (40 mM citric acid, 120 mM disodium hydrogen
orthophosphate, 1.35 M sorbitol, 1 mM EGTA, pH
5.8). Lyticase was added at 3 mg/ml, and the cells were incubated at
30 °C for exactly 30 min. Subsequent steps were at 4 °C.
Spheroplasts were pelleted, washed twice in 40 ml of buffer containing
10 mM Tris-maleate, 0.75 M sorbitol, 0.4 M mannitol, 2 mM EGTA, 0.1% bovine serum
albumin, pH 6.8, then resuspended in 25 ml of mitochondrial isolation
buffer (30) (0.6 M mannitol, 10 mM
Tris/maleate, 0.5 mM Na2HPO4, 1%
bovine serum albumin, pH 6.8, with protease inhibitor tablets
(Complete®; Roche Molecular Biochemicals) added at 1 tablet/40 ml immediately prior to use). The spheroplasts were
homogenized by 12 passes with a Wesley Coe homogenizer. The homogenate
was centrifuged at 800 × g for 10 min. The
supernatants were removed by pipette, to prevent disruption of the
pellet, and centrifuged at 11,000 × g for 10 min.
Mitochondrial pellets were washed in buffer containing 10 mM Tris-maleate, 0.65 M mannitol, 2 mM EGTA, pH 6.8, then resuspended in a small volume of this
buffer and assayed for protein content (31).
Respiration with NADH as Substrate--
Respiration was measured
at 30 °C immediately following mitochondrial isolation. Mitochondria
were suspended at 0.15 mg of protein/ml in 2 ml of electrode buffer (10 mM Tris/maleate, 0.6 M mannitol, 0.5 mM EGTA, 2 mM MgCl2, 10 mM K2HPO4, 0.1% bovine serum
albumin, pH 6.8) containing 3 mM NADH in a Rank oxygen
electrode. Respiratory control ratios (rate with FCCP/rate without) for
control mitochondria were around 7 (Table II), comparable to published studies (1, 14, 16). Additions were made as solutions in water (NADH,
GDP), or methanol (FCCP, fatty acids).
Proton Conductance--
Complex III was inhibited with
myxothiazol, and ascorbate in the presence of the artificial electron
carrier N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was used as a well defined respiratory substrate whose oxidation
could be titrated conveniently. The oxygen electrode was fitted with a
methyltriphenylphosphonium (TPMP)-sensitive electrode to allow
simultaneous measurements of membrane potential (32) and oxygen
consumption rate (which is equal to proton leak rate divided by the
H+/O ratio of 4.0). The dependence of oxygen
consumption rate on membrane potential gives the kinetic response of
the proton leak to its driving force. The proton conductance at each
membrane potential can be read from the proton leak curves.
Mitochondria were suspended in electrode buffer at 0.5 mg/ml, 30 °C.
Oligomycin (1 µg/ml) was added to inhibit the ATP synthase, so that
all oxygen consumption was attributable to the leak pathway and not ATP
synthesis. Nigericin (100 ng/ml) was added to clamp the pH gradient
across the inner membrane. Myxothiazol (3 µM) was added
to inhibit electron transport at respiratory complex III because some
additions were in ethanol, which can be oxidized by yeast mitochondria
through an NADH-linked pathway. 2 mM ascorbate was used as
respiratory substrate. Ascorbate oxidation is through cytochrome
c oxidase and is linearly dependent on TMPD, which catalyzes
electron transport to mitochondrial cytochrome c.
The TPMP electrode was then calibrated with four additions, each of 1 µM TPMP. Ascorbate oxidation was increased sequentially by adding TMPD to cumulative concentrations of 6.25, 12.25, 25, 37.5, 50, and 75 µM. At each TMPD concentration, steady-state oxygen consumption and membrane potential were measured. Membrane potentials were calculated from TPMP concentrations outside the mitochondria, as described in Ref. 32, assuming a TPMP binding correction of 0.4 (µl/mg) Statistics--
Means were compared using Student's
t test.
Chemicals--
Chemicals were purchased from Sigma, unless
otherwise stated.
Expression Levels of UCP2 in Yeast Mitochondria--
The levels of
UCP2 expression in mitochondria isolated from transfected yeast were
determined by Western blot using solubilized UCP2 inclusion bodies as
calibration standards (Fig. 1). The two antibodies (C-14 and N-19) gave virtually identical results. UCP2 was
expressed in yeast mitochondria at three levels that ranged from 33 ng/mg of mitochondrial protein in UCP2low yeast to 4 µg/mg in
UCP2high yeast (Table I).
UCP2low Yeast--
Induction of UCP2 expression in UCP2low yeast
had no effect on yeast growth rates. The mean doubling time of UCP2low
yeast in selective galactose medium was 1.83 ± 0.07 h,
compared with 1.81 ± 0.03 h (S.E., n = 6) in
paired controls grown under identical conditions.
Respiration rates with NADH as substrate, both coupled and uncoupled
with FCCP, were not different between UCP2low mitochondria and their
paired controls (Table II). Palmitate (50 µM) stimulated respiration equally in UCP2low and control
mitochondria (data not shown).
The proton leak kinetics were determined in mitochondria isolated from
UCP2low yeast and paired controls (Fig.
2a). Over the range of
membrane potentials (driving force for proton leak), no differences in
proton conductance were observed.
UCP2mid Yeast--
Induction of UCP2 expression in UCP2mid yeast
had no effect on growth. The doubling time of UCP2mid yeast growing in
exponential phase following induction was 1.75 ± 0.06 h,
compared with 1.77 ± 0.06 (S.E., n = 6) for
paired controls.
Respiration rates with NADH as substrate, both coupled and uncoupled
with FCCP, were not different in mitochondria isolated from UCP2mid
yeast and paired controls (Table II). Palmitate (50 µM)
and all-trans-retinoic acid (45 µM, buffer, pH
7.3) stimulated respiration equally in UCP2mid and control mitochondria
(Fig. 3, a and
b).
The proton leak kinetics of UCP2mid mitochondria were similar to
control mitochondria (Fig. 2b). UCP2mid mitochondria had identical, or perhaps slightly lower, proton conductance than controls
at all measured values of membrane potential.
UCP2high Yeast--
Induction of UCP2 expression in UCP2high yeast
significantly inhibited growth rate in the exponential phase. The
doubling time of UCP2high yeast in exponential phase following
induction with 1% D-galactose was 4.1 ± 0.1 h,
compared with 2.6 ± 0.1 h (S.E., n = 7) for
paired controls.
Mitochondria isolated from UCP2high yeast had significantly higher
rates of respiration with NADH as substrate, slightly (not significantly) lowered FCCP uncoupled rates and significantly lowered
respiratory control (Table II and Fig. 3c). GDP did not inhibit respiration with NADH as substrate in UCP2high mitochondria or
their paired controls (Fig. 3c). 3 mM GDP
slightly stimulated respiration in UCP2high mitochondria (Fig.
3c), perhaps through the nucleotide inducible proton
conductance pathway (33). Our assay conditions were designed to
minimize the proton leak through this pathway (33), but may not have
abolished it entirely.
UCP2high yeast mitochondria had altered proton leak kinetics (Fig.
2c). At all measured membrane potentials, proton conductance was greater in UCP2high mitochondria. Membrane potentials and oxygen
consumption rates can be compared for each concentration of TMPD;
UCP2high mitochondria achieved a lower membrane potential, but did not
respire faster than control mitochondria. Increased proton conductance
normally lowers membrane potential and stimulates respiration (34).
Thus, substrate (ascorbate/TMPD) oxidation was impaired in UCP2high
mitochondria. Indeed, fully FCCP-uncoupled rates of NADH oxidation in
UCP2high mitochondria became progressively impaired as the time between
UCP2 induction and mitochondrial isolation increased (Fig.
4). This was also apparent when the oxidized substrate was ascorbate/TMPD (data not shown).
UCP2 Expression Levels in Mammalian Mitochondria--
Antibodies
to UCP2 are typically able to detect the presence of the protein
expressed in yeast mitochondria, but the same antibodies often fail to
detect UCP2 in mitochondria from mammalian tissues. Recently, this has
been shown to be due to two limitations: low levels of UCP2 in
mitochondria from most mammalian tissues, and cross-reactivity of the
commercially available UCP2 antibodies with other proteins with
apparent molecular masses of about 32 kDa.
Nonetheless, UCP2 levels in mammalian mitochondria have recently been
quantified, using an antibody whose specificity has been verified using
mitochondria from the tissues of wild-type and UCP2 knockout mice (35).
The highest levels of UCP2 are apparently found in spleen mitochondria,
which contain 160-fold less UCP2 than there is UCP1 in mouse brown
adipose mitochondria. If mouse brown adipose tissue mitochondria
contain ~50 µg of UCP1/mg of mitochondrial protein, then spleen
mitochondria contain 50/160, or 313 ng, of UCP2/mg of protein. UCP2
levels are reported as 4 times lower in lung (78 ng/mg), 10 times
lower in stomach (31 ng/mg), and undetectable in heart, muscle,
liver, brain, and brown adipose tissue mitochondria.
These findings of low levels of UCP2 are consistent with a number of
other observations. Most other members of the mitochondrial transporter
protein superfamily are also present in very low amounts (with the
notable exceptions of the adenine nucleotide transporter, the phosphate
carrier, and, in brown fat mitochondria, UCP1) (36); purification of
native UCP2 from mammalian sources has not been reported, and no UCP2
band is visible on SDS gels of mammalian mitochondria.
Effect of Physiological Concentrations of UCP2 on Proton Leak in
Yeast Mitochondria--
When UCP2 was expressed in yeast at 33 ng of
UCP2/mg of mitochondrial protein, similar to levels in stomach and
greater than the levels present in most other mouse tissues, the proton
leak kinetics of mitochondria isolated from UCP2low and paired controls were not different, indicating no change in proton conductance. Similarly, rates of respiration with and without the uncoupler FCCP
were not different between UCP2low and paired control mitochondria. Thus, this amount of UCP2 expressed in yeast mitochondria did not
uncouple respiration.
We increased UCP2 expression in yeast mitochondria by an order of
magnitude (UCP2mid) by removing a 5'-untranslated nucleotide region
that was present in the UCP2low plasmid, and inserting a Kozak sequence
(27) around the initiation ATG (see "Experimental Procedures").
UCP2 expression in mitochondria isolated from these yeast was 283 ng/mg
of protein, similar to levels reported for spleen mitochondria (35,
37). However, no increase in proton conductance was observed in
mitochondria isolated from UCP2mid yeast. Rates of NADH oxidation in
the coupled, and FCCP-uncoupled, states were unaffected by this level
of UCP2 expression. Two putative effectors of UCP2 activity, palmitate
(13) and all-trans-retinoic acid (at pH 7.3) (16), failed to
stimulate UCP2 activity. Thus, when expressed at 283 ng/mg of protein,
approximately equal to the highest level measured in mammalian
mitochondria, UCP2 did not uncouple yeast mitochondria.
Is Our Assay Sensitive Enough to Detect Uncoupling by Physiological
Concentrations of Active UCP2?--
When UCP1 was expressed in yeast
at levels (900 ng/mg of protein) similar to UCP2mid (283 ng/mg),
specific palmitate-activated and GDP-inhibitable mitochondrial
uncoupling was readily measurable using our experimental methods (22,
23). If UCP2 had similar uncoupling activity, then it would have been
readily observed under our experimental conditions. As it was not seen,
we conclude that the native uncoupling activity of UCP2 expressed in
yeast mitochondria is less than that of UCP1 (or indeed zero). The
specific activity of UCP1 in yeast mitochondria is comparable to its
native activity in brown adipose tissue mitochondria, indicating good insertion and folding of the protein to give a native, functional molecule (22, 23). There is no reason to suppose that, when expressed
at similar levels, UCP2 is not also folded correctly in the yeast system.
Effect of Supraphysiological Concentrations of UCP2 on Proton Leak
in Yeast Mitochondria--
UCP2 caused increased proton conductance in
yeast only when expressed at about 4 µg/mg of mitochondrial protein.
This level is more than an order of magnitude higher than in UCP2mid
yeast and mouse spleen mitochondria. Mitochondria isolated from
UCP2high yeast were partially uncoupled. This uncoupling was apparent
in the proton leak kinetics and the rates of coupled respiration of
mitochondria isolated from UCP2high yeast compared with paired controls. Growth rates of UCP2high yeast were also impaired.
From Fig. 2c, the increased respiration due to UCP2 in
UCP2high yeast mitochondria was 300 nmol of O/min/mg of protein at 160 mV. Thus the proton cycling rate caused by UCP2 expression was about
1.2 µmol of H+/min/mg of yeast mitochondrial protein at
160 mV. As UCP2high yeast mitochondria have 4 µg of UCP2/mg of
protein, the specific activity of UCP2 in these yeast mitochondria can
be calculated as 1.2/4, or 0.3 µmol of H+/min/µg of
UCP2, at 160 mV. The proton cycling due to UCP2 activity (assayed under
identical conditions) in UCP2mid yeast mitochondria with 300 ng of
UCP2/mg of mitochondrial protein should be 90 nmol of
H+/min/mg of mitochondrial protein. Given a
H+/O ratio for TMPD respiration of 4, the additional
respiration attributable to UCP2 activity in these mitochondria should
be 22.5 nmol of O/min/mg of protein. However, no increase in
respiration was observed in UCP2mid mitochondria, suggesting that the
uncoupling seen in UCP2high mitochondria was probably not due to UCP2
activity per se, but rather to an artifactual effect of high
protein expression.
When UCP1 was expressed in yeast mitochondria at levels (~11 µg/mg
of protein) similar to those of UCP2 in UCPhigh, an artifactual proton
conductance that was insensitive to GDP was observed (22, 23). This
uncoupling is not attributable to native UCP1 function, which is fully
inhibitable by GDP. The artifactual GDP-insensitive proton conductance
caused by 11 µg/mg UCP1 was nearly twice the proton conductance
caused by 4 µg/mg UCP2 (Fig. 5), as
shown by the roughly doubled respiration rate at the same membrane
potential of 145 mV. Thus the uncoupling caused by high expression of
UCP2 was quantitatively almost the same as the artifactual uncoupling caused by the same quantity of UCP1, strongly suggesting that the
uncoupling caused by UCP2 was also an artifact of high expression.
In UCP2high mitochondria, a secondary effect of UCP2 expression was
observed that could not be attributed simply to an uncoupling activity;
mitochondrial substrate oxidation was inhibited. This can be seen in
the proton leak kinetics (Fig. 2c); increased proton conductance should increase oxygen consumption at any concentration of
substrate (34). UCP2high mitochondria were uncoupled, but at each TMPD
concentration they did not respire faster. It can also be seen in fully
FCCP uncoupled rates of NADH oxidation (Fig. 4), which decreased as
UCP2 expression was increased by longer periods of galactose induction.
A similar effect occurs at high levels of UCP1 (22, 23) (Fig. 5).
Inhibition of substrate oxidation similarly accompanies UCP2 or
UCP3-mediated uncoupling in a number of studies where the UCP1
homologues have been overexpressed (15, 16, 18, 24). There is no reason
related to a simple uncoupling activity for this to occur, and it
suggests that mitochondrial integrity was compromised at this level of
UCP2 expression.
The uncoupling observed in UCP2high yeast mitochondria was insensitive
to GDP. UCP1 is fully inhibited by millimolar concentrations of
nucleotides (38), and it has been reported that homologues of UCP1
share this property (13, 24, 39, 40). The inability of GDP to inhibit
proton conductance in UCP2high yeast mitochondria is in agreement with
evidence from mammalian mitochondria. Even high concentrations of
nucleotides do not lower the proton conductance of rat muscle
mitochondria in which UCP2 and UCP3 are present (41, 42). In brown
adipose tissue from UCP1 knockout mice, UCP2 and UCP3 mRNA rise but
basal proton leak remains insensitive to nucleotides (43, 44).
If the Uncoupling Measured in UCP2high Mitochondria Was a Native
Function of UCP2, Could It Make a Major Contribution to Proton Cycling
in Mammalian Mitochondria?--
Levels of UCP2 have been reported for
spleen, lung, and stomach mitochondria. As calculated above, these are
313, 78, and 31 ng of UCP2/mg of protein, respectively. The rates of
proton cycling that would be catalyzed by these amounts of UCP2 are 94, 23, and 9 nmol of H+/min/mg of protein. UCP2 protein is
undetectable in heart, muscle, liver, brain, and brown adipose tissue
mitochondria (35). UCP2 levels in these tissues are, therefore, lower
than the lowest measurable amounts of 30 ng/mg, or indeed perhaps zero.
Based on the uncoupling seen in mitochondria from UCP2high yeast,
proton cycling due to UCP2 would, in mitochondria from these tissues, be less than 9 nmol of H+/min/mg of protein. In comparison,
the proton cycling rates measured in mitochondria from rat liver,
brain, and muscle (corrected to 30 °C assuming a Q10 of
2) are about 21, 84, and 240 nmol of H+/min/mg of protein
(45). Thus, UCP2-catalyzed proton cycling might constitute less than
4% of the total rate observed in muscle, less than 11% of the rate in
brain, and less than 43% of the rate in liver (where in fact UCP2
expression is believed to be restricted to Kupffer cells) mitochondria.
In all cases, these are, of course, upper limits to the UCP2
contribution to proton cycling.
Only in mitochondria from spleen could the observed activity of UCP2
from mitochondria of UCP2high yeast play a significant role in proton
conductance, even assuming that this activity is not artifactual.
However, under standard conditions of proton leak assay, there is no
evidence that spleen mitochondria have greater proton conductance than
kidney, liver, or muscle mitochondria.2
Similar calculations can be made for the proton leak rate caused by
UCP2 in proteoliposomes (13), with a reported
Vmax of 10-30 µmol of H+/min/mg
of UCP2, or about 20 nmol of H+/min/µg of UCP2. From the
data reported in Ref. 13, the proteoliposome membrane potential can be
estimated to be about 150 mV during the measurements. If the
UCP2-dependent proton leak seen in proteoliposomes was a
native function of the protein, it would account for only 20 nmol of
H+/min/mg of protein × 0.3 µg of UCP2/mg of
protein, or about 6 nmol of H+/min/mg of protein, of proton
cycling in spleen mitochondria. This is comparable to the leak through
the phospholipid bilayer (46) and not great enough to be the major
contributor to proton cycling in mammalian mitochondria.
From both sets of calculations, it is obvious that, even if the proton
cycling observed in mitochondria from UCP2high yeast is a true
activity, it is not great enough to account for the basal
proton leak observed in mitochondria. It may, or may not, catalyze an
inducible, regulated, leak, as does UCP1.
In summary, UCP2 uncouples yeast mitochondria only at
supraphysiological levels of the protein, when symptoms of
mitochondrial damage appear. When expressed in yeast mitochondria at
levels similar to those found in mitochondria from mouse tissues, UCP2 does not uncouple. The increased proton conductance caused by high
levels of UCP2 expression is GDP-insensitive and insufficient to
explain the basal proton conductance of mammalian mitochondria, although UCP2 could catalyze an inducible leak pathway. The uncoupling caused by UCP2 expression in yeast probably represents compromised mitochondrial function, and not a native UCP2 activity. This may apply
also to other UCP1 homologues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside. After 2 h cells
were harvested by centrifugation at 3000 × g for 15 min. All centrifugation steps were carried out at 4 °C. Cell pellets
were stored at
85 °C.
85 °C.
), were transformed (26)
with one of three different UCP2 expression constructs.
1. A different
TPMP binding correction would affect all the measured values of
membrane potential but would not significantly affect our conclusions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (55K):
[in a new window]
Fig. 1.
Identification and quantification of
UCP2. a, identification and quantification of UCP2 in
E. coli inclusion bodies. SDS-polyacrylamide gel stained
with Coomassie Blue. Lane 1, protein molecular
size markers; lane 2, 20 µg of E. coli with pET-empty vector induced for 2 h; lane
3, 20 µg of E. coli with pET-UCP2 induced for
2 h; lanes 4-8, UCP2 inclusion bodies
isolated from 2-h induced pET-UCP2 E. coli, loaded at 20, 15, 10, 5, and 2 µg of protein, respectively. b,
quantification of UCP2 expression in mitochondria isolated from
UCP2high and UCP2mid expression yeast using N-19 antibody.
Lanes 1-4, UCP2 inclusion body preparation
loaded at 7, 13, 70, and 130 ng, respectively; lane
5, mitochondria from yeast transfected with pkV49-empty
vector; lanes 6-8, mitochondria from different
UCP2high expression yeast cultures, loaded at 15 µg; lanes
9-11, mitochondria from different UCP2mid expression yeast
cultures loaded at 30 µg of protein.
UCP2 expression levels in mitochondria isolated from yeast containing
UCP2 expression constructs
Respiration with NADH as substrate in mitochondria isolated from yeast
containing UCP2 expression constructs
View larger version (18K):
[in a new window]
Fig. 2.
Proton leak kinetics of mitochondria isolated
from the three yeast UCP2 expression constructs. a,
UCP2low and paired control; b, UCP2mid and paired control;
c, UCP2high and paired control. For details, see
"Experimental Procedures." Values are means ± S.E. of three
to five separate experiments with two to three yeast
transformants.
View larger version (16K):
[in a new window]
Fig. 3.
Effects of putative effectors of UCP2
activity on mitochondrial respiration with NADH as substrate.
a, effect of palmitate (50 µM) on respiration
rates of mitochondria isolated from UCP2mid (filled
bars) and paired control yeast (open
bars). Bars represent means ± S.E. of three
separate experiments. b, effect of 45 µM
all-trans-retinoic acid (RA) on respiration rates
of mitochondria isolated from UCP2mid (filled
bars) and paired control yeast (open
bars). Bars represent means ± S.E. of two
experiments. c, effect of GDP on respiration rates of
mitochondria isolated from UCP2high (filled bars)
and control yeast (open bars). *, significantly
different from paired empty vector control (p < 0.01).
+, significantly different from UCP2high mitochondria with NADH
(without GDP) (p < 0.01). Bars represent
means ± S.E. of four separate experiments. Effectors and
uncoupler were added sequentially in each set of experiments.
View larger version (15K):
[in a new window]
Fig. 4.
Effect of induction time on uncoupled
respiration in UCP2high mitochondria. Respiration with NADH as
substrate was measured in the presence of FCCP in mitochondria isolated
from UCP2high yeast at different times after induction of UCP2
expression. Open circles, pKV49-empty vector
control; filled squares, UCP2high mitochondria.
Data at 4 h are from Table II. Data points at 7 and 15 h are
means of two or three separate measurements from one mitochondrial
preparation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 5.
Comparison of the proton leak kinetics of
mitochondria from UCP2high yeast with the GDP-insensitive proton leak
kinetics of mitochondria from yeast expressing similar levels of
UCP1. Data for UCP2 are from Fig. 2c; data for UCP1 are
from Ref. 23.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Mike Runswick at the Medical Research Council Dunn Human Nutrition Unit (Cambridge, United Kingdom) for advice and assistance in the synthesis of some of the UCP2 constructs used in this study.
![]() |
FOOTNOTES |
---|
* This research was supported by The Wellcome Trust, BBSRC/CASE, Knoll Pharmaceuticals and the Medical Research Council, UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 44-1223-252800; Fax: 44-1223-252805; E-mail: martin.brand@mrc-dunn.cam.ac.uk.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M011566200
2 K. Echtay, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: UCP1, uncoupling protein 1; UCP2, uncoupling protein 2; UCP3, uncoupling protein 3; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; PCR, polymerase chain reaction; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; TPMP, methyltriphenylphosphonium; SL, selective lactate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Fleury, C., Neverova, M., Collins, S., Raimbault, S., Champigny, O., Levi-Meyrueis, C., Bouillaud, F., Seldin, M. F., Surwit, R. S., Ricquier, D., and Warden, C. H. (1997) Nat. Genet. 15, 269-272[Medline] [Order article via Infotrieve] |
2. | Gimeno, R. E., Dembski, M., Weng, X., Deng, N., Shyjan, A. W., Gimeno, C. J., Iris, F., Ellis, S. J., Woolf, E. A., and Tartaglia, L. A. (1997) Diabetes 46, 900-906[Abstract] |
3. | Boss, O., Samec, S., Paoloni-Giacobino, A., Rossier, C., Dulloo, A., Seydoux, J., Muzzin, P., and Giacobino, J. P. (1997) FEBS Lett. 408, 39-42[CrossRef][Medline] [Order article via Infotrieve] |
4. | Vidal-Puig, A., Solanes, G., Grujic, D., Flier, J. S., and Lowell, B. B. (1997) Biochem. Biophys. Res. Commun. 235, 79-82[CrossRef][Medline] [Order article via Infotrieve] |
5. | Klingenberg, M., and Echtay, K. S. (2001) Biochim. Biophys. Acta 1504, 128-143[Medline] [Order article via Infotrieve] |
6. | Kozak, L. P., and Harper, M.-E. (2000) Annu. Rev. Nutr. 20, 339-363[CrossRef][Medline] [Order article via Infotrieve] |
7. | Jezek, P., and Garlid, K. D. (1998) Int. J. Biochem. Cell Biol. 30, 1163-1168[CrossRef][Medline] [Order article via Infotrieve] |
8. | Lowell, B. B., and Spiegelman, B. M. (2000) Nature 404, 652-660[Medline] [Order article via Infotrieve] |
9. | Nedergaard, J., Matthias, A., Golozoubova, V., Jacobsson, A., and Cannon, B. (1999) J. Bioenerg. Biomembr. 31, 475-491[CrossRef][Medline] [Order article via Infotrieve] |
10. | Ricquier, D., and Bouillaud, F. (2000) Biochem. J. 345, 161-179[CrossRef][Medline] [Order article via Infotrieve] |
11. | Brand, M. D., Brindle, K. M., Buckingham, J. A., Harper, J. A., Rolfe, D. F. S., and Stuart, J. A. (1999) Int. J. Obes. 23 Suppl. 6, S4-S11[Medline] [Order article via Infotrieve] |
12. | Stuart, J. A., Cadenas, S., Jekabsons, M. B., Roussel, D., and Brand, M. D. (2001) Biochim. Biophys. Acta 1504, 144-158[Medline] [Order article via Infotrieve] |
13. |
Jaburek, M.,
Varecha, M.,
Gimeno, R. E.,
Dembski, M.,
Jezek, P.,
Zhang, M.,
Burn, P.,
Tartaglia, L. A.,
and Garlid, K. D.
(1999)
J. Biol. Chem.
274,
26003-26007 |
14. | Hinz, W., Gruninger, S., De Pover, A., and Chiesi, M. (1999) FEBS Lett. 462, 411-415[CrossRef][Medline] [Order article via Infotrieve] |
15. | Zhang, C. Y., Hagen, T., Mootha, V. K., Slieker, L. J., and Lowell, B. B. (1999) FEBS Lett. 449, 129-134[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Rial, E.,
Gonzalez-Barroso, M.,
Fleury, C.,
Iturrizaga, S.,
Sanchis, D.,
Jimenez-Jimenez, J.,
Ricquier, D.,
Goubern, M.,
and Bouillaud, F.
(1999)
EMBO J.
18,
5827-5833 |
17. | Clapham, J. C., Arch, J. R., Chapman, H., Haynes, A., Lister, C., Moore, G. B., Piercy, V., Carter, S. A., Lehner, I., Smith, S. A., Beeley, L. J., Godden, R. J., Herrity, N., Skehel, M., Changani, K. K., Hockings, P. D., Reid, D. G., Squires, S. M., Hatcher, J., Trail, B., Latcham, J., Rastan, S., Harper, A. J., Cadenas, S., Buckingham, J. A., Brand, M. D., and Abuin, A. (2000) Nature 406, 415-418[CrossRef][Medline] [Order article via Infotrieve] |
18. | Cadenas, S., Buckingham, J. A., Clapham, J. C., and Brand, M. D. (2000) Int. J. Obes. 24, S187 |
19. | Cadenas, S., Buckingham, J. A., Samec, S., Seydoux, J., Din, N., Dulloo, A. G., and Brand, M. D. (1999) FEBS Lett. 462, 257-260[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Yu, X. X.,
Barger, J. L.,
Boyer, B. B.,
Brand, M. D.,
Pan, G.,
and Adams, S. H.
(2000)
Am. J. Physiol.
279,
E433-E446 |
21. |
Jekabsons, M. B.,
Gregoire, F. M.,
Schonfeld-Warden, N. A.,
Warden, C. H.,
and Horwitz, B. A.
(1999)
Am. J. Physiol.
277,
E380-E389 |
22. | Stuart, J. A., Harper, J. A., Brindle, K. M., and Brand, M. D. (2000) Int. J. Obes. 24, S187 |
23. | Stuart, J. A., Harper, J. A., Brindle, K. M., Jekabsons, M. B., and Brand, M. D. (2001) Biochem. J., in press |
24. | Heidkaemper, D., Winkler, E., Muller, V., Frischmuth, K., Liu, Q., Caskey, T., and Klingenberg, M. (2000) FEBS Lett. 406, 1-6[CrossRef] |
25. | Way, M., Pope, B., Gooch, J., Hawkins, M., and Weeds, A. G. (1990) EMBO J. 9, 4103-4109[Abstract] |
26. | Hinnen, A., Hicks, J. B., and Fink, G. R. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1929-1933[Abstract] |
27. | Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148[Abstract] |
28. | Bouillaud, F., Arechaga, I., Petit, P. X., Raimbault, S., Levi-Meyrueis, C., Casteilla, L., Laurent, M., Rial, E., and Ricquier, D. (1994) EMBO J. 13, 1990-1997[Abstract] |
29. | Guerin, B., Labbe, P., and Somlo, M. (1979) Methods Enzymol. 55, 149-159[Medline] [Order article via Infotrieve] |
30. | Arechaga, I., Raimbault, S., Prieto, S., Levi-Meyrueis, C., Zaragoza, P., Miroux, B., Ricquier, D., Bouillaud, F., and Rial, E. (1993) Biochem. J. 296, 693-700[Medline] [Order article via Infotrieve] |
31. |
Gornall, A. G.,
Bardawill, C. J.,
and David, M. M.
(1949)
J. Biol. Chem.
177,
751-766 |
32. | Brand, M. D. (1995) in Bioenergetics: A Practical Approach (Brown, G. C. , and Cooper, C. E., eds) , pp. 39-62, Oxford University Press, New York |
33. | Prieto, S., Bouillaud, F., and Rial, E. (1996) Arch. Biochem. Biophys. 334, 43-49[CrossRef][Medline] [Order article via Infotrieve] |
34. | Brand, M. D. (1990) Biochim. Biophys. Acta 1018, 128-133[Medline] [Order article via Infotrieve] |
35. |
Pecqueur, C.,
Alves-Guerra, M.-C.,
Gelly, C.,
Levi-Meyrueis, C.,
Couplan, E.,
Collins, S.,
Ricquier, D.,
Bouillaud, F.,
and Miroux, B.
(2001)
J. Biol. Chem.
276,
8705-8712 |
36. | Palmieri, F. (1994) FEBS Lett. 346, 48-54[CrossRef][Medline] [Order article via Infotrieve] |
37. | Arsenijevic, D., Onuma, H., Pecqueur, C., Raimbault, S., Manning, B. S., Miroux, B., Couplan, E., Alves-Guerra, M.-C., Goubern, M., Surwit, R., Bouillaud, F., Richard, D., Collins, S., and Ricquier, D. (2000) Nat. Genet. 26, 435-439[CrossRef][Medline] [Order article via Infotrieve] |
38. | Nicholls, D. G. (1979) Biochim. Biophys. Acta 549, 1-29[Medline] [Order article via Infotrieve] |
39. | Echtay, K. S., Liu, Q., Caskey, T., Winkler, E., Frischmuth, K., Bienengraber, M., and Klingenberg, M. (1999) FEBS Lett. 450, 8-12[CrossRef][Medline] [Order article via Infotrieve] |
40. | Hagen, T., Zhang, C. Y., Vianna, C. R., and Lowell, B. B. (2000) Biochemistry 39, 5845-5851[CrossRef][Medline] [Order article via Infotrieve] |
41. | Cadenas, S., and Brand, M. D. (2000) Biochem. J. 348, 209-213[CrossRef][Medline] [Order article via Infotrieve] |
42. | Jekabsons, M. B., and Horwitz, B. A. (2001) Biochim. Biophys. Acta 1503, 314-328[Medline] [Order article via Infotrieve] |
43. |
Matthias, A.,
Jacobsson, A.,
Cannon, B.,
and Nedergaard, J.
(1999)
J. Biol. Chem.
274,
28150-28160 |
44. |
Monemdjou, S.,
Kozak, L. P.,
and Harper, M. E.
(1999)
Am. J. Physiol.
276,
E1073-E1082 |
45. | Rolfe, D. F., Hulbert, A. J., and Brand, M. D. (1994) Biochim. Biophys. Acta 1188, 405-416[Medline] [Order article via Infotrieve] |
46. | Brookes, P. S., Hulbert, A. J., and Brand, M. D. (1997) Biochim. Biophys. Acta 1330, 157-164[Medline] [Order article via Infotrieve] |