THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 21, Issue of May 25, pp. 18633–18639, 2001
Printed in U.S.A.
Physiological Levels of Mammalian Uncoupling Protein 2
Do Not Uncouple Yeast Mitochondria*
Received for publication, December 21, 2000, and in revised form, February 20, 2001
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M011566200
Jeff A. Stuart‡§, James A. Harper‡§, Kevin M. Brindle§, Mika B. Jekabsons‡, and
Martin D. Brand‡¶
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
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 ␮g/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 ␮g/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 repre* 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. This 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: [email protected].
1
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.
This paper is available on line at http://www.jbc.org
sent 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.
EXPERIMENTAL PROCEDURES
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 ␮g/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-␤-D-thiogalactopyranoside. After 2 h cells
were harvested by centrifugation at 3000 ⫻ g for 15 min. All centrifu-
18633
18634
UCP2 Uncoupling of Yeast Mitochondria
gation steps were carried out at 4 °C. Cell pellets were stored at ⫺85 °C.
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 ␮g/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 ⫺85 °C.
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 ␮g (Fig. 1a).
For staining with silver (Bio-Rad) and SYPRO Orange (Bio-Rad), 0.1–
1.0 ␮g 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)
Marvel娂 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/␣), were transformed (26) with one of three
different UCP2 expression constructs.
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 ␮g/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 ␮M) 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 ␮M TPMP. Ascorbate oxidation was increased sequentially by adding
TMPD to cumulative concentrations of 6.25, 12.25, 25, 37.5, 50, and 75
␮M. 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 (␮l/mg)⫺1. A
different TPMP binding correction would affect all the measured values
of membrane potential but would not significantly affect our
conclusions.
Statistics—Means were compared using Student’s t test.
UCP2 Uncoupling of Yeast Mitochondria
18635
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 ␮g of E.
coli with pET-empty vector induced for
2 h; lane 3, 20 ␮g of E. coli with pETUCP2 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 ␮g 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 ␮g; lanes 9 –11, mitochondria from different UCP2mid
expression yeast cultures loaded at 30 ␮g
of protein.
Chemicals—Chemicals were purchased from Sigma, unless otherwise stated.
RESULTS
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 ␮g/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
␮M) 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 ␮M) and all-trans-retinoic acid (45 ␮M, 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 conduct-
ance 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).
DISCUSSION
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
18636
UCP2 Uncoupling of Yeast Mitochondria
TABLE I
UCP2 expression levels in mitochondria isolated from yeast containing
UCP2 expression constructs
UCP2 concentrations were determined using N-19 antibody calibrated with inclusion body UCP2 (see “Experimental Procedures”).
Values represent means ⫾ S.E. from three to five separate experiments
with two or three transformants for each construct.
Yeast expression
construct
Induction regime
UCP2 expression
level
(ng UCP2/mg
mitochondrial
protein)
UCP2low
Overnight 2%
D-galactose
UCP2mid
Overnight 2%
D-galactose
UCP2high
32.5 ⫾ 3.3
283 ⫾ 22
4066 ⫾ 1274
4-h 1% Dgalactose
TABLE II
Respiration with NADH as substrate in mitochondria isolated from
yeast containing UCP2 expression constructs
Values are in nmol of O/min/mg of mitochondrial protein, and represent means ⫾ S.E. of five to seven separate experiments with two or
three different transformants for each construct. Respiratory control
ratio (RCR) ⫽ (NADH ⫹ FCCP rate)/NADH rate.
UCP2low
Paired control
UCP2mid
Paired control
UCP2high
Paired control
NADH
NADH ⫹ FCCP
RCR
165 ⫾ 23
161 ⫾ 24
290 ⫾ 64
284 ⫾ 39
501 ⫾ 54*
260 ⫾ 34
1311 ⫾ 245
1256 ⫾ 202
1849 ⫾ 317
1878 ⫾ 151
1351 ⫾ 81
1522 ⫾ 157
7.9 ⫾ 0.8
7.8 ⫾ 0.4
6.9 ⫾ 0.6
6.8 ⫾ 0.4
2.8 ⫾ 0.1*
6.0 ⫾ 0.4
* Significantly different from paired control (p ⬍ 0.01).
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 wildtype 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 ␮g 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.
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.
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
UCP2 Uncoupling of Yeast Mitochondria
18637
FIG. 3. Effects of putative effectors of UCP2 activity on mitochondrial respiration with NADH as substrate. a, effect of palmitate (50
␮M) 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 ␮M 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.
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.
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.
UCP2mid (283 ng/mg), specific palmitate-activated and GDPinhibitable 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 ␮g/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 ␮mol of H⫹/min/mg of yeast
mitochondrial protein at 160 mV. As UCP2high yeast mitochondria have 4 ␮g of UCP2/mg of protein, the specific activity
of UCP2 in these yeast mitochondria can be calculated as 1.2/4,
or 0.3 ␮mol of H⫹/min/␮g 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
18638
UCP2 Uncoupling of Yeast Mitochondria
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 ␮g/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
␮g/mg UCP1 was nearly twice the proton conductance caused
by 4 ␮g/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-cata-
lyzed 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 ␮mol of H⫹/min/mg of UCP2, or about 20 nmol of
H⫹/min/␮g 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 ␮g 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 GDPinsensitive 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.
Acknowledgment—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.
REFERENCES
1. Fleury, C., Neverova, M., Collins, S., Raimbault, S., Champigny, O., LeviMeyrueis, C., Bouillaud, F., Seldin, M. F., Surwit, R. S., Ricquier, D., and
Warden, C. H. (1997) Nat. Genet. 15, 269 –272
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
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
4. Vidal-Puig, A., Solanes, G., Grujic, D., Flier, J. S., and Lowell, B. B. (1997)
Biochem. Biophys. Res. Commun. 235, 79 – 82
5. Klingenberg, M., and Echtay, K. S. (2001) Biochim. Biophys. Acta, 1504,
128 –143
6. Kozak, L. P., and Harper, M.-E. (2000) Annu. Rev. Nutr. 20, 339 –363
7. Jezek, P., and Garlid, K. D. (1998) Int. J. Biochem. Cell Biol. 30, 1163–1168
8. Lowell, B. B., and Spiegelman, B. M. (2000) Nature 404, 652– 660
9. Nedergaard, J., Matthias, A., Golozoubova, V., Jacobsson, A., and Cannon, B.
(1999) J. Bioenerg. Biomembr. 31, 475– 491
10. Ricquier, D., and Bouillaud, F. (2000) Biochem. J. 345, 161–179
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
12. Stuart, J. A., Cadenas, S., Jekabsons, M. B., Roussel, D., and Brand, M. D.
(2001) Biochim. Biophys. Acta, 1504, 144 –158
2
K. Echtay, personal communication.
UCP2 Uncoupling of Yeast Mitochondria
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
15. Zhang, C. Y., Hagen, T., Mootha, V. K., Slieker, L. J., and Lowell, B. B. (1999)
FEBS Lett. 449, 129 –134
16. Rial, E., Gonzalez-Barroso, M., Fleury, C., Iturrizaga, S., Sanchis, D., JimenezJimenez, 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
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
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
25. Way, M., Pope, B., Gooch, J., Hawkins, M., and Weeds, A. G. (1990) EMBO J.
9, 4103– 4109
26. Hinnen, A., Hicks, J. B., and Fink, G. R. (1978) Proc. Natl. Acad. Sci. U. S. A.
75, 1929 –1933
27. Kozak, M. (1987) Nucleic Acids Res. 15, 8125– 8148
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,
18639
1990 –1997
29. Guerin, B., Labbe, P., and Somlo, M. (1979) Methods Enzymol. 55, 149 –159
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
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
34. Brand, M. D. (1990) Biochim. Biophys. Acta 1018, 128 –133
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
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
38. Nicholls, D. G. (1979) Biochim. Biophys. Acta 549, 1–29
39. Echtay, K. S., Liu, Q., Caskey, T., Winkler, E., Frischmuth, K., Bienengraber,
M., and Klingenberg, M. (1999) FEBS Lett. 450, 8 –12
40. Hagen, T., Zhang, C. Y., Vianna, C. R., and Lowell, B. B. (2000) Biochemistry
39, 5845–5851
41. Cadenas, S., and Brand, M. D. (2000) Biochem. J. 348, 209 –213
42. Jekabsons, M. B., and Horwitz, B. A. (2001) Biochim. Biophys. Acta 1503,
314 –328
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
46. Brookes, P. S., Hulbert, A. J., and Brand, M. D. (1997) Biochim. Biophys. Acta
1330, 157–164