Life, 52: 175–179, 2001
c 2001 IUBMB
Copyright °
1521-6543/01 $12.00 + .00
IUBMB
Critical Review
Uncoupling Proteins—How Do They Work
and How Are They Regulated
Martin Klingenberg
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, D-80336, Munich, Germany
Summary
Uncoupling proteins (UCPs) are regulated H+ transporters and
a subfamily of the mitochondrial carrier family. Whereas UCP1 in
brown adipose tissue has a well-deŽ ned role in thermogenesis, the
roles of other UCPs are still tentative, such as in control of immune
response, oxygen radical formation, and insulin secretion. The popular overexpression in yeast did not yield a functional form of UCP3
and possibly of other UCPs in mitochondria with the exception of
UCP1. Whereas UCP1 can be isolated in native form, the isolation
of other native UCPs from tissues or from overexpression in yeast
failed. UCPs (UCP1, 2, and 3) expressed in E. coli as inclusion bodies can be reconstituted to yield H+ transport only in the presence
of CoQ requiring fatty acids as native UCP1. The rates are similar
to native UCP1 and are inhibited by low nucleotide concentrations.
Native UCP1 is activated by endogenous CoQ. Differences between
UCPs may reside in the regulation, such as by the ATP/ ADP ratio
in accordance with the speciŽ c cellular requirements.
IUBMB Life, 52: 175–179, 2001
Keywords
Uncoupling proteins; mitochondria; coenzyme Q; e constitution; Ht transport.
INTRODUCTION
Uncoupling proteins are suggested to uncouple oxidative
phosphorylation (see ref. 1). Whereas there was little doubt for
the uncoupling function of UCP1 from brown adipose tissue,
for other UCPs (e.g., UCP2 to UCP5), the uncoupling and the
synonymous HC transport function were less clearly deŽ ned and
subject to a wider range of proposed functions (reviewed in 2–5).
In addition to thermogenesis, these comprise obesity, fatty acid
metabolism, and control of oxygen radical (ROS) levels. The
main source for these suppositions were responses of mRNA
expression to various stress conditions, such as staring, exercise,
thyroid hormone injection, and cold. However, it was then realized that changes in mRNA do not necessarily re ect the levels of
Received 7 August 2001; accepted 7 September 2001.
Address correspondence to Martin Klingenberg. Fax: 089-5996473. E-mail: [email protected]
UCP protein expression. Genetically modiŽ ed mice where UCPs
genes are deleted, ablated, or the expression of UCPs is transgenically enhanced provided a new forum for studying a variety
of physiological responses (6–12). Here, UCP1 was conŽ rmed
as the sole UCP responsible for nonshivering thermogenesis,
whereas UCP2 was associated with the immune response and
insulin secretion.
The problems with elucidating the function of UCPs variants on a biochemical level were low expression levels in their
natural host cells, and thus studies on isolated mitochondria
and the isolated native proteins were not possible. Heterologous
expressions of the UCP variants Ž rst in yeast and then in
E. coli were therefore applied (13–19). However, in yeast the
state of UCPs was not well deŽ ned and problems in the interpretation of the functional assays of uncoupling arose. Here, we
deal with these problems and offer a solution to the understanding of UCPs function on the level of their transport function and
regulation.
It has been our view (1) that UCP1 is not only historically,
but also in a practical working hypothesis, a paradigm for other
UCPs variants, although this view is not shared by several investigators in the Ž eld. This working hypothesis has turned out
to be very fruitful. Next, we summarize experimental evidence
for the regulated uncoupling function of UCPs, Ž rst by pointing
out the fallacies of the yeast expression system and then by reviewing new methods to renature and to activate HC transport
in UCPs. In the course of these pursuits, coenzyme Q was discovered as an obligatory cofactor for HC transport. In the last
section, the mechanism of HC transport is discussed, that is, the
long-standing problem of mechanism of fatty acid involvement
and, in view of new results, of a cofactor role of CoQ.
The Expression of UCPs in Yeast
For the recombinant expression of the UCPs, S. cerevisiae has
been a preferred host. First, UCP1 from brown adipose tissue
was introduced into yeast cells and was quite abundantly incorporated into yeast mitochondria without inhibiting the growth of
the yeast cells (21, 24). This expression system was extensively
175
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KLINGENBERG
used as a vehicle for elucidating the structure–function relationship in UCP1 by directed mutagenesis (1). Following this lead,
the discoveries of UCP variant genes, UCP2, UCP3, UCP4, and
BCMP (UCP5) also initiated their expression in
S. cerevisiae (5–11). The uncoupling was measured by
 uorescence-activated cell sorting (FACS) using a  uorescent
probe for the mitochondrial membrane potential originally introduced for measuring the effect of UCP1 mutation (25). In
yeast cells expressing UCP2 and UCP3, a stronger decrease of
membrane potential was reported than in cells expressing UCP1.
However, by this method it cannot be decided a priori whether the
observed downshift of the  uorescence maximum in the FACS
proŽ le was caused by a decrease of the membrane potential, that
is, an uncoupling effect, or merely by a decrease of the amount
of mitochondria. The uncoupling function of UCP2 and UCP3
was then investigated at the level of isolated mitochondria from
yeast in various laboratories (13–19). In most cases, by measuring respiration, the responses to fatty acids and nucleotide
were determined. It was concluded that uncoupling by UCP3 is
not (or only poorly) activated by fatty acids and not inhibited
by nucleotides. Differently, isolated mitochondria from UCP2expressing yeast showed no basal uncoupling nor any response
to fatty acids (26). Only on high overexpression, a fatty acid
and nucleotide unresponsive uncoupling was observed.
Our contribution in analysing the function of UCP3 was to
compare the uncoupling and HC transport properties of UCP1
and UCP3 expressed in yeast mitochondria by measuring the
membrane potential, the HC transport, and the ATP synthesis
rates in cells (27, 28). Using different vectors, varying levels of
expression were achieved. UCP1 consistently, even under very
high expression, did not cause a major uncoupling as measured
in cells. In isolated yeast mitochondria, UCP1 showed a regulated uncoupling, of activation by fatty acids and, inhibition
by nucleotides, as in mitochondria from brown fat. Obviously,
UCP1 in yeast cells must be inhibited. A Ž nal proof came when
we were able to show in yeast cells harboring UCP1 mutants
with defective nucleotide binding that only the mutant UCP1 responded to fatty acid additions, indicating that in vivo wt UCP1
is inhibited by nucleotides (29).
In contrast, in cells harbouring UCP3, growth and ATP synthesis were drastically inhibited and the yield of mitochondria
was low (27, 28). The mitochondria exhibited no regulated uncoupling, but they were already uncoupled without addition of
fatty acids. Also, they could be only marginally recoupled with
nucleotides. The deleterious effect on cells and mitochondria
increased strongly with the expression level of UCP3. At high
levels, a gelatinous pellet surrounding the few mitochondria in
the centrifugal sediment was found, which contains the inclusion
type aggregate of UCP3 insoluble in Triton X100 but soluble in
ionic detergents such as sarcosyl. Also with low expression level,
UCP3 turned out to be largely refractory to Triton solubilisation. In contrast, UCP1 at low or very high expression level was
Triton-soluble. Because solubility in nonionic detergent should
only re ect insertion into the mitochondrial membrane as op-
posed to solubilisation by ionic detergents such as sarcosyl, we
concluded that the bulk of UCP3 is not incorporated into the
mitochondria but rather present in extramitochondrial inclusion
body-type aggregates.
The sequence of events is depicted as follows: Ž rst, a small
fraction of UCP3 is incorporated into the mitochondria, mostly
in a deranged form unable to bind nucleotides but still able to
transport HC . The resulting unregulated uncoupling and low 1Ã
prevents further UCP3 from being imported into the
mitochondria. Apparently, subtle structural differences between
UCP1 and UCP3 are the reason for allowing the native folding
of UCP1 (but not of UCP3) in the foreign environment of the
yeast mitochondria. The overexpression of UCP3 then causes
extramitochondrial aggregation. The insolubility in Triton provides a simple assay for the state of heterologously expressed
UCP3, not only for UCP3 but also for UCP2, UCP4, and UCP5.
It can be expected on the basis of the observed uncoupling characteristics of yeast cells expressing UPC2, that also UCP2 is not
fully folded in the yeast mitochondria. This reasoning implies
that, similar to UCP1, other UCPs are controlled by nucleotides
and argues against the conclusions drawn by other groups from
measuring uncoupling by UCPs in isolated yeast mitochondria
that UCP3 is not regulated by nucleotides and does not require
fatty acids (30–33).
Expression in E. coli and Reconstitution of H + Transport
Expression in E. coli has been a favored way of obtaining
sizable protein amounts of mitochondrial carriers and for subsequent reconstitution to measure their transport functions. The
proteins are deposited into inclusion bodies and thus do not
strongly affect growth of the cells. From there they can be further puriŽ ed and solubilised.
Also, the expression of UCPs in E. coli results in their deposition into inclusion bodies. Solubilisation with the anionic
detergent sarcosyl is the preferred method, but reconstitution
attempts with UCP1 did not lead to HC transport with characteristics comparable to that of native UCP1. Fatty acid-induced
HC transport was much lower, but only inhibited by 100-fold
higher concentration of nucleotides than native UCP1 (34, 35).
When taking into account the measuring temperature (25± C)
and the calibration of the HC transport scale, the reported HC
transport rates were 20 to 40 times lower than those measured
with reconstituted native UCP1. However, on reconstituting Cl¡
transport with UCP1 and UCP3 from E. coli inclusion bodies,
a high sensitivity to nucleotides was observed (36). Taking into
account these results, the lack of HC transport regulated as in native UCP1 seemed to be an artefact of the reconstitution system
and demanded scrutiny of the possible defect.
With the aim of achieving similar HC transport as with native
protein, we converted sarcosyl-solubilised UCP1 from
E. coli into the nucleotide-binding form by replacing sarcosyl
with digitonin (20). In this detergent, UCP1 assumed a stable
nucleotide binding form, which was used for incorporation into
phospholipid vesicles. Also, with this nucleotide binding form of
UNCOUPLING PROTEINS: FUNCTION AND REGULATION
UCP1, no HC transport could be reconstituted. After eliminating
some possible causes for the defect, we pursued the potential existence of a cofactor necessary for HC transport. With various
soluble extracts from brown fat or UCP-expressing yeast mitochondria, no activation of HC transport was obtained (37). We
prepared lipid extracts from mitochondria that failed when using the usual solvents for extracting phospholipids. Only with
extracts of neutral lipids from lyophilised mitochondria, an activation of HC transport beyond the basic rate was obtained (20).
This additional HC transport had to fulŽ l our criteria of speciŽ c
UCP-linked HC transport, that is, inhibition by low concentrations of nucleotides. On further improvement, HC transport activities reaching the same rates as with native UCP1 were measured. The working hypothesis that the activating component is
associated speciŽ cally only with UCP1 was invalidated when
it was found that not only extracts from brown adipose tissue
and from UCP1-expressing yeast mitochondria, but also from
bovine heart and normal yeast mitochondria had activation potency. Fractionation on thin layer was optimised as to Ž nally
identify one small neutral lipid fraction responsible for the activation. The analysis by NMR showed mostly triglycerides in this
fraction. An absorption spectrum with a weak band at 280 nm
may have indicated ®-keto fatty acids in the triglycerides, but a
suspicion of coenzyme Q presence led us to test the response of
the UV absorption to the reductant NaBH4 . The resulting strong
decrease in absorption gave clear evidence of CoQ presence.
Commercial CoQ migrated in the thin layer assay with identical
Rf as the activating fraction from mitochondria. The Ž nal step
was reached when, on addition of CoQ, full activation of HC
transport was obtained, which was dependent on fatty acid and
inhibited by nucleotides, and thus fulŽ lled our criteria for UCP
involvement.
After the discovery of CoQ as the activating component for
UCP1 reconstituted from E. coli inclusion bodies, the question
arose as to why the native UCP1 did not require addition of CoQ.
We found that native UCP1 isolated with Triton X100 contained
CoQ at a molar ratio of ¼3 to UCP1. Further, after mild extraction of native UCP1 with ether, reconstitution of HC transport
required the same procedure as with UCP1 from E. coli, for
example, the addition of CoQ. This experiment eliminated the
presence of a covalently bound cofactor in native UCP1 necessary for HC transport. When the excess amount of CoQ contained in Triton-solubilised UCP1 was decreased by a sucrose
gradient to a molar ratio of 0.8 to UCP1, a requirement for CoQ
addition emerged also for the native UCP1. These results show
that CoQ is also an activating cofactor in native UCP1.
The speciŽ city for CoQ was examined using CoQ with varying isoprenoid or alkyl chain lengths. A minimum of 13 carbon
atoms was required, indicating that a strong interaction of CoQ
with the membrane lipids is required. Besides benzoquinone, as
in CoQ, also naphthoquinone as in Vit K1 , can serve as an activating headgroup. Importantly, reduced CoQ is unable to serve
as activator, as was shown both with added CoQ for the UCP1
from E. coli and for the endogenous CoQ in native UCP1. The
177
dependence of HC transport on the amount of CoQ, fatty acids,
and on the nucleotide concentration for the inhibition was determined. The K1 of 0.05 to 0.08 for ATP and ADP was unusually
low, probably partially due to the foreign lipid environment. Saturation of HC transport requires a CoQ content at a molar ratio
of 80 to 1. The afŽ nity seems low, but in mitochondria the molar density of CoQ in the membrane is 1 to 100 phospholipids,
whereas in the reconstituted vesicles at saturation, it is 1 to 300.
With UCP1, clear criteria for a regulated HC transport to be
achieved with reconstituted UCP1 from inclusion bodies were
set by previous data obtained with the native UCP1. These
were Ž nally met with the discovery of CoQ as an obligatory
cofactor for HC transport (20). Now it seemed obvious to apply
the same reconstitution conditions to other UCPs expressed in
E. coli. Here, the fundamental difference to UCP1 is the lack of
native UCPs isolated from the parent tissues and, as discussed
previously, also from transformed yeast. The same methods of
renaturation and reconstitution as established for UCP1 were
applied to UCP2 and UCP3 harvested from E. coli. Again, nucleotide binding was recovered by the replacement of sarcosyl
with digitonin as assayed by the dansyl GTP binding. Reconstitution with those preparations allowed us to achieve high HC
transport rates on addition of CoQ (37). This HC transport was
dependent on fatty acid and inhibited by nucleotide. With both
UCP2 and UCP3, the rates reached the same level as with UCP1.
These Ž ndings established also UCP2 and UCP3 as highly active HC transporters. The doubts about an uncoupling function
of UCP2 and UCP3 in vivo now appeared to be less plausible.
Interesting is the difference of nucleotide sensitivity between
UCP3 versus UCP1 and UCP2. It is a Ž rst case of a welldeŽ ned biochemical difference between UCP variants and was
Ž rst noted with reconstituted UCP1 and UCP3, by comparing
the inhibition of Cl¡ transport by ADP and GDP with that by
ATP and GTP. Also for the HC transport, the sensitivity towards
ADP versus ATP is inverted between UCP1 and UCP3. Whereas
HC transport by UCP1 and UCP2 has a higher sensitivity toward
ATP than to ADP, the inverse is true for UCP3. Based on the
Ž ndings, we deduced a regulation not only by the nucleotide
concentration but also by the ATP/ADP ratio. In a physiological
context, these differences between UCP1 and UCP3 are visualised to correspond to the different thermogenetic requirements
of brown adipose tissue, where UCP1 is the major compound,
and of skeletal muscle, where UCP3 is prevalent. In brown adipose tissue, thermogenesis is the major purpose of respiration
and is associated with a high level of uncoupling and lower
ATP/ADP ratio. In skeletal muscle, only in the resting state is
supplementary thermogenesis required when the ATP/ADP ratio
is high.
The Role of CoQ and Fatty Acids
The mechanism of HC transport by UCPs is intimately linked
to the role of fatty acids, which are obligatory activators of HC
transport. Different views exist on the way fatty acids serve in
HC transport (1). Originally, an allosteric activation of UCP1 by
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KLINGENBERG
FA was discussed with an option of a more active role of FA (38).
Then, more speciŽ cally, the FA carboxyl group was proposed
(39) to serve as a catalyst of HC translocation, similar to carboxyl groups in other HC -transferring proteins. Given the possibility by free FA to withdraw or insert the carboxyl group in UCP,
an inactivation of activation of HC transport can be achieved.
Within the translocation pathway, the FA can function in concert with resident carboxyl groups, Ž lling a carboxyl-deŽ cient
gap. In a more speciŽ c application, FA might interact with UCP
only from the cytosolic side functioning as a HC capturer and
injector.
An alternative model proposes that UCP is a FA anion transporter, as re ected in the ability to transport small anions such
as Cl¡ (40). In this model, FAs  ip freely across the membrane
in the undissociated form and return as anions via UCP. Thus,
HC ions are picked up by FAs on the cytosyl side and released
on the matrix side. Evidence supporting this model is based on
the transport of alkyl sulfonates by UCP in mitochondria. However, the rates of this transport are slow as compared to the HC
transport rates measured by FA. Another objection to this anion
transport model comes from using FA derivatives that contain at
the omegaposition a hydrophilic substituent, such as glucose or
carboxyl. These substituents should have difŽ culty in  ipping
though the membrane.
With the advent of CoQ as a cofactor of HC transport, a
new twist has arisen in the model of HC transport. Both CoQ
and long-chain FAs are primarily imbedded in the lipid bilayer
and therefore can be visualised to be reconstituted by UCP as
diffusable compounds from the lipid phase (37). Further, the interaction of both compounds by their headgroups seems feasible
because the undissociated carboxyl groups may form a hydrogen bond with the oxo-group of CoQ. Through this interaction,
the abstraction of HC from the FA, with a high pK when bound
in the lipid layer, may be facilitated. With this mechanism, as
illustrated in Fig. 1, FAs are visualised to transfer HC from the
aqueous phase into the UCP with the intervention of CoQ. In
an alternative model, the transfer of FA to the HC channel is
facilitated by CoQ, again through hydrogen bond interaction.
In conclusion, the discovery of a quinone as a cofactor for HC
transport illustrates the enormous versatility used by nature to
accomplish the elementary HC transport function. HC transport
by UCP seemed at Ž rst to be about the simplest HC transport
known, but now it turns out to require a cofactor that was previously known to have only a redox function in electron transport.
The cofactor role of CoQ deserves the greatest attention in future
research.
OUTLOOK
Uncoupling of oxidative phosphorylation is a multifaceted
phenomenon implicated in the very nature of the electrochemical
coupling between electron transport and ATP synthesis. UCPs
furnish only one mechanism of lowering the efŽ ciency of ATP
synthesis and are apparently engaged with different physiological requirements in the respective cells. These are served by
different UCPs in which regulation may differ gradually, but
should have the same basic mechanism of transport. A beginning of elucidating the functional difference was the Ž nding of
different regulation mechanisms by the ADP/ATP ratio between
UCPs. It may turn out that other factors such as the in uence of
CoQ may also vary between the UCP variants.
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Figure 1. Models of the role of fatty acids and coenzyme Q in
catalysing HC transport by UCPs. Cooperation of both lipophilic
compounds by forming a hydrogen bond between their headgroups. A) CoQ facilitates the transfer of protonated FA to the
HC channel of UCP. B) CoQ acts as a mediator of HC transfer
into the HC channel of UCP.
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