Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells Danilo Guerini 1 , Elisabetta Prati 1 , Urvi Desai 2 , Hans Peter Nick 1 , Rolf Flammer 1 , Stephan Gru¨ ninger 1 , Frederic Cumin 1 , Machael Kaleko 2 , Sheila Connelly 2 and Michele Chiesi 1 1 Metabolic and Cardiovascular Diseases, Novartis Pharmaceuticals Ltd, Basel, Switzerland; 2 Genetic Therapy Inc., Gaithersburg, MD, USA The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abun- dantly expressed in skeletal m uscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of c ell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding o f the prote in and not to its intrinsic uncoupling activity. In the present study, we investigated the properties o f UCP3 overexpressed in a relevant mammalian host system such a s the rat myoblast L6 cell line . UCP3 was expressed in r elatively low levels (< 1 lgÆmg )1 membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that i ts staining pa ttern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating condi- tions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respir atory capacity of mito- chondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubi- lize m embrane bound proteins. Thus, UCP3 can be uniformly overexpressed in the mitochondria of a r elevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in a n incompetent c onformation. Keywords: uncoupling protein; mitochondria; respiration; thermogenesis; aden ovirus. In the last few years several novel proteins homologous to thermogenin have been identified. Thermogenin is the uncoupling protein (UCP) first identified in brown fat mitochondria and is now referred to as UCP1. While the role of UCP1 in thermoregulatory thermogenesis is undis- puted, t here is still uncertainty concerning th e physiological role of the other homologues. The ubiquitous UCP2 [1,2] could play an important role in modulating insulin secretion by b-cells [3,4], in mediating fever du ring infection [ 5] or, as a general mechanism, in protecting cells from oxidative stress by limiting the mitochondrial production of reactive oxygen species [6]. Another protein belonging to the same family, UCP3, has received much attention because of its restricted expression in skeletal muscle [7,8], the major thermogenic tissue in higher m ammals [9]. The uncoupling properties of this protein, in fact, could explain the high level of nonphosphorylating oxygen consumption of skeletal muscle mitochondria. UCP3 is considered a promising target for pharmacological intervention in the obese state, as a controlled i ncrease in skeletal muscle thermogenesis could safely correct for the energy imbalance. In addition to this possible role in thermogenesis, it has been proposed that UCP3, whose expression is strongly induced by free fatty acids (FFAs), could facilitate or be beneficial when the energy utilization in the muscles shifts from carbohydrates to lipids [10]. To gather i nformation abou t t he potential r ole of U CP2 and UCP3, many laboratories h ave investigated their properties after overexpression in various host cell systems or in transgenic animals. The first indications that UCP2 and UCP3 could play a role in mitochondrial uncoupling were o btained using yeast expression sys tems [1,2,11,12]. In contrast to observations of UCP1 overexpression in yeast, the uncoupling activity of UCP2 and UCP3 has not been shown to be regulated by FFA or purine nucleotides [13,14]. Mammalian cell lines such as L6, C2C12 or human primary muscle cells have also been used to analyse the properties of UCP3 [15,16]. In these cells, UCP3 decreased the mito- chondrial m embrane poten tial [15] but also caused changes in substrate flow, such as a n increased l actate secretion [16]. UCP2 and UCP3 have also been overexpressed in an insulinoma cell line and found to uncouple respiration in association with increased lipid oxidation [17]. R econstitu- tion experiments with recombinant UCPs have clearly Correspondence to M. Chiesi, Metabolic and Cardiovascular Diseases, Novartis Pharmaceuticals Ltd, 4000 Basel, Switzerland. Fax: + 4 1 61 696 3783, Tel.: + 41 61 696 4 485, E-mail: michele.chiesi@phama.novartis.com Abbreviations: UCP, uncoupling protein; KO, knock-out; DMEM, Dulbecco’s modified Eagle’s medium; m.o.i., multiplicity of infection; DABCO, 2,4-diazabicyclo-(2,2,2)-octane; RU, Ru[dpp(SO 3 Na) 2 ] 3 ; MTP, mitochondrial import stimulation factor; FFA, free fatty acid. (Received 1 8 October 2001, revised 28 D ecember 2001, ac cepted 9 January 200 2) Eur. J. Biochem. 269, 1373–1381 (2002) Ó FEBS 2002 shown that, similar to UCP1, UCP2 and UCP3 also transport protons across lipid membranes [18,19], strongly suggesting that their major physiological function is to increase the mitochondrial proton leak. The role of UCP3 in the control of energy homeostasis became apparent after the generation of mice with a disrupted UCP3 gene [20,21]. The knock-out (KO) animals did not show any evident obese phenotype thus indicating that UCP3 does not play an essential role in whole body basal energy expenditure in rodents. However, oxidative phosphorylation in the muscles of KO mice was found to be much more efficient and the rate of AT P production markedly increased [ 22]. Therefore, although the lack of UCP3 has strong effects on the efficiency of oxidative phosphorylation, these latter effects must be masked by the induction of compensatory mech- anisms th at increase ATP consumption. Finally, transgenic mice overexpressing UCP3 in skeletal muscle have been generated and characterized [23]. The transgenic animals had uncoupled muscle mitochondria and remained lean despite their hyperphagia. The properties of UCP2 and UCP3 overexpressed in yeast mitochondria have been recently questioned [24,25]. Careful analysis has shown that only nonphysiological, high concentrations of UCP2 and UCP3 i nduced the changes in the mitochondrial p roton permeability previously observed. These uncoupling effects were the result of overexpression of the recombinant proteins leading to a compromised mitochondrial integrity rather then to an intrinsic property of the proteins. T his could e xplain the lack of r egulation by free fatty acids and purine nucleotides reported by the previous investigations [13,14]. Such artefacts might not be restricted to yeast but could also occur in more relevant expression systems such as m uscle-derived cell lines or even in transgenic animals. In the present study, we addressed this problem by analysing the characteristics o f muscle-derived L 6 cells that express U CP3 u nder the control o f the relatively weak rous sarcoma virus prom oter. It was observed that, at expression levels giving rise to an uncoupling phenotype, the mito- chondrial respiratory activity became impaired, the mito- chondrial membrane potential showed no sp ecific response to FFA or nucleotides, a nd most of the U CP3 could n ot be extracted by nonionic detergents. These results strongly indicate that in mammalian cells, recombinant UCP3 is expressed in a n on-native state. Therefore, extreme care is necessary when interpreting the results of a forced overex- pression of UCP3 in any host system. EXPERIMENTAL PROCEDURES Cloning of hUCP3 cDNA and construction of the adenovirus shuttle plasmid The h UCP3 cDNA was obtained from P. M uzzin (Univer- sity of Geneva, Switzerland). T he h UCP3 cDNA in pBlue- script II S K(+) plasmid (Stratagene, La Jolla, CA, USA) was digested with SpeIandClaI, purified by gel electrophor- esis and ligated into SpeI/ClaI-digested adenoviral shuttle plasmid pAvS6alx [26] to generate pAvhUCP3lx. pAvhUCP3lx contains a constitutive Rous Sarcoma V irus (RSV) promoter, a 198-bp fragment containing the a deno- virus serotype 5-tripartite leader s equence, the hUCP3 cDNA, a nd an SV40 early polyadenylation signal. Construction and in vitro characterization of recombinant adenovirus The recombinant adenovirus encoding human UCP3 (Av3hUCP3) was c onstructed by a rapid vector generation protocol using Cre recombinase-mediated recombination [27] of two plasmids, one containing th e r ight hand portion of the adenoviral vector genome and a lox site, and the other plasmid, pAvhUCP3lx, containing the left portion of the viral genome, the UCP3 expression cassette, and a lox site as described previously [26]. Both plasmids and the C re- encoding plasmid were cotransfected into AE1-2a cells (A549 cells stably transfected with E1/E2a regions under the control of d examethasone inducible promoters [ 28]). Vector genome integrity was verified by viral DNA restriction analysis. Vector concentrations were determined by spectro- photometric analysis [29]. Titers are stated as particles per mL. The control vector, Av3Null, was identical to the Av3hUCP3 vector except that it lacked a transgene, but retained the RSV promoter and SV40 poly(A)+ signal. Cell culture and adenoviral infection L6 rat skeletal muscle cells [30] were culture d in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37 °C und er 5% CO 2 . C ells were seede d at adensityof1.5· 10 4 cellsÆcm )2 in order to reach 80% confluency the next day. Then cells were infected with the recombinant hUCP3 or control b- Gal viral st ocks at different multiplicity of infection (m.o.i.) by keeping the total final amount of adenovirus particles constant (i.e. 10 4 ). This was achieved by combining hUCP3 and the control b-Gal adenovirus particles at t he moment of cells transduc- tion. Infection o f L 6 cells with the adenovirus particles was performed with the help of the transfection reagent Lipo- fectamine P lus (Life Technologies) following the supplier’s instruction. The e fficacy of infec tion f or varying viral loads was determined by staining for b-Gal (data not shown). Metabolic [ 35 S]methionine labeling and membrane preparation L6 cells were infected with hUCP 3, b-Gal or empty (Av3Null) recombinant aden oviruses for 48 h. The cu lture medium was replaced with methionine-free MEM (Gibco- BRL) and the cells were left at 37 °C for 20 min. Then the cells were incubated with methionine-free M EM containing 120 lCiÆmL )1 [ 35 S]methionine (1000 CiÆmmol )1 ,Amer- sham) for 2 h at 37 °C. After labeling, the cells were washed twice with DMEM, resuspended in 200 lL10m M Tris/HCl, pH 8.0, 1 m M EDTA, 0.25 m M dithiothreitol and disrupted by three cycles of freezing-thawing. The mem- branes were sedimented at 20 800 g for 5 min (Eppendorf centrifuge) and solubilized in 200 lL1%SDSin10m M Tris/HCl, pH 8.0, 1 m M EDTA. The samples were separ- ated on a 10–15% SDS/polyacrylamide gel, stained with Coomassie Brilliant Blue and dried, prior to exposure to autoradiographic films (24–72 h). Immunoblotting Ten m icrogra ms o f protein we re s ep arate d o n 1 2. 5% S DS/ polyacrylamide gels a nd electroblotted to nitrocellulose 1374 D. Guerini et al. (Eur. J. Biochem. 269) Ó FEBS 2002 membranes (Bio-Rad Laboratories). Blots were block ed with 3% BSA in NaCl/P i with 0.1% Tween-20 (NaCl/P i / Tween) for 1 h and incubated overnight at 4 °Cwith an affinity-purified rabbit anti-hUCP3 Ig (UCP3-2A, Alpha Diagnostic International Inc.; 1 lgÆmL )1 )orwithan affinity chromatography-purified mouse anti-prohibitin Ig (0.5 lgÆmL )1 ; NeoMarkers). B lots were washed with NaCl/ P i /Tween and exposed to horseradish peroxidase-conjugat- ed second ary anti-(rabbit I gG) I g or a nti-(mouse I gG) Ig a t a 1 : 10000 dilution in NaCl/P i /Tween for 1 h at room temperature. Blots were washed again an d developed by enhanced chemiluminescence using a standard kit (ECL, Amersham Pharmacia Biotech.). Immunofluorescence Unless otherwise stated, all steps were performed at room temperature in a humidified chamber. L6 cells (1.5 · 10 4 cellsÆcm )2 ) were seede d on poly- L -lysine-coated slides and they were i nfected 24 h l ater w ith the adenovirus particles for hUCP3 and b-Gal, as described above. After 48 h, the cells were washed three times in NaCl/P i ,thenfixed in 4% (w/v) paraformaldehyde/NaCl/P i for 60 min. The cells were w ashed four times in NaCl/P i and then i ncubated for 60 min in NaCl/P i containing 0.1 M glycine, pH 8.6. After three washes with NaCl/P i , the cells were permeabi- lized using 0.1% ( v/v) Triton X-100 in NaCl/P i for 3 min, followed by four NaCl/P i washes. The slides were then incubated in blocking buffer containing 5% (v/v) fetal bovine serum, 0.1% (w/v) BSA, 5% (v/v) glycerol, and 0.04% NaN 3 in NaCl/P i for 60 min. The slides were overlaid with anti-hUCP3 Ig (UCP3–2 A , Alpha Diagnostic Intl Inc.) diluted 1 : 100 in blocking buffer and kept under gentle rocking for 90 min at room temperatu re. After fi ve washes (5 min each) in blocking buffer, the slides were incubated for 60 min with secondary antibodies [goat a nti-(rabbit IgG) Ig coupled to A LEXA 594, Molecular P robes] dilut ed 1 : 100 in blocking buffer. The slides were washed twice in b locking buffer, twice in NaCl/P i and finally mounted in a medium containing 80% g lycerol, 2.5% 2,4-diazabicyclo-(2,2,2 )- octane (DABCO) in NaCl/P i , pH 8 .0. The cells were observed in an AXIOVERT 10 microscope (Carl Zeiss) equipped with epifluorescence illumination using a 10x, 20x, 40x and 63x oil immersion plan-neofluor objective. Images were collected with a cleavage coupled device (CCD) camera. Extraction of membrane bound proteins L6 cells were harves ted a nd membranes prepared as described above. Proteins were extracted from the mem- branes as described p reviously [31]. B riefly, the m embranes were suspended in a buffer containing 3.6% Triton X-100, 1.2 M ammonium acetate, 1 m M EDTA, 5 m M phenyl- methanesulfonyl flu oride and 1 m M dithiothreitol at a concentration of 3.6 mg detergent per mg protein. The suspension was sonicated and incubated for 20 min at 0 °C. The solubilized m aterial was separated by centrifugation a t 100 000 g for 20 m in. Measurements of oxygen consumption Measurements of oxygen consumption of L6 cells were performed by using Ru[dpp(SO 3 Na) 2 ] 3 (RU), a water soluble oxygen sensor. The probe was synthesized as des- cribed previously [32]. The principle of measurement was based on fluorescence quenching of the sensor by oxygen dissolved in the reaction medium. The quantum yield of the fluorescence, was shown to be linear with oxygen concen- tration as p redicted by the Stern–Volmer E quation. L6 cells were cultured and transfected as described above. The medium was r emoved an d c ells were washed with NaCl/P i . Cells were trypsinized and s uspended i n a solution contain- ing 5.5 m M glucose, 120 m M NaCl, 4 m M KCl, 1 m M KH 2 PO 4 ,1m M MgSO 4 ,1.3m M CaCl 2 ,10m M Hepes, pH 7.4 (medium A) supplemented with 10% fetal bovine serum. Aliquots containing 1.5 · 10 6 cells wer e centrifuged, resuspended in 100 lL medium A supplemented with 20 l M RU at 32 °C, an d added to a cuvette containing 1.9 mL o f t he same medium that had b een preincubated at 32 °C in a temperature adjustable cuvette holder of a PerkinElmer LS50B fluorimeter. E xcitation wavelength was 470 nm and e mission was measured at 600 nm. Measurement of membrane potential DY The membrane potential was r ecorded using the fluorescent probe 3,3¢-dihexyloxacarbocyanine iodide DiOC6 obtain ed from Molecular Probes. L6 cells expressing either hUCP3 or b-Gal or the combination of the two different types of adenovirus particles were first subjected to digitonin treat- ment in order to permeabilize the plasma membrane. This was p erformed by incubating L6 cells at a concentration o f 1 · 10 6 cellsÆmL )1 into Na Cl/P i containing 50 l M digitonin on ice f or 5 min. Cells were washed once i n buffer A (1 m M EGTA, 2 m M MgCl 2 ,5m M phosphate, 5 m M Hepes pH 8.0, 20 m M sucrose, 20 m M mannitol, 120 m M KCl). To ensure removal of endogenous substrates and tightly bound nucleotides, permeabilized cells were subsequently treated f or 30 min at room temperature by gently shaking with Dowex 21K (Fluka) in 2 10 m M sucrose, 70 m M mannitol and 10 m M Hepes, pH 7.4. This procedure was proven to be effective in removing tightly bound nucleotides to UCP1 in isolated mitochondria [33]. Cells were then incubated w ith 150 n M of DiOC6 in buffer A at pH 7.4 f or 15 min at room temperature. After a subsequent centrifuga- tion step, the mitochondrial membrane potential of the permeabilized cells resuspended in buffer A at pH 7.4 ( 2 · 10 6 cellsÆmL )1 ) was measured at room temperature using a n e xcitation a nd an emission wavelength pair of 485 and 530 nm. RESULTS The aim of this study was to evaluate t he properties of hUCP3 e xpressed in a relevant system, such as the muscle cell line L6, at a much lower level than in yeast. L6 cells have lost the ability to express endogenous UCP3 (only trace amounts o f UCP2 mRNA can be found) and display well coupled mitochondrial respiration (see below). These char- acteristics make L 6 myoblasts particularly suited for studies aimed at analysing small changes in their mitochondrial characteristics that m ight be induced by the overexpression of recombinant h UCP3. I n p reliminary attempts to e xpress hUCP3, stable and inducible expression systems have been tried. None of these efforts could successfully produce levels of hUCP3 e xpression sufficient to give a measurable change Ó FEBS 2002 Uncoupling properties of UCP3 in L6 cells (Eur. J. Biochem. 269) 1375 in the r espiration pr operties of the cells. Transient transfec- tion methods based on adenovirus particles proved to be more successful. L6 myoblasts, however, were found to be extremely resistant to adenoviral infection. Only after application of viral particles in great number (> 1000 m.o.i.) in combination with a transfection agent such as Lipofectamine Plus, an homogeneous infection o f the majority of the cells could be achieved. This is illustrated in Fig. 1 in w hich immunocytochemistry using anti-hUCP3 Ig was u sed to v isualize h UCP3 expression. In control cells transduced with b-galactosidase recombinant adenovirus, the antibody reaction produced only a faint background staining. On the other hand, almost every cell transduced with hUCP3 recombinant adenovirus showed a strong signal after the treatment with the UCP3 antibody. Inter- estingly, discrete subcellular s tructures with a punctuated or slightly elongated appearance were visible, which strongly resembled mitochondria (Fig. 1E). [ 35 S]Methionine labell- ing experiments were carried out to verify if UCP3 was expressed without perturbing the normal p rotein expression pattern of the L6 cells. Figure 2 shows that, in fact, the protein expression patterns of cells infected with either control (empty) viruses or hUCP3 expressing viruses w ere identical except f or a single major protein band d isplaying a molecular weight corresponding to that of the hUCP3. Figure 3 shows the level of hUCP3 expression after infecting L6 cells with increasing amounts of UCP3 recombinant adenovirus. The total number of virus particles was kept constant (i.e. 10 000 mo.i.) by adding a corres- ponding amount of control virus expressing the b-Gal protein. Two days after infection, the respiratory character- istics of L6 cells were analysed and then the level of recombinant h UC P3 expressed was determined by Western blotting (Fig. 3A, upper and medium panels). Quantifica- tion was based on a standard curve obtained with recom- binant hUCP3 expressed in inclusion bodies from E. coli (Fig. 3B). As the purity of hUCP3 in the inclusion bodies was  80% (as estimated by Coomassie Brilliant Blue staining), and the crude mitochondrial preparation used in the analysis was still contaminated by other membrane fractions, the levels of hUCP3 per mg mitochondrial protein given in F ig. 3B represent an underes timation o f t he actual levels. In t he absence of hUCP3 expression, the addition of the mitochondrial H + -ATPase inhibitor oligomycin strongly reduced the oxygen c onsumption. As the oligomy- cin-resistant portion of respiration reflects the level of proton leak of mitochondria, the low l evel of respiration in the presence of oligomycin of L6 cells showed that they were very well coupled. The oligomycin resistant respiratory activity of the cells could be strongly stimulated by the addition of the protonophore carbonyl cyanide m-chloro- phenyl hydrazone (CCCP) (Fig. 3A, upper panel). Stimu- lation of oxygen consumption by CCCP was maximal at concentrations between 0.5 and 1 l M while higher concen- trations were inhibitory (not shown). In t he presence of an uncoupler, such as C CCP, the respiratory activity o f Fig. 1. Localization of hUCP3 in L6 cells by immunofluorescence. L6 cells were seeded at a density of 1.5 · 10 4 cellsÆcm )2 .Infectionwascarriedout 24 h later by incubating the cells f or 6 h in the presence of the transfection r eagent Lipofectamine Plus combined w ith adenoviruses contain ing hUCP3 or b-gal cDNA (hUCP3 and control cells, r espectively) at a multiplicity of infection of 10 4 . For details see t he Materials and methods section. After 2 days cells were stained with specific hUCP3 antibodies (UCP3–2 A from Alpha Diagnostic Intl Inc.). (A,B) Control L6 cells infected with b-Gal adenoviruses under phase contrast (A) and fluorescent light (B), respectively. Panel C and D show L6 cells infected with hUCP3 adenoviruses under phase contrast and fluorescent light, respectively. Panel E illustrates a single L6 cell infected with hUCP3 viral particles at higher magnification. 1376 D. Guerini et al. (Eur. J. Biochem. 269) Ó FEBS 2002 mitochondria is pushed to i ts maximal c apability. While the basal oxygen consumption of the cells expressing UCP3 was not affected, the portion of respiration measured in the presence of oligomycin increased concomitantly to the levels of hUCP3 expressed (Fig. 3 upper p anel, black bars). This suggested that hUCP3 increased the uncoupling of mito- chondria. It was noted, however, that in cells expressing UCP3 the maximal respiration levels obtained i n the presence of CCCP were clearly reduced (Fig. 3 upper panel, grey bars). The amount of the membrane protein prohibitin, a marker of the mitochondria inner membrane, was f ound to remain constant also in cells expressing the highest amounts of hUCP3 thus indicating that the t otal number of mitochondria/cell was not appreciably affected (Fig. 3A, lower panel). It has been previously reported that the bulk of hUCP3 expressed in yeast is aggregated. In fact, most of the recombinant UCP3 remained insoluble after extraction with high concentrations of nonionic detergents such as Triton X-100 that normally fully solubilize UCP1 or other membrane bound proteins [31]. We applied a similar procedure to membranes isolated from L6 cells expressing recombinant hUCP3. Prohibitin that is localized in the inner membrane of mitochondria could be fully solubilized by the extraction procedure (see Fig. 4). On the other hand, a consistent portion of the recombinant h UCP3 was r esistant to solubilization thus indicating that it was presumably in an aggregated form. The proton transport activity of recombinant hUCP3 refolded and reconstituted in proteoliposomes requires FFA and is strongly inhibited by p urine nucleotides [18,19]. In an attempt to analyse whether the activity of the recombinant hUCP3 i n L 6 cells was also r egulated by these c ompounds, we measured the membrane potential of mitochondria in situ after selective p ermeabilization o f t he plasma m emb rane of the cells with a m ild digitonin treatment. The mitochondrial potential was analysed using the probe DiOC6, whose fluorescence becomes quenched at h igh membrane potentials. The left trace of Fig. 5A illustrates a typical Fig. 2. [ 35 S]Met pulse ex periment o f infected L6 cells. Infection of L6 cells with hUCP3 or Null adenovirus vectors was carried out as des- cribed in the legend to Fig. 1. Infected cells were grown for 2 days and then a [ 35 S]Met pulse experiment was performed as described in the Experimental procedure s section. Cells were harvested, lysed and analysed on a 12.5% SDS/polyacrylamide gel. The dried gel was exposed for 24 h to autoradiographic film. The amount of sample loaded to ea ch lane s c orresponded t o 1 50 000 –200 0 00 c pm. L ane 1 , cells infected with Null adenovirus; lane 2, cells infected with UCP3 recombinant adenovirus. T he position of t he probable UCP3 band is indicated o n the right side o f the pan el. Fig. 3. Effect of hUCP3 expression on mitochondrial uncoupling. (A) L6 cells were infected with hUCP3 adenoviruses at various multiplic ity of infection (m.o.i.) as indicated. The total number of moi was kept constant by adding correspondin g amounts of control viruses (b-G al). After 2 days, the effect of hUCP3 ex pression on the mitochondrial respiration w as investigated (upper panel). Cellular o xygen consump- tion was m easured using the fluorescent oxygen sensor R uCP ( white bars). O ligomycin (oligo), wa s used a t a co ncentration o f 10 lgÆmL )1 to inhibit the portion o f the total cellular respiration co upled to oxidative phosphorylation (black bars). T he uncoupler CCCP (1 l M ) was added after oligomycin to achieve maximal respiratory rates (grey bars).The expression levels of hUCP3 (middle panel) and of the typical inner-mitochondrial membrane mark er prohibitin (lo wer panel) were assessed by Western blot analysis using specific antibodies. The graphs represent mean values ± SEM (n ¼ 5). (B) Immunoreactivity of recombinant hUCP3 from inclusion bodies u sed as calibration to quantify hUCP3 expression levels. Ó FEBS 2002 Uncoupling properties of UCP3 in L6 cells (Eur. J. Biochem. 269) 1377 control experiment s howing that, after d igitonin treatmen t, endogenous substrates delivering NADH to the mito- chondrial complex I were still sufficient to energise the organelles. Addition of rotenone that blocks the u tilization of NADH by complex I, was needed to fully depolarize mitochondria. A subsequent addition of succinate that delivers electrons directly to complex III thus bypassing rotenone inhibition, re-energised mitochondria. Finally, addition of the K + ionophore valinomycin fully depolarized mitochondria. This control experiment showed that mito- chondria remained functional after skinning of the c ells and that sufficient endogenous small molecular weight compo- nents were retained to support mitochondrial respiration. As nucleotides could also remain trapped and inhibit the intrinsic uncoupling activity of hUCP3, the digitonin treated cells were extensively incubated with the resin Dowex-K21. This extracting procedure was found to effectively remove endogenous substrates so that, after about 30 min incuba- tion, mitochondria were completely de-energised (see level of right trace in Fig. 5A before the addition of rotenone and succinate). Hence, this procedure that has been originally developed t o s trip nu cleotides t ightly bound to UCP1 from isolated brown fat mitochondria [33] could be applied also to skinned cells. As Dowex treatment p resumably r emoved most of the purine nucleotides from skinned L6 cells, the effect of exogenously added GDP on the membrane potential of mitochondria could be investigated. Figure 5B shows t hat m mol c oncentrations of GDP did not affect the membrane potential in both c ontrol cells and in cells expressing hUCP3 (subsequent quantification gave values in the order of 1 lghUCP3permgmembraneprotein).To exclude the possibility that the recombinant hUCP3 was inactive because t he cells have been s tripped also of endogenous FFAs, lauric acid was added. Reconstitution experiments h ave p reviously shown that l auric a cid i nduces optimal activation of hUCP3 [18,19]. No effect on the membrane potential of mitochondria by lauric acid in cells expressing hUCP3 could be noticed. At concentrations above 10–20 l M a drop in the membrane potential was observed (see Fig. 5B). This effect, however, was identical in both, control and UCP3 expressing cells, and was not reverted by the subsequent addition of GDP. DISCUSSION Heterologous yeast expression systems have proven to be very useful to study the properties of UCP1 [34]. Once expressed in yeast mitochondria, the protein was found to be fully functional and to be regulated by FFAs and nucleotides, s imilarly to when expressed i n i ts native location, the mitochondria of the brown adipose tissue. Analogous strategies have been used to characterize the function of two novel, recently discovered UCP1 homo- logues such as UCP2 and UCP3. A general finding has been Fig. 4. Extraction of prohibitin and hUVP3. L6 cells were transduced with hUCP3 a denoviruses a t 1 0 4 multiplicities of infection, and wer e harvested 2 days later. After the isolation of the membrane fraction , proteins were extracted in the presence of 3.5% Triton X-100 and separated f rom the insoluble material b y centrifugation. The proteins in the vario us fractions ( T, total b efore extraction; S, solubilized p ro- tein; P, i nsoluble protein aggregates i n the pellet) we re then separated by SDS/polyacrylamide gel electrophoresis. Prohibitin and hUCP3 were visualized using specific antibodies after Western blotting. F or details see Experimental p rocedures section. Fig. 5. Effect of hUCP3 on mitochondrial DY in skinned cells. Recording of the m itochondrial membrane potential DY was performed with t he flu orescent p robe 3,3 ¢-dihexyloxacarbocyanine DiOC6. L6 cells expressing either hUCP3 or b-Gal (control) were subjected to digitonin treatment in o rder to permeabilize the plasma membrane. To ensure removal of endogenous su bstrates and tightly bound nucleotides, permeabilized cells were treated for 30 min with Dowex K21. Where indicated, additions were: rotenone 5 l M (rot), succina te 5 m M (suc), GDP 0.5 m M ,lauricacid50l M (LA), valino- mycin 10 n M (val). (A) Control skinned cells before (left) and after Dowex K21 treatment (right) (untreated and treated, respectively). (B) Skinned c ells overexpressing hUCP3 or b-gal (control) were treated with Dowex K 21 before measuring the membrane potential. 1378 D. Guerini et al. (Eur. J. Biochem. 269) Ó FEBS 2002 that these latter UCPs display much stronger uncoupling effects than UCP1 on the yeast cells, while their activity does not seem to be reg ulated by nucleotides [11–14,35]. The lack of regulation of UCP2 and U CP3 was quite unexpected a s both proteins share with UCP1 an highly conserved putative nucleotide binding domain. Moreover, the proton transport activity of UCP2 and UCP3, once refolded from inclusion bodies and reconstituted in p roteoliposomes, w as shown to require FFAs such as lauric acid [18,19] and to be highly sensitive to purine nucleotides [19]. Recent s tudies from two different laboratories have s hed some light on the possible reasons underlyin g these controversial findings, by showing that the expression of UCPs in yeast can lead to nonselective damage of m itochondrial integrity. T his damage c aused a n increase in the proton leak that is not regulated by, for example, nucleotides [24,25]. Relatively low levels of expression of UCP2 and U CP3 ( sublg per mg mitochond- rial protein) are sufficient to cause unregulated uncoupling. The ÔclassicalÕ U CP1 can b e e xpressed a t concentrations up to 1 lgÆmg –1 without causing any nonselective damages of the mitochondria thereby retaining its physiological, regu- lated function. However, when expressed at levels higher than 10 lgÆmg )1 , UCP1 was also found to promote a nonregulated uncoupling of yeast mitochondria [36]. It remains unclear why UCP1 attains a native confor- mation once expressed in yeast while UCP2 and UCP3 do not. Our data strongly suggest that this phenomenon may not be restricted to yeast but occurs also in other host cells. In the present study, low to moderate levels of UCP3 were expressed in the rat m uscle-derived L6 cell line. When the expression of UCP3 was about 0.1–0.2 lgÆmg )1 membrane protein, i.e. a concentration similar t o that f ound in skeletal muscle, no significant uncoupling could be detected. A clear increase of the nonphosphorylating respiratory activity (i.e. uncoupling) was apparent only at the highest levels of expression that resulted in a reduction of the maximal cellular respiratory capability (as measured in the presence of a strong uncoupling agent such as CCCP). Since the amount o f prohibitin per mg of membrane pr otein w as not affected, one can presume that the expression of hUCP3 did not influence the number of mitochondria per cell. It is likely therefore that the lower maximal cellular respiration reflected a general impairment of the mitochondrial func- tion caused by the presence of a noncompetent hUCP3, rather than a decrease in mitochondrial number. This hypothesis is supported by the lack of a specific effect of lauric acid and GDP, even at m M concentrations, on the membrane potential of mitochondria in cells expressing UCP3. This observation is very similar to what was previously reported using p ermeabilized yeast cells expres- sing UCP3 [25]. The membrane potential of mitochondria in permeabilized yeast cells expressing UCP1, on the other hand, was reported to be strongly inhibited b y FFAs and the effects were fully reversed by GDP [25]. To obtain further evidence t hat the UCP3 is not proper ly folded w hen expressed in L6 cells, membrane proteins of the infected cells were extracted with high concentrations of the nonionic detergent Triton X-100. This procedure h as been proposed to be a simple and stringent assay to evaluate the state of proteins localized in the inner mitochondrial membrane [31]. While hUCP3 was shown to be associated to the mitochondria by immunocyto chemical staining, the protein was resistant to solubilization by h igh nonionic detergents. Another typical mitochondrial membrane protein such as prohibitin was fully solubilized demonstrating the efficacy of the extraction procedure. What is the cause of the improper folding of UCP2 and UCP3 into the mitochondria, when their expression is forced in a host cell? A search for interacting proteins u sing a yeast-two hybrid system revealed that the C-terminals of UCP2 and UCP3 (but not that of UCP1) specifically interact with members of the 14.3.3 protein family [37]. The 14.3.3 proteins are located in the cytosol where they regulate various aspects o f cell p hysiology. The 14.3.3 proteins h ave also been named mitochondrial import stimulation factors (MSFs) because of their ability to chaperone the insertion in the mitochondrial membrane of some of the anion trans- porters. Specifically, MSFs have been shown to facilitate the docking of the precursor proteins of the mitochondrial P i and the ADP/ATP carriers on the Tom70–Tom37 complex, an import receptor localized on the outer mitochondrial membrane. One could hypothesize th erefore that UCP1 is inserted into the mitochondria without any special transport mechanism w hile UCP2 and U CP3 m ight require a specific import machinery. In the absence of sufficient amounts of specific 14.3.3 proteins, UCP2 and UCP3 might not be able to cross the intermembrane space and reach the inner mitochondrial membrane in a properly folded state. A recent in vivo experiments (based on NMR analysis) comparing UCP3 K O with w ild-type m ice, has s hown that the a mount of UCP3 exp ressed in skeletal muscle of wild- type animals, i.e. about 50–100 ngÆmg )1 mitochondrial protein, strongly affects the efficiency of oxidative phos- phorylation. It was therefore surprising that the expression of similar amounts in L6 myoblasts did not cause any relevant change in the proton leak. One could argue that, even at the lowest expression levels, the protein cannot be properly inserted into the mitochondria without a corres- ponding coexpression of some specific ancillary protein(s). Alternatively, it is possible that certain cofactors necessary for the uncoupling are missing in the cell c ulture system. 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Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells Danilo Guerini 1 , Elisabetta. esults and phenotypes obtained after overexpression of UCP3, and possibly also UCP2, in any host system (i.e. not on ly cellular but also in tissue systems and