Reconstitution of Novel Mitochondrial ... - Bioscience Reports

Bioscience Reports, Vol. 22, No. 1, February 2002 ( 2002)

MINI REVIEW

Reconstitution of Novel Mitochondrial Uncoupling Proteins UCP2 and UCP3 ˇ a´cˇkova1 and Petr Jezˇek2,3 Marke´ta Z Receiûed September 13, 2001 Reconstitution of novel mitochondrial uncoupling proteins, human UCP2 and UCP3, expressed in yeast, was performed to characterize fatty acid (FA)-induced H+ efflux in the resulted proteoliposomes. We now demonstrate for the first time that representatives of physiologically abundant long chain FAs, saturated or unsaturated, activate H+ translocation in UCP2- and UCP3-proteoliposomes. Efficiency of lauric, palmitic or linoleic acid was roughly the same, but oleic acid induced faster H+ uniport. We have confirmed that ATP and GTP inhibit such FA-induced H+ uniport mediated by UCP2 and UCP3. Coenzyme Q10 did not further significantly activate the observed H+ efflux. In conclusion, careful instant reconstitution yields intact functional recombinant proteins, UCP2 and UCP3, the activity of which is comparable with UCP1. KEY WORDS: Fatty acid; mitochondria; UCP2 & 3; uncoupling protein. ABBREVIATIONS: FA, fatty acid; FCCP, carbonyl cyanide trifluoro-methoxyphenylhydrazone; HTP, hydroxylapaptite; MES, 2-[N-morpholino]ethanesulfonic acid; OctylPOE, octylpentaoxyethylene; PN, purine nucleotides; UCP1, uncoupling protein of brown adipose tissue mitochondria; UCP2, ‘‘ubiquitous’’ uncoupling protein; UCP3, skeletal muscleand brown adipose tissue-specific uncoupling protein; SPQ, 6 methoxy-N-(3sulfopropyl)quinolinium; TEA, tetraethyl ammonium; TES, N-tris [hydroxymethyl]-2amino-ethanesulfonic acid.

INTRODUCTION Novel mitochondrial uncoupling proteins UCP2, UCP3, UCP4, and BMCP (or alternatively UCP5) have attained a great interest of scientific community, since the discovery of ubiquitous UCP2 by Tartaglia’s (Gimeno et al., 1997) or Ricquier’s and Warden’s groups in 1997 (Fleury et al., 1997). Predominantly muscle-specific UCP3 was described the same year (Vidal–Puig et al., 1997; Boss et al., 1997). It was followed by recognition of two brain-specific proteins, BMCP (or UCP5, Sanchis et al., 1998) and UCP4 (Mao et al., 1999), respectively. Novel UCPs undoubtedly form 1

Institute of Physiology, Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, 14220 Prague 4, Czech Republic. 2 Depart. No. 375, Membrane Transport Biophysics, Institute of Physiology, Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, 14220 Prague 4, Czech Republic; E-mail: [email protected]. Fax: 011–4202–4752488 or 44472269 3 To whom correspondence should be addressed. 33 0144-8463兾02兾0200-0033兾0  2002 Plenum Publishing Corporation

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ˇ a´ cˇ kova and Jezˇ ek Z

a gene sub-family within the mitochondrial anion carrier gene family together with ‘‘classic’’, brown fat-specific, UCP1, and at least three distinct plant UCPs (Jezˇ ek and Urba´ nkova´ , 2000; Hana´ k and Jezˇ ek, 2001). Their common phylogenetic precursor seems to be the ancestral UCP4-like protein (Hana´ k and Jezˇ ek 2001). However, detailed physiological role of novel UCPs is still rather unknown. Participation of UCP2 or UCP3 in the regulation of body weight and in fever or other adaptive thermogenic processes has been suggested on the basis of their mRNA screening (for review see Boss et al., 2000; Ricquier and Bouillaud, 2000; Bouillaud et al., 2001). Screening revealed numerous transcriptional upregulations of novel UCPs such as by leptin (Zhou et al., 1997; Gong et al., 1997), TNFα (Faggioni et al., 1998), thyroid hormones (Jekabsons et al., 1999; Gong et al., 1997), or by fatty acids (FAs) and related compounds. The latter act via nuclear orphan receptors PPARγ (Camirand et al., 1998) or PPARβ (Chevillotte et al., 2001) and in concert with retinoic receptors (Carmona et al., 1998). Direct β 2-adrenergic transcriptional stimulation was also reported by Nagase et al. (2001). But, translational down-regulation could lead to a dispaired relationship between mRNA and protein amount in tissues (Pecqueur et al., 2001). Recently, a possible UCP2 involvement in diabetes was reported (Zhang et al., 2001; Lameloise et al., 2001; Chan et al., 2001), as well as its participation in the apoptotic pathway (Voehringer et al., 2000) or importance for cardiac function (Noma et al., 2001). Several findings on the level of organism indicated that UCP2 (Hong et al., 2001) and UCP3 (Clapham et al., 2000, Jucker, et al., 2000a, b) can indeed provide a protonophoric function in mitochondria, so they are really uncoupling proteins. On the other hand, demonstrations of uncoupling ability of novel UCPs in ûitro have not been so straightforward and putative purine nucleotide (PN) inhibition has been often sought without success. Only reports of groups of Garlid (Jabu˚ rek et al., 1999) and Klingenberg (Echtay et al., 2001) describing reconstitution of E. coliexpressed UCP2 or UCP3 have demonstrated substantial activity. E. coli-expressed UCP2 or UCP3 exhibited reconstituted uncoupling, i.e. protonophoric, activity in the presence of FAs and this was weakly inhibited by ATP (Ki of 0.6 mM) and by GTP (Ki of 0.7 mM (Jabu˚ rek et al., 1999). Alkylsulfonate uniport was also observed. Kis measured by Echtay et al. (2001) were lower (∼0.1 mM) since the opposite direction of H+ uniport (uptake) was followed in essentially salt free media. It is known that sulfate used by the former group diminishes nucleotide binding to UCP1 (Klingenberg and Huang 1999). Each group is also interpreting differently the mechanism, how H+ are translocated via UCPs. Garlid and colleagues have brought several evidences to support the fatty acid cycling hypothesis (for UCP1 see Garlid et al., 1996; Jabu˚ rek et al., 2001), originally developed by Skulachev (1991). UCPs are supposed to mediate a uniport of anionic fatty acid. After its translocation, FA anion is protonated on a trans-side and flips back across the membrane in the protonated form, so it carries H+ across the membrane but not across the UCP molecule. Klingenberg’s group is promoting the local buffering mechanism, when FAs accumulated by not yet defined manner at the mouth of a putative H+-conducting pathway help to focus H+ to enter (exit) the pathway (Klingenberg and Huang 1999). Echtay et al. (2001) also claim

Mitochondrial UCP2 and UCP3

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that cofactors like Coenzyme Q10 (Ubiquinon 50) are required to keep the conformation or stability of the recombinant UCP2 and UCP3 which are expressed in E. coli. These proteins are indeed obtained by lauroylsarcosinate solubilization from inclusion bodies, in which their conformation might not match the native one. It is also considered that CoQ10 might even be a part of a translocation mechanism (Echtay et al., 2000). Taking into account only the above reports, one would consider novel UCPs as quite similar to UCP1. On the contrary yeast expression system for UCP2 and UCP3 encountered some obstacles and many contradicting results were reported with yeast expressed UCPs. Stuart et al. (2001) even concluded that UCP2 does not possess uncoupling function. Higher uncoupling was reported for short splicing isoform UCP3S (Hinz et al., 1999); little effects of UCP3L on the state 4 mitochondrial respiration was observed (Zhang et al., 1999), as well as no stimulatory effects of FAs (Hagen et al., 1999; 2000; Hagen and Lowell, 2000). FA activation was reported to be restored in chimeric UCP3 having its 2nd repeat replaced by the corresponding UCP1 sequence (Hagen and Lowell, 2000). Rial et al. (1999) claimed that UCP2 is specifically activated (state 4 respiration in yeast mitochondria is stimulated) by all-trans-retinoic acid and that it is insensitive to palmitic acid, 9-cis-retinoic acid and other FAs tested, including arachidonic, linoleic, docosahexaenoic acid, and COOH-bearing prostaglandin E2. It is difficult to imagine a physiochemical basis for such a strange FA specificity, which is incompatible with previous surveys of various FAs interacting with UCP1 (Jezˇ ek et al., 1997a, b). A dearranged state of recombinant UCP3 was supposed to result from yeast expression. Winkler et al. (2001) have demonstrated that with highly expressing vectors extramitochondrial like-inclusion bodies are formed also in yeast cells which makes the use of the expressed proteins difficult. In our work we have tried to resolve these controversial issues and employed a classic low-expression vector, pCGS110 (Murdza-Inglis et al., 1991), while attempting to study reconstituted recombinant yeast-expressed UCP2 and UCP3 in their partially purified (but highly active) form. Recombinant UCP2 or UCP3 were purified on hydroxylapatite with lipid protection, hence any stimulating factors, if required, were transferred from yeast mitochondria. We found that all tested natural long chain FAs activate H+ translocation in UCP2- or UCP-3 proteoliposomes. This was inhibited up to 50% by ATP and GTP and completely inhibited by undecanesulfonate and hexafluorophosphate.

EXPERIMENTAL PROCEDURES Most of the chemicals were purchased from Sigma, St Louis, MO, USA. Hydroxylapatite, BIO-GEL HTP and Bio-Beads SM2 were from Bio-Rad, Richmond, VA, USA. Octylpentaoxyethylene (OctylPOE) was from Bachem Feinchemikalien, Bubendorf, Switzerland. Materials for reconstitution were the same as described elsewhere (Zˇ a´ cˇ kova´ et al., 2000), materials for yeast fermentation were from Difco, Zymolyase 100T was from ICN. All other chemicals were of a reagent grade.


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Reconstitution of Yeast-expressed UCP2, UCP3 W303 yeast containing pCGS110 (or pYES) vectors with inserted cDNAs coding for human UCP2 and human UCP3 under control of inducible Gal 1 promoter and ura− selection were donated by Ruth E. Gimeno and Louis A. Tartaglia (Millennium Pharmaceuticals, Inc., Cambridge, MA; see Gimeno et al., 1997). cDNA coding for UCP1 in pCGS110 vector contained in JB516 yeast was from Prof. Karl Freeman (McMaster University, Hamilton, Ontario, Canada; MurzdaInglis et al., 1991). Yeast were grown on ura− selective plates and were inoculated into 4 to 8 Erlenmayer 250 ml flasks with medium containing 203 mM (1.5 vol%) lactate, 0.05% glucose, 0.17% yeast nitrogen base (Difco) 0.5% ammonium sulfate, 0.005% each of L-amino acids (tryptophan, methionine, arginine, leucine, histidine) and adeninesulfate. After 24 hours, 0.2% galactose was added and cells were shaken for another 24–28 h until the optical density of 1 was achieved. Mitochondria were prepared immediately after terminating fermentation using Zymolyase 100T (ICN) to cleave the cell wall (Murzda-Inglis et al., 1991). Reconstitution with lipid protection was adopted from Klingenberg and Winkler (1985) to comply with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) fluorescent monitoring of ion fluxes (Jezˇ ek et al., 1994; Garlid et al., 1996; Zˇ a´ cˇ kova´ et al., 2000). It included OctylPOE extraction of yeast mitochondria (usually 30 mg protein), isolation step on HTP, detergent removal on Bio-Beads SM2 and external probe washing on Sephadex G25-300. The total amount of added lipid was 40.7 mg (egg yolk lecithin, type XI-E, Sigma, 4% cardiolipin and 1.6% L-α -phosphatidic acid). Additional lipids, up to 12 mg, could originate from mitochondria in 1 ml of final suspension. The internal medium in liposomes contained 84.4 mM TEA2SO4, 28.8 mM TEA-TES with 9.2 mM TEA, pH 7.2, 0.6 mM TEA-EGTA. The Amidoblack method served to estimate the liposomal protein content (Kaplan and Pedersen, 1985). The UCP2 (UCP3) content in HTP-passthrough was verified by MALDI-mass spectroscopy analysis when peptide mapping was performed after ingel trypsine cleavage of the PAGE-separated acetone-precipitated samples. Valinomycin-induced H+ fluxes in the presence of various FAs were monitored by the SPQ quenching method (Jezˇ ek et al., 1994; Garlid et al., 1996; Zˇ a´ cˇ kova´ et al., 2000) with 2 mM SPQ internally. Ethanolic solutions of FAs were added to 25 µl (∼1.3 mg) of vesicles in 2 ml of external medium (84.4 mM K2SO4, 28.8 mM TEATES with 9.2 mM TEA, pH 7.2, and 0.6 mM TEA-EGTA) and H+ efflux was initiated by 0.1 µM valinomycin. For decreasing or increasing external pH the external medium was replaced with either 84.4 mM K2SO4, 0.6 mM TEA-EGTA, 28.8 mM TEA-MES (9.2 mM TEA), pH 6; or with 84.4 mM K2SO4, 0.6 mM TEAEGTA, 28.8 mM Tris (TES salt), pH 8.2, respectively. Fluorescence was monitored on a fluorometer RF5301 PC (Shimadzu, Japan), equipped with a Polaroid polarization filters in cross-orientation in order to decrease light scattering. Fluorescence was calibrated to [H+] and H+ flux rates were calculated as previously described (Zˇ a´ cˇ kova´ et al., 2000). The internal volume was calculated from SPQ volume distribution (Jezˇ ek et al., 1994; Garlid et al., 1996) assuming the total lipid content of 52.7 mg (∼12 mg lipids originated from 30 of mitochondrial protein) in 1 ml (1.3 mg in the assay).


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RESULTS +

H Efflux Induced by Lauric Acid Proteoliposomes Containing UCP2 Figure 1 illustrates monitoring of H+ fluxes induced by K+ diffusion potential in the presence of 200 µM lauric acid in proteoliposomes containing the recombinant uncoupling protein UCP2. Upon addition of 200 µM lauric acid and 0.1 µM valinomycin, UCP2 was able to mediate H+ efflux observed as quenching of SPQ fluorescence. Figure 1 shows intraliposomal H+ monitoring by SPQ, i.e., the time courses of internal [H+] changes (in mM — ‘‘H+ traces’’ — obtained from the fluorescence traces by calibration. The lauric acid addition leads to the interior acidification of vesicles, called flip-flop acidification, reflecting the redistribution of FA molecules in both leaflets of lipid bilayer (Jezˇ ek et al., 1997a, b). On the contrary, 12hydroxylauric acid, which is unable to flip-flop across the lipid bilayer (Jezˇ ek et al., 1997a, b) induced no flip-flop acidification neither any significant H+ efflux (not shown). Slow basal H+ efflux was observed, when no fatty acid but 0.1 µM valinomycin was added (Fig. 1b). Similar slow H+ efflux is observed in liposomes lacking UCP2 (Table 1). Due to different volume of liposomes, flux densities should be compared and these are smaller for basal FA-induced H+ efflux in liposomes than for reconstituted UCP2 (Table 1). Inhibition of UCP2-mediated H+ Transport by Hydrophobic Anions Undecanesulfonate as one of alkylsulfonates translocated by UCP1 (Garlid et al., 1996, Jabu˚ rek et al., 2001) or by UCP2 or UCP3 (Jabu˚ rek et al., 1999) inhibited the lauric-acid-induced H+ efflux in UCP2-containing proteoliposomes with Ki around 250 µM, but not the FCCP-mediated H+ efflux (not shown). The mutual competition of undecanesulfonate and FAs was previously indicated for UCP1 by kinetic experiments (Garlid et al., 1996). However, in higher concentrations (>0.5 mM), undanesulfonate effect might be non-specific, as found for the reconstituted phosphate carrier (Engstova´ et al., 2001). Another translocation substrate of UCP1, hexafluorophosphate (F6Pi), (Jezˇ ek and Garlid, 1990) was found to completely inhibit at 0.5 mM concentration the UCP2-mediated FA-induced H+ efflux (Fig. 1a). However, we should consider this inhibition by such a hydrophobic anion as nonselective, since hexafluorophosphate was able to completely block the FAinduced H+ efflux mediated not only by UCP2, but also by the other carriers tested, such as UCP1 (natural hamster brown adipose tissue UCP1, or recombinant UCP1, yeast expressed), the recombinant yeast phosphate carrier or the partially isolated ADP兾ATP carrier. In turn, methylene-diphosphonate (MDPh), a specific inhibitor of the phosphate carrier-mediated FA cycling (Zˇ a´ cˇ kova´ et al., 2000; Engstova´ et al., 2001), did not inhibit presumed FA cycling via UCP2 (not shown). Nucleotide Inhibition of UCP2 — Mediated Fatty Acid-induced H+ Uniport Higher concentrations of ATP or GTP inhibited up to about 50% the lauric acid-induced H+ efflux in proteoliposomes containing UCP2 (Fig. 1c, d, e, f). As for UCP1 we explain this on the basis of uniform distribution of UCPn molecules with


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Fig. 1. Lauric acid-induced H+ fluxes in proteoliposomes with reconstituted UCP2. (a) control and inhibition by hexafluorophosphate (‘‘CF6Pi’’); (b) absence of lauric acid; (c) inhibition by 1.25 mM ATP or (d) by 1.25 mM GTP; (e) measured at external pH 6 or (f) pH 8.2. Traces of intraliposomal H+ monitoring by SPQ are shown in proteoliposomes equilibrated with 200 µM lauric acid (‘‘LA’’). Lauric acid addition leads to so-called flip-flop acidification, resulting from establishing equilibrium with regards to both membrane and acidbase-equilibrium (Jezˇ ek et al., 1997a, b). Subsequent addition of 0.1 µM valinomycin (’Val’) leads to internal alkalinization indicating H+ efflux. This H+ efflux was fully inhibited by hexafluorophosphate (a) and up to ∼50% by ATP (c, e, f) and GP (d). Flux densities in 10−6 pmol H+ s−1 µm−2 were estimated as follows: (a) control: 75; CF6Pi: 6; (b) no FA: 11; (c) CATP: 40; (d) CGTP: 36; (e) pH 6 control: 55 CATP: 26; (f) pH 8.2 control: 68 CATP: 44.


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Table I. Comparison of Typical Fatty Acid-induced H+ Fluxes in Liposomes and in Proteoliposomes with Reconstituted UCP2 or UCP3

Fatty acid: Lauric acid Palmitic acid Oleic acid Linoleic acid No fatty acid

Valinomycin-induced H+ efflux without protein: flux density at 200 µM FA (10−6 pmol H+兾s−1兾µm−1

Valinomycin-induced H+ efflux with UCP2: flux density at 200 µM FA (10−6 pmol H+兾s−1兾µm−1)

Valinomycin-induced H+ efflux with UCP3: flux density at 200 µM FA (10−6 pmol H+兾s−1兾µm−1

27 17 39 19 —

70 69 160 66 13

80 98 173 n.d. 12

nucleotide binding sites exposed outside and inside (Jezˇ ek et al., 1990, 1994; Garlid et al., 1996). Indeed nucleotides added to both sides inhibit nearly completely (not shown). Stock solutions of nucleotides, that are usually acidic, had to be buffered by Tris base and assay pH was carefully checked to be constant at pH 7.2 (pH of the usual external medium), otherwise, the artificial inhibition’’ was observed due to the decrease in rates by acidic pH. This could be even an origin of a discrepancy, when for example Echtay et al., 2001) consider nucleotide inhibition as 100% and orientation of UCP molecules as uniformly oriented outward, but they use non-buffered external media in which a part of the effect could be due to the decrease of transport rate caused by a pH change. Indeed, the observed H+ efflux rates were lower in any external pH that differed from the internal pH 7.2. Thus, at external pH 6 or 8.2 (while the interior pH was kept at 7.2) slower rates of H+ efflux were observed (Fig. 1e, f). Nevertheless, 1 mM ATP inhibited by the same strength at pH 6 and with lower strength at pH 8.2 (Fig. 1e, f). No Effect of Coenzyme Q10 on UCP2-mediated H+ Efflux Induced by Lauric Acid We have also tested the effect of presumed activating cofactor, Coenzyme Q10, on the observed lauric acid-induced H+ fluxes mediated by UCP2. No significant effect of oxidized CoQ10 (1 to 5 µM) was observed when added directly to the assay, nor when oxidized CoQ10 was added to lipids during extraction and formation of vesicles as compared to control vesicles. These results do not entirely exclude the activating effect of CoQ10 on UCPs, since in our own experiments when using E. coliexpressed proteins 1 µM CoQ10 at 100 µM lauric acid activated UCP2 to 132%. Nevertheless, when using yeast expression, the effective CoQ10 dose could be extracted from yeast mitochondria together with recombinant proteins (Echtay et al., 2000). All these facts document that in our reconstituted system the recombinant UCP2 protein is intact and does not need further activation other than by fatty acids. The used lipid-protection seems to have a key role in this. Activation of UCP-2 mediated H+ Uniport by Various Fatty Acids The H+ fluxes were also observed in UCP2-proteoliposomes when activated by other FAs such as palmitic acid, oleic acid, linoleic acid, or heptylbenzoic acid (Fig.


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Fig. 2. H+ efflux in UCP-2 proteoliposomes activated by various fatty acids. Activation of H+ fluxes by 200 µM fatty acids in proteoliposomes (1.3 mg) containing UCP2 and their inhibition by 2.5 mM ATP is illustrated for palmitic, oleic, linoleic, lauric and heptylbenzoic (‘‘HBA’’) acid. The arrows indicate additions of FA and 0.1 µM valinomycin, respectively. Flux densities in 10−6 pmol H+ s−1 µm−2 and lower estimates of turnover per dimer (in parentheses) were as follows: palmitic acid: 70 (50 −1) or 37 (27 s−1) with ATP; oleic acid: 124 (85 s−1) or 82 (52 s−1) with ATP; linoleic acid: 60 (83 s−1) or 27 (37 s−1) with ATP; lauric acid: 73 (52 s−1); and heptylbenzoic acid: 39 (27 s−1). The protein in 1 ml of vesicles was originating from 30 mg of yeast mitochondrial protein.

2). The calculated flux densities for UCP2-mediated transport were higher than flux densities observed for the same FAs in liposomes lacking protein (Table 1). Considering our interpretation describing the observed FA-induced H+ efflux as FA cycling, we can conclude that it is not restricted to a specific FA, exclusively interacting with UCP2 as claimed for all-trans-retinoic acid by Rial et al. (1999), but to all FAs which are able to flip-flop across the lipid bilayer. Indeed, we can assume that all unipolar hydrophobic anions such as anionic FAs could be translocated via UCP2 and while flipping back across the membrane in a protonated form they could conduct H+.


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Fig. 3. Lauric acid-induced H+ fluxes in proteoliposomes with reconstituted UCP3. Control and inhibition by 1.25 mM ATP. Intraliposomal H+ monitoring as in Fig. 1 is illustrated for proteoliposomes (1.3 mg) containing UCP3 (of 30 mg yeast protein passed via HTP) with 200 µM lauric acid (‘‘LA’’) and 0.1 µM valinomycin (‘‘Val’’). This H+ efflux was inhibited up to 50% by ATP. Flux densities in 10−6 ppmol H+ s−1 µM−2 and lower estimates of turnover per dimer (in parentheses) were: in control 87 (58 s−1); with ATP: 55 (37 s−1). HBA, hepthylbenzoic acid.

H+ Uniport Activated by Various Fatty Acids in UCP3-proteoliposomes Similarly as for UCP2, we have observed FA-activated H+ efflux in proteoliposomes containing UCP3 when induced by K+ diffusion potential in the presence of 200 µM lauric acid (Fig. 3) or other FAs such as palmitic and oleic acid (Table 1). The observed H+ efflux was also inhibited up to about 50% by ATP (Fig. 3) or GTP. DISCUSSION In this work we have elucidated transport biochemistry of novel uncoupling proteins UCP2 and UCP3. Our data obtained with the reconstituted recombinant UCP2 and UCP3, expressed in yeast, represent their estimated phenotypes. We have confirmed that ATP and GTP inhibit the FA-induced H+ uniport mediated by these proteins. We have also shown that representatives of physiologically abundant long chain FAs, saturated or unsaturated, activate H+ translocation in UCP2- and UCP3proteoliposomes. The efficiency of lauric, palmitic, and linoleic acid was roughly the same, oleic acid induced faster H+ uniport. Coenzyme Q10 did not further significantly activate the observed H+ uniport. All these findings suggest that in our reconstituted system the recombinant UCP2 and UCP3 proteins are intact and do not need further activation other than by fatty acids. The used lipid-protection had most probably the key role in preservation of their activity. We have partially elucidated the major contradictions in the research of novel UCPs which frequently reported the opposite results with reconstituted recombinant UCPs and in studies on mitochondrial or cellular level. For example, no purine nucleotide inhibition and binding has been detected in presumably UCP2- or UCP3 rich mitochondria from UCP1-knockout mice (Matthias et al., 1999), which were otherwise uncoupled by FAs. Discrepancies related to mitochondria studies could be explained by the existing translational down-regulation (Pecqueur et al., 2001) — simply the expected UCP2 or UCP3 content as judged from mRNA amount might not be present. On the contrary, positive evidences were found for protonophoric role of UCP2 (Hong et al., 2001) or UCP3 (Clapham et al., 2000, Jucker et al., 2000a, b) in ûiûo. On the other hand, Brand and colleagues (Stuart et al., 2001) even concluded that UCP2 does not possess uncoupling function. Problems could be introduced by the use of highly overexpressing vectors, since even in yeast the

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inclusion-like bodies were formed (Winkler et al., 2001). Also the first reconstitutions of UCP3 by Klingenberg’s group did not exhibit a FA-induced H+ transport (Echtay et al., 1999). Later, they claimed that with E. coli-expressed UCP2 and UCP3, only activation by Coenzyme Q10 can result in active proteins (Echtay et al., 2001), whereas Jabu˚ rek et al. (1999) did not need such a cofactor to observe the substantial ATP and GTP-sensitive H+ uniport. Our results have shown that phenotypes of UCP2 and UCP3 concerning with protonophoric activity do not differ qualitatively from the UCP1 phenotype. Evaluated minimum turnovers per dimer (based upon the total protein present) fall into the range of 50–100 s−1. It is complying with the turnover of 94 s−1 that can be derived from the rate of 86.7 µmol−1 (mg protein)−1 for lauric acid at 25°C and CoQ10-activated E. coli-expressed UCP2 (Echtay et al., 2001). Also maximum turnover per dimer reported for UCP1 as 133 s−1 (Echtay et al., 2000) is comparable, since we did not estimate Vmax. Similar values were found for plant UCP (PUMP) — 99 s−1 (Jezˇ ek et al., 1997c). The inhibition by PN required higher doses in our reconstituted system. We must admit that sulfate used in our assay media strongly decreases the nucleotide affinity to UCP2 (Klingenberg and Huang, 1999), perhaps also to UCP2, UCP3, which might explain discrepancies between our measurements and those made by Klingenberg’s group (Echtay et al., 2001). The latter authors also study H+ uptake and not the H+ efflux. We chose sulfate to ensure that the anion used is membrane impermeant and does not quench the SPQ probe. Sacrificing the efficiency of PN inhibition, we ensured high K+ diffusion potential and the correct probe response. Our media may, however, better simulate in ûiûo conditions and exclude any artefactual inhibition by pH change. Our data have also shown that representatives of physiologically abundant FAs are able to activate both UCP2 and UCP3. Hence, there is most likely no preferential FA as a substrate and兾or activator for these proteins. We cannot explain why Rial et al. (1999) could not find any response with all their tested FAs but all-transretinoic acid in yeast mitochondria of yeast expressing UCP2. Nevertheless, we have undoubtedly demonstrated that for example palmitic acid activates both UCP2 and UCP3 in the reconstituted system as well as oleic and linoleic acid. Our findings have implications for molecular physiology of novel UCPs. First, the cellular ATP concentrations are within the range in which UCP2- or UCP3mediated FA-induced uncoupling can be blocked. At present, we do not know why PN-sensitive FA-induced uncoupling was not found in mitochondria from the tissues where UCP2 or UCP3 expression is indicated, such as brain and skeletal muscle (Jezˇ ek et al., unpublished); or from UCP1-depleted brown fat (Matthias et al., 1999). Either, a very low amount of actually expressed protein could explain this, or we might speculate about the existence of other types of regulations, which can disable PN inhibition of UCP2 or UCP3. These speculative regulations could have character of a ligand-gated regulation, regulation by a cofactor such as Coenzyme Q10 (Echtay et al., 2000, 2001), or covalent modifications (phosphorylation, glycosylation, etc.). The three main physiological roles can be predicted for novel UCPs in nonthermogenic tissues, similarly as for plant UCPs. The first one lies in the ability of UCPs to partially uncouple mitochondria in the presence of FAs and thus slightly enhance


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respiration rate and the concomitant metabolic rate. This is beneficial to many physiological processes and for prevention of reactive oxygen species (Skulachev, 1998). The latter represents the second main role. The third main role lies in the inevitable mild thermogenesis concomitant to the mild uncoupling. We have clearly shown that the recombinant UCP2 and UCP3 were able to mediate H+ transport in the presence of FAs that proceeds most probably by the FA cycling mechanism. Under circumstances when not all FAs are metabolized, certain non-zero uncoupled state in all types of mitochondria containing non-zero amounts of expressed UCP2 or UCP3 can exist. Such a weak uncoupling state is enabled by the lower affinity of UCP2 and UCP3 to purine nucleotides, namely to ATP. When the yet unknown regulatory mechanisms allow, UCPs could be fully activated (PN inhibition could be completely released). This activation should lead to the more coupled state or to the complete uncoupling, that would represent the specific physiological roles of UCP2 or UCP3 in different tissues, such as tissue-specific adaptive thermogenesis. It is, however, unlikely that PN inhibition is the only mechanism that restricts for example UCP2 function in the beating heart, since under certain physiological situations leading to full uncoupling, the consequences would be fatal. On the contrary, one could speculate that in order to reach the open state of UCPs, a putative modulator of PN inhibition would be required. ACKNOWLEDGMENTS Excellent technical assistance of Jana Brucknerova´ and Jana Kosˇarˇova´ is gratefully acknowledged as well as help of Petr Hana´ k, M.S., with yeast expression and of Eva Urba´ nkova´ , M.S., with the computer program. The project was supported by the grants of the program Kontakt from the Czech Ministry of Education (ME 389) and by the Internal Grant Agency of the Academy of Sciences of the Czech Republic (No. A5011106). Fluorometer was purchased from the funds of the CzechU.S. Science and Technology Program, grant No. 86043. REFERENCES Boss, O. et al. (1997) Uncoupling protein-3: a new member of mitochondrial carrier family with tissue specific expression. FEBS Lett. 408:39–42. Boss, O., Hagen, T., and Lowell, B. B. (2000) Uncoupling proteins 2 and 3: potential regulators of mitochondrial energy metabolism. Diabetes 49:143–156. Bouillaud, F., Couplan, E., Pecqueur, C., and Ricquier, D. (2001) Homologues of the uncoupling protein from brown adipose tissue (UCP1): UCP2, UCP3, BMCP1 and UCP4, Biochim. Biophys. Acta 1504:107–119. Camirand, A., Marie, V., Rabelo, R., and Silva, J.E. (1998) Thiazolinediones stimulate uncoupling protein-2-expression in cell lines representing white and brown adipose tissue and skeletal muscle. Endocrinol. 139:428–431. Carmona, M. C. et al. (1998) 9-cis Retinoic acid induces the expression of the uncoupling protein-2 gene in brown adipocytes. FEBS Lett. 441:447–450. Chan, C. B. et al. (2001) Increased uncoupling protein-2 levels in β-cells associated with impaired glucosestimulated insulin secretion. Mechanisms of action, Diabetes 50:1302–1310. Chevillote, E., Rieusset, J., Roques, M., Desage, M., and Vidal, H. (2001) The regulation of uncoupling protein-2 gene expression by ω-6 polyunsaturated fatty acids in human skeletal muscle cells involves

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