Reconstitution of Recombinant Uncoupling Proteins

UCP1, -2, AND -3 HAVE SIMILAR AFFINITIES FOR ATP AND ARE UNAFFECTED BY COENZYME Q10*

Martin Jaburek {ddagger} and Keith D. Garlid §

From the Department of Biology, Portland State University, Portland, Oregon 97207

Received for publication, February 28, 2003 , and in revised form, April 29, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The successful development of recombinant expression and reconstitution protocols has enabled a detailed study of the transport properties and regulation of the uncoupling proteins (UCP). We optimized conditions of isolation and refolding of bacterially expressed uncoupling proteins and reexamined the transport properties and regulation of bacterially expressed UCP1, -2, and -3 reconstituted in liposomes. We show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. The Ki values for ATP inhibition were 50 µM (UCP1), 70 µM (UCP2), and 120 µM (UCP3) at pH 7.2. These affinities for ATP are similar to those obtained with native UCP1 isolated from brown adipose tissue mitochondria (Ki = 65 µM at pH 7.2). The Vmax values for proton transport were also similar among the UCPs, ranging from 8 to 20 µmol·min1·mg1, depending on experimental conditions. We also examined the effect of coenzyme Q on fatty acid-catalyzed proton flux in liposomes containing recombinant UCP1, -2, and -3. We found that coenzyme Q had no effect on the fatty acid-dependent proton transport catalyzed by any of the UCPs nor did it affect nucleotide regulation of the UCPs. We conclude that coenzyme Q is not a cofactor of UCP-mediated proton transport.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Uncoupling protein 1 (UCP1)1 of BAT mitochondria is known to dissipate energy and generate heat by catalyzing back-flux of protons into the mitochondrial matrix. This most likely occurs by a protonophoretic mechanism in which UCP transports the FA carboxylate by a flippase mechanism, and the protonated FA completes the cycle by spontaneous flip-flop through the bilayer (15). UCP2 and -3 were identified based on their sequence similarities with UCP1 (69). Evidence from in vivo studies implicates the UCPs in the etiology of type 2 diabetes (1012), in the mitigation of metabolic syndrome (13, 14), and in the mitigation of cellular damage due to reactive oxygen species (1519).

Direct evidence shows that skeletal muscle UCP3 uncouples in vivo (20); however, biochemical studies have so far failed to reveal uncoupling by UCP2 and -3 in isolated mitochondria (21, 22). Further advances in this field will require this conflict to be resolved. Our approach has been to express the UCPs in Escherichia coli followed by isolation, reconstitution, and assays of activity in liposomes. This has proven to be a useful approach for the study of mitochondrial transporters, including the oxoglutarate (23), tricarboxylate (24), citrate (25), phosphate (26), and ADP/ATP carriers (23, 27).

We used this approach in the first demonstration of fatty acid-dependent uncoupling by UCP2 and -3 (28). The anionic detergent sarcosyl was used to extract the proteins from inclusion bodies, and reconstitutively active UCP2 and -3 were obtained by dialysis of the extract in the presence of non-ionic detergent (octylpentaoxyethylene) and phospholipids. We showed that UCP2 and -3 were qualitatively identical in their transport and regulatory behavior to native UCP1. We noted, however, that the affinities of UCP2 and -3 for ATP were 5–10-fold lower than those for reconstituted, native UCP1 (28). This raised the question of whether our protocols were yielding UCPs with true native function. To address this question, we turned our attention to recombinant UCP1. The properties of native UCP1 are well established, and a successful reconstitution protocol for recombinant proteins should reproduce those properties.

We now report conditions of isolation and refolding of bacterially expressed UCP1 that lead to reconstitutively active protein that behaves identically to native UCP1. Using these protocols, we show for the first time that ATP inhibits UCP1, -2, and -3 with similar affinities. These experiments were carried out in the absence of coenzyme Q10, which has been reported to be "obligatory" for UCP function (29, 30). We examined the effects of CoQ and its solvent, dichloromethane, on FA-catalyzed proton flux in liposomes containing UCP1, -2, and -3. We found that CoQ had no effect whatever on FA-dependent proton transport catalyzed by the UCPs or on ATP regulation of the UCPs. Dichloromethane, however, had profound effects on the liposomes, as is expected of such a strong solvent.2


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of UCP1, -2, and -3 in E. coli—Rat UCP1 cDNA open reading frames were amplified by PCR and inserted into the NdeI and XhoI sites of the pET21a vector (Novagen), and human UCP2 and UCP3 containing plasmids were prepared as described previously (28). From DNA sequencing, the constructs are predicted to encode proteins with amino acid sequence identical to the wild type UCP1 (31), UCP2 (6, 7), and UCP3 (8, 9). Plasmids were transformed into the bacterial strain BL21 (Novagen). Transformed cells were grown in the presence of carbenicillin (0.1 mg/ml) at 37 °C for several hours until A600 reached 0.3–0.4. The expression of UCPs was induced with 1 mM isopropyl-{beta}-D-thiogalactopyranoside, and incubation was continued at 30 °C for 3 h. Cells were lysed in a French press, and inclusion bodies were isolated and stored at –80 °C as previously described (28).

Extraction of UCP1, -2, and -3 from Inclusion Bodies—The pelleted inclusion bodies (about 2 mg of protein) were washed 2 times in sodium salts of 0.15 M phosphate, 25 mM EDTA, 10 mM dithiothreitol, 0.2% sodium lauroylsarcosinate (sarcosyl), pH 7.8, and centrifuged 14,000 x g for 10 min. The resulting pellet was resuspended in 4 ml of 50 mM CAPS, 25 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 10% glycerol, 2% sarcosyl, pH 10.0, adjusted by Tris. After 30 min of incubation at room temperature, the extract was centrifuged 14,000 x g for 10 min to remove unsolubilized particles. The supernatant was diluted rapidly in 6 ml of 10% glycerol, 1% Triton X-114, and 1 mM ATP. This mixture was incubated for 2 h at 4 °C with constant mixing. To remove sarcosyl, the extract was either dialyzed (see "Results") or passed through a 2.5-ml Dowex 11A8 column (flow rate 0.5 ml/min). The collected protein was supplemented with 5 mg/ml phosphatidylcholine and 1 mM ATP. After 2 h of incubation at 4 °C, the extract was concentrated 2-fold in an Ultrafree-15 centrifugal filter device (Millipore). 1-ml aliquots were stored at –20 °C.

Purification of Recombinant UCP1, -2, and -3 on Hydroxyapatite (HTP) Column—The extracted protein was dialyzed twice for 3 h and once overnight at 1:100 dilution against the internal medium (potassium salts of 50 mM TES, 80 mM ,2mM EDTA, pH 7.5). The dialyzed protein extract was centrifuged at 14,000 x g for 10 min to remove any precipitate, and the supernatant was passed through 0.5 ml (0.12 g) of HTP (Bio-Gel column, Bio-Rad) that had been pre-equilibrated with internal medium. The flow-through was expected to contain properly folded uncoupling proteins.

Isolation of UCP1 from Brown Adipose Tissue Mitochondria—UCP1 was purified and reconstituted into proteoliposomes using previously described procedures (4, 32, 33). Briefly, frozen BAT mitochondria were first washed with additional bovine serum albumin (5 mg/ml) and then extracted with octylpentaoxyethylene in the presence of phospholipids. UCP1 was purified on the HTP column in a medium composed of potassium salts of 50 mM TES, 25 mM , and 2 mM EDTA. The composition of phospholipids and internal medium were adjusted for subsequent reconstitution.

Reconstitution of UCPs into Liposomes—Recombinant UCPs and native UCP1 were reconstituted in liposomes using previously described procedures (4, 28, 33). Briefly, phospholipids (asolectin supplemented with 5% cardiolipin or 100% phosphatidylcholine) were dried under nitrogen and stored under vacuum overnight. The dried phospholipids were solubilized with detergent (C8E5), and protein and fluorescent probe were added. The protein/lipid mixture was incubated with Bio-Beads SM-2 (Bio-Rad) to remove detergent and form vesicles. The vesicles were passed through a Sephadex G-50–300 column to remove external probe. Protein-free liposomes were prepared using the same protocol.

Fluorescence Measurements of Ion Fluxes—Ion fluxes in proteoliposomes were measured using an SLM Aminco 8000C spectrofluorometer. H+ ion fluxes were measured as changes in intraliposomal acid concentration, obtained from changes in SPQ fluorescence due to quenching by the anion of TES buffer (34). Transport was driven by a K+ gradient initiated by the addition of 50 nM valinomycin. The proteoliposomes were studied at 0.5 mg/ml phospholipid in 2 ml of assay media at 25 °C. Each preparation was individually calibrated for fluorescence probe response, and the internal volume of vesicles was estimated from the volume of distribution of the fluorescent probe (33). The protein content in proteoliposomes was estimated by the Amido Black procedure (35).

Chemicals and Reagents—SPQ was purchased from Molecular Probes, Inc. (Eugene, OR). Asolectin (45% L-{alpha}-phosphatidylcholine) was purchased from Avanti Polar Lipids, Inc. Sulfuric acid was purchased from Fisher. All other chemicals were from Sigma.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Optimizing Expression-Reconstitution Protocols for Recombinant UCPs—Our previous protocols enabled us to demonstrate that UCP2 and -3 catalyze FA-dependent, electrophoretic proton flux as well as electrophoretic flux of the FA analog, undecanesulfonate (28). However, ATP inhibition occurred with affinities that were10-fold lower than that for native UCP1 (28). Because there are no standards of native activity for UCP2 and -3 in intact mitochondria, it was not possible to determine whether the altered affinities are due to biochemical differences in the protein or to artifacts arising from the method. We decided that the best way to address this question was to demonstrate that our protocols gave native function for recombinant UCP1, whose activities and regulation are well characterized.

We expressed UCP1 in E. coli and carried out extraction-reconstitution using protocols adapted from those originally used for UCP2 and -3 (28). Fig. 1 (squares) shows that this preparation also gave abnormally high Ki values for ATP inhibition, about 480 µM. We therefore devoted our efforts to developing a protocol that would yield normal ATP affinity for bacterially expressed reconstituted UCP1. We found that the affinity for ATP improved with extended dialysis, the Ki decreasing to 190 µM after a total of 44 h of dialysis (Fig. 1, circles). This finding suggested that sarcosyl may decrease the affinity of UCPs for nucleotides and that residual sarcosyl was being removed by prolonged dialysis, albeit with low efficiency. Therefore we introduced an additional treatment with a Dowex anion exchange column to improve sarcosyl removal. This approach was successful. The affinities for ATP of Dowex-treated, bacterially expressed UCP1 were nearly identical to those of native UCP1, with observed Ki values of 50 µM (Fig. 1, triangles) and 65 µM (Fig. 1, crosses), respectively.



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FIG. 1.
Effect of sarcosyl removal on the affinity of UCP1 for ATP. The dependence of UCP1-catalyzed H+ transport on [ATP] was measured in the presence of 100 µM laurate and 50 nM valinomycin at pH 7.2. Recombinant UCP1 expressed in E. coli was solubilized with sarcosyl and dialyzed for 20 h ({square}, Ki = 480 µM), 44 h ({circ}, Ki = 190 µM), or treated with Dowex ({triangleup}, Ki = 50 µM) to remove sarcosyl. Native UCP1 was isolated from BAT mitochondria and was not exposed to sarcosyl (x, Ki = 65 µM). Proton efflux was driven by a K+ gradient in the presence of valinomycin and monitored as changes in SPQ fluorescence. 100% refers to H+ flux in the absence of ATP, and 0% refers to flux in the presence of 5 mM ATP. The solid curve was plotted by fitting all data to the Hill equation with the Hill coefficient = 1. The assay medium contained tetraethylammonium cation salts of MOPS buffer (50 mM), (80 mM), and EDTA (2 mM), pH 7.2. The data are representative of three independent experiments.

 

We also added an HTP column purification step to meet recent criteria for isolation of bacterially expressed UCPs (36). Based on the known behavior of native BAT UCP1, the properly folded UCPs were expected to pass through the HTP and collect in the flow-through. The recovery of protein after HTP treatment is summarized in Table I. The yield of the flow-through fraction after HTP treatment ranged from 40 to 60%. Our previous protocol (28) yielded about 20% recovery of UCP2 and UCP3 after the HTP treatment (not shown). The HTP treatment had no additional effect on the affinity of UCP1 for ATP, but it improved the apparent purity of all three UCPs, as judged by a 50% increase in the protein-specific transport rates (not shown).


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TABLE I
Protein recovery after dialysis and HTP purification

Inclusion bodies were solubilized, treated with Dowex, and concentrated as described under "Experimental Procedures." An aliquot of the protein (Starting protein) was passed through an HTP column after adjusting its medium composition by dialysis.

 

ATP Regulation of UCP1, -2, and -3—We next applied the new protocol to reconstitution of recombinant UCP2 and -3. We measured inhibition by ATP under the same conditions as those used for the UCP1 experiments of Fig. 1. Fig. 2 shows that the protocol applied to UCP2 (Fig. 2, circles) and UCP3 (Fig. 2, triangles) now yields proteins with similar affinities for ATP. In three independent experiments, the Ki values were 70 µM ± 8 µM (mean ± S.D.) for UCP2, 114 µM ± 17 µM for UCP3, and 56 µM ± 8 µM for bacterially expressed UCP1, respectively. This new result shows that UCP1, -2, and -3 have similar affinities for ATP.



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FIG. 2.
The Ki for ATP inhibition of recombinant UCP2 and -3. The dependence of UCP2- and UCP3-catalyzed H+ transport on [ATP] was measured in the presence of 100 µM laurate and 50 nM valinomycin at pH 7.2. The Ki value obtained in this experiment was 70 µM for UCP2-catalyzed H+ transport (circles) and 120 µM for UCP3-catalyzed H+ transport (triangles). The transport was measured in the presence of 100 µM laurate at pH 7.2. The experimental conditions and media composition are as described in the legend of Fig. 1. The data are representative of three independent experiments.

 

Fatty Acid-dependent Proton Transport Mediated by UCP1, -2, and -3—It is important to note that the new protocols had no effect on H+ flux catalyzed by UCP2 and -3, and we obtained the same results as were reported previously (28). Our previous experiments were carried out at pH 7.2 in vesicles composed of soy bean phospholipids supplemented with 5% cardiolipin, which provide a complex lipid environment. Because we wished to evaluate the effects of CoQ, we also carried out experiments at pH 6.8 in phosphatidylcholine vesicles, conditions similar to those used by Echtay et al. (29, 30). The representative ion flux traces in Fig. 3 show that bacterially expressed UCP1 catalyzes FA-dependent, electrophoretic proton flux in the absence of CoQ when reconstituted in the phosphatidylcholine vesicles. Laurate induces a strong H+ flux (Fig. 3, trace a) that is sensitive to ATP (trace b), being maximally inhibited by ATP on both sides of the membrane (trace c). The partial inhibition (about 50%) by external ATP demonstrates the random orientation of the nucleotide binding sites of reconstituted UCP1, which we always observe. The transport rates with 100 µM laurate in the absence of ATP were about 10 µmol·min1·mg1 protein. This rate was about half of that observed with native UCP1 when flux was measured in the opposite direction (2). This effect is due to a smaller gradient for K+ efflux than for K+ influx. Entirely similar results were obtained with UCP2 and -3 (not shown). Importantly, these experiments, like all our previous experiments, were carried out without added CoQ. They show that CoQ is not obligatory for UCP function and confirm our published finding that bacterially expressed uncoupling proteins catalyze FA-dependent, ATP-sensitive H+ transport in the absence of CoQ.



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FIG. 3.
Fatty acid-dependent H+ efflux from vesicles containing recombinant UCP1. Shown are fluorescence traces from proteoliposomes containing UCP1. SPQ fluorescence increases in response to internal acidification, because TES buffer anion quenches SPQ fluorescence. 100 µM laurate was present in each assay, and UCP-mediated uncoupling was initiated by the addition of 50 nM valinomycin. FA-dependent, electrophoretic proton flux (trace a) was partially inhibited by 5 mM external ATP (trace b) and completely inhibited when 5 mM ATP was present on both sides of the membrane (trace c). Trace d is a record from a parallel experiment in protein-free liposomes, showing that trace c can be completely accounted for by proton leak through the bilayer. The phospholipid composition of the vesicles was 100% phosphatidylcholine. Note that UCP1-mediated uncoupling did not require the addition of CoQ. The assay medium contained tetraethylammonium cation salts of MOPS buffer (50 mM), (82 mM), and EDTA (2 mM), pH 6.8. The data are representative of five independent experiments.

 

Our flux measurements are supported by extensive control experiments. We routinely measured fluxes in pure liposomes (Fig. 3, trace d), and we observed that these fluxes were nearly identical with fluxes observed in proteoliposomes containing UCP with ATP present on both sides of the membrane (Fig. 3, trace c). We also controlled for valinomycin concentration by titrating valinomycin at constant concentrations of fatty acid in both liposomes and in proteoliposomes containing UCP. This allows us to quantitate nonspecific proton transport and to choose the correct amount of valinomycin that supports the maximal ATP-sensitive rate. We find this value to be 40–80 nM (80–160 pmol·mg1 lipid). This value is more than 20 times lower than the valinomycin concentrations used by Echtay et al. (29, 30). Under our experimental conditions, concentrations of valinomycin above 200 pmol·mg1 lipid resulted in massive protein-independent fluxes, which may be due to ion pair transport of the FA anion-K+-valinomycin complex.

The ATP-sensitive proton flux rates observed in our laboratory had a Vmax for UCP1 up to 20 µmol of H+ min1·mg1 protein (4), a number that is in excellent agreement with observations in BAT mitochondria. Sundin et al. (37) measured 42 µg of UCP1/mg of hamster BAT mitochondria. The UCP1-dependent respiration when oxidizing FA was 85 ng atoms of O min1·mg1 protein, which converts to 850 nmol of H+ min1·mg1 protein. Thus, hamster BAT mitochondria also exhibit a Vmax of about 20 µmol of H+ min1·mg1 protein.

The effects of Coenzyme Q10 on UCP1, -2, and -3 Transport and Regulation—The traces in Fig. 4 show the effects of adding CoQ dissolved in dichloromethane to the assay, as was done by Echtay et al. (29). Trace a is from a control in the absence of CoQ or dichloromethane. Trace b shows the effect of CoQ addition, which indeed caused a large increase of H+ flux. Trace c shows that an identical increase in proton flux was caused by adding the solvent without CoQ. We obtained identical results with reconstituted recombinant UCP2 and -3 (not shown).



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FIG. 4.
The effects of coenzyme Q10 and dichloromethane on UCP-mediated uncoupling. Shown are fluorescence traces from proteoliposomes containing UCP1. Trace a, control trace without added CoQ or dichloromethane. Trace b, CoQ (10 nmol·mg1 lipid) dissolved in dichloromethane was added before the addition of 100 µM laurate and 50 nM valinomycin (VAL). Trace c, an equal amount of dichloromethane (4 µl) was added without CoQ. The assay conditions were otherwise identical to those described in the legend to Fig. 3. The data are representative of five independent experiments.

 

Dichloromethane is very hydrophobic and almost insoluble in water. Most of the added solvent will go into the lipid bilayer, and it is not surprising that it has profound effects. It is surprising that Echtay et al. (29) did not report any controls for solvent effects.

To determine whether CoQ had any effects on UCP activity, we compared the ATP-sensitive FA-dependent H+ fluxes in the presence and absence of CoQ and dichloromethane. We varied the concentration of CoQ from 2.5 to 25 nmol·mg1 lipid and the corresponding amount of solvent from 0.05 to 0.5% in the 2 ml of assay. As shown in Fig. 5, there was no difference between the presence (Fig. 5, filled symbols) and absence (Fig. 5, open symbols) of CoQ in these assays; all of the effect was caused by the solvent at all doses tested. Moreover, the results show that CoQ-dichloromethane caused a partial inhibition of ATP-sensitive proton flux at concentrations higher than 10 nmol CoQ·mg1 lipid. These results show that the increased fluxes in the presence of CoQ or dichloromethane were not caused by an increased activity of the protein but most likely by a solvent-induced increase in the proton permeability of the vesicles.



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FIG. 5.
CoQ does not activate uncoupling catalyzed by UCP1, UCP2, or UCP3. ATP-sensitive H+ flux is plotted versus the concentration of CoQ dissolved in dichloromethane (filled symbols) or the corresponding amount of dichloromethane only (open symbols). 100 µM laurate and 50 nM valinomycin were used to initiate H+ flux. 100% flux was determined as flux in the absence of ATP and CoQ. 0% flux was determined as flux in the presence of 5 mM external ATP. These values were determined at each concentration of CoQ.

 

We further evaluated the effects of dichloromethane in the experiments of Fig. 6, which were carried out in protein-free liposomes. It can be seen that small amounts of the solvent caused large increases in the proton permeability of vesicles when FA and valinomycin were present. This liposome control of the effect of dichloromethane was also not reported in Echtay et al. (29). Note that the assay volume used in Echtay et al. (29) was only 0.33 ml, and thus, the amount of solvent we tested was in the range of solvent amount used in their experiments.



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FIG. 6.
Dichloromethane induces H+ flux in protein-free liposomes. Traces follow changes in internal acidification, which was determined from changes in SPQ fluorescence in protein-free liposomes composed of 100% phosphatidylcholine. The addition of dichloromethane was followed by the addition of 50 µM laurate and 50 nM valinomycin (VAL). The volume of dichloromethane added to the 2 ml of assay medium was varied as noted in the figure. The assay conditions were otherwise identical to those described in the legend to Fig. 3.

 

We wanted to determine whether CoQ affects the kinetics of laurate-induced H+ fluxes. To avoid the complicating effects of adding dichloromethane to the assay, we carried out a series of experiments in which CoQ was incorporated into the phospholipids used for reconstitution. Fig. 7 contains Eadie-Hofstee plots of transport rates. For each UCP, rates were corrected for proton leak measured in protein-free liposomes, and the net rates were normalized to measured protein contents. The presence of CoQ (5 nmol·mg1 lipid) had a negligible effect on the Km or Vmax values for H+ transport catalyzed by bacterially expressed UCP1, with Km values of 37 and 36 µM in the absence or presence of CoQ, respectively (Fig. 7A). The Vmax values were 10–12 µmol·min1·mg1 protein. Similar values were obtained with native UCP1 studied under identical experimental conditions (Fig. 7A), with Km and Vmax values of 31 µM and 9 µmol·min1·mg1 protein. The presence of CoQ also had a negligible effect on the Km or Vmax values for transport catalyzed by bacterially expressed UCP2 and UCP3. For UCP2, the Km values were 75 and 78 µM in the absence or presence of CoQ, respectively, and the Vmax values were about 12 µmol·min1·mg1 protein (Fig. 7B). For UCP3, the Km values were 36 and 37 µM in the absence or presence of CoQ, respectively, and the Vmax values were about 8 µmol·min1·mg1 protein (Fig. 7C). These experiments confirm that CoQ has no effect on H+ transport mediated by UCP1, -2, or -3. Finally, we examined the effects of CoQ on the affinities of UCP1, -2, and -3 for ATP and found that there was no effect (data not shown).



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FIG. 7.
The kinetics of UCP-mediated proton transport in the presence or absence of CoQ. Concentration dependence of laurate-dependent H+ fluxes mediated by UCP1, -2, and -3 in the absence (filled symbols) or presence (open symbols) of CoQ. CoQ (5 nmol·mg1 lipid) was incorporated in the phosphatidylcholine before the formation of proteoliposomes. Panel A, UCP1. Native UCP1 isolated from BAT mitochondria was added as a control (x). Panel B, UCP2. Panel C, UCP3. Eadie-Hofstee plots were constructed for the net fluxes corrected for leak values estimated in protein-free liposomes. The Km and Vmax values obtained are given in the text. The assay conditions were otherwise identical to those described in the legend to Fig. 3.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Reconstitution of Bacterially Expressed Uncoupling Proteins—Successful reconstitution of bacterially expressed proteins is difficult, as is well illustrated by a series of papers from Klingenberg and Echtay; in 1998, they reported that proton transport by UCP1 depends on a histidine pair that is absent in UCP2 and -3, and the authors concluded that these UCPs transport protons poorly or not at all (38). In 1999, they reported that recombinant UCP3 does not catalyze proton transport upon reconstitution (39). These results led the authors to speculate that a cofactor from mitochondria was required that was missing in bacteria. Echtay et al. (29, 30) then reported that these UCPs are highly active when activated by CoQ. Moreover, they found that CoQ was obligatory for UCP function in their hands.

Our laboratory succeeded in observing UCP-mediated uncoupling by recombinant UCP2 and -3 (28); however, the apparent affinities for ATP were abnormally low. We decided to address this problem of isolation and refolding of bacterially expressed UCPs by examining the effects of protocol conditions on the Ki for ATP inhibition of UCP1-mediated proton transport, which was determined to be about 65 µM for native UCP1 at pH 7.2 (Fig. 1). When the conditions for obtaining native properties from recombinant UCP1 were achieved, we applied the same protocols to UCP2 and -3. This led to functional behavior of reconstituted recombinant UCP1, -2, and -3, which reflects all known properties of native UCP1; they pass through an HTP column, they catalyze FA-dependent H+ transport, they catalyze undecanesulfonate transport (28), and they are inhibited by ATP. We observed 50% inhibition by external ATP, indicating random orientation of bacterially expressed UCPs in the liposomal membrane, a result routinely observed with reconstituted native UCP1 isolated from BAT mitochondria (4, 40). The first important finding of this effort is that UCP1, -2, and -3 are inhibited by ATP with affinities similar to those obtained with native UCP1. This removes the uncertainties surrounding ATP inhibition of UCP-mediated uncoupling and fulfills expectations based on predictions from sequence identities in the nucleotide binding domain of the UCPs.

Table I shows that the recovery of bacterially expressed UCP1, -2, and -3 after HTP treatment is 40–50%, which may be compared with the results of Jekabsons et al. (36), who observed 20% recovery from HTP of UCP2 treated with polyoxyethylene ether detergent C12E9. This difference may be caused by differences in refolding protocols, such as the technique of detergent exchange after the solubilization of inclusion bodies by sarcosyl, the type of detergent used, or by the presence of phospholipids and ATP in our protocols, which may promote refolding.

Does Coenzyme Q10 (CoQ) Affect UCP Function?—Echtay et al. (29, 30) report that UCP2 and -3 are highly active proton transporters and are highly sensitive to nucleotides when activated by CoQ. These workers designated CoQ as an ``obligatory cofactor'' for the UCPs. This finding was in conflict with our own results because we had observed normal proton transport by UCP2 and -3 in the absence of CoQ (28); therefore, we undertook a detailed study of the effects of CoQ on UCP1, -2, and -3. We used conditions similar to those used by Echtay et al. (29, 30) but with our techniques of protein refolding and reconstitution. As summarized in Figs. 4 and 5, the apparent effects of CoQ on the FA-dependent H+ fluxes were indistinguishable from the effects of its solvent, dichloromethane. Indeed, the stimulation of H+ flux was shown to be caused by a solvent-dependent increase in H+ permeability of the vesicles, as demonstrated in Fig. 6. This control was not reported by Echtay et al. (30). In their experiments the solvent effect would be amplified by their use of excessive concentrations of valinomycin, which is more than 20-fold higher than we use. We also observed that dichloromethane is a partial inhibitor of UCP-mediated H+ transport (Fig. 5). To avoid the complications caused by adding the solvent to the assay, we also examined CoQ effects after its incorporation into the lipids used for reconstitution. Again, we observed that CoQ was without effect on transport or regulation (Fig. 7). Therefore, we conclude that CoQ is not a cofactor of UCP-mediated H+ transport. These results suggest that the previously reported effects of CoQ may be caused by solvent effects and excessive doses of valinomycin. However, the proton fluxes reported by Echtay et al. (29, 30) were found to be inhibited by nucleotides, albeit at extremely low ionic strengths. Moreover, these authors observed a 10-fold difference between CoQ0 and CoQ10. If the same volumes of solvent were used in both assays, this finding would argue against a solvent artifact. Therefore, we speculate that the need for CoQ may reflect specific experimental conditions used in Echtay et al. (29, 30), such as the use of digitonin for refolding of these bacterially expressed proteins. It was reported by Jekabsons et al. (36) that digitonin-treated bacterially expressed UCP2 did not pass through an HTP column, suggesting that digitonin treatment may not lead to proper folding of the UCPs. According to this scenario, the addition of CoQ or dichloromethane might have promoted structural changes that enabled the digitonin-treated proteins to support FA-dependent H+ transport.

Does Sarcosyl Activate UCP-mediated Uncoupling?—We infer that restoration of normal ATP inhibition by long dialysis or anion exchange chromatography is due to sarcosyl removal, although we have not demonstrated this directly. Our initial impression was that residual sarcosyl prevented the UCPs from achieving a native configuration; however, this explanation seemed unlikely in view of the fact that proton transport itself was unaffected by sarcosyl removal. Only the ATP affinity was affected. We have subsequently found that sarcosyl is a specific activator of UCP1, -2, and -3.3 That is, sarcosyl causes a reduction in ATP affinity due to specific interaction of sarcosyl with the nucleotide binding site on UCP. Thus, sarcosyl appears to be a true activator of UCP-mediated uncoupling in that it causes UCP-mediated uncoupling in the presence of inhibitory concentrations of ATP. This is a physiologically relevant definition of activation, because it addresses the question, How can the UCPs uncouple in the presence of normal cellular ATP? This effect and UCP activation by biological compounds with related functional groups will be reported in detail in subsequent communications.

Conclusions—Our results confirm that UCP2 and -3 are true uncoupling proteins and show that they are qualitatively identical in all their biophysical properties with UCP1. This result supports studies that have demonstrated uncoupling by UCP3 in vivo (20). Importantly, we find that UCP1, -2, and -3 have similar apparent affinities for ATP as an inhibitor of proton transport. We also find that coenzyme Q10 is without effect on UCP1, -2, and -3.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK56273. 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. Back

{ddagger} Present address: Dept. of Membrane Transport Biophysics, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague, Czech Republic. Back

§ To whom correspondence should be sent: Dept. of Biology, Portland State University, P. O. Box 751, Portland, OR 97207-0751. Tel.: 503-725-8969; Fax: 503-725-3888; E-mail: garlid{at}pdx.edu.

1 The abbreviations used are: UCP, uncoupling protein; BAT, brown adipose tissue; CoQ, coenzyme Q10; FA, fatty acid; HTP, hydroxyapatite; MOPS, 3-(N-morpholino)propanesulfonic acid; sarcosyl, sodium lauroylsarcosinate; SPQ, 6-methoxy-N-(3-sulfopropyl) quinolinium; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid. Back

2 A preliminary report of these results was presented in abstract form (41). Back

3 M. Jaburek and K. D. Garlid, unpublished results. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Hongfa Zhu for expert assistance.



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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