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. 2001 Feb 13;98(4):1416-21.
doi: 10.1073/pnas.98.4.1416.

Uncoupling proteins 2 and 3 are highly active H(+) transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

K S Echtay  1 , E Winkler, K Frischmuth, M Klingenberg
Affiliations

Uncoupling proteins 2 and 3 are highly active H(+) transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

K S Echtay et al. Proc Natl Acad Sci U S A. .

Abstract

Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H(+) transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609-613] we show here that UCP2 and UCP3 are also highly active H(+) transporters and require CoQ and fatty acid for H(+) transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H(+) from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2'-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl(-) but no H(+) are transported. The addition of CoQ initiates H(+) transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H(+) transporters.

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Figures

Figure 1
Figure 1
Fluorescence response of [2′-O-(dimethylamino)-naphthaline-1-sulfonyl]-GTP (dansyl-GTP) binding to digitonin-treated IB-UCP2 and IB-UCP3. Dansyl-GTP (5 μM) was added to a solution of 45 μg protein/ml in 10 mM Mes/Hepes buffer containing 0.3% digitonin (pH 6.5) at 10°C. To differentiate the UCP-linked binding, 0.5 mM ATP was added to displace the fluorescent ligand. For a further discrimination, the binding site was inactivated by Woodward reagent K (WRK). WRK at a concentration of 10 μM was incubated with the protein for 30 min before the addition of dansyl-GTP. Fluorescence was observed at λexc = 350 nm and λem = 525 nm.
Figure 2
Figure 2
Activation of H+ transport in UCP2 and UCP3 by CoQ10. H+ influx into phospholipid vesicles reconstituted with digitonin-treated IB-UCP2 and IB-UCP3 was recorded. (A) Recordings of H+ uptake in reconstituted UCP liposomes in the presence of CoQ10 (2 nmol) and absence of LA, with LA (125 μM) and without CoQ10, with CoQ10 and LA, and the inhibition with 20 μM ATP. (B) Evaluated H+ transport rates. H+ influx was measured as the change in external pH monitored by pyranine fluorescence at λex = 467 nm and λem = 510 nm in the presence of 125 μM LA. In all experiments (A and B) a 50-μl portion of vesicles containing 1.3 μg protein and 420 μg phospholipid was added to 0.5 mM Hepes buffer (pH 7.3) containing 1 μM pyranine, 0.5 mM EDTA, and 280 mM sucrose to a final volume of 330 μl at pH 6.8 and 10°C. H2SO4 was added in steps of 10 nmol H+ to adjust the pH to 6.8, and valinomycin (Val) was added to a concentration of 2.5 μM. Uncoupling by 1 μM carbonylcyanide m-chlorophenylhydrazone determined the capacity of H+ uptake of the vesicles. Results are presented as the initial H+ transport rate (V: μmol/min mg protein) divided by the capacity of the vesicles (C: μmol/ml).
Figure 3
Figure 3
Dependence of H+ transport by UCP2 and UCP3 on the dose of CoQ and FA. H+ transport activity measured with IB-UCP1, -2, and -3. (A) Titration of activity with CoQ10 (nmol/0.4 mg phospholipid). H+ transport of IB-UCP in reconstituted vesicles is measured as shown in Fig. 2 with an increasing amount of CoQ at saturating concentration of lauric acid (125 μM). (B) Titration with lauric acid at 2 nmol CoQ. The net transport activity given was obtained by subtracting the activity in the presence of 20 μM ATP.
Figure 4
Figure 4
Inhibition of H+ transport by UCP1, -2, and -3 from E. coli by ATP and ADP after stimulation with CoQ and FA. H+ transport measurements were performed as described in the legend to Fig. 2 at fixed amounts of lauric acid (125 μM) and CoQ (2 nmol). The KI values for ATP and ADP inhibition are contained in Table 1. The values for UCP1 are from (42).
Figure 5
Figure 5
Tentative mechanism of the cofactor role of CoQ as a catalyst of FA-mediated H+ transfer into UCP. FAH forms a H-bond with the quinone oxo group bound to UCP. The FAH-CoQ complex facilitates the delivery of the FA carboxyl to UCP, where it donates H+ to an acceptor group. The FA− returns to the surface and diffuses back to the membrane, where it collects another H+. The light shaded area corresponds to phospholipids and the dark shaded area to UCP protein.
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