Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and import into mitochondrial membrane

Vol. 167, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 16, 1990 Pages 784-789

EXPRESSION OF THE BROWN FAT MITOCHONDRIA UNCOUPLING PROTEIN IN XENOPUS

OOCYTES AND IMPORT INTO MITOCHONDRIAL MEMBRANE

Susanne Klaus, Louis Casteilla, Frederic Bouillaud, Serge Raimbault, and Daniel Ricquier

Centre National de la Recherche Scientifique

Centre de Recherches sur la Nutrition, 9, rue Jules Hetzel

F-921 90 Meudon-Bellevue, France

Received January 23, 1990

Non shivering thermogenesis of brown adipose tissue is due to the uncoupling protein (UCP), located in the inner mitochondrial membrane, which functions as a proton translocator and can thus uncouple mitochondrial respiration. We describe here the expression of UCP in Xenopus laevis oocytes after injection of UCP mRNA, which was transcribed in vitro. UCP seems to be correctly transported into mitochondria and integrated into the membrane, but we were not able to establish definitely the functionality of this UCP. We conclude that this expression system could be suitable for the study of the mitochondrial import mechanism but not for the examination of physiological properties of UCP. 01990 Academic mess, 1°C.

The mitochondrial uncoupling protein (UCP) is responsible for the heat dissipating ability of

brown adipose tissue (BAT) by its function as a proton translocator and thus its ability to

uncouple mitochondrial respiration from ATP synthesis. This 33 kD protein is located ex-

clusively in mitochondria of BAT and spans as a dimer the inner mitochondrial membrane. Its

proton translocating activity is regulated positively by fatty acids and negatively by purine

di- or triphosphate nucleotides (1). UCP is coded for by a nuclear gene which seems to exist

as only one copy and is expressed only in BAT (2,3). Its primary sequence (4,5,6) shows

that UCP belongs to a protein family including the ADP/ATP carrier (AAC)(G) and the

mitochondrial phosphate carrier (7), which are mitochondrial carrier proteins, and a very

recently identified protein called hML-7, whose function is not yet known (8). Like AAC and

hML-7 this mitochondrial protein does not possess a precursor with a transient, targeting

signal sequence (4,5) and despite some studies on AAC and UCP import (9,iO) the regions

implicated in the import are not yet well determined. Moreover, the molecular mechanism of

the proton translocation and its regulation by nucleotides and fatty acids are not yet well

understood and no molecular study has been made to characterize them. The integration of

UCP into the inner mitochondrial membrane as a prerequisite for its physiological function

complicates the study of functional domains of UCP. We have tried to find an expression

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system for UCP, able to process correctly the protein and containing numerous

mitochondria, in order to study UCP import in vitro and eventually to elucidate the

relationships between UCP structure and function. Among different expression sytems,

Xenopus oocytes are a useful tool for the study of protein expression and function. They do

not only translate injected mRNAs very efficiently, but they also carry out correctly

numerous post-translational modifications of proteins and can direct them into the correct

intracellular membranes (11). In this study we describe the expression of UCP in Xenopus

oocytes and its import into oocyte mitochondrial membranes. To our knowledge this system

has not yet been used for the study of mitochondrial membrane carrier proteins. It is also the

first report of UCP expression in an eukaryotic expression system.

MATERIALS AND METHODS

uncoupling orotein mRNA: The complete UCP cDNA was cloned into pTZ 19 (Pharmacia) behind the promoter T7 (fig. la). The plasmid was linearized by Eco Rl and transcription in vitro was performed with T7 polymerase using the mCAP kit from Stratagene according to the instructions. Functionality of the in vitro transcription product was tested by per- forming a cell-free in vitro translation using a kit from Bethesda Research Laboratories. The translation products were analyzed by SDS PAGE and autoradiography. Isolation and injection of oocytes; Oocytes were obtained from female Xenopus lawis kept in a watertank at 15 “C. Animals were put in icewater for anaesthesia, and lobes of ovaries were removed according to need. Ovaries were washed and stored in a modified Barth’s saline (OR2: 82.5 mM NaCI, 2.5 mM KCI, 1 mM CaCl2, 1 mM MgCI2, 1 mM Na2HP04, 5 mM Hepes, 100 U/ml Streptomycin and Penicillin G, pH 7.5). Individual, mature oocytes were dissected manually. For UCP expression 50 nl RNA (lpg/trI) were microinjected into the vegetative pole, and oocytes were incubated individually at 17 “C in OR2 which was renewed every day. Control oocytes were injected with 50 nl of sterile water and treated in the same way. Isolation of mitochondria; Mitochondria were isolated by differential centrifugation in 250 mM Sucrose, 10 mM TRIS and 1 mM EDTA, pH 7.2. Oocytes were crushed and suspended in a large volume of isolation buffer. Two subsequent 5 min. centrifugations at 8009 and 25009, respectively, were performed after which the pellet was discarded. After a final 20 min. centrifugation at 90009 the pellet was resuspended in a small volume of isolation buffer. Purification of mitochondria was followed by monitoring cytochrome c oxidase activity spectrophotometrically (12). Routinely a lo- to 15- fold purification and a yield between 10 and 20% were obtained by this method. Immunoblou For Western blotting proteins were separated by SDS PAGE at 12% acrylamide and electrotransfered onto a nitrocellulose membrane. lmmunoblotting was performed using an antibody raised against purified rat UCP, with a second antibody coupled to peroxidase (13).

RESULTS AND DISCUSSION

The in vitro transcription of the UCP cDNA by means of the T7 polymerase produced a mRNA

of the expected size (about 1.2 kb), which was translated into a protein of 33 kD (fig 1).

Routinely between 12 and 15 trg RNA were obtained from1 ug plasmid. Injected Xenopus

oocytes were able to translate this mRNA into uncoupling protein, as revealed by immuno-

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Vol. 167, No. 2, 1990 BIOCHEMICAL AND RIOPHYSICAL RESEARCH COMMUNICATIONS

a

-Em Rl

. 1.2 kb

- 69 kD

- 46 kD

- 30 kD

I - 21 kD

1 2 . . . . .

Fiaure 1. In vitro transcrlDtlon its vehmm a: Construction of pTZ 55, a pTZ 19 containing the complete cDNA of rat UCP. The black area represents the complete coding region of UCP cDNA, the arrow points to the 3’ end. ORI= origin of transcription, AmpR= gene for ampicillin resistance. b: Northern blot of the mRNA obtained by in vitro transcription of the linearized pTZ 55. A cDNA probe of UCP was used for hybridisation. c: Autoradiography of cell free translation product of UCP mRNA, using 0.5 ug (lane 1) or 1 ug (lane 2) mRNA. kb= kilobase, kD= kilodalton

blotting. After separation of cell compartments by differential centrifugation, the strongest

immunoreactive response was found in the mitochondrial fraction (data not shown). A

weaker response was detected in the other fractions, but we have not been able to determine

if these signals were due to contamination with mitochondria, or if some UCP was also in-

corporated into other cell compartments. After several assays we choose to incubate the

injected oocytes at a temperature of 17 “C in order to keep them in good condition. Under

these conditions, maximal incorporation of UCP into mitochondria was obtained only after 7

days as shown in fig. 2. It is important to notice that the first detectable presence of UCP in

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- 33 kD

2 4 7 7c BAT days

lmmunoblotting of mitochondria of oocytes injected with 50 ng UCP mRNA and incubated for 2, 4, or 7 days, respectively. Each lane corresponds to mitochondria of eight oocytes. c= control oocytes, injected with water, BAT= 4 f.rg mitochondrial protein of brown adipose tissue of cold-acclimated rats.

mitochondria was found after 2 days. This latency apparently corresponds to the time

necessary for translation of the mRNA and processing of the protein. In order to investigate

the correct targeting of UCP into mitochondrial membranes, mitochondria were disrupted

either by suspension in a hypoosmotic medium or by sonication (followed in both cases by

freezing and rethawing). The membranes were then collected by ultracentrifugation and

subjected to immunoblotting (fig. 3a). We still found a strong immunoreactive signal of the

disintegrated membranes. We therefore concluded that nearly all UCP detected in the mito-

chondrial fraction was integrated into membranes. Further evidence was obtained by

trypsin, i.e. proteolytic treatment of the mitochondria. After mitochondria were incubated

with trypsin for 30 or 60 minutes, UCP could still be detected (fig 3b). This shows clearly

that the whole information for the targeting of UCP into mitochondria is still present in

artificially produced mRNA and that no mechanism specific for brown adipocytes is

necessary for integration of UCP into mitochondrial membranes. It is not yet clear which

exact signal sequences are involved in the targeting of UCP or AAC into mitochondria and

their insertion into the inner membrane. Recent findings (14) point to the amino terminal

one third of UCP as being essential for both import and membrane insertion. The authors

obtained this information from transcription of plasmids containing complete or partially

deleted UCP cDNA, followed by translation in vitro and incorporation of the obtained protein

into isolated heart muscle mitochondria. They also suggested the presence of a second import

signal within the carboxyl-terminal two thirds, which does not mediate membrane insertion.

As for AAC, different groups suggest that domains distant from the amino terminus may carry

specific targeting information (9,lO). The expression of modified UCP in an eucaryotic

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a b

- 33 kD

M H S 0 30 60 6Oc

minutes

Ewe 3: lntearation of UCP IntO mltochondrial lmmunoblotting of mitochondrial membranes of oocytes injected with 50 ng UCP mRNA. a: mitochondria were dissrupted by suspension in hypotonic medium (TRIS lOmM, EDTA 1 mM, pH 7.2) followed by freezing and rethawing (H) or sonicated 2x20 set followed by freezing and rethawing (S). Membranes were then collected by ultracentrifugation at 150000g for 20 min. M= intact mitochondria. Each lane corresponds to mitochondria of 15 oocytes. b: mitochondria were incubated at 37 “C with trypsin (0.25 pg/kg protein) for 0, 30, or 60 minutes. The reaction was stopped by adding trypsin inhibitor (0.5 pgglpg protein). 6Oc = mitochondria incubated for 60 min without trypsin. Each lane corresponds to mitochondria of 13 oocytes.

expression system could prove valuable for further examination of mitochondrial import

mechanism.

We tried to examine the functionality of UCP, expressed in Xenopus oocytes, by doing

respiration studies on isolated mitochondria. We did not succeed because of several

experimental problems. The purification of oocyte mitochondria is made difficult by the high

content of vitellogenin which seems to trap mitochondria, resulting in a very low yield. We

also observed that oocyte mitochondria hardly ever showed a coupled respiration, which

would be necessary to study the uncoupling properties of UCP. Regarding the nucleotide

binding capacity of UCP, we performed photoaffinity labeling of oocyte mitochondria (data

not shown). The results seemed to indicate that UCP expressed in oocytes is able to bind ATP

and GDP. However, these assays were hindered by the contamination of the mitochondrial

fraction with vitellogenin, and were thus not conclusive.

CONCLUSIONS

We describe here for the first time the expression of UCP, a mitochondrial carrier protein,

in an eukaryotic expression system, the Xenopus oocyte. This system is not only able to

express UCP, but also to direct it into mitochondria and insert it into the membrane.

However, examination of the functionality of UCP in oocyte mitochondria was not successful.

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We conclude that this system might be useful to study the import mechanism of the protein

into mitochondria, but it does not seem suitable for the study of UCP function. We recently

obtained the expression of UCP in an other eukaryotic expression system, a fibroblast cell

line, in which tests of UCP functionality have been positive (Ricquier et al, in preparation).

We thus have a second eukaryotic expression system avaible, which seems to be more

promising for the study of protein domains involved in nucleotide binding and proton

ACKNOWLEDGMENTS

We wish to thank Dr. Raymond T. Kado for his advise concerning the oocyte injection, as well as for his comments and discussion. This study was supported by CNRS and MRES. S. Klaus was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft.

REFERENCES

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783-792. 3. Kozak, L..P., Britton, J.H., Kozak, U.C., Wells, J.M. (1988) J. Biol. Chem. 263, 12274-

12277 4. Bouillaud, F., Weissenbach, J., and Ricquier, D. (1986) J; Biol. Chem. 261, 1487-1490. 5. Aquila, H., Link, T.A., Klingenberg, M. (1985) EMBO J. 4, 2369-2376. 6. Ridley, R.G., Patel, H.V., Gerber, G.E., Morton, R.C., and Freeman, K.B. (1986) Nucl. Acid

Res. 14, 4025-4035. 7. Runswick, M.J., Powell, J.T., Nyren, P., and Walker, J.E. (1987) EMBO J. 6,1367-1373. 8. Zarrilli, R., Oates, E.L., McBride, O.W., Lerman, M.I., Chart, J.Y., Santisteban, P., Ursini,

M.V., Notkins, A.L., and Kohn, L.D. (1989) Mol. Endocrinol. 3, 1498-1508 9. Pfanner, N., Hoeben, P. ,Tropschung, M., and Neupert, W. (1987) J. Biol. Chem. 31,

14851-14854. 10. Smagula, C. and Douglas, M.G. (1988) J. Biol. Chem. 263, 6783-6790. 11. Colman, A. (1984) Transcription and translation. A practical approach, IRL Press. Oxford,

Washington DC. pp 271-302. 12. Wharton, D.C., and Tzagoloff, A. (1967) Meth. Enzymol. 10, 245-250. 13. Ricquier, D., Barlet, J.P., Garel, J.M., Combes-George, M., Dubois, M.P. (1983)

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Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and import into mitochondrial membrane