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

Vol. 167, No. 2, 1990 March 16, 1990

EXPRESSION

OF

BIOCHEMICAL

THE

OOCYTES

Susanne

BROWN

FAT

AND

MITOCHONDRIA

IMPORT

Klaus, Louis Casteilla,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 784-789

INTO

Frederic

UNCOUPLING

Bouillaud,

January

23,

and Daniel Ricquier

Scientifique

sur la Nutrition,

9, rue Jules Hetzel

F-921 90 Meudon-Bellevue, Received

IN XENOPUS

MEMBRANE

Serge Raimbault,

Centre National de la Recherche Centre de Recherches

PROTEIN

MITOCHONDRIAL

France

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 Academicmess, 1°C. The mitochondrial brown

adipose

uncouple

uncoupling tissue (BAT)

mitochondrial

translocating

di- or triphosphate

mitochondrial recently hML-7

identified

nucleotides

this mitochondrial

signal sequence implicated

understood

carrier

0006-291X/90 Copyright All rights

and thus its ability to

This 33 kD protein

called

hML-7,

protein

including

the ADP/ATP

are mitochondrial

whose function

does not possess

ex-

membrane.

Its

by purine

sequence carrier

carrier

(AAC)(G)

proteins,

is not yet known

a precursor

(4,5,6)

shows and the

and a very

(8). Like AAC and

with a transient,

targeting

some studies on AAC and UCP import (9,iO) the regions

in the import are not yet well determined. and its regulation

and no molecular

is located

by fatty acids and negatively

only in BAT (2,3). Its primary

(7), which

Moreover,

by nucleotides

the molecular

membrane

the study of functional

$1.50 0 1990 by Academic Press, Inc. of reproduction in any form reserved.

domains

as a prerequisite of UCP.

784

mechanism

of

and fatty acids are not yet well

study has been made to characterize

UCP into the inner mitochondrial complicates

positively

family

(4,5) and despite

the proton translocation

translocator

ability of

(1). UCP is coded for by a nuclear gene which seems to exist

to a protein

protein

for the heat dissipating

of BAT and spans as a dimer the inner mitochondrial

activity is regulated

phosphate

as a proton

from ATP synthesis.

as only one copy and is expressed that UCP belongs

(UCP) is responsible

by its function

respiration

clusively in mitochondria proton

protein

them. The integration

for its physiological

of

function

We have tried to find an expression

Vol.

BIOCHEMICAL

167, No. 2, 1990

system

for

UCP,

mitochondria, relationships

able

to process

in order between

to study

AND BIOPHYSICAL

correctly

UCP

UCP structure

import

the

protein

in vitro

and function.

and

Among

RESEARCH COMMUNICATIONS

and

eventually different

Xenopus

oocytes are a useful tool for the study of protein expression

not only

translate

numerous

injected

post-translational

intracellular

membranes

mRNAs

very efficiently,

modifications

of proteins

first report of UCP expression

AND

in an eukaryotic

numerous

to elucidate

the

expression

sytems,

and function.

They do

also

carry

out correctly

and can direct them into the correct the expression

membranes.

has not yet been used for the study of mitochondrial

MATERIALS

but they

(11). In this study we describe

oocytes and its import into oocyte mitochondrial

containing

membrane

expression

of UCP in Xenopus

To our knowledge

this system

carrier proteins.

It is also the

system.

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 performing 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% onto a nitrocellulose membrane. lmmunoblotting was acrylamide and electrotransfered 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 expected Routinely oocytes

between

of the UCP cDNA by means of the T7 polymerase

size (about

1.2 kb), which

was translated

12 and 15 trg RNA were obtained

were able to translate

from1 ug plasmid.

this mRNA into uncoupling 785

into a protein

protein,

produced

a mRNA

of 33 kD (fig 1). Injected

as revealed

Xenopus by immuno-

Vol.

167,

No.

BIOCHEMICAL

2, 1990

AND

RIOPHYSICAL

RESEARCH

COMMUNICATIONS

a

-Em

Rl

- 69 kD - 46 kD - 30 kD

. 1.2 kb

I

- 21 kD

1 2 .. . its vehmm

. transcrlDtlon Fiaure 1.. In vitro of pTZ 55, a pTZ 19 containing a: Construction

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

After separation

blotting.

of cell compartments

immunoreactive

response

weaker

was detected

response

was found

in the mitochondrial

in the other fractions,

if these signals were due to contamination corporated

into other cell compartments.

injected oocytes these conditions,

at a temperature maximal

by differential

centrifugation, fraction

(data

the strongest not shown).

but we have not been able to determine

with mitochondria, After several

or if some UCP was also in-

assays we choose

to incubate

of 17 “C in order to keep them in good condition.

incorporation

days as shown in fig. 2. It is important

of UCP into mitochondria

was obtained

to notice that the first detectable 786

A

presence

the

Under

only after 7 of UCP in

Vol.

BIOCHEMICAL

167, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

- 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 necessary

was found for translation

the correct

targeting

chondrial trypsin,

in a hypoosmotic

was

i.e. proteolytic

with trypsin

We therefore integrated treatment

information

artificially

produced

necessary

for integration

their insertion

deleted

evidence

of the mitochondria.

After

mitochondria

UCP could still be detected

and

are involved

membrane.

Recent

by translation

signal within the carboxyl-terminal

targeting

information

findings

(9,lO).

by

incubated

is still present

insertion.

is

does not mediate

and

terminal

The authors

complete

or partially

of the obtained

the presence

of modified

in

adipocytes

protein

of a second import membrane

distant from the amino terminus

expression 787

were

(14) point to the amino

containing

They also suggested

The

was obtained

It is not yet clear which

in vitro and incorporation

two thirds, which

in the mito-

of UCP or AAC into mitochondria

of plasmids

As for AAC, different groups suggest that domains

signal of the

for brown

for both import and membrane

from transcription

UCP cDNA, followed

specific membranes.

in the targeting

and

(fig 3b). This shows clearly

of UCP into mitochondria

that no mechanism

into isolated heart muscle mitochondria.

specific

in both cases by

that nearly all UCP detected

of UCP into mitochondrial

into the inner

this information

concluded

disrupted

by ultracentrifugation

Further

one third of UCP as being essential obtained

were

(followed

then collected

to the time

In order to investigate

mitochondria

or by sonication

were

for the targeting

mRNA

exact signal sequences

of the protein.

into membranes.

for 30 or 60 minutes,

that the whole

corresponds

(fig. 3a). We still found a strong immunoreactive

membranes. fraction

apparently

membranes,

medium

The membranes

to immunoblotting

disintegrated

latency

of the mRNA and processing

and rethawing).

subjected

This

of UCP into mitochondrial

either by suspension freezing

after 2 days.

insertion. may carry

UCP in an eucaryotic

Vol.

BIOCHEMICAL

167, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

b

a

- 33 kD

M

H

S

0

30 60 minutes

6Oc

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 1mM, 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 respiration

studies

experimental

the functionality on isolated

problems.

also observed would binding

capacity

We did

mitochondria

not succeed

properties

photoaffinity

these assays were hindered

fraction with vitellogenin,

because

labeling

by doing of several

in a very low yield.

a coupled

respiration,

of UCP. Regarding

not shown). The results seemed to indicate that UCP expressed and GDP. However,

oocytes,

is made difficult by the high

resulting

hardly ever showed

to study the uncoupling

of UCP, we performed

in Xenopus

of oocyte mitochondria

which seems to trap mitochondria,

that oocyte

be necessary

mitochondria.

The purification

content of vitellogenin

of UCP, expressed

of oocyte

We which

the nucleotide

mitochondria

(data

in oocytes is able to bind ATP

by the contamination

of the mitochondrial

and were thus not conclusive.

CONCLUSIONS

We describe

here for the first time the expression

in an eukaryotic

expression

express

UCP,

However,

examination

system, the Xenopus

but also to direct

of UCP, a mitochondrial

and insert

it into the membrane.

of UCP in oocyte mitochondria 788

protein,

oocyte. This system is not only able to

it into mitochondria

of the functionality

carrier

was not successful.

Vol. 167,

No.

2, 1990

We conclude

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

that this system might be useful to study the import mechanism

into mitochondria, obtained

BIOCHEMICAL

but it does not seem suitable

the expression

line, in which

of UCP in an other eukaryotic

tests of UCP functionality

expression

have been positive

We thus have a second

eukaryotic

expression

promising

of protein

domains

for the study

for the study of UCP function.

system involved

We recently

system, a fibroblast

(Ricquier

avaible,

of the protein

which

in nucleotide

cell

et al, in preparation). seems to be more 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

1. Nicholls, D.G., and Locke, R.M. (1984) Physiol. Rev. 64, l-64. 2. Bouillaud, F., Raimbault, S., and Ricquier, D. (1988) Biochem. Biophys. Res. Comm. 157, 783-792. 3. Kozak, L..P., Britton, J.H., Kozak, U.C., Wells, J.M. (1988) J. Biol. Chem. 263, 1227412277 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) Biochem. J. 210, 859-866. J. Cell Biol. 107, 503-509. 14. Liu, X., Bell, A.W., Freeman, K.B., and Shore, G.C. (1988)

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