Cloning, ontogenesis, and localization of an atypical uncoupling protein 4 in Xenopus laevis
Patrick A. Keller1,
Lorenz Lehr1,
Jean-Paul Giacobino1,
Yves Charnay2,
Françoise Assimacopoulos-Jeannet1 and
Natalia Giovannini1
1 Department of Cell Physiology and Metabolism, University Medical Center 1, Geneva
2 Division of Neuropsychiatry, Belle-Idée, Geneva University Hospital, Chene-Bourg, Switzerland
 |
ABSTRACT
|
---|
Uncoupling protein 1 (UCP1) is the first UCP described. It belongs to the family of mitochondrial carrier proteins and is expressed mainly in brown adipose tissue. Recently, the family of the UCPs has rapidly been growing due to the successive cloning of UCP2, UCP3, UCP4, and UCP5, also called brain mitochondrial carrier protein 1. Phylogenetic studies suggest that UCP1/UCP2/UCP3 on one hand and UCP4/UCP5 on the other hand belong to separate subfamilies. In this study, we report the cloning from a frog Xenopus laevis (Xl) oocyte cDNA library of a novel UCP that was shown, by sequence homology, to belong to the family of ancestral UCP4. This cloning provides a milestone in the gap between Drosophila melanogaster or Caenorhabditis elegans on one hand and mammalian UCP4 on the other. Xl UCP4 is already expressed in the oocyte, being the first UCP described in germ cell lineage. During development, it segregates in the neural cord, and, in the adult, in situ hybridization shows its expression in the neurons and also in the choroid plexus of the brain. By RT-PCR analysis, it was found that Xl UCP4 is present in all the subdivisions of the brain and also that it differs from mammalian UCP4 by a very high relative level of expression in peripheral tissues such as the liver and kidney. The peripheral tissue distribution of Xl UCP4 reinforces the hypothesis that UCP4 might be the ancestral UCP from which other UCPs diverged from.
cDNA; cloning
 |
INTRODUCTION
|
---|
UNCOUPLING PROTEIN 1 (UCP1) is the first UCP described. It belongs to the family of mitochondrial carrier proteins and is expressed mainly in brown adipose tissue. UCP1 uncouples oxidative phosphorylation by dissipating the proton gradient generated by the activity of the respiratory chain and is considered as the main effector of adaptative thermogenesis in rodents (7).
Recently, the family of the UCPs has rapidly been growing due to the successive cloning of UCP2 (9), UCP3 (6), UCP4 (22), and UCP5 (also called brain mitochondrial carrier protein 1) (32, 40). Sequence alignment and analysis of energy transfer protein signatures suggested that UCP1/UCP2/UCP3 on one hand and UCP4/UCP5 on the other hand belong to separate subfamilies (2, 12). In rodents and humans, UCP2 was found to be expressed in almost all of the tissues studied (9), whereas UCP3 was expressed mostly in skeletal muscle (6). Several other UCPs also belong to this subfamily: carp and zebrafish UCP2, whose tissue distribution and possible role were not studied (35); an avian UCP that shares about 70% homology with mouse UCP2 and UCP3 and is expressed mostly in skeletal muscle (29, 37) but also to a lesser extent in the liver and heart (38); and plant UCP1 and UCP2 (3). Recently, marsupial UCP2 and UCP3 were cloned and found to be expressed ubiquitously for UCP2 and in skeletal muscle for UCP3, thus resembling the expression pattern found in rodents (14).
UCP4 mRNA in mammalians is exclusively expressed in the brain (22). UCP5 mRNA in humans and rodents is predominantly expressed in the brain (17, 32, 40), but, in humans, it was found to be also expressed in the testis, kidney, uterus, and heart (40). In rodents, it was found to be expressed in the testis, white adipose tissue, kidney, and heart (15, 40). UCP5 protein in rodents was the highest in the brain, where it was found to be almost exclusively neuronal (15). Recently, an UCP5 homolog has been cloned in Drosophila melanogaster and found to be predominantly expressed in the adult head (10). In addition, quantitative RT-PCR analysis has shown that UCP4 and UCP5 are the most abundant of all the UCP isoforms in the mouse brain cortex (20). Therefore, all the data available until now suggest that UCP4 is exclusively and UCP5 essentially expressed in the brain.
What about the biological function of UCPs? UCP2 and UCP3 do not seem to be involved in adaptative thermogenesis in opposition to UCP1 (4, 26, 31). On the other hand, Solanum tuberosum plant UCP (18, 21) and avian muscle UCP (29, 37, 38) have been found to be upregulated by cold exposure. Rodent spinal cord UCP2 and human brain UCP4 and UCP5 have also been shown to be upregulated by cold exposure (24, 40). Therefore, some UCPs might play a role in the protection against a cold environment. UCP2 and UCP3, considered as minor players in thermoregulation, have alternatively been proposed to be involved in the prevention of reactive oxygen species (ROS) accumulation (4, 26, 31). This role was also postulated in the brain for UCP2 (13) and UCP5 (15). Finally, UCP3 has been proposed to protect the mitochondria against lipotoxicity by functioning as an exporter of fatty acids outside the mitochondria (34).
Phylogenetically, it was postulated that UCP4 represents an ancestral UCP from which the other UCPs diverged and that UCP1, UCP2, and UCP3 have developed later during evolution. D. melanogaster and Caenorhabditis elegans are the closest UCP4 analogs described until now (12).
It is still unknown how the ancestral UCP4 evolved into a brain-specific protein. For this reason, the possible existence of UCP4 in a species that is intermediary between D. melanogaster or C. elegans and mammals was examined. The frog, being a poikilotherm, i.e., a vertebrate animal essentially devoid of thermoregulatory needs, seemed an interesting model for this study. We searched expressed sequence tag (EST) databases and found a Xenopus laevis (Xl) oocyte EST (Accession No. AW147976) coding for a segment of putative UCP4. The present study describes the cloning of the Xl oocyte UCP as well as its fate during development and tissue distribution.
 |
MATERIALS AND METHODS
|
---|
Animals.
All experiments carried out conformed to the guidelines set by the Swiss Federal Veterinary Office. Animal protocols were approved by André Solaro (responsible for X. laevis frogs at the Univ. of Geneva). Xl frogs were obtained from the Station de Zoologie in Geneva, and in vitro fertilization was performed according to Thiebaud et al. (36). The fertilized oocytes were incubated in OR2 ficoll medium (39) with antibiotics (50 U/ml of streptomycin-penicillin). The stages were determined according to Nieuwkoop and Faber (27). Adult brains were carefully removed from the skull and frozen on dry ice for hybridization in situ and real-time PCR studies. Tissue blocks of four different representative regions of the brain (see Fig. 1) were separated and used for real-time PCR studies. Samples of the liver, heart, kidney, and leg skeletal muscle were also collected.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1. Schematic representation of the delineation of the four blocks of brain tissues used for the estimation of uncoupling protein 4 (UCP4) mRNA levels by real-time PCR. Cx, brain cortex; TeO, optical tegmentum; Ce, cerebellum; SP, spinal cord.
|
|
Cloning of the novel UCP gene.
An incomplete Xl potential UCP4 EST was identified in GenBank (Accession No. AW147976). The cDNA encoding full-length Xl UCP4 was cloned by a nested PCR from a Xl oocyte cDNA library in pBluescript generously provided by Dr. Nigel Garrett (Wellcome/CRC Institute, Cambridge, UK). Primers were chosen on the vector and on the incomplete UCP clone. For the first round, the primers were 5'-tcagtgccatttagtcttatttac-3' (insert) and 5'-tgagcggataacaatttcac-3' (vector), and for the second round primers were 5'-tgctttcattttctctgtaactgggcggg-3' (insert) and 5'-acagctatgaccatgattacgccaagcg-3' (vector). Three cDNAs stemming from independent PCRs were cloned in pCR 2.1 TOPO (Invitrogen; Leek, The Netherlands) and sequenced (Univ. Medical Center Sequencing Unit, Univ. of Geneva, Switzerland). The overlaps were confirmed to match with each other and with the AW147976 sequence. The consensus cDNA was then cloned in pcDNA 3.1 (Invitrogen) using BstXI as the restriction site.
Northern blot analyses.
Total RNA was purified by the method of Chomczynski and Sacchi (8), and 1220 µg were electrophoresed on a 1.2% agarose gel containing formaldehyde, as described by Lehrach et al. (19), and transferred to Electran Nylon Blotting membranes (BDH Laboratory Supplies; Poole, UK) by vacuum blotting. The probe used, obtained by PCR (upstream primer: 5'-tggttgtttctcagtgccat-3', downstream primer: 5'-gaagcgattgggcagtttt-3'), had a size of
600 bp and was controlled by sequencing. It was labeled by random priming with [
-32P]dCTP (Amersham; Bucks, UK) to a specific radioactivity of
1 x 109 disintegrations·min1·µg1 DNA. Northern blots were performed as previously described (5). Housekeeping mRNA levels are subject to changes during embryogenesis. They cannot therefore be used as internal references. The ß-actin level, for instance, was found to be 3.2-fold higher in the tadpole than in the oocyte (results not shown). For this reason, we present the UCP4 results during development as arbitrary values obtained in the known amount of RNA loaded on the gel. The membrane was colored with methylene blue to check RNAs for degradation. The signals on the autoradiograms were quantified by scanning photodensitometry using ImageQuant Software version 3.3 (Molecular Dynamics; Sunnyvale, CA).
Real-time PCR.
Oligo-dT primed first-strand cDNAs were synthesized using the Superscript II Reverse Transcriptase kit (Invitrogen) according to the manufacturer's instructions. Real-time PCR was performed using a ABI rapid thermal cycler system and a SYBR green PCR master mix for the quantification of UCP4 according to the manufacturer's instructions. An assay performed without reverse transcriptase was always included to check for possible contamination of the RNA preparation by genomic DNA. GAPDH was used as a control to account for any variations in the efficiencies of the reverse transcription and PCR. The Xl UCP4 oligonucleotide primers used were upstream 5'-ggaacgccagctgatgttatc-3' and downstream 5'-gcccccgtccatgctt-3'. This primer pair covered nucleotides 724790 of the Xl UCP4 cDNA (GenBank Accession No. AY166600). All samples were analyzed in duplicate. The specificity of the reaction was shown by cloning of the PCR product in the vector pCR 2.1 TOPO and by sequencing. The GAPDH oligonucleotide primers used were upstream 5'-catcaccgtcttccaggagc-3' and downstream 5'-gcatctccccacttaatgctg-3'. This primer pair covered nucleotides 256307 of the GAPDH cDNA (GenBank Accession No. XlU41753.
In situ hybridization of whole mount embryos.
The PCR fragment described above for the Northern blot analysis was inserted in a TOPO II vector from Invitrogen and subcloned in the EcoRI site of the pBluescript KSII+ plasmid from Stratagene (La Jolla, CA) for RNA digoxigenine (DIG) probe transcription. Plasmid DNA was linearized with BamHI or XhoI for the production of antisense or sense probes with T3 or T7 RNA polymerase, respectively (Promega; Madison, WI). The incorporation of the DIG nucleotides was monitored by spotting on Nylon N+ membranes followed by hybridization with an anti-DIG antibody coupled to alkaline phosphatase (AP) and staining with BM purple (Roche; Rothkreus, Switzerland) according to Peng (28). Entire Xl embryos were fixed in methanol. The sense and antisense probes for Xl UCP4 were hybridized at 42°C to the embryos and were detected with the anti-DIG antibody and BM as described below.
In situ hybridization of brain slices.
The pCR 2.1 TOPO Xl UCP4 plasmid was used as a cDNA template for PCR in a ReadyMix PCR (Sigma). The oligonucleotides primers used were upstream 5'-aacccgcctacagattca-3' and downstream 5'ccatctgcatctgaactt-3'. This primer pair covered nucleotides 123- 460 of the Xl UCP4 cDNA. The PCR product was then directly cloned in the plasmid vector pCR4-TOPO (Invitrogen). Plasmid DNA was linearized with NotI or PmeI for the production of antisense and sense probes with T3 or T7 polymerase, respectively (Invitrogen). Labeled riboprobes were obtained by the incorporation of DIG-UTP (Roche Diagnostics) during an in vitro transcription reaction according to the manufacturer's instructions (Maxiscript T7/T3, Ambion). The riboprobes were purified by Sephadex G50 (Quickspin columns, Roche) and checked by agarose-formaldehyde gel electrophoresis with ethidium bromide staining.
Xl frogs were deeply anesthetized by immersion in 0.2% 3-aminobenzoïc acid ethyl ether methanesulfonate salt before decapitation. The brains were carefully removed, and sagittal and frontal cryostat sections (15 um) of representative brain regions (from the cortex to the medulla) were collected onto poly-L-lysine-coated glass slides. They were fixed by immersion in a fixative containing 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 15 min at 4°C and washed in 2x standard saline citrate (SSC) acetylated in 0.5% acetic anhydride in 0.9% NaCl containing 100 mM triethanolamine (pH 8). Sections were then dehydrated in an ascending series of ethanol washes, delipidated in chloroform, and air dried before hybridization.
Probes were heat denatured (5 min at 65°C) and then placed on ice. They were added to a final concentration of 0.5 µg/ml to the hybridizing buffer containing 1x Denhardt's solution, 50% formamide, 20 mM Tris·HCl (pH 7.4), 300 mM NaCl, 1 mM EDTA, 10% dextran sulfate, 50% formamide, 100 µg/ml salmon testes DNA, and 250 µg/ml yeast tRNA. After hybridization overnight at 60°C, the sections were washed at room temperature (RT) 4 x 5 min in 4x SSC, incubated with RNase A (50 µg/ml, Sigma) for 30 min at 37°C, and then washed successively 2 x 5 min in 2x SSC, 10 min in 1x SSC, 10 min in 0.5x SSC at RT, 30 min in 0.1x SSC at 50°C, and finally 15 min in 0.1x SSC at RT.
Slides were washed in Tris saline buffer (150 mM Tris·HCl and 100 mM NaCl, pH 7.5) and incubated for 1 h in blocking solution (Roche). They were then incubated for 1 h at 4°C in AP-conjugated anti-DIG antibodies (working dilution 1:100, DAKO Diagnostics). The immunoreactivity was revealed by a dark blue purple staining in the presence of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) (Dakocytomation).
Phylogenetic analysis.
The amino acid sequence of Xl UCP4 was aligned with other known UCPs, and the phylogenetic tree (Megalign of the Lasergene 5.0 sequence analysis program) was constructed using the Jotun Hein method as described by Hanak and Jezek (12).
Statistical analysis.
Results are given as means ± SE. For statistical comparison, one-way ANOVA followed by an unpaired Student's t-test were done on Origin 7.0 (OriginLab).
 |
RESULTS
|
---|
An EST coding for a segment of a putative UCP4 was found in Genbank, and the full-length cDNA was cloned and sequenced as described in MATERIALS AND METHODS. As shown in Fig. 2A, Xl UCP displays structural features of mitochondrial carriers, i.e., six putative transmembrane domains and three mitochondrial energy transfer protein signatures that can be identified at the border and downstream of the first, third, and fifth potential transmembrane domains. It also shares a purine nucleotide-binding domain with other UCPs. Xl UCP was found to display the highest level of amino acid identity with mouse and human UCP4 (70.8 and 70.6%, respectively). The identities of the Xl UCP with mouse (m)UCP5, mUCP3, and mUCP2 were 32.6, 31.7, and 29.1%, respectively. The presence of the amino acids (position in brackets) valine (96), serine (98), glutamine (194), alanine (196), proline (298), serine (300), and phenylalanine (303) suggests that the Xl UCP belongs to the UCP4 family (12). The phylogenetic analysis of Fig. 2B, which compares Xl UCP4 to other UCPs, shows that Xl UCP4 is positioned at the basis of its mammalian orthologs. Despite a high phylogenetic distance between amphibian and mammals, UCP4 orthologs are relatively similar (about 70% amino acid identity). It is noteworthy that at the same phylogenetic distance, UCP2 orthologs are more similar (80% amino acid identity) (14). These differences reveal different evolutionary speeds of the UCP4 and UCP2 families.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 2. A: amino acid sequence alignments of rat (rUCP4), human (hUCP4), and Xenopus laevis (XlUCP4; GenBank Accession No. AY166600) obtained with the ClustalW Multiple Sequence Alignment program (K.C. Worely, Human Genome Center, Baylor College of Medicine). The sequences are presented in single letter codes. Gaps introduced into the sequences to optimize alignments are indicated by a dash. Identical and similar amino acids are highlighted in black or shaded, respectively. The potential purine-nucleotide binding domain (PNBD) is underlined. *Three mitochondrial energy transfer protein signature domains. The six putative transmembrane domains are underlined and labeled as IVI. B: amino acid phylogenetic tree of known and predicted UCPs (12) comparing the evolutionary rank of XlUCP4 to other UCPs using the Jotun Hein method. UCP sequences were obtained from Genbank. Abbreviations, species, and accession numbers are as follows: huUCP1, huUCP2, huUCP3, huUCP4, and huUCP5 are human (Homo sapiens) UCPs with Accession Nos. NM_021833, NM_003355, NM_003356, AF110532, and AF155809, respectively; XlUCP4 is X. laevis UCP4 with Accession No. AY166600; avUCP is chicken UCP (Gallus gallus) with Accession No. AF287144; StPUMP1 is potato (Solanum tuberosum) pump with Accession No. Y11220; AtUCP4 is Arabidopsis thaliana UCP4 with Accession No. AAD39301 DmUCP4A, DmUCP4B, and DmUCP4C are Drosophila melanogaster UCPs with Accession Nos. CG6492, CG18340, and CG9064, respectively; and CeUCP4 is predicted Caenorhabditis elegans UCP4 with Accession No. AF003384.
|
|
What is the fate of UCP4 during development? To answer this question, the UCP4 mRNA level was measured at various stages of Xl development from the oocyte to tadpole. As shown in Fig. 3A, UCP4 mRNA is already present in the oocyte. The level of UCP4 mRNA is not modified by fertilization, decreased by 50% in the gastrula stage, and increased 3.3-fold in the tadpole stage. Thus the basal level of expression of UCP4 in the oocyte increases during development. Does UCP4 mRNA expression colocalize with a peculiar structure during development? In situ hybridization of the embryo showed that, at the neurula stage, UCP4 mRNA expression segregates in the neural cord (Fig. 3B).

View larger version (64K):
[in this window]
[in a new window]
|
Fig. 3. A: UCP4 mRNA expression during X. laevis development. Levels of XlUCP4 in laid and fertilized oocytes as well as gastrula (stages 11 and 12), neurula (stages 1618), and tadpole (stages 22 and 23). Total RNA was extracted and Northern blots were performed as described in MATERIALS AND METHODS. A photodensitometric comparison of signals obtained from total RNA hybridized with a 32P-labeled Xl UCP4 probe is shown. The results were obtained from two different Northern blots. They were expressed in each blot as percentages of the mean laid oocyte value. The results are expressed as percentages of the laid oocytes values taken as 100%. Values are means ± SE of 47 experiments. ***P < 0.005 and **P < 0.002 vs. laid oocytes;°°P < 0.02 vs. gastrula. B: in situ hybridization of neurula (stages 1618). Antisense (left) and sense (right) probes were hybridized and detected as described in MATERIALS AND METHODS.
|
|
In the adult Xl frog, UCP4 mRNA expression was detected by in situ hybridization in most of the subdivisions of the brain. As illustrated in Fig. 4, the hybridizing signal given by the antisense probe was clearly detected in nerve cells (A, C, and E) and in the choroid plexus (C). No significant signal was observed in tissue sections incubated with the sense probe (Fig. 4, B, D, and F). Considering the size and the morphology of the UCP4-positive nerve cells, it can be assumed that most of them represent neuronal cells.

View larger version (143K):
[in this window]
[in a new window]
|
Fig. 4. UCP4 mRNA expression in the brain of X. laevis frogs. The antisense digoxigenin (DIG)-labeled probe revealed UCP4 mRNA expressing neurons (dark labeling) in most of the brain subdivisions. A: representative result throughout the thalamic region. C: high hybridization signal in the brain choroid plexus. Note also the hybridization signal in the neurons of the cerebral cortex (arrow). E: superficial layer of the parietal cortex. Note the high density of cortical neurons expressing UCP4. The sense DIG-labeled probe did not reveal any significant labeling in the respective adjacent sections (B, D, and F). Scale bars = 260 µm in AD and 100 µm in E and F.
|
|
In a first approach to get a quantification of UCP4 mRNA expression in the brain, four blocks of brain including the cerebral cortex, optical tegmentum, cerebellum, and spinal cord (Fig. 1) were dissected, and UCP4 mRNA was measured in each of them by real-time PCR. UCP4 mRNA was expressed in the four regions and is therefore expressed in the whole brain. UCP4 mRNA was also found in peripheral tissues. Figure 5 shows the distribution of UCP4 mRNA in the brain subdivisions and peripheral tissues tested. The level of expression of UCP4 in the heart has been defined as representing one arbitrary unit. The level of UCP4 mRNA in the cortex is 6.4-fold, in the optical tegmentum 6.3-fold, in the spinal cord 5.2-fold, in the cerebellum 7.2-fold, in the liver 38.1-fold, and in the kidney 45.5-fold that of in the heart. No UCP4 mRNA was detected in skeletal muscle and adipose tissue (data not shown).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5. UCP4 mRNA expression in four blocks of brain and in some peripheral tissues of X. laevis frogs. Quantitative real-time PCR determinations were performed as described in MATERIALS AND METHODS. The results are expressed as arbitrary units, with the heart value being considered as 1. Values are means ± SE of 6 samples for each tissue. ***P < 0.001 for peripheral tissues (liver and kidney or heart) vs. brain regions.
|
|
To answer the question of a possible thermoregulatory role of Xl UCP4, we studied the effect of an exposure to 4°C during 30 h in Xl oocytes or during 36 h in the adult Xl frog. These treatments did not change UCP4 mRNA expression in oocytes or in the adult Xl brain (data not shown).
 |
DISCUSSION
|
---|
The novel Xl UCP that we describe in this study is the first member of the UCP4 family identified in the evolution between D. melanogaster or C. elegans and mammals (12). The absence of an UCP4 mRNA upregulation in oocytes and the Xl brain upon exposure to cold is in contrast with the results obtained in the rodent whole brain, where UCP4 was found to be increased in response to cold exposure (40).
UCP2, UCP3, and UCP5 have been proposed to be involved in the prevention of ROS accumulation (4, 13, 15, 27, 32). It is therefore interesting to discuss the possible role of Xl UCP4 in the context of this prevalent hypothesis.
Xl UCP4 is the first UCP found to be expressed in germ cell lineage. This finding suggests a new role for UCPs in oocyte maturation and preimplantation embryo development. What could be the role of UCP4 in Xl during development? ROS, like H2O2, O2, and OH, are chemical mediators that act on signaling pathways to modulate, among others, growth and differentiation (33). Metabolic gradients are established at very early stages of development that, by inducing the formation of redox and ROS gradients, influence the expression and activity of proteins involved in development (1). Although ROS are necessary during embryogenesis, they can also induce defective embryo development. The internal protection against deleterious ROS accumulation comprises antioxidant enzymes like superoxide dismutase, glutathione peroxidase, and
-glutamylcysteine synthetase, whose transcripts are already present in oocytes and embryos (11). UCP4 could constitute an additional protection mechanism against potential deleterious molecules like ROS during oocyte maturation and embryogenesis. Thus the variation of Xl UCP4 expression during development could be explained by the fact that the embryo may display different sensitivities to ROS at different developmental stages (25). Recently, UCP2 was found to be expressed in mouse ovarian cells and postulated to contribute to minimize the inflammatory response accompanying changes in follicular structure (30). The possible role of UCPs at early stages of development is a field that deserves further study.
UCP4 is localized not only in the neurons, as reported for UCP5 (15), but also in the choroid plexus, and this suggests a protective role against endogenous accumulation in brain vital cells or against penetration into the brain of potential deleterious molecules like ROS. This hypothesis is supported by the recent findings that in the mouse another UCP, UCP2, might act as a cell death-suppressing protein (13) and prevent ischemic neuronal death by decreasing ROS accumulation (23).
The tissue distribution of the Xl UCP4 is atypical compared with that of mammalian UCP4 (22, 40). Whereas in mammals UCP4 mRNA is expressed only in the brain, it was also found in Xl in other tissues. In adult Xl, indeed, very high levels of expression of UCP4 mRNA compared with the brain are observed in the liver and kidney. Our study therefore shows that during phylogenesis, UCP4 disappears from the periphery, where it might be redundant with ubiquitous UCP2, also present in Xl (16). It would be interesting to determine whether UCP4 is also expressed in the periphery in invertebrates, like D. melanogaster and C. elegans.
In conclusion, we showed the existence in Xl of UCP4 already present in the oocyte, which is also expressed in the adult animal in the choroid plexus and neurons of the brain as well as in the periphery. The exact function of UCP4 remains to be determined; it might play a role in the protection against ROS accumulation during development and in the adult animal.
 |
GRANTS
|
---|
This study was supported by Swiss National Science Foundation Grant 31-65431.01.
 |
ACKNOWLEDGMENTS
|
---|
We are indebted to André Solaro for the invaluable help in the Xl development experiments and to Patrizia Arboit, Francine Califano, and Brigitte Greggio for expert technical assistance.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: P. Keller, Dept. of Cell Physiology and Metabolism, Univ. Medical Center 1, rue Michel-Servet, 1211 Genève 4, Switzerland (E-mail: Patrick.Keller{at}medecine.unige.ch)
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
10.1152/physiolgenomics.00012.2005
 |
REFERENCES
|
---|
- Allen RG. Oxygen-reactive species and antioxidant responses during development: the metabolic paradox of cellular differentiation. Proc Soc Exp Biol Med 196: 117129, 1991.[Abstract]
- Borecki IB and Suarez BK. Linkage and association: basic concepts. Adv Genet 42: 4566, 2001.[Medline]
- Borecky J, Maia IG, and Arruda P. Mitochondrial uncoupling proteins in mammals and plants. Biosci Rep 21: 201212, 2001.[CrossRef][ISI][Medline]
- Boss O, Hagen T, and Lowell BB. Uncoupling proteins 2 and 3: potential regulators of mitochondrial energy metabolism. Diabetes 49: 143156, 2000.[Abstract]
- Boss O, Samec S, Kuhne F, Bijlenga P, Assimacopoulos-Jeannet F, Seydoux J, Giacobino JP, and Muzzin P. Uncoupling protein-3 expression in rodent skeletal muscle is modulated by food intake but not by changes in environmental temperature. J Biol Chem 273: 58, 1998.[Abstract/Free Full Text]
- Boss O, Samec S, Paoloni-Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P, and Giacobino JP. Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression. FEBS Lett 408: 3942, 1997.[CrossRef][ISI][Medline]
- Cannon B and Nedergaard J. The biochemistry of an inefficient tissue: brown adipose tissue. Essays Biochem 20: 110164, 1985.[ISI][Medline]
- Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156159, 1987.[CrossRef][ISI][Medline]
- Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, and Warden CH. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 15: 269272, 1997.[CrossRef][ISI][Medline]
- Fridell YW, Sanchez-Blanco A, Silvia BA, and Helfand SL. Functional characterization of a Drosophila mitochondrial uncoupling protein. J Bioenerg Biomembr 36: 219228, 2004.[CrossRef][ISI][Medline]
- Guerin P, El Mouatassim S, and Menezo Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod 7: 175189, 2001.
- Hanak P and Jezek P. Mitochondrial uncoupling proteins and phylogenesisUCP4 as the ancestral uncoupling protein. FEBS Lett 495: 137141, 2001.[CrossRef][ISI][Medline]
- Horvath TL, Diano S, and Barnstable C. Mitochondrial uncoupling protein 2 in the central nervous system: neuromodulator and neuroprotector. Biochem Pharmacol 65: 19171921, 2003.[CrossRef][ISI][Medline]
- Jastroch M, Withers K, and Klingenspor M. Uncoupling protein 2 and 3 in marsupials: identification, phylogeny, and gene expression in response to cold and fasting in Antechinus flavipes. Physiol Genomics 17: 130139, 2004.[Abstract/Free Full Text]
- Kim-Han JS, Reichert SA, Quick KL, and Dugan LL. BMCP1: a mitochondrial uncoupling protein in neurons which regulates mitochondrial function and oxidant production. J Neurochem 79: 658668, 2001.[CrossRef][ISI][Medline]
- Klein SL, Strausberg RL, Wagner L, Pontius J, Clifton SW, and Richardson P. Genetic and genomic tools for Xenopus research: the NIH Xenopus initiative. Dev Dyn 225: 384391, 2002.[CrossRef][ISI][Medline]
- Kondou S, Hidaka S, Yoshimatsu H, Tsuruta Y, Itateyama E, and Sakata T. Molecular cloning of rat brain mitochondrial carrier protein-1 cDNA and its up-regulation during postnatal development. Biochim Biophys Acta 1457: 182189, 2000.[ISI][Medline]
- Laloi M, Klein M, Riesmeier JW, Muller-Rober B, Fleury C, Bouillaud F, and Ricquier D. A plant cold-induced uncoupling protein. Nature 389: 135136, 1997.[CrossRef][ISI][Medline]
- Lehrach H, Diamond D, Wozney JM, and Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16: 47434751, 1977.[CrossRef][ISI][Medline]
- Lengacher S, Magistretti PJ, and Pellerin L. Quantitative rt-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle. J Cereb Blood Flow Metab 24: 780788, 2004.[CrossRef][ISI][Medline]
- Maia IG, Benedetti CE, Leite A, Turcinelli SR, Vercesi AE, and Arruda P. AtPUMP: an Arabidopsis gene encoding a plant uncoupling mitochondrial protein. FEBS Lett 429: 403406, 1998.[CrossRef][ISI][Medline]
- Mao W, Yu XX, Zhong A, Li W, Brush J, Sherwood SW, Adams SH, and Pan G. UCP4, a novel brain-specific mitochondrial protein that reduces membrane potential in mammalian cells. FEBS Lett 443: 326330, 1999.[CrossRef][ISI][Medline]
- Mattiasson G, Shamloo M, Gido G, Mathi K, Tomasevic G, Yi S, Warden CH, Castilho RF, Melcher T, Gonzalez-Zulueta M, Nikolich K, and Wieloch T. Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma. Nat Med 9: 10621068, 2003.[CrossRef][ISI][Medline]
- Mizuno T, Miura-Suzuki T, Yamashita H, and Mori N. Distinct regulation of brain mitochondrial carrier protein-1 and uncoupling protein-2 genes in the rat brain during cold exposure and aging. Biochem Biophys Res Commun 278: 691697, 2000.[CrossRef][ISI][Medline]
- Morales H, Tilquin P, Rees JF, Massip A, Dessy F, and Van Langendonckt A. Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos. Mol Reprod Dev 52: 149157, 1999.[CrossRef][ISI][Medline]
- Muzzin P, Boss O, and Giacobino JP. Uncoupling protein 3: its possible biological role and mode of regulation in rodents and humans. J Bioenerg Biomembr 31: 467473, 1999.[CrossRef][ISI][Medline]
- Nieuwkoop PD and Faber J.The Normal Development Table of Xenopus laevis. Amsterdam: Elsevier, 1967.
- Peng XR, Hsueh AJ, LaPolt PS, Bjersing L, and Ny T. Localization of luteinizing hormone receptor messenger ribonucleic acid expression in ovarian cell types during follicle development and ovulation. Endocrinology 129: 32003207, 1991.[Abstract]
- Raimbault S, Dridi S, Denjean F, Lachuer J, Couplan E, Bouillaud F, Bordas A, Duchamp C, Taouis M, and Ricquier D. An uncoupling protein homologue putatively involved in facultative muscle thermogenesis in birds. Biochem J 353: 441444, 2001.[CrossRef][ISI][Medline]
- Rousset S, Alves-Guerra MC, Ouadghiri-Bencherif S, Kozak LP, Miroux B, Richard D, Bouillaud F, Ricquier D, and Cassard-Doulcier AM. Uncoupling protein 2, but not uncoupling protein 1, is expressed in the female mouse reproductive tract. J Biol Chem 278: 4584345847, 2003.[Abstract/Free Full Text]
- Russell AP and Giacobino JP. Old and new determinants in the regulation of energy expenditure. J Endocrinol Invest 25: 862866, 2002.[ISI][Medline]
- Sanchis D, Fleury C, Chomiki N, Goubern M, Huang Q, Neverova M, Gregoire F, Easlick J, Raimbault S, Levi-Meyrueis C, Miroux B, Collins S, Seldin M, Richard D, Warden C, Bouillaud F, and Ricquier D. BMCP1, a novel mitochondrial carrier with high expression in the central nervous system of humans and rodents, and respiration uncoupling activity in recombinant yeast. J Biol Chem 273: 3461134615, 1998.[Abstract/Free Full Text]
- Sauer H, Wartenberg M, and Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 11: 173186, 2001.[CrossRef][ISI][Medline]
- Schrauwen P and Hesselink MK. The role of uncoupling protein 3 in fatty acid metabolism: protection against lipotoxicity? Proc Nutr Soc 63: 287292, 2004.[CrossRef][ISI][Medline]
- Stuart JA, Harper JA, Brindle KM, and Brand MD. Uncoupling protein 2 from carp and zebrafish, ectothermic vertebrates. Biochim Biophys Acta 1413: 5054, 1999.[ISI][Medline]
- Thiebaud CH, Colombelli B, and Muller WP. Diploid gynogenesis in Xenopus laevis and the localization with respect to the centromere of the gene for periodic albinism ap. J Embryol Exp Morphol 83: 3342, 1984.[ISI][Medline]
- Toyomizu M, Ueda M, Sato S, Seki Y, Sato K, and Akiba Y. Cold-induced mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in chicken skeletal muscle. FEBS Lett 529: 313318, 2002.[CrossRef][ISI][Medline]
- Vianna CR, Hagen T, Zhang CY, Bachman E, Boss O, Gereben B, Moriscot AS, Lowell BB, Bicudo JE, and Bianco AC. Cloning and functional characterization of an uncoupling protein homolog in hummingbirds. Physiol Genomics 5: 137145, 2001.[Abstract/Free Full Text]
- Wallace RA, Jared DW, Dumont JN, and Sega MW. Protein incorporation by isolated amphibian oocytes. 3. Optimum incubation conditions. J Exp Zool 184: 321333, 1973.[CrossRef][ISI][Medline]
- Yu XX, Mao W, Zhong A, Schow P, Brush J, Sherwood SW, Adams SH, and Pan G. Characterization of novel UCP5/BMCP1 isoforms and differential regulation of UCP4 and UCP5 expression through dietary or temperature manipulation. FASEB J 14: 16111618, 2000.[Abstract/Free Full Text]
Copyright © 2005 by the American Physiological Society.