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J. Biol. Chem., Vol. 276, Issue 49, 46160-46164, December 7, 2001

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Ubiquinone Is Necessary for Mouse Embryonic Development but Is Not Essential for Mitochondrial Respiration^*

Françoise Levavasseur, Hiroko Miyadera^§, Jacinthe Sirois^¶, Michel L. Tremblay^¶, Kiyoshi Kita^§, Eric Shoubridge^**, and Siegfried Hekimi

From the  Department of Biology, McGill University, 1205 Avenue Dr. Penfield, Montréal, Québec H3A 1B1, Canada, ^§ Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan, ^¶ Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec H3G 1Y6, Canada, and  Montreal Neurological Institute and Department of Human Genetics, McGill University, 3801 University Street, Montréal, Québec H3A 2B4, Canada

Received for publication, September 17, 2001, and in revised form, October 2, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1/ embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of / embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1/ embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

clk-1 mutants of Caenorhabditis elegans are being studied for their pleiotropic phenotype, in which the rates of many biological processes are deregulated and slowed down on average (1, 2). clk-1 encodes a highly conserved (3, 4) mitochondrial (5, 6) protein that is required for ubiquinone (UQ)¹ biosynthesis in yeast (7) and worms (8, 9). Recent evidence suggests that CLK-1 is a hydroxylase that converts demethoxyubiquinone (DMQ) into 5-hydroxy-UQ (10). Indeed, a bacterial CLK-1 homologue is capable of replacing the function of UbiFp, the unrelated enzyme that carries out this function in Escherichia coli. Consistent with this finding, clk-1 mutants in yeast and worms accumulate DMQ₉ instead of producing UQ₉ (7, 9) (the subscript refers to the length of the isoprenoid side chain). In E. coli, DMQ₈ is able to sustain respiration in isolated membranes although at a lower rate than Q₈ (11). Similarly, DMQ₉ also appears to be capable of sustaining electron transport in clk-1 mutants at almost wild-type levels (6, 9). Furthermore, synthetic DMQ₂ can function as a co-factor for electron transport from Complex I and, albeit more poorly, from Complex II (9).

It is not clear how the absence of UQ relates to the other phenotypes of clk-1 mutants as there is no correlation between the biochemical phenotype and the severity of the overall phenotype. Indeed, the quinone phenotype is identical for all three known clk-1 alleles (e2519, qm30, and qm51); UQ₉ is undetectable in the mitochondria in all three cases, and all three accumulate the same amount of DMQ. Yet, most of the features affected in clk-1 mutants are slowed down much more severely in the putative null alleles qm30 and qm51 than they are in the partial loss of function allele e2519 (1, 6).

Recently, it has been found that clk-1 mutants are unable to grow on a UQ-deficient bacterial strain despite the presence and the activity of DMQ₉ (8). This has been interpreted to suggest that DMQ₉ is insufficient for normal mitochondrial function and that dietary bacterial UQ₈ can reach the mitochondria and function there in trace amounts (8). However, dietary UQ is generally not capable of reaching mitochondria, at least in vertebrates (reviewed in Ref. 12), and DMQ appears to be capable of functionally replacing UQ in the respiratory chain. An alternative possibility is that UQ is necessary for viability at other sites than the respiratory chain. To investigate further the function of CLK-1 and the biological roles of UQ, we have generated a knockout mutation of the murine orthologue of clk-1. Our study of ubiquinone biosynthesis and function in mutant embryos and ES cell lines allowed us to resolve some of the questions raised by the work on clk-1 in worms and yeast.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of the Targeting Vector and ES Cell Transfection-- An lFIX II genomic library from mouse strain 129/SvJ DNA (Stratagene) was screened with a genomic mclk1 fragment, and six overlapping genomic clones were obtained. Genomic DNA fragments from two clones were subcloned into Bluescript SK and characterized in detail. A 7-kb NotI-BamHI fragment containing part of the mclk1 promoter and exons I, II, and III was subcloned into Bluescript SK (pL5). A 1.6-kb fragment containing part of exons II and III was removed from pL5 by StuI-BamHI digestion and replaced with a neomycin cassette consisting of a 1.1-kb XhoI blunted-BamHI fragment from pMC1Neo poly(A) to produce pL5+Neo. A 2.8-kb PstI-SacI genomic fragment containing introns IV and V and 500 bp from the 5'-untranslated region was subcloned in Bluescript (pL15). A 2.5-kb EcoRV-XhoI fragment from pL15 was inserted into the SmaI-XhoI sites of pL5+Neo to produce the final replacement targeting vector pL17. A KpnI fragment from the targeting vector was isolated and electroporated into R1 ES cells. Two thousand G418-resistant clones were analyzed by Southern blot analysis. Genomic DNA was digested with BglII and then hybridized with a 3'-external probe flanking the 3' region of the targeting vector (SacI-XhoI fragment). After analyzing the promising neomycin-resistant clones by extensive Southern blot, four clones showed homologous recombinants. We used two independently targeted ES cell clones with the correct karyotype to generate mclk1/ mice.

Genotype Determination-- DNA was prepared from tails of adult mice or yolk sacs of embryos. Southern blot analysis was done as described above. Polymerase chain reaction was done for 30 cycles (95 °C, 30 s; 58 °C, 30 s; 72 °C, 30 s). The primers used to detect the wild-type mclk1 allele were as follows: forward (KO5), 5'-ggt gaa gtc ttt tgg gtt tga gca t-3'; reverse (KO6), 5'-tgt cta agg tca tcc ccg aac tgt g-3'. They amplify a product of 302 bp. The targeted mclk1 allele was detected with the primers KO7 (5'-gcc agc gat atg act cag tgg gta a-3') and KO8 (5'-cac ctt aat atg cga agt gga cct g-3'), which give a product of 397 bp.

Northern and Western Blotting-- Northern analysis was performed using the full-length mclk1 cDNA as a probe as described (13). Western blots were performed using monoclonal antibodies against cytochrome c oxidase subunits I (1D6-E1-A8) and IV (20E8-C12) from Molecular Probes and against human porin 31HL from Calbiochem.

Preparation of Cell-free Extracts for Quinone Analysis and Enzyme Activity Measurements-- The samples were homogenized in 50 mM potassium phosphate buffer (pH 7.4) and centrifuged at 1,000 × g for 5 min at 4 °C. The supernatants were used to determine quinone content and to measure enzyme activity. Protein concentration was determined with bovine serum albumin as the standard (14).

Identification of Quinones-- Quinones were extracted as described (9), with slight modifications. Briefly, the quinones extracted in n-hexane/EtOH were dried under nitrogen gas, dissolved in acetone, and left at 80 °C. After 30 min, the samples were centrifuged at 17,000 × g for 15 min at 4 °C, and the supernatant was dried under nitrogen gas. The residue was dissolved in EtOH, vortexed for 2 min, and applied to HPLC equipped with a guard column, an analytical column (Inertsil ODS-3, C-18, 5 µm, 4.6 × 250 mm, GL Science, Tokyo), and a reduction column (Type RC-10, Irica, Kyoto, Japan). The mobile phase was 50 mM sodium percholorate in tetrahydrofuran/MeOH/H₂O (107/125/18, v/v) with a flow rate of 0.5 ml/min. The elution was monitored by a photodiode array UV-visible detector (Shimadzu SPD10-A) at 275 nm and an amperometric electrochemical detector (ECD, Nanospace SI-2/3005, Shiseido, Tokyo). The oxidation potential for ECD was 600 mV on a glassy carbon electrode. The concentration of quinones was determined spectrophotometrically as described (9) or by ECD. The data were analyzed by Shimadzu Class-VP software (version 5.03).

Enzyme Assays-- All of the assays were performed in 50 mM potassium phosphate buffer (pH 7.4) at 25 °C. NADH-cytochrome c reductase activity and succinate-cytochrome c reductase activity were assayed as described (9). NADH-ferricyanide reductase activity was measured in the presence of 200 µM NADH and 1.3 mM potassium ferricyanide (Sigma) by monitoring the absorbance change of potassium ferricyanide at 420 nm using an extinction coefficient of 1.0 mM¹ cm¹.

Establishment of ES Cell Lines-- Wild-type, heterozygous, and homozygous ES cell lines were established using a standard procedure (15).

Polarography/Oxygen Consumption-- Oxygen consumption was measured as described previously (16) in mclk1 +/+, +/, and / ES cells.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To further study the function of CLK-1 and the biological roles of ubiquinone, we disrupted the mclk1 locus in murine ES cells by homologous recombination and produced heterozygous and homozygous mice using standard methods (see "Experimental Procedures" and Figs. 1 and 2, c and d). Heterozygous +/ mice are viable and fertile. They show no obvious anatomical or behavioral defects. However, after crossing heterozygous male and female mice, no newborn / mice were observed in more than 81 offspring (Table I), indicating that homozygous disruption of mclk1 results in embryonic lethality. To determine the timing of the lethality, embryos from heterozygous intercrosses were analyzed at different days of gestation (Table I). mclk1 / embryos were present at expected Mendelian frequencies at day 8.5 postcoitum (E8.5). By E13.5, however, all mclk1 / embryos detected were in the process of being resorbed. The homozygous embryos showed a developmental delay that is clearly evident by E9.5 (Fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Maps of the wild-type mclk1 locus and of the targeting vector. The black boxes represent exons. The targeting vector consists of the replacement of a part of exon II and exons III and IV by the neomycin gene (white box). A, BamHI; B, BglII; E, EcoRI; K, KpnI; R, EcoRV; S, SacI; X, XhoI.

View larger version (53K): [in this window] [in a new window]   Fig. 2.   Targeted disruption of the mouse mclk1 gene. a, Northern blot analyses of total RNA levels in tissues from mclk1 +/+ and +/ mice and from E11.5 mclk1 +/+, +/, and / littermates. The expression level of the mitochondrially encoded cox1, which codes for a subunit of cytochrome c oxidase (Complex IV), is shown as one of the controls. The expression level of cox1 serves as a measure of the capacity for oxidative phosphorylation in a given tissue. The decreased level observed in homozygous embryos is likely to be due to the beginning of the resorption process. b, Western blot analyses of protein levels in tissues from mclk1 +/+ and +/ mice. Total protein extracts from the liver and brain of 2 -day-old mice were probed with antibodies against mCLK1, COX1, and porin. Porin is a protein of the outer mitochondrial membrane encoded in the nucleus. c, genotype analysis of embryos by polymerase chain reaction using two mixes of primers (see "Experimental Procedures"). The upper band (397 bp) is specific for the wild-type mclk1 allele, and the lower band (302 bp) is specific for the mutant allele. Mut, mutant; Wt, wild type. d, Southern blot analysis. DNA of E9.5 embryos from intercross of mclk1 +/ mice was digested with BglII, electrophoresed on agarose gel, blotted, and hybridized with a 3'-external probe (XhoI-SacI fragment). This probe hybridized to a 3.7-kb band in BglII digests of the native gene and an 8.2-kb band in the disrupted gene.

View larger version (56K): [in this window] [in a new window]   Fig. 3.   Severe developmental delay in mclk1 mutant embryos. a, gross morphology of +/ and / embryos. The embryos are at E10.5 and are littermates. T, telencephalon; M, mesencephalon; Aa, forelimb bud; So, somites; Vg, visceral groove; H, heart; Oc, otic vesicle; 1, first arch; 2, second arch. b, c, and d, histological sections of mclk1 / embryos at E10.5 stained with hematoxylin and eosin. No obvious specific developmental defects were observed. At E10.5, wild-type embryos have 30-35 somites, and many internal structures can be discerned such as the visceral arches, the heart, the liver, and a rudimentary mesonephros. The size, the number of somites (15-20), and the structural organization of mclk1 / embryos resemble those of the wild-type embryos at earlier stages of development (here, around E9).

Northern blot analysis of total E11.5 embryo RNA showed that the amount of mclk1 mRNA was reduced by ~50% in heterozygous embryos compared with normal embryos and could not be detected in mclk1 / embryos (Fig. 2a). A ~50% decrease of mclk1 transcript was observed in several tissues of 44-day-old mclk1 +/ mouse compared with wild-type littermates (Fig. 2a). Immunoblotting with a polyclonal antibody (13) revealed a band of ~ 21 kDa in liver and brain extracts from +/+ and +/ mice. This signal was reduced by 50% in extracts from +/ mice compared with that from +/+ mice (Fig. 2b). The results confirm that the mclk1 mutation is a null mutation and demonstrated a gene-dosage effect on protein levels in +/ mice.

The amounts of ubiquinone-9 (UQ₉) and -10 (UQ₁₀) in homogenates of mclk1 +/+, +/, and +/ embryos were determined by HPLC (Fig. 4a). For mclk1 +/+ embryos, a major peak elutes at 42.4 min and is identical to standard UQ₉ for elution time. A smaller peak that elutes around 57 min corresponds to UQ₁₀. The quinone profile of mclk1 +/ embryos is indistinguishable from that of the wild type. The amounts of UQ₉ and UQ₁₀ were similar in wild-type and heterozygous embryos (Table II). However, neither UQ₉ nor UQ₁₀ was detected in mclk1 / embryos (Fig. 4a and Table II). These mutant embryos instead exhibited a major peak eluting 2.3 min earlier than UQ₉. This component co-eluted with DMQ₉ purified from clk-1 mutant C. elegans (9).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Quinone content in embryos and ES cell lines. Elution profiles of quinones from embryos (a) and ES cell lines (b). Reverse-phase HPLC chromatograms show the elution of quinones from cell-free extracts of embryos and ES cells as monitored by ECD. The elution of standard UQ9 and DMQ9 purified from C. elegans clk-1 (qm51) (9), lipid extracts from ES-2, ES-8, and ES-7 cell lines (200 µg of protein) and from wild-type (200 µg), heterozygous (101.5 µg), and homozygous (42.8 µg) embryos.

mclk1 +/+, +/, and / ES cell lines were derived from E3.5 blastocysts obtained from heterozygous matings. The profiles and concentrations of quinones in these lines were very similar to those observed in the genetically equivalent embryos (Fig. 4b; Table II). In particular, only DMQ₉ was detected in the mclk1 / ES cell line (ES-7) (Fig. 4b).

We used the ES cell lines to study the effect of the UQ deficiency on the function of mitochondrial enzymes and overall cellular oxygen consumption (Table III). The activity of succinate cytochrome c reductase, for which UQ is a required co-factor and involves respiratory Complexes II and III, is severely reduced (15% of wild type). However, the activity of NADH-cytochrome c reductase, which involves the activity of Complex I and Complex III, is not very strongly affected (63% of wild type). As a control, we measured the activity of NADH-ferricyanide reductase, which involves subunits of Complex I but is not believed to require UQ (17, 18). As expected, NADH-ferricyanide reductase remains unaffected in the mutant cells (Table III). Consistent with the above observations, we find that the overall level of oxygen consumption in the mutants is reduced relatively little, to 65% of wild type, which parallels the moderate reduction in the activities of Complexes I and III (Table III).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our results show that UQ is not made in mclk1 knockout embryos and ES cells. Instead, DMQ accumulates in levels very similar to those of UQ in wild-type embryos or cells. UQ is a co-factor in the function of the respiratory chain in transporting electrons from Complexes I and II to Complex III. However, we find that the functions of Complexes I and III, as well as overall respiration, are relatively mildly impaired in mclk1 cells. In contrast, the function of Complex II in electron transport is much more severely affected. Similar observations have been made in other organisms such as E. coli and C. elegans, in which DMQ is capable of replacing UQ in Complexes I and III but is a relatively poor acceptor of electrons from succinate (9, 11). Note, however, that in worm clk-1 mutants that produce only DMQ, succinate cytochrome c reductase appears to be less affected (6, 9) than what we observed in the ES cell lines.

Thus, as in the case of the clk-1 mutants in C. elegans, the DMQ produced in mclk1 mutants appears to be sufficient for the maintenance of a relatively high level of mitochondrial function (65% of wild-type oxygen consumption). In C. elegans, it has been argued that the maintenance of mitochondria activity could be because of dietary UQ₈ acquired from the bacterial food source (8). This hypothesis is based on the observation that clk-1 mutant worms cannot grow on bacteria that do not synthesize UQ. However, this cannot be the explanation for the high oxygen consumption in the mclk1 ES cells, as we used charcoal-depleted serum and could not find any trace of UQ in this serum by HPLC (data not shown). It is highly unlikely therefore that any exogenous UQ is responsible for the level of oxygen consumption we observed in the mclk1 cells.

It is surprising that the levels of mitochondrial function found in the mclk1 mutant cells are insufficient to carry out embryogenesis. This observation is similar to the unexpected finding that dietary UQ is necessary for C. elegans clk-1 mutant development despite the presence of high level of active mitochondrial DMQ. One possibility is that embryogenesis in both organisms requires UQ at other sites in addition to the respiratory chain. UQ is found in almost all biological membranes (12) and is known to be a co-factor of the uncoupling proteins in the mitochondria (19, 20), to regulate the permeability transition pore (21), and to function in plasma membrane and lysosomal oxidoreductase systems (22, 23). UQ is also a co-factor for a number of cellular enzymes, including glycerol 3-phosphate dehydrogenase, dihydroorotate reductase, and ETF (electron-transferring flavoprotein):ubiquinone reductase. Furthermore, it has recently been discovered that, in bacteria, respiratory quinones are the primary signal for the regulation of growth in response to oxygen availability (24). Given the conservation between prokaryotes and eukaryotes of crucial molecular mechanisms that sense environmental signals (e.g. the PAS domain proteins) (25), the complete UQ deficiency in mclk1 mutants could directly affect the regulation of embryonic growth. Note that although quinone contents are higher in tissues like heart, there is no evidence for tissue-specific abnormalities in early development. Had development proceeded further, it is possible that such effects would have been exposed. In conclusion, although we have shown that DMQ can replace UQ in the respiratory chain, it is possible that DMQ is unable to replace UQ for one or more of its functions at other sites.

	ACKNOWLEDGEMENTS

We thank Abdelmadjid Hihi, Claire Bénard, and Robyn Branicky for critically reading the manuscript and Josée Breton for expert technical assistance.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** A Montreal Neurological Institut Killam scholar.

A Canadian Institutes of Health Research scientist. To whom correspondence should be addressed. Tel.: 514-398-6440; Fax: 514-398-1674; E-mail: Siegfried.Hekimi@McGill.ca.

Published, JBC Papers in Press, October 3, 2001, DOI 10.1074/jbc.M108980200

	ABBREVIATIONS

The abbreviations used are: UQ, ubiquinone; DMQ, demethoxyubiquinone; ES cell, embryonic stem cell; kb, kilobase pair(s); bp, base pair(s); HPLC, high pressure liquid chromatography; ECD, electrochemical detector; E, embryonic day (e.g. E10.5).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 276/49/46160    most recent M108980200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Levavasseur, F. Articles by Hekimi, S. Search for Related Content PubMed PubMed Citation Articles by Levavasseur, F. Articles by Hekimi, S. Social Bookmarking                      What's this?