Quinones in long-lived clk-1 mutants of Caenorhabditis elegans

Ubiquinone (UQ) (coenzyme Q) is a lipophilic redox-active molecule that functions as an electron carrier in the mitochondrial electron transport chain. Electron transfer via UQ involves the formation of semiubiquinone radicals, which causes the generation of superoxide radicals upon reaction with oxygen. In the reduced form, UQ functions as a lipid-soluble antioxidant, and protects cells from lipid peroxidation. Thus, UQ is also important as a lipophilic regulator of oxidative stress. Recently, a study on long-lived clk-1 mutants of Caenorhabditis elegans demonstrated that biosynthesis of UQ is dramatically altered in mutant mitochondria. Demethoxy ubiquinone (DMQ), that accumulates in clk-1 mutants in place of UQ, may contribute to the extension of life span. Here we elucidate the possible mechanisms of life span extension in clk-1 mutants, with particular emphasis on the electrochemical property of DMQ. Recent findings on the biochemical function of CLK-1 are also discussed.     

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.     

Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration

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.     

Erratum

Ubiquinone (UQ), also known as coenzyme Q (CoQ), is a redox-active lipid present in all cellular membranes where it functions in a variety of cellular processes. The best known functions of UQ are to act as a mobile electron carrier in the mitochondrial respiratory chain and to serve as a lipid soluble antioxidant in cellular membranes. All eukaryotic cells synthesize their own UQ. Most of the current knowledge on the UQ biosynthetic pathway was obtained by studying Escherichia coli and Saccharomyces cerevisiae UQ-deficient mutants. The orthologues of all the genes known from yeast studies to be involved in UQ biosynthesis have subsequently been found in higher organisms. Animal mutants with different genetic defects in UQ biosynthesis display very different phenotypes, despite the fact that in all these mutants the same biosynthetic pathway is affected. This review summarizes the present knowledge of the eukaryotic biosynthesis of UQ, with focus on the biosynthetic genes identified in animals, including Caenorhabditis elegans, rodents, and humans. Moreover, we review the phenotypes of mutants in these genes and discuss the functional consequences of UQ deficiency in general.     

Molecular mechanism of maternal rescue in the clk-1 mutants of Caenorhabditis elegans

The clk-1 mutants of Caenorhabditis elegans display an average slowing down of physiological rates, including those of development, various behaviors, and aging. clk-1 encodes a hydroxylase involved in the biosynthesis of the redox-active lipid ubiquinone (co-enzyme Q), and in clk-1 mutants, ubiquinone is replaced by its biosynthetic precursor demethoxyubiquinone. Surprisingly, homozygous clk-1 mutants display a wild-type phenotype when issued from a heterozygous mother. Here, we show that this maternal effect is the result of the persistence of small amounts of maternally derived CLK-1 protein and that maternal CLK-1 is sufficient for the synthesis of considerable amounts of ubiquinone during development. However, gradual depletion of CLK-1 and ubiquinone, and expression of the mutant phenotype, can be produced experimentally by developmental arrest. We also show that the very long lifespan observed in daf-2 clk-1 double mutants is not abolished by the maternal effect. This suggests that, like developmental arrest, the increased lifespan conferred by daf-2 allows for depletion of maternal CLK-1, resulting in the expression of the synergism between clk-1 and daf-2. Thus, increased adult longevity can be uncoupled from the early mutant phenotypes, indicating that it is possible to obtain an increased adult lifespan from the late inactivation of processes required for normal development and reproduction.     

Reproductive fitness and quinone content of Caenorhabditis elegans clk-1 mutants fed coenzyme Q isoforms of varying length

Caenorhabditis elegans clk-1 mutants lack coenzyme Q9 and accumulate the biosynthetic intermediate demethoxy-Q9. A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q8 isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q7, Q8, Q9, or Q10. Wild-type nematodes showed no change in reproductive fitness regardless of the Qn isoform fed. clk-1(e2519) fed the Q9 diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q7-containing bacteria. The content of Q7 in the mitochondria of clk-1 animals was decreased relative to Q8, suggesting less effective transport of Q7 to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q7-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.     

Development and fertility in Caenorhabditis elegans clk-1 mutants depend upon transport of dietary coenzyme Q8 to mitochondria

The Caenorhabditis elegans clk-1 mutants lack coenzyme Q(9) and instead accumulate the biosynthetic intermediate demethoxy-Q(9) (DMQ(9)). clk-1 animals grow to reproductive adults, albeit slowly, if supplied with Q(8)-containing Escherichia coli. However, if Q is withdrawn from the diet, clk-1 animals either arrest development as young larvae or become sterile adults depending upon the stage at the time of the withdrawal. To understand this stage-dependent response to a Q-less diet, the quinone content was determined during development of wild-type animals. The quinone content varies in the different developmental stages in wild-type fed Q(8)-replete E. coli. The amounts peak at the second larval stage, which coincides with the stage of arrest of clk-1 larvae fed a Q-less diet from hatching. Levels of the endogenously synthesized DMQ(9) are high in the clk-1(qm30)-arrested larvae and sterile adults fed Q-less food. Comparison of quinones from animals fed a Q-replete or a Q-less diet establishes that the Q(8) present is assimilated from the E. coli. Furthermore, this E. coli-specific Q(8) is present in mitochondria isolated from fertile clk-1(qm30) adults fed a Q-replete diet. These results suggest that the uptake and transport of dietary Q(8) to mitochondria prevent the arrest and sterility phenotypes of clk-1 mutants and that DMQ is not functionally equivalent to Q.     

Demethoxy-Q, an intermediate of coenzyme Q biosynthesis, fails to support respiration in Saccharomyces cerevisiae and lacks antioxidant activity

Caenorhabditis elegans clk-1 mutants cannot produce coenzyme Q(9) and instead accumulate demethoxy-Q(9) (DMQ(9)). DMQ(9) has been proposed to be responsible for the extended lifespan of clk-1 mutants, theoretically through its enhanced antioxidant properties and its decreased function in respiratory chain electron transport. In the present study, we assess the functional roles of DMQ(6) in the yeast Saccharomyces cerevisiae. Three mutations designed to mirror the clk-1 mutations of C. elegans were introduced into COQ7, the yeast homologue of clk-1: E233K, predicted to disrupt the di-iron carboxylate site considered essential for hydroxylase activity; L237Stop, a deletion of 36 amino acid residues from the carboxyl terminus; and P175Stop, a deletion of the carboxyl-terminal half of Coq7p. Growth on glycerol, quinone content, respiratory function, and response to oxidative stress were analyzed in each of the coq7 mutant strains. Yeast strains lacking Q(6) and producing solely DMQ were respiratory deficient and unable to support (6)either NADH-cytochrome c reductase or succinate-cytochrome c reductase activities. DMQ(6) failed to protect cells against oxidative stress generated by H(2)O(2) or linolenic acid. Thus, in the yeast model system, DMQ does not support respiratory activity and fails to act as an effective antioxidant. These results suggest that the life span extension observed in the C. elegans clk-1 mutants cannot be attributed to the presence of DMQ per se.     

Comparison of a coq7 deletion mutant with other respiration-defective mutants in fission yeast

Among the steps in ubiquinone biosynthesis, that catalyzed by the product of the clk-1/coq7 gene has received considerable attention because of its relevance to life span in Caenorhabditis elegans. We analyzed the coq7 ortholog (denoted coq7) in Schizosaccharomyces pombe, to determine whether coq7 has specific roles that differ from those of other coq genes. We first confirmed that coq7 is necessary for the penultimate step in ubiquinone biosynthesis, from the observation that the deletion mutant accumulated the ubiquinone precursor demethoxyubiquinone-10 instead of ubiquinone-10. The coq7 mutant displayed phenotypes characteristic of other ubiquinone-deficient Sc. pombe mutants, namely, hypersensitivity to hydrogen peroxide, a requirement for antioxidants for growth on minimal medium, and an elevated production of sulfide. To compare these phenotypes with those of other respiration-deficient mutants, we constructed cytochrome c (cyc1) and coq3 deletion mutants. We also assessed accumulation of oxidative stress in various ubiquinone-deficient strains and in the cyc1 mutant by measuring mRNA levels of stress-inducible genes and the phosphorylation level of the Spc1 MAP kinase. Induction of ctt1, encoding catalase, and apt1, encoding a 25 kDa protein, but not that of gpx1, encoding glutathione peroxidase, was indistinguishable in four ubiquinone-deficient mutants, indicating that the oxidative stress response operates at similar levels in the tested strains. One new phenotype was observed, namely, loss of viability in stationary phase (chronological life span) in both the ubiquinone-deficient mutant and in the cyc1 mutant. Finally, Coq7 was found to localize in mitochondria, consistent with the possibility that ubiquinone biosynthesis occurs in mitochondria in yeasts. In summary, our results indicate that coq7 is required for ubiquinone biosynthesis and the coq7 mutant is not distinguishable from other ubiquinone-deficient mutants, except that its phenotypes are more pronounced than those of the cyc1 mutant.     

Suppression of Glutamic Acid Codons by Mutant Glycine Transfer Ribonucleic Acid

In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.     

Coenzyme Q is irreplaceable by demethoxy-coenzyme Q in plasma membrane of Caenorhabditis elegans

A procedure was developed to isolate fractions enriched in plasma membrane from Caenorhabditis elegans. Coenzyme Q9 (Q9) was found in plasma membrane isolated from either wild-type or long-lived qm30 and qm51 clk-1 mutant strains of Caenorhabditis elegans, along with dietary coenzyme Q8 (Q8) and the biosynthetic intermediate demethoxy-Q9 (DMQ9). NADH was able to reduce both Q8 and Q9, but not DMQ9. Our results indicate that DMQ9 cannot achieve the same redox role of Q9 in plasma membrane, suggesting that proportion of all these Q isoforms in plasma membrane must be an important factor in establishing the clk-1 mutant phenotype.     

Sensitivity of Caenorhabditis elegans clk-1 mutants to ubiquinone side-chain length reveals multiple ubiquinone-dependent processes

Ubiquinone (coenzyme Q, or Q) is a membrane constituent, whose head group is capable of accepting and donating electrons and whose lipidic side chain is composed of a variable number of isoprene subunits. A possible role for Q as a dietary antioxidant for treating conditions that involve altered cellular redox states is being intensely studied. Mutations in the clk-1 gene of the nematode Caenorhabditis elegans affect numerous physiological rates including behavioral rates, developmental rates, reproduction, and life span. clk-1 encodes a protein associated with the inner mitochondrial membrane that is necessary for Q biosynthesis in C. elegans. clk-1 mutants do not synthesize Q but accumulate demethoxyubiquinone, a Q synthesis intermediate that is able to partially sustain mitochondrial respiration in worms as well as in mammals. Recently, we and others have found that exogenous Q is necessary for the fertility and development of clk-1 mutants. Here, we take advantage of the clk-1 genetic model to identify structural features of Q that are functionally important in vivo. We show that clk-1 mutants are exquisitely sensitive to the length of the side chain of the Q they consume. We also identified differential sensitivity to Q side-chain length between null alleles of clk-1 (qm30 and qm51) and the weaker allele e2519. This allows us to propose a model where we distinguish several types of Q-dependent processes in vivo: processes that are very sensitive to Q side-chain length and processes that are permissive to Q with shorter chains.     

The submitochondrial distribution of ubiquinone affects respiration in long-lived Mclk1+/? mice

Mclk1 (also known as Coq7) and Coq3 code for mitochondrial enzymes implicated in the biosynthetic pathway of ubiquinone (coenzyme Q or UQ). Mclk1^+/− mice are long-lived but have dysfunctional mitochondria. This phenotype remains unexplained, as no changes in UQ content were observed in these mutants. By producing highly purified submitochondrial fractions, we report here that Mclk1^+/− mice present a unique mitochondrial UQ profile that was characterized by decreased UQ levels in the inner membrane coupled with increased UQ in the outer membrane. Dietary-supplemented UQ₁₀ was actively incorporated in both mitochondrial membranes, and this was sufficient to reverse mutant mitochondrial phenotypes. Further, although homozygous Coq3 mutants die as embryos like Mclk1 homozygous null mice, Coq3^+/− mice had a normal lifespan and were free of detectable defects in mitochondrial function or ubiquinone distribution. These findings indicate that MCLK1 regulates both UQ synthesis and distribution within mitochondrial membranes.     

Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5' and 3', respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q(10). Here we show that the Q(9) content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q(9) content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.     

Intragenic and Extragenic Suppressors of Temperature Sensitive Mutations in the Replication Initiation Genes dnaD and dnaB of Bacillus subtilis

Background

The Bacillus subtilis genes dnaD and dnaB are essential for the initiation of DNA replication and are required for loading of the replicative helicase at the chromosomal origin of replication oriC. Wild type DnaD and DnaB interact weakly in vitro and this interaction has not been detected in vivo or in yeast two-hybrid assays.

Methodology/Principal Findings

We isolated second site suppressors of the temperature sensitive phenotypes caused by one dnaD mutation and two different dnaB mutations. Five different intragenic suppressors of the dnaD23ts mutation were identified. One intragenic suppressor was a deletion of two amino acids in DnaD. This deletion caused increased and detectable interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction caused by a missense mutation in dnaB that is an extragenic suppressor of dnaD23ts. We isolated both intragenic and extragenic suppressors of the two dnaBts alleles. Some of the extragenic suppressors were informational suppressors (missense suppressors) in tRNA genes. These suppressor mutations caused a change in the anticodon of an alanine tRNA so that it would recognize the mutant codon (threonine) in dnaB and likely insert the wild type amino acid (alanine).

Conclusions/Significance

The intragenic suppressors should provide insights into structure-function relationships in DnaD and DnaB, and interactions between DnaD and DnaB. The extragenic suppressors in the tRNA genes have important implications regarding the amount of wild type DnaB needed in the cell. Since missense suppressors are typically inefficient, these findings indicate that production of a small amount of wild type DnaB, in combination with the mutant protein, is sufficient to restore some DnaB function.     

C. elegans knockouts in ubiquinone biosynthesis genes result in different phenotypes during larval development

Ubiquinone is an essential molecule in aerobic organisms to achieve both, ATP synthesis and antioxidant defence. Mutants in genes responsible of ubiquinone biosynthesis lead to non-respiring petite yeast. In C. elegans, coq-7/clk-1 but not coq-3 mutants live longer than wild type showing a 'slowed' phenotype. In this paper we demonstrate that absence in ubiquinone in coq-1, coq-2 or coq-8 mutants lead to larval development arrest, slowed pharyngeal pumping, eventual paralysis and cell death. All these features emerge during larval development, whereas embryo development appeared similar to that of wild type individuals. Dietary coenzyme Q did not restore any of the alterations found in these coq mutants. These phenomena suggest that coenzyme Q mutants unable to synthesize this molecule develop a deleterious phenotype leading to lethality. On the contrary, phenotype of C. elegans coq-7/clk-1 mutants may be a unique phenotype than can not generalize to mutants in ubiquinone biosynthesis. This particular phenotype may not be based on the absence of endogenous coenzyme Q, but to the simultaneous presence of dietary coenzyme Q and the its biosynthesis intermediate demethoxy-coenzyme Q.     

tRNA(2Gln) mutants that translate the CGA arginine codon as glutamine in Escherichia coli.

We present a novel missense suppression system for the selection of tRNA(2GIn) mutants that can efficiently translate the CGA (arginine) codon as glutamine. tRNA(2Gln) mutants were cloned from a partially randomized synthetic gene pool using a plasmid vector that simultaneously expresses the tRNA gene and, to ensure efficient aminoacylation, the glutamine aminoacyl-tRNA synthetase gene (glnS). tRNA mutants that insert glutamine at CGA were selected as missense suppressors of a lacZ mutant (lacZ625(CGA)) that contains CGA substituted for an essential glutamine codon. Preliminary characterizations of four suppressors is presented. All of them contain two anticodon mutations: C-->U at position 34 and U-->C at position 35, which allow for cognate translation of CGA. U35 was previously shown to be an important determinant for glutaminylation of tRNA(2Gln) in vitro; suppression in vivo requires overexpression of the glutaminyl-tRNA synthetase gene (glnS). One tRNA variant contains no further mutations and has the highest missense suppression activity (8%). Three other isolates each contain an additional point mutation that alters suppression efficiency. This system will be useful for further studies of tRNA structure and function. In addition, because relatively efficient translation of the rare CGA codon as glutamine is not toxic for Escherichia coli, it may be possible to translate this sense codon with other alternate meanings, a property which could greatly facilitate protein engineering.