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.     

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.     

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.     

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.     

coq7/clk-1 regulates mitochondrial respiration and the generation of reactive oxygen species via coenzyme Q

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, and shows sluggish behaviours and an extended lifespan. In C. elegans and Saccharomyces cerevisiae, coq7/clk-1 is required for the biosynthesis of coenzyme Q (CoQ), an essential co-factor in mitochondrial respiration. The clk-1 mutant contains dietary CoQ(8) from Escherichia coli and demethoxyubiquinone 9 (DMQ9) instead of CoQ(9). In a previous study, we generated COQ7-deficient mice by targeted disruption of the coq7 gene and reported that mouse coq7/clk-1 is also essential for CoQ synthesis, maintenance of mitochondrial integrity and neurogenesis. In the present study, we rescued COQ7-deficient mice from embryonic lethality and established a mouse model with decreased CoQ level by transgene expression of COQ7/CLK-1. A biochemical analysis showed a concomitant decrease in CoQ(9), mitochondrial respiratory enzyme activity and the generation of reactive oxygen species (ROS) in the mitochondria of CoQ-insufficient mice. This implied that the depressed activity of respiratory enzymes and the depressed production of ROS may play a physiological role in the control of lifespan in mammalian species and of C. elegans.     

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.     

Caenorhabditis elegans ubiquinone biosynthesis genes

Ubiquinone (coenzyme Q, Q) is an essential lipid electron carrier in the mitochondria respiratory chain, and also functions as antioxidant and participates as a cofactor of mitochondrial uncoupling proteins. Caernorhabditis elegans synthesize Q9, but both dietary Q8 intake and endogenous Q9 biosynthesis determine Q balance. Thus, it is of current interest to know the regulatory mechanisms of Q9 biosynthesis in this nematode. Here we review results that leaded to identification of genes involved in Q9 biosynthesis in this nematode using the RNA interference technology. C. elegans coq genes were silenced and depletion of Q content was observed, indicating that the genes related here participate in Q9 biosynthesis. Silenced populations showed an extension of adult life span, probably by the decrease of endogenous oxidative stress produced in mitochondria. We also report the heterologous complementation of C. elegans coq-5 and coq-7 genes in their homologue yeast coq null mutants, leading to restore its ability to growth in non-fermentable sugars. These complemented yeast strains accumulated Q6 but also the intermediate demethoxy-Q6. These findings support the conservative functional homology of these genes.     

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.     

Mitochondrial oxidative stress alters a pathway in Caenorhabditis elegans strongly resembling that of bile acid biosynthesis and secretion in vertebrates

Mammalian bile acids (BAs) are oxidized metabolites of cholesterol whose amphiphilic properties serve in lipid and cholesterol uptake. BAs also act as hormone-like substances that regulate metabolism. The Caenorhabditis elegans clk-1 mutants sustain elevated mitochondrial oxidative stress and display a slow defecation phenotype that is sensitive to the level of dietary cholesterol. We found that: 1) The defecation phenotype of clk-1 mutants is suppressed by mutations in tat-2 identified in a previous unbiased screen for suppressors of clk-1. TAT-2 is homologous to ATP8B1, a flippase required for normal BA secretion in mammals. 2) The phenotype is suppressed by cholestyramine, a resin that binds BAs. 3) The phenotype is suppressed by the knock-down of C. elegans homologues of BA-biosynthetic enzymes. 4) The phenotype is enhanced by treatment with BAs. 5) Lipid extracts from C. elegans contain an activity that mimics the effect of BAs on clk-1, and the activity is more abundant in clk-1 extracts. 6) clk-1 and clk-1;tat-2 double mutants show altered cholesterol content. 7) The clk-1 phenotype is enhanced by high dietary cholesterol and this requires TAT-2. 8) Suppression of clk-1 by tat-2 is rescued by BAs, and this requires dietary cholesterol. 9) The clk-1 phenotype, including the level of activity in lipid extracts, is suppressed by antioxidants and enhanced by depletion of mitochondrial superoxide dismutases. These observations suggest that C. elegans synthesizes and secretes molecules with properties and functions resembling those of BAs. These molecules act in cholesterol uptake, and their level of synthesis is up-regulated by mitochondrial oxidative stress. Future investigations should reveal whether these molecules are in fact BAs, which would suggest the unexplored possibility that the elevated oxidative stress that characterizes the metabolic syndrome might participate in disease processes by affecting the regulation of metabolism by BAs.     

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.     

Uncoupling the pleiotropic phenotypes of clk-1 with tRNA missense suppressors in Caenorhabditis elegans

clk-1 encodes a demethoxyubiquinone (DMQ) hydroxylase that is necessary for ubiquinone biosynthesis. When Caenorhabditis elegans clk-1 mutants are grown on bacteria that synthesize ubiquinone (UQ), they are viable but have a pleiotropic phenotype that includes slowed development, behaviors, and aging. However, when grown on UQ-deficient bacteria, the mutants arrest development transiently before growing up to become sterile adults. We identified nine suppressors of the missense mutation clk-1(e2519), which harbors a Glu-to-Lys substitution. All suppress the mutant phenotypes on both UQ-replete and UQ-deficient bacteria. However, each mutant suppresses a different subset of phenotypes, indicating that most phenotypes can be uncoupled from each other. In addition, all suppressors restore the ability to synthesize exceedingly small amounts of UQ, although they still accumulate the precursor DMQ, suggesting that the presence of DMQ is not responsible for the Clk-1 phenotypes. We cloned six of the suppressors, and all encode tRNA(Glu) genes whose anticodons are altered to read the substituted Lys codon of clk-1(e2519). To our knowledge, these suppressors represent the first missense suppressors identified in any metazoan. The pattern of suppression we observe suggests that the individual members of the tRNA(Glu) family are expressed in different tissues and at different levels.     

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.     

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.     

Mouse homologue of coq7/clk-1, longevity gene in Caenorhabditis elegans, is essential for coenzyme Q synthesis, maintenance of mitochondrial integrity, and neurogenesis.

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, which showed sluggish behavior and an extended life span. Mouse coq7 is homologous to Saccharomyces cerevisiae coq7/cat5 that is required for biosynthesis of coenzyme Q (CoQ), an essential cofactor in mitochondrial respiration. Here we generated COQ7-deficient mice to investigate the biological role of COQ7 in mammals. COQ7-deficient mouse embryos failed to survive beyond embryonic day 10.5, exhibiting small-sized body and delayed embryogenesis. Morphological studies showed that COQ7-deficient neuroepithelial cells failed to show the radial arrangement in the developing cerebral wall, aborting neurogenesis at E10.5. Electron microscopic analysis further showed the enlarged mitochondria with vesicular cristae and enlarged lysosomes filled with disrupted membranes, which is consistent with mitochondriopathy. Biochemical analysis demonstrated that COQ7-deficient embryos failed to synthesize CoQ(9), but instead yielded demethoxyubiquinone 9 (DMQ(9)). Cultured embryonic cells from COQ7-deficient mice were rescued by adding bovine fetal serum in vitro, but exhibited slowed cell proliferation, which resembled to the phenotype of clk-1 with delayed cell divisions. The result implied the essential role of coq7 in CoQ synthesis, maintenance of mitochondrial integrity, and neurogenesis in mice.     

Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants.

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q(6) by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains. Q(6) supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains. Following culture in medium containing dextrose, the added Q(6) was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q(6) in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q(6). These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q(6) into the plasma membrane. The two coq7 mutant strains rescued by Q(6) supplementation for growth on ethanol contained mitochondrial Q(6) levels similar to wild type. However, the content of Q(6) in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q(6), succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q(6) content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c(1) polypeptide. These studies show that transport of extracellular Q(6) to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc(1) complex is unstable. These results indicate that delivery of exogenous Q(6) to mitochondria is required fore activity and stability of the bc(1) complex in yeast coq mutants.     

Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q

Coenzyme Q(n) is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Caenorhabditis elegans fed Escherichia coli devoid of Q(8) have a significant lifespan extension when compared to C. elegans fed a standard 'Q-replete'E. coli diet. Here we examine possible mechanisms for the lifespan extension caused by the Q-less E. coli diet. A bioassay for Q uptake shows that a water-soluble formulation of Q(10) is effectively taken up by both clk-1 mutant and wild-type nematodes, but does not reverse lifespan extension mediated by the Q-less E. coli diet, indicating that lifespan extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q-less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q-less E. coli, and in fact requires E. coli viability. Q-less E. coli have defects in respiratory metabolism. C. elegans fed Q-replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced lifespan extension, although not as dramatic as those fed the respiratory deficient Q-less E. coli diet. The data suggest that feeding respiratory incompetent E. coli, whether Q-less or Q-replete, produces a robust life extension in wild-type C. elegans. We believe that the fermentation-based metabolism of the E. coli diet is an important parameter of C. elegans longevity.     

Conservation of the Caenorhabditis elegans timing gene clk-1 from yeast to human: a gene required for ubiquinone biosynthesis with potential implications for aging

Mutations in the Caenorhabditis elegans gene clk-1 have a major effect on slowing development and increasing life span. The Saccharomyces cerevisiae homolog COQ7 encodes a mitochondrial protein involved in ubiquinone biosynthesis and, hence, is required for respiration and gluconeogenesis. In this study, RT-PCR and 5′ RACE were used to isolate both human and mouse clk-1/COQ7 homologs. Human CLK-1 was mapped to Chr 16(p12–13.1) by Radiation Hybrid (RH) and fluorescence in situ hybridization (FISH) methods. The number and location of human CLK1 introns were determined, and the location of introns II and IV are the same as in C. elegans. Northern blot analysis showed that three different isoforms of CLK-1 mRNA are present in several tissues and that the isoforms differ in the amount of expression. The functional equivalence of human CLK-1 to the yeast COQ7 homolog was tested by introducing either a single or multicopy plasmid containing human CLK-1 cDNA into yeast coq7 deletion strains and assaying for growth on a nonfermentable carbon source. The human CLK-1 gene was able to functionally complement yeast coq7 deletion mutants. The protein similarities and the conservation of function of the CLK-1/clk-1/COQ7 gene products suggest a potential link between the production of ubiquinone and aging. Received: 15 March 1999 / Accepted 11 June 1999     

CLK-1 controls respiration, behavior and aging in the nematode Caenorhabditis elegans

Mutations in the clk-1 gene of the nematode Caenorhabditis elegans result in an average slowing of a variety of developmental and physiological processes, including the cell cycle, embryogenesis, post-embryonic growth, rhythmic behaviors and aging. In yeast, a CLK-1 homologue is absolutely required for ubiquinone biosynthesis and thus respiration. Here we show that CLK-1 is fully active when fused to green fluorescent protein and is found in the mitochondria of all somatic cells. The activity of mutant mitochondria, however, is only very slightly impaired, as measured in vivo by a dye-uptake assay, and in vitro by the activity of succinate cytochrome c reductase. Overexpression of CLK-1 activity in wild-type worms can increase mitochondrial activity, accelerate behavioral rates during aging and shorten life span, indicating that clk-1 regulates and controls these processes. These observations also provide strong genetic evidence that mitochondria are causally involved in aging. Furthermore, the reduced respiration of the long-lived clk-1 mutants suggests that longevity is promoted by the age-dependent decrease in mitochondrial function that is observed in most species.