From the Department of Biomedical Chemistry, Graduate
School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan, the
§ Department of Ecological Engineering, Toyohashi University
of Technology, Toyohashi 441-8580, Japan, the ¶ Central Laboratory
of Medical Sciences, Juntendo University School of Medicine, Tokyo
113-8421, Japan, the
Division of Applied Life Sciences, Graduate
School of Agriculture, Kyoto University, Kyoto 606-8502, Japan, the
** Department of Molecular Life Science, Tokai University School of
Medicine, Kanagawa 259-1193, Japan, and the
Department of Biology, McGill University,
Montréal, Québec H3A 1B1, Canada
Received for publication, December 18, 2000
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ABSTRACT |
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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
(UQ9) biosynthesis and show that clk-1 mutants
mitochondria do not contain detectable levels of UQ9.
Instead, the UQ9 biosynthesis intermediate,
demethoxyubiquinone (DMQ9), is present at high levels. This
result demonstrates that CLK-1 is absolutely required for the
biosynthesis of UQ9 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 DMQ9 can function as an electron carrier in the
respiratory chain. To test this possibility, the short side chain
derivative DMQ2 was chemically synthesized. We find that
DMQ2 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-UQ2, cannot.
The accumulation of DMQ9 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.
The understanding of the biological pathways that control life
span can be studied in Caenorhabditis elegans through the
identification of genes that alter the length of life when mutated (1).
For example, mutations in clk-1 are known to cause an
extended life span, as well as the slowing of a variety of
developmental and physiological events, including the cell cycle,
embryogenesis, post-embryonic development, and rhythmic adult behaviors
(2, 3). Thus, CLK-1 is expected to play a unique biological role that
is necessary to determine the life span and to coordinate these various
biological processes. However, the biochemical differences between
clk-1 mutants and the wild-type strain, which might indicate the function of CLK-1, have yet to been identified (1, 4-7).
clk-1 encodes a 187-residue polypeptide that is homologous
to yeast coq7/cat5 (8). COQ7/CAT5 is located in the inner
membrane of yeast mitochondria and is necessary for the biosynthesis of ubiquinone (UQ)1 in yeast (9,
10). Therefore, yeast coq7/cat5 mutants, which lack
UQ6, are unable to grow on nonfermentable carbon sources (9). Orthologs of clk-1/coq7/cat5 have also been reported
from mammals, including human (11-13), and appear to be highly
conserved among species.
Recently, a green fluorescent protein fusion to C. elegans CLK-1 was shown to localize to the mitochondria of all the
somatic cells of the worm (14). However, in contrast to the situation in yeast, which is defective in respiratory growth, C. elegans clk-1 mutants are able to respire almost normally. In fact, the metabolic capacities and the ATP levels of adult clk-1
mutants are unchanged or even higher than those of the wild-type strain (15), and clk-1 mutants mitochondria exhibit
succinate-cytochrome c reductase activity that is comparable
with that of wild-type mitochondria (14). These observations suggest
that CLK-1 is not exclusively involved in UQ biosynthesis in
C. elegans.
In this report, we analyzed the quinone composition of clk-1
mutants mitochondria, to elucidate the effect of clk-1
mutation on the biosynthesis of UQ in C. elegans, and found
that UQ biosynthesis is dramatically altered in clk-1
mutants. That is, clk-1 mutants mitochondria do not possess
detectable levels of UQ9 and instead contain a UQ
biosynthesis intermediate, demethoxy ubiquinone (DMQ9). We
further analyzed the respiratory activities of mutants mitochondria and
found that DMQ can functionally replace UQ to maintain active respiration in clk-1 mutant mitochondria, despite the
absence of UQ9.
Nematode Strains--
The wild-type strain used was the N2
(Bristol) strain. Mutant strains used were CB4876 clk-1
(e2519), MQ438 clk-1 (qm51), and MQ50
clk-1 (qm30). MQ50 clk-1
(qm30) was supplied from the Caenorhabditis Genetics Center.
Mitochondria Preparation--
Nematodes were grown at 20 °C
on NGA plates, which contain 3-fold bacto-peptone with the
supplement of Escherichia coli OP50. Nematodes
were collected in M9 buffer and were sedimented in a 200-ml cylinder.
The sedimented worms were washed with M9 buffer until the buffer become
clear and were applied to Baermann's Device and left overnight. The
nematodes were collected and further purified by centrifugation on 30%
(w/v) sucrose, at 750 × g for 5 min at 4 °C (16).
The worms were homogenized in 0.21 M mannitol, 0.07 M sucrose, 0.1 mM EDTA (MSE), containing 1 mM phenylmethylsulfonyl fluoride, using glass-glass
homogenizer (Iwaki, Tokyo) with the inclusion of glass beads
(0.10-0.11 mm, B. Brown Melsungen AG). The degree of breakage was
checked under a light microscope. The homogenates were then centrifuged
at 1,080 × g for 10 min at 4 °C. The pellet,
containing glass beads, was washed with MSE and was centrifuged at
1,080 × g for 10 min. The supernatants were then
centrifuged at 23,500 × g for 10 min, and the pelleted
mitochondrial fraction was resuspended in MSE.
Identification of Quinones--
Quinones were extracted from
lyophilized mitochondria (3.0 mg of protein). The mitochondria were
vortexed in EtOH/n-hexane (2/5, v/v) for 10 min and
centrifuged at 15,000 rpm for 5 min at room temperature. The
supernatants were pooled, and the extraction of quinones was repeated
two times. After drying the pooled extracts under a stream of nitrogen
gas, the residue was redissolved in ethanol and analyzed by HPLC.
Quinones were applied to a reverse-phase column (Inertsil ODS-3, C-18,
5 µm, 4.6 × 250 mm, GL Science, Tokyo) and was eluted in
isocratic condition (1 ml/min), with diisopropyl ether/MeOH (1/4, v/v)
as described previously (17). The eluted quinones were
identified by comparing their retention times with authentic
UQ9 (Sigma). The spectral characteristics of each quinone
were monitored using photodiode array UV-visible detector
(Shimadzu SPD10-A). The concentration of quinones was determined
spectrophotometrically using coefficients of E Enzyme Assays--
NADH-cytochrome c reductase
activity and succinate-cytochrome c reductase activity were
assayed as described previously (20) in 50 mM
potassium phosphate buffer (pH 7.7), 200 µM NADH, or 10 mM potassium succinate, 2 mM KCN, and 50 µM horse heart ferricytochrome c. NADH-quinone
reductase activity was assayed in 50 mM potassium phosphate
buffer (pH 7.7), 200 µM NADH, 2 mM KCN, and
90 µM quinone analogues. The oxidation of NADH was
monitored at 340 nm, using a millimolar extinction coefficient of 6.2 for NADH. Succinate-quinone reductase activities were measured as
described previously (21). DMQ2 and
3-hydroxy-UQ2 were synthesized as described previously (22). All the assays were performed at 20 °C.
Quinones were extracted from the mitochondria of N2 and
clk-1 mutant strains, and the quinone composition was
directly analyzed by reverse-phase HPLC (Fig.
1). Three different mutant strains, including a missense mutant (e2519), a deletion mutant
(qm30), and a splice acceptor mutant
(qm51), were used for the analysis. The major peak at 18.3 min from N2 mitochondria is identical to standard UQ9 for
both elution time and absorption property (Fig. 1, A and
B, and Fig. 2A).
However, a corresponding peak was not observed in clk-1
mutants. The mutant mitochondria instead exhibited a major peak eluting
1 min earlier than UQ9 (Fig. 1, C-E). The slightly polar nature and the absorption property (absorption peak at
270 nm, Fig. 2B) of this compound coincide well with those reported for the ubiquinone biosynthesis intermediate, DMQ9
(see Fig. 3) (19, 23). A mass
spectrometry analysis of the accumulated quinone in clk-1
(qm51) mutant detected a molecular ion peak at m/z 765, which corresponds to the molecular mass of
DMQ9 (theoretical mass
[C53H80O3] = 765.2005) (Fig.
4). For comparison, the mass spectrum of
the standard UQ9 (Sigma) showed a molecular ion peak at
m/z 795 (theoretical mass
[C54H82O4] = 795.2264) (data not shown).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1
cm
1 at 271 nm for DMQ (19). The mass spectrum
of the quinone accumulated in clk-1 (qm51) was
analyzed by Hitachi M-8000 LC/MS 3DQ system with atmospheric pressure
chemical ionization.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Elution profiles of quinones from N2 and
clk-1 mutants mitochondria. Reverse-phase HPLC
chromatograms of quinones eluted in the isocratic condition
(diisopropyl ether/MeOH (1/4, v/v, 1 ml/min) are shown. The
elution of standard UQ9 (A), lipid extracts from
N2 (B), clk-1 missense mutant (e2519)
(C), deletion mutant (qm30) (D), and
splice acceptor mutant (qm51) (E) mitochondria
were monitored at 275 nm. The elution time of the major peak is
indicated.
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Fig. 2.
Absorption spectra of quinones in N2 and
clk-1 mutants mitochondria. The spectra of the
quinones from N2 (eluted at 18.31 min in Fig. 1B)
(A) and clk-1 mutant (qm51) (eluted at
17.11 min in Fig. 1E) (B) are monitored by
photodiode array UV-visible detector. Maximum absorption of each
compound ( max) is indicated.
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Fig. 3.
Pathway for biosynthesis of
UQ9. The proposed pathway of UQ biosynthesis in
eukaryotes (9) is shown. The accumulation of DMQ9 in
clk-1 mutants indicates that CLK-1 is necessary for the step
converting DMQ9 to 3-hydroxy-UQ9. The
intermediates indicated are (from the top)
3-nonaprenyl-4-hydroxybenzoate, DMQ9,
3-hydroxy-UQ9, and UQ9.
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Fig. 4.
Mass spectrum of the quinone biosynthesis
intermediate from clk-1 (qm51) mutant
mitochondria. The mass spectrum of the quinone accumulated in
clk-1 (qm51) mitochondria, which corresponds to
the peak eluting at 17.1 min in Fig. 1E, is shown.
The molecular structure of DMQ9 is also shown. The peak at
m/z 765 corresponds to the molecular ion of
DMQ9.
The amount of DMQ9 in all the three mutant strains was in the same range as UQ9 content in N2 mitochondria (Table I). In all clk-1 mutants, the peak corresponding to UQ9 was undetectable by UV absorbance, indicating that the levels of UQ9 in clk-1 mutants mitochondria are less than 0.1 nmol/mg. Since clk-1 mutants show normal levels of oxygen consumption (15) and succinate-cytochrome c reductase activity (14), it has been suggested that CLK-1 may not be critically involved in UQ biosynthesis in nematodes (4-7). Our findings, however, clearly demonstrate that clk-1 encodes a protein that is absolutely required for the biosynthesis of UQ in C. elegans (Fig. 3).
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UQ is known as an essential component of the respiratory electron transfer chain and is required for mitochondrial aerobic respiration. In fact, UQ deficiency in humans causes mitochondrial encephalomyophathy (24), and yeast coq7-1 mutant, which contains DMQ6 but not UQ6, is defective in aerobic growth (9). Given this, the apparent absence of UQ9 (Fig. 1) and the normal level of respiration observed in clk-1 mutants (7, 14, 15) raise the question of how electron transfer is carried out in clk-1 mutants. Since a large amount of DMQ9 is accumulated in clk-1 mutants mitochondria, one possibility might be that DMQ9 serves as an electron carrier in mutants mitochondria. To test this possibility, we measured various electron transfer activities in clk-1 mutants mitochondria (Table I). Succinate-cytochrome c reductase activity of mutants mitochondria was measured and found to be only slightly affected, confirming the previous report (14). Similarly, the activity of NADH-cytochrome c reductase in clk-1 mutants was comparable with that in the wild-type strain (Table I). These results indicate the possibility that the electron transfer between complex I and complex III in clk-1 mutants mitochondria might be mediated by endogenous DMQ9, with almost the same efficiency as UQ9.
To further investigate the activity of DMQ as an electron acceptor, we chemically synthesized a short side chain DMQ2 and measured the activities of NADH-quinone reductase and succinate-quinone reductase. As shown in Table II, DMQ2 was able to accept reducing equivalent from complex I with a rate comparable with UQ2. DMQ2 was also capable of serving as electron acceptor of complex II, although the activity was lower than that with UQ2. In contrast to DMQ2, 3-hydroxy-UQ2, which is a direct precursor of UQ (see Fig. 3), was unable to serve as efficient electron acceptor neither at complex I nor at complex II (Table II), indicating that not all the quinone biosynthesis intermediates are recognized as functional substrates by respiratory complexes. Interestingly, DMQ appears to be a more efficient substrate for complex I than for complex II (Tables I and II). This tendency has been also reported for E. coli ubiF mutants, which accumulate DMQ8 (25), suggesting that the structure and the redox potential of DMQ might be more favorable for reduction by complex I than by complex II. The active respiration in clk-1 mutants mitochondria raises the question as to why yeast coq7-1 mutants, which also contain DMQ6, are defective in respiratory growth (9). Our results suggest that this is probably due to the relatively small amount of DMQ6 accumulated in coq7-1 mutants (9) and the inherent lack of complex I in the S. cerevisiae respiratory chain.
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The finding of an altered quinone composition in clk-1 mutants is the first indication of a biochemical difference between clk-1 mutants and wild-type strains and shows that CLK-1 is absolutely required for the step converting DMQ9 to 3-hydroxy-UQ9. However, there are reasons to believe that CLK-1 may not directly participate in the hydroxylation of DMQ9, since clk-1 and its homologues do not possess any monooxygenase/hydroxygenase motifs in their primary structure (8, 9, 11-13), in contrast to E. coli ubiF gene, which has been recently identified to be responsible for the synthesis of 3-hydroxy-UQ8 from DMQ8 (23). The fact that a gene homologous to E. coli ubiF does exist in the genome of C. elegans (GenBankTM accession number O01884) suggests that it, rather than CLK-1, catalyzes the hydroxylation of DMQ9 in C. elegans. In addition, Hsu et al. (26) recently reported that yeast CLK-1 homologue, COQ7/CAT5, is necessary for the stable expression of Coq3p, which participates in the O-methylation steps of the UQ biosynthesis pathway. These observations suggest that CLK-1/COQ7/CAT5 may participate in a fundamental regulatory mechanism in the UQ biosynthesis of eukaryotes and that the hydroxylation of DMQ9 is one of the major reactions under the control of CLK-1.
What is the relation between UQ biosynthesis and the overall phenotype of clk-1 mutants? The normal rate of respiration observed in clk-1 mutants by distinct methodologies (present study and Ref. 15) strongly implies that the phenotype of clk-1 mutants is not the direct consequence of decreased energy metabolism, as has been discussed previously (27). Another observation that suggests that the phenotype of clk-1 mutants is not explained solely by the accumulation of DMQ9 in the adult mitochondria is the absence of correlation between the severity of the overall mutant phenotype and the severity of the biochemical phenotype in the three clk-1 alleles. Indeed, we could not find a quantitative difference in the amount of DMQ9 between the weaker missense mutant (e2519) and the more severe mutants (qm30 and qm51) (Table I). These observations suggest that UQ biosynthesis might be only one of the processes that is regulated by clk-1.
One phenotype of clk-1 mutants that is not very different in
the different alleles is the increase in life span (2, 3). The
alteration of the content of different quinones, which is similar in
all alleles, might thus contribute to slower aging. Reactive oxygen
species (ROS) produced as a by-product of electron transport are widely
believed to be an important determinant of aging (28-30). An important
source of ROS is the ubisemiquinone radical, which is a reaction
intermediate during the reduction and the oxidation of UQ in complex I
and complex III (31). Possibly, the chemical properties of the
semiquinone produced from DMQ9 allow for a lesser level of
ROS production, and thus to a slower rate of oxidative damage
accumulation, which in turn could promote a long life span.
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ACKNOWLEDGEMENTS |
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We acknowledge the C. elegans Genetics Center for supplying MQ50 clk-1 (qm30), which is funded by the National Institutes of Health National Center for Research Resources (NCRR). We thank Dr. Mogi (University of Tokyo) for supplying authentic UQ8.
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FOOTNOTES |
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* This work was supported by Grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan (No. 11470065) and by the Iwadare Foundation (to H. M.).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.
§§ To whom correspondence should be addressed: Dept. of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-3526; Fax: 81-3-5841-3444; E-mail: kitak@m.u-tokyo.ac.jp
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.C000889200
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ABBREVIATIONS |
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The abbreviations used are: UQ, ubiquinone; DMQ9, demethoxy ubiquinone or 2-methoxy-5-methyl-6-nonaprenyl-1,4-benzoquinone; 3-hydroxy-UQ9, 2-methoxy-3-hydroxy-5-methyl-6-nonaprenyl-1,4-benzoquinone; UQ9, 2,3-methoxy-5-methyl-6-nonaprenyl-1,4-benzoquinone; ROS, reactive oxygen species; HPLC, high performance liquid chromatography.
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