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J. Biol. Chem., Vol. 277, Issue 3, 2202-2206, January 18, 2002
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§,From the Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada
Received for publication, September 18, 2001, and in revised form, November 6, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Ubiquinone (UQ) is a lipid co-factor that is
involved in numerous enzymatic processes and is present in most
cellular membranes. In particular, UQ is a crucial electron carrier in
the mitochondrial respiratory chain. Recently, it was shown that
clk-1 mutants of the nematode worm Caenorhabditis
elegans do not synthesize UQ9 but instead accumulate
demethoxyubiquinone (DMQ9), a biosynthetic precursor of
UQ9 (the subscript refers to the length of the isoprenoid side chain). DMQ9 is capable of carrying out the function
of UQ9 in the respiratory chain, as demonstrated by the
functional competence of mitochondria isolated from clk-1
mutants, and the ability of DMQ9 to act as a co-factor for
respiratory enzymes in vitro. However, despite the presence
of functional mitochondria, clk-1 mutant worms fail to
complete development when feeding on bacteria that do not produce
UQ8. Here we show that clk-1 mutants cannot
grow on bacteria producing only DMQ8 and that worm
coq-3 mutants, which produce neither UQ9 nor
DMQ9, arrest development even on bacteria producing
UQ8. These results indicate that UQ is required for nematode development at mitochondrial and non-mitochondrial sites and
that DMQ cannot functionally replace UQ at those non-mitochondrial sites.
Ubiquinone (UQ)1 is a
prenylated benzoquinone that is an essential co-factor in the
mitochondrial respiratory chain, where its function is best
characterized. UQ is also found in many other locations in the cell,
such as the lysosome and Golgi membranes, as well as in nuclear and
plasma membranes (1). The exact role of UQ at these extramitochondrial
sites is being actively explored (e.g. Refs. 2 and 3).
The gene clk-1 of the nematode Caenorhabditis
elegans affects many physiological rates, including embryonic and
post-embryonic development, rhythmic behaviors, reproduction, and life
span (4). clk-1 encodes a 187-amino acid protein that is
localized in mitochondria (5) and that is homologous to the yeast
protein Coq7p, which has been shown to be required for UQ biosynthesis
(6). clk-1 has also been shown to be necessary for UQ
biosynthesis in worms (7, 8) and in the mouse (9). Indeed,
UQ9 is entirely absent from mitochondria purified from worm
and mouse clk-1 mutants (8, 9) (the subscript refers to the
length of the isoprenoid side chain). Instead, these mitochondria
accumulate demethoxyubiquinone (DMQ9), which is an
intermediate in the synthesis of UQ9 (8, 9). Consistently,
recent evidence suggests that clk-1 encodes a DMQ
hydroxylase (10), which converts DMQ to ubiquinol. In Escherichia
coli, DMQ8 is able to sustain respiration in isolated membranes although at a lower rate than Q8 (11). Similarly, DMQ9 is capable of sustaining electron transport in
eukaryotic mitochondria, as the function of purified mitochondria (5), and mitochondrial enzymes (8), from clk-1 worm mutants
appears to be almost intact compared with the wild type. In addition, synthetic DMQ2 has been shown to function in
vitro as a co-factor for electron transport from worm complex I
and, albeit more poorly, from complex II (8). Finally, it was found
that in the absence of any exogenous UQ the oxygen consumption of mouse
embryonic stem cells with a deleted mclk1 gene is only
reduced by 35% (9).
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 (7). Although, dietary UQ is generally not
capable of reaching mitochondria (reviewed in Ref. 1), 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 (7).
To resolve these issues, we have generated a strain carrying a knockout
mutation in the nematode gene coq-3, which encodes a
methyltransferase required for UQ synthesis, and whose inactivation does not lead to DMQ accumulation in yeast (12). We find that coq-3 worms are not able to complete development even on
bacteria that contain UQ8. These results indicate that 1)
dietary UQ cannot complement a UQ deficiency in the absence of DMQ, 2)
the growth impairment of clk-1 mutants without a dietary
supply of UQ is due to a non-mitochondrial requirement for UQ, and 3)
DMQ cannot functionally replace UQ at the non-mitochondrial sites.
Nematode and Bacterial Strains--
We used the wild type N2
(Bristol) strain. Mutant strains analyzed include daf-2(e1370),
eat-2(ad465), dpy-9(e12), and mau-2(qm160). None of
these strains had any difficulty in growing on bacteria that do not
produce UQ. A detailed analysis was performed using clk-1(qm30),
clk-1(e2519), and clk-1(qm51). Standard procedures were
used for bacterial and worm cultures (13), except that the nematode
growth medium plates contained 0.5% glucose, to minimize the
reversion of UQ-deficient strains. The genotypes of the bacterial strains used are described in Table I.
Production of the coq-3 Knockout Mutation--
A null mutation
in the C. elegans gene coq-3 was generated according to the
protocol previously established by Molder and Barstead
(snmc01.omrf.uokhsc.edu/revgen/RevGen.html). A detailed description of
the manipulations performed, including the primer sequences used for
PCR, can be obtained upon request. See Fig. 2 for a schematic
representation of the coq-3 gene (which is part of an
operon), the extent of the deletion in coq-3 (qm188)
mutants, the positions of the primers used to screen for the mutation, and the positions of the primers used to amplify a rescuing fragment of
the gene. To verify the genotype of coq-3, we
performed PCR analysis and used sets of primers whose priming regions
are either outside of the coq-3 gene or inside the
region corresponding to the qm188 deletion mutation.
Phenotypic Characterization of coq-3--
We analyzed the gonads
of the coq-3 mutants using DIC microscopy. Quantitative
measurements of the growth rate and the brood size were performed as
described previously (14). For brood size measurements, we counted the
entire progeny of 10 worms. The experiments were performed twice.
Transformation Rescue--
We followed the usual micro-injection
procedure to generate standard extrachromosomal arrays (15). A PCR
fragment (50 ng/µl) comprising the coq-3 genomic sequence
was injected to assay for rescue. We used the pRF4 plasmid (120 ng/µl), which contains a dominant mutation rol-6 (su1006d)
as a co-injection marker to screen for transgenic worms.
coq3/dpy-4 heterozygous worms were utilized for injection,
since coq-3 is homozygous lethal. We selected the homozygous
coq-3 rescued lines by picking the lines in which the Dpy
phenotype was absent in the progeny and confirmed the genotype by PCR
analysis. The primer sequences can be obtained upon request (see also
Fig. 2).
Growth and Brood Size Analysis--
To evaluate the development
on various bacterial strains, we first placed adult worms in a drop of
bleach on a plate containing the test bacteria. This step ensures that
no OP50 bacteria is present in the test plate. L1 larvae that hatched
from the bleached eggs were transfered to a fresh test plate, and the
growth of the worms was examined. For brood size measurements, we
counted the entire progeny of 10 worms. The experiments were performed twice for N2 and four times for the clk mutants.
Ubiquinone Is Necessary for C. elegans Development and
Fertility--
It was recently reported that clk-1 mutants
are incapable of completing development when fed on a ubiG E. coli mutant strain (7). The ubiG gene product is required at two
steps of the UQ biosynthesis pathway, and ubiG mutants do
not produce any UQ (16). We tested whether this growth phenotype
resulted from a specific toxicity of the ubiG strain (GD1)
for clk-1 mutants or from the absence of UQ. For this
purpose, we systematically analyzed the growth of clk-1
mutant worms on a variety of E. coli mutants that are
defective for UQ biosynthesis (ubi mutants). Nine E. coli enzymes have been described as participating in UQ biosynthesis (see Fig. 1) (16). They are
all membrane-bound, except the first one, ubiC, which is a
soluble chorismate lyase (17). The next enzyme in the pathway is the
prenyltransferase ubiA that attaches the isoprenoid side
chain to the quinone ring (eight subunits in E. coli) (18).
The other enzymes are grouped in three categories: decarboxylases
(ubiD and ubiX), monooxygenases (ubiB,
ubiH, ubiF), and methyltransferases (ubiG, ubiE)
(19-24). We examined the growth of the three clk-1 mutant
strains on strains of bacteria mutant for each of these genes, except
ubiC (Table I). The three
clk-1 mutant alleles are qm30 and
qm51, which are putative nulls, and e2519, a
point mutation that results in a milder phenotype (14, 25).
We find that on all the bacterial ubi- (mutant)
strains tested, L1 larvae from the wild type strain N2 are capable of
completing development to adulthood and that these adults have a brood
size of around 320, which is similar to their brood size on
ubi+ bacteria (OP50) (Table
II). This indicates that endogenously synthesized Q is sufficient to maintain a wild type phenotype, without
a requirement for dietary UQ. We have also examined a number of worm
mutants that are not known to be involved in UQ synthesis (dpy-9,
eat-2, mau-2), including long lived mutants (daf-2 and
a number of strains that show a clk-1-like phenotype that
have not yet been fully characterized). In no case was the growth of
the mutants impaired on ubi Endogenous Ubiquinone Is Necessary for C. elegans Development and
Fertility--
To test whether dietary UQ is sufficient for C. elegans development, we produced a knockout mutation of the worm
gene coq-3. coq-3 encodes a methyltransferase whose
homologues (Coq3p and UbiG) have been extensively characterized in
S. cerevisiae and in E. coli, respectively. The
enzyme acts at two different steps of Q synthesis, and neither UQ nor
DMQ are produced in the yeast and bacterial mutants (24, 26). The worm
COQ-3 protein is 29% identical to S. cerevisiae Coq3p and
28% to E. coli UbiG. We used a method of random mutagenesis
and PCR-based screening to identify a deletion in coq-3 (see
"Experimental Procedures" and Fig.
2). The deletion qm188 removes
exons 3 and 4, such that no functional COQ-3 protein can be produced
(Fig. 2A). Self-fertilizing coq-3(qm188)/+
hermaphrodites produce one-quarter of homozygous coq-3(qm188)/coq-3(qm188) progeny, as verified by PCR (see
"Experimental Procedures" and Fig. 2B). These
coq-3 homozygotes develop slowly and appear substantially
smaller than wild type worms. Most are sterile (Fig. 2C),
but ~25% (n = 31) produce some progeny (5-10 eggs)
that arrest at the L1 stage and die quickly thereafter. These
observations are consistent with a partial maternal effect from the
heterozygous mothers of coq-3 homozygotes, as the phenotype of the first homozygous generation (slow development to adulthood) is
less severe than that of the second homozygous generation (arrest at
the L1 stage). The maternal effect could be due to UQ provided to the
embryo by the mother or to maternal deposits of coq-3
mRNA or protein.
To ascertain whether the observed phenotypes are solely due to the
mutation in the coq-3 gene, we introduced the genomic
fragment corresponding to the coq-3 gene into
coq-3/+ heterozygotes using the dominant rol-6
(su1006d) transformation marker by germ line transformation.
Homozygous coq-3 transgenic animals (displaying the marker
phenotype, Rol) develop normally and are fertile, indicating that the
phenotype we observe is indeed due to the coq-3 deletion. However, the extrachromosomal array carrying the coq-3
and rol-6 sequences is incapable of producing a strong
maternal effect. Indeed, homozygous animals without the array
(phenotypically non-Rol) issued directly from mothers carrying the
array (phenotypically Rol) did not develop beyond the L2 stage. The
expression of genes from extrachromosomal arrays is sometimes silenced
and is generally poor in the C. elegans germline (27). The
observation of a maternal effect suggests that the mother deposits an
essential product in the oocytes, which here could be UQ or
coq-3 mRNA. In either case, proper expression of
coq-3 in the germ line appears to be necessary for the effect.
We also observed that the brood size of heterozygous
coq-3/dpy-4 worms (243 ± 38; n = 10)
was similar to that of dpy-4/+ worms (248 ± 23;
n = 10) (Fig. 2D), suggesting that the
expression level of coq-3 in the heterozygotes might not be
limiting for UQ biosynthesis. The lethal phenotype of coq-3
mutants on ubi+ indicates that dietary UQ is not
sufficient for the growth and development of worms. This is consistent
with findings in other systems that indicate that dietary UQ cannot
reach the mitochondrial compartment or only in extremely small amounts
(1). The possibility that dietary UQ could be sufficient for worms was
proposed to account for the viable phenotype of clk-1
mutants grown on ubi+ bacteria and their lethal
phenotype when grown on ubi- mutant bacteria.
However, the phenotype of coq-3 mutants indicates clearly that
even in the presence of dietary bacterial UQ8, a total
absence of endogenous UQ9 and DMQ9 (as in
coq-3 mutants) is not equivalent to the replacement of
endogenous UQ9 by endogenous DMQ9 (as in
clk-1 mutants).
In this context, it is of particular interest that clk-1
mutants cannot thrive by feeding on ubiF mutants (see
above). Indeed, UQ biosynthesis in ubiF mutants is blocked
at the same level as in clk-1 mutants, and ubiF
bacteria thus produce DMQ8 (22). As DMQ9
performs efficiently in the mitochondrial respiratory chain (8), our
findings indicate that neither endogenous nor dietary DMQ can replace
UQ at non-mitochondrial sites of UQ requirement.
Our results suggest that UQ is necessary for C. elegans
growth and development at different subcellular locations, in
particular it appears to be necessary at sites distinct from the
mitochondrial respiratory chain (Fig. 3).
Indeed, for its respiratory function in the mitochondria, endogenous
DMQ9 can functionally replace endogenous UQ9,
as indicated by the observation that clk-1 mutant mitochondria do not appear to contain UQ9 but are
functionally competent (8). On the other hand, coq-3
mutants, in which a failure to manufacture UQ9 and
DMQ9 is expected, display a much more severe phenotype than
clk-1 mutants. Thus, at some still unknown site or sites,
distinct from the respiratory chain, endogenous DMQ9, or
dietary DMQ8, cannot functionally replace endogenous UQ9, while dietary UQ8 can. In fact,
clk-1 mutants, which have functional mitochondria and make
DMQ9, cannot develop and grow without dietary
UQ8, even in the presence of dietary DMQ8 from ubiF bacteria.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
The ubiquinone biosynthesis pathway. The
pathway of ubiquinone biosynthesis is schematically depicted and
corresponds to the synthesis steps that are common to prokaryotes and
eukaryotes. The metabolites that lead to ubiquinone are shown in
boxes. They are derived from chorismate, and they contain a
polyprenyl chain (eight isoprenoid units in E. coli and nine
in C. elegans), added by the prenyltransferase
ubiA to the 4-hydroxybenzoate. The prokaryotic genes that
encode the enzymes involved in the pathway are on the left
of the arrows (ubi genes). The C. elegans genes described in this study (clk-1 and
coq-3) are on the right of the arrows.
The prokaryotic enzymes can be grouped as following, considering their
described enzymatic activities: chorismate lyase (ubiC),
prenyltransferase (ubiA), decarboxylases (ubiD
and ubiX), monooxygenases (ubiB, ubiH, ubiF), and
methyltransferases (ubiG, ubiE). All these enzymes are
membrane-bound, except ubiC, which is a soluble enzyme. The
ubiquinone precursor accumulated in C. elegans clk-1 mutants
is DMQ (depicted in bold).
E. coli strains used in the analyses presented
bacteria. In
contrast, all three clk-1 mutants behave identically on most
ubi bacterial strains tested: they develop
very slowly, or not at all, and produce no progeny (Table II). However,
the clk-1 mutants can develop and produce some progeny on
ubiD, ubiX, and ubiH mutant strains, which are
point mutants that produce residual amounts of ubiquinone (around 15%
of the wild type) (16). It appears therefore that relatively low levels
of bacterial UQ8 are sufficient to allow for the growth of
clk-1 mutants.
Growth and brood size analysis of wild type and Clk worms on
ubi+ and ubi bacteria
strain is
mutant in a gene implicated in the ubiquinone biosynthesis pathway
except RKP1452, which has knockouts in the two first genes in the
pathway (ubiC and ubiA). The order of the
ubi bacteria in the table reflects the ordering of
the ubi genes in the ubiquinone biosynthesis pathway.
View larger version (41K):
[in a new window]
Fig. 2.
Characterization of the coq-3
deletion mutant. A, genomic structure of the
coq-3 gene. coq-3 is located on linkage
group IV of C. elegans and is part of an operon,
comprising the gdi-1 gene and a gene (Y57G11C.12) encoding a
subunit of NADH-ubiquinone oxidoreductase. coq-3 contains
five predicted exons. The deletion in coq-3 (qm188) mutants
removes 2456 bp, eliminating exons 3 and 4, and consequently no
functional protein can be produced. Primers (SHP) used for PCR
screening of the deletion library and individual worms are indicated.
B, PCR analysis of genomic DNA from N2,
coq-3/dpy-4, and coq-3/coq-3 worms. To check the
presence of a deletion in the coq-3 gene, PCR analyses were
carried out using genomic DNA from single worms. Each DNA preparation
was simultaneously tested with primers recognizing sequences either
outside the coq-3 gene (SHP 1774 and SHP 1775, lanes
2-4) or inside the obtained deletion (SHP 1840 and SHP 1865, lanes 5-7). See A for the primer localization.
When using primers amplifying the whole coq-3 gene, a band
of 4.3 kb was obtained from a wild type worm (lane 2). In
contrast, a mutant band was amplified at 1.8 kb from a
coq-3/coq-3 worm. DNA from heterozygotes only amplified the
mutant band (lane 2), probably because the PCR conditions
are in favor of the shorter amplicon. When using primers annealing in
the deletion region, both wild type and heterozygote worms gave a PCR
product of 1.1 kb (lanes 5 and 6), while no band
was detected from a coq-3/coq-3 homozygote worm, which
confirmed that the mutants were homozygous. C, photographs
of adult gonads of N2 and coq-3/coq-3 worms. In contrast to
the wild type (N2), no embryos are present in most
coq-3/coq-3 adult worms, which underlies their sterile
phenotype. D, brood size analysis. Experiments were
performed twice, yielding similar results. The sample size is indicated
in parentheses. Growth is scored as the capacity to reach adulthood and
be fertile. We analyzed coq-3/dpy-4 heterozygotes,
maternally rescued coq-3/coq-3
(m+z ), coq-3/coq-3 rescued with a
transgene carrying the wild type sequence of the coq-3 gene
(coq-3/coq-3 rescue), and dpy-4/dpy-4 as well as
dpy-4/+ as controls. The results show that
coq-3/dpy-4 heterozygotes have a brood size comparable with
dpy-4/+ or dpy-4/dpy-4 animals. coq-3
homozygotes rescued with a wild type version of the coq-3
gene are fertile, but their brood-size is low.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
[in a new window]
Fig. 3.
A model for the subcellular targets of
UQ. This model hypothesizes at least two spatially distinct
functional requirements for ubiquinone in C. elegans. It
distinguishes between UQ at mitochondrial and non-mitochondrial
locations based on the phenotype of clk-1 mutants on
ubi- bacteria and that of coq-3
mutants on ubi+ bacteria. Endogenous
UQ9 is able to reach all of the locations where UQ is
necessary, while dietary UQ8 only accesses
non-mitochondrial sites. On the other hand, endogenous DMQ9
does not function at those sites, but is active in mitochondrial
respiration. Mitochondria are depicted in red, and the
undetermined non-mitochondrial locations are represented in
blue. Arrows point to locations within the cell
where a given quinone is functional.
This model is consistent with the findings by numerous studies on UQ uptake and metabolism in other systems, such as rodents (1). What has been found is that dietary UQ appears to be taken up only poorly (2-3% of the initially ingested ubiquinone), and the majority is then distributed to the plasma membrane, the lysosomes, and the Golgi, with only minute quantities, if any at all, appearing in the mitochondria. Given that every cell endogenously produces UQ, it is possible that no active uptake system exists to assimilate this rather complex lipid.
Our studies clarify the roles of endogenous and dietary UQ in the worm's biology. Also, we demonstrate for the first time the functional importance of UQ at non-mitochondrial locations for an organism's viability. Action of dietary UQ at non-mitochondrial sites could underlie the beneficial effects of dietary UQ for patients with mitochondrial diseases (1). For example, UQ has been found to participate in reactions that regulate the redox state of the cell at the plasma membrane (28). Disease states that arise from deficient mitochondria are often found to increase cellular oxidative stress, and dietary UQ could stimulate a protective function at the plasma membrane (28). In addition, in bacteria, quinones have recently been found to act as the primary signal of the redox state of the cell (29). In E. coli, UQ negatively modulates the phosphorylation status and function of ArcB, an important global regulator of gene expression. The eventual discovery of additional roles for UQ in eukaryotes, and in particular as a signaling cue, will help to better understand the pleiotropic effects of mutations in genes that affect UQ, including clk-1.
Finally, we note that the coq-3 and clk-1 mutant
strains provide genetic models to identify compounds that could
selectively replace ubiquinone at the mitochondria and/or at
non-mitochondrial sites. The development of such bio-available
ubiquinone mimetics could be of great medical interest.
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ACKNOWLEDGEMENTS |
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We thank Claire Bénard and Robyn Branicky for careful reading of the manuscript and Robert Poole, Georges Javor, Philip Rather, Catherine Clarke, David Clark, Bernard Lemire, and the E. coli stock center at Yale for sharing bacterial clones.
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FOOTNOTES |
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* 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.
Fellow of the Swiss National Foundation.
§ These two authors contributed equally to the work presented.
¶ Canadian Institute of Health Research Scientist. To whom correspondence should be addressed: Dept. of Biology, McGill University, 1205 Ave. Docteur Penfield, Montreal, Quebec H3A 1B1, Canada. Tel.: 514-398-6440; Fax: 514-398-1674; E-mail: Siegfried.Hekimi@McGill.ca.
Published, JBC Papers in Press, November 8, 2001, DOI 10.1074/jbc.M109034200
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ABBREVIATIONS |
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The abbreviations used are: UQ, ubiquinone; DMQ, demethoxy- ubiquinone.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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