From the Department of Chemistry and Biochemistry and
the Molecular Biology Institute, §§ Department of
Biological Chemistry, University of California, Los Angeles, Los
Angeles, California 90095 and the ¶ Institut fur Mikrobiologie der
Johann Wolfgang Goethe-Universitat Frankfurt, Biozentrum Niederusel,
Marie-Curie Str. 9, D-60439 Frankfurt am Main, Germany
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ABSTRACT |
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Mutations in the clk-1 gene result in slower development and increased life span in Caenorhabditis elegans. The Saccharomyces cerevisiae homologue COQ7/CAT5 is essential for several metabolic pathways including ubiquinone biosynthesis, respiration, and gluconeogenic gene activation. We show here that Coq7p/Cat5p is a mitochondrial inner membrane protein directly involved in ubiquinone biosynthesis, and that the defect in gluconeogenic gene activation in coq7/cat5 null mutants is a general consequence of a defect in respiration. These results obtained in the yeast model suggest that the effects on development and life span in C. elegans clk-1 mutants may relate to changes in the amount of ubiquinone, an essential electron transport component and a lipid soluble antioxidant.
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INTRODUCTION |
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Research into the components responsible for controlling longevity have uncovered both environmental effects and genetic determinants (1, 2). The nematode Caenorhabditis elegans has been used as a model for many such studies. Multiple life-extension mutants have been identified which affect various aspects of development (3). One of the genes identified in determination of life span was identified as Clock-1 (clk-1). clk-1 mutants exhibit a pleiotropic phenotype, characterized by delayed embryonic and postembryonic development, a slowing of adult behaviors such as swimming, pharyngeal pumping, and defecation, and an extended life span (4). The clk-1 mutants also have an increased resistance to stress induced by UV treatment (5). Recently the C. elegans clk-1 gene was characterized and found to be conserved among eukaryotes, including humans, rodents, and the yeast Saccharomyces cerevisiae (6).
The yeast clk-1 homologue was independently isolated as COQ7 and CAT5 (7, 8). The COQ7 gene is required for the synthesis of ubiquinone (coenzyme Q or Q),1 an isoprenylated benzoquinone that functions in the respiratory electron transport chain in the inner mitochondrial membrane of eukaryotes (9). Like other yeast coq mutants (10), the coq7/cat5 mutants lack Q, are respiration defective, and are incapable of growing on nonfermentable carbon sources (8, 10). A yeast mutant harboring the coq7-1 allele (encoding the substitution of Asp for Gly104) was found to accumulate both 3-hexaprenyl-4-hydroxybenzoate (HHB) and a small amount of 2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone, two intermediates in Q biosynthesis (8). However, mutants with deletions in the COQ7 gene produce only HHB. HHB is the predominant Q intermediate that accumulates in yeast mutants with deletions in any one of six COQ genes (COQ3-COQ8) (11). Transformation of either the coq7-1 point mutant or the coq7 null mutant with the yeast COQ7 gene restored both growth on nonfermentable carbon sources and the synthesis of Q. These results led to the development of two models for Coq7p function in Q biosynthesis: (i) Coq7p may itself act in one or more monooxygenase steps in the pathway, and (ii) Coq7p provides a component of a multisubunit complex that is required for the conversion of HHB to Q (8, 11). Since the amino acid sequence shares no similarity to any known monooxygenase or hydroxylase proteins, there is little support for the first model. The COQ7/CAT5 homologue from either rat or C. elegans rescued the yeast coq7/cat5 mutant for growth on nonfermentable carbon sources, suggesting a conservation of function from yeast to animals (6, 12).
The yeast COQ7 gene was independently isolated as CAT5, a gene required for the release of gluconeogenic genes from glucose repression (7). Glucose repression is a global regulatory system in S. cerevisiae that affects the transcription of genes involved in gluconeogenesis, alternative sugar metabolism, and respiration (13-15). Upon deletion of CAT5, binding of gene activators to the upstream activating sequences within gluconeogenic promoters was abolished resulting in a complete loss of gluconeogenic gene activation (7). These data provided support for a role of Cat5p in the cascade regulating gluconeogenic gene activation. Other genes necessary for the release from glucose repression were identified by the characterization of glucose derepression mutants cat1 (snf1) (16, 17), cat3 (snf4) (18, 19), and cat8 (20). Expression of gluconeogenic genes requires the pleiotropic Cat1p·Cat3p protein kinase complex (21, 22) and the zinc cluster-transcriptional activator Cat8p (7, 20, 23). Since strains with mutations in genes mediating glucose repression (cat1, cat3, or cat8) were defective in activation of a CAT5-lacZ reporter gene, a coregulation of respiratory chain elements and gluconeogenesis was postulated (7).
Elucidation of the function of Coq7/Cat5p in yeast should provide insight regarding the function of clk-1 in aging and development in C. elegans. The apparent dual function of Coq7p/Cat5p in yeast Q biosynthesis and glucose derepression raised the question of whether the observed defect in Q biosynthesis resulted from a defect in glucose derepression, or vice versa. In the present study the relationship between these functions is further investigated.
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EXPERIMENTAL PROCEDURES |
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Strains and Growth Media-- The strains of S. cerevisiae used in this study are described in Table I. Strains were grown in standard media as described (24). Growth and in vivo labeling of Q6 with p-[U-14C]hydroxybenzoic acid (365 Ci/mol) was as described (25). For derepression experiments cells were grown in glucose-containing medium to mid-log phase and then transferred to the respective ethanol containing medium for 6 h.
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Rescue of coq Mutants with Exogenous Q6--
Yeast
strains W303.1B (wild-type), W303COQ7 (coq7
/cat5
),
CC303 (coq3
), CC304 (atp2
), and W303
COR1
(cor1
) were grown overnight in 15 ml of YPD to stationary
phase and then diluted into 40 ml of YPE (OD600 nm = 0.6)
with or without Q6 supplementation (Sigma). Growth was
monitored by OD600 nm measurements, and at the same time
samples were taken for enzymatic assays.
Enzyme Assays-- Yeast crude extracts were prepared with glass beads (26) in 0.1 M potassium phosphate buffer and the protein concentration was determined using the bicinchoninic acid protein assay method (Pierce). Phosphoenolpyruvate carboxykinase and isocitrate lyase activities were measured as described (27, 28).
Plasmid Constructions--
Two yeast expression plasmids, one
single copy and one multiple copy, were constructed to express the Coq7
polypeptide containing a carboxyl-terminal peptide (MYPYDVPDYASLDGPMST)
corresponding to the carboxyl terminus of the influenza hemagglutinin
(HA) viral protein, an epitope for the 12CA5 monoclonal antibody (29). Construction was begun by directional cloning using SalI and
NotI sites in the plasmid pADCL (30). The COQ7
yeast nucleotide sequence corresponding to the open reading frame was
amplified by polymerase chain reaction with oligonucleotides containing
SalI and NotI linkers to allow for the in-frame
ligation of the COQ7 sequence. The sequences encoding the
ADH promoter, COQ7 sequence, HA-epitope, and termination
site were then liberated from pADCL by BamHI partial restriction enzyme digestion and subsequently ligated into the BamHI site of two plasmids pRS316 and pRS426 (31) to give
psHA71 and pmHA71 providing single and multiple copy plasmid
maintenance in yeast, respectively. pNMQ71 is maintained in single copy
and contains the COQ7 nucleotide sequence and 414 base pairs
of upstream sequence, constructed as described previously (8). Yeast
cells were transformed with pNMQ71, psHA71, pmHA71, or pRS316 (32). Transformants were selected for the presence of the URA3
gene on SD-Ura plates. The Ura+ colonies were subsequently
replica plated to YPG plate media. The Coq7-HA epitope fusion protein
retains activity as assayed by the ability of either the single or
multicopy plasmid construct to rescue coq7/cat5 null mutant
yeast strains for growth on media containing a nonfermentable carbon
source (YPG plates, data not shown). One of the coq7 null
mutants used in the complementation above, JM43COQ7 (8), was used
for subcellular localization by Western analysis.
Cell Lysis and Fractionation-- Cell cultures (1 liter) were grown in semisynthetic lactate media to saturation density. Spheroplasts were prepared and lysed by Dounce homogenization with a tight fitting pestle as described (33) with one exception: protease inhibitors were prepared in dimethyl sulfoxide and added prior to cell lysis. Final concentrations of the protease inhibitors were as follows: benzamidine 1 mM, leupeptin 1 µg/ml, pepstatin 2 µg/ml, chymostatin 1 µg/ml, aprotinin 1 µg/ml, antipain 1 µg/ml. Purified mitochondria were isolated from a linear Nycodenz gradient as described (33).
Subfractionation of Purified Mitochondria-- The outer mitochondrial membrane was broken by adding 5 volumes of ice-cold 20 mM HEPES-KOH, pH 7.4, to 2 mg of purified mitochondria. After a 10-min incubation on ice, the mixture was centrifuged in a microcentrifuge at 4 °C for 10 min. The supernatant contained the intermembrane space components while the pellet consisted of mitoplasts and disrupted outer membrane. The pellet was resuspended in 1 ml of 20 mM HEPES-KOH, pH 7.4. Integral and peripheral membrane proteins were separated via two methods: 1) alkaline carbonate extraction (34) or 2) extraction with the same conditions as the first method, but with 2 M urea in place of alkaline carbonate as the extracting agent (35).
Western Analysis-- Fractions were assayed for protein concentration by the bicinchoninic acid assay (Pierce). Equal amounts of protein from the mitochondrial fractions of cells containing the plasmids pNMQ71, psHA71, and pmHA71 were analyzed by electrophoresis on 12% Tris glycine gels and subsequently transferred to Hybond ECL Nitrocellulose (Amersham). Western analysis and membrane stripping were performed as described by Amersham. An exception to the stated protocol was the use of Western washing buffer: 10 mM Tris, pH 8.0, 154 mM NaCl, 0.1% Triton X-100.
Polyclonal rabbit antisera were generated to detect Coq7p/Cat5p in a wild-type strain CEN.PK2-1C, and in both crude and purified mitochondria fractions. The COQ7/CAT5 reading frame was amplified by polymerase chain reaction and inserted into pGEX-CS1 (Pharmacia, Piscataway, NJ) allowing for the isopropyl-1-thio- ![]() |
RESULTS |
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Defects in Glucose Derepression Do Not Impair Q Biosynthesis-- To assay whether Q biosynthesis is affected by mutations in the glucose derepression system, yeast strains harboring mutations in cat1, cat3, or cat8 were tested for their ability to synthesize Q. A total lipid extract prepared from wild-type yeast grown in the presence of p-[U-14C]hydroxybenzoic acid and separated by normal phase high performance liquid chromatography gives rise to a peak of radiolabeled material that co-elutes with a Q6 standard (fractions 6 and 7, Fig. 1A). When this same procedure is performed on radiolabeled lipid extracts from the cat1, cat3, or cat8 mutants, production of Q6 is still observed (Fig. 1, B, C, and E). Thus neither the pleiotropic Cat1p/Cat3p (Snf1p/Snf4p) protein kinase involved in glucose derepression nor the transcriptional activator of gluconeogenic genes Cat8p are essential for Q biosynthesis. However, the coq7/cat5 null mutant fails to produce Q6 (Fig. 1D), and instead accumulates HHB, the predominant intermediate found in the yeast Q mutants coq3-coq8 (8, 11). This finding suggests that the inability to produce Q is a very specific defect and cannot be caused by a lack of glucose derepression in the examined mutant strains.
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Gluconeogenic Derepression Is Defective in Both coq and atp2
Mutants--
To determine whether a general loss of respiration
affects glucose derepression of gluconeogenic genes, activation of a
PCK1-lacZ reporter fusion containing the entire phosphoenolpyruvate
carboxykinase promoter was assayed in a variety of respiratory yeast
mutants. As shown in Table II, all of the
wild-type strains used, although differing in absolute specific
activities, reveal a dramatic increase in specific -galactosidase
activity upon the shift to nonfermentable growth conditions. In
contrast, such induction is completely absent in the
coq7/cat5 mutant and in another Q-deficient mutant
(coq3). A yeast strain containing a deletion in the
ATP2 gene (encoding the
subunit of the
F1-ATPase) also fails to activate the phosphoenolpyruvate carboxykinase promotor, although Q is still produced in this strain (11). The lack of ATPase activity causes a pleiotropic effect that
results in a suppression of the bc1 complex and a severe reduction of respiration (10). There is a similar lack of
-galactosidase activation in four other mutants with defects in
respiration. The mutants either affect the respiratory chain in a
structural component (Cox7p, Ref. 36), its synthesis (Cox10p, affecting heme a synthesis, Ref. 37), its assembly (Cox15p, Ref. 38), or in
general mitochondrial gene expression (Mtf2, specifically needed
in COX1 expression but also affecting overall mitochondrial gene expression, Ref. 39). These results indicate that glucose derepression of gluconeogenic enzymes is dependent on intact
respiratory metabolism.
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Rescue by Supplementation with Q6--
We tested
whether respiration (as assayed by growth on ethanol) and derepression
of gluconeogenic genes could be restored by exogenous Q. As shown in
Fig. 2 the coq7/cat5 mutant
fails to grow on media containing a nonfermentable carbon source (3% ethanol). This is also true for another coq mutant,
coq3, and two respiratory mutants, atp2
,
and cor1
(completely lacks the bc1 complex,
Ref. 40). This growth defect was corrected in the two coq
mutants by supplementation with 15 µM Q6, and
after a brief lag compared with the wild-type strain, the rescued
coq7/cat5 and coq3 strains grew to the same
stationary cell titer. Neither the atp2 nor the
cor1 mutants could be rescued by exogenously added
Q6. The effect of the addition of Q6 on the
derepression of gluconeogenesis was simultaneously investigated.
Addition of Q6 fully restored gluconeogenic enzyme
activities in both the coq7/cat5 mutant and the
coq3 mutant (Table III).
However, such addition of Q6 failed to restore induction of
these activities in the atp2 and cor1 mutants.
Addition of Q6 also increased both the NADH dehydrogenase
activity and the rate of oxygen consumption of the coq
mutants; the latter showed a 2-3-fold increase from their average
baseline in YPE alone of 10 µl of O2/min/OD (data not
shown). However, oxygen consumption rates in the atp2 and cor1 mutants remained very low in the presence or absence of
exogenous Q (baseline rates in YPE and YPE + Q6 were
approximately 4 µl of O2/min/OD, data not shown). These
data show that the loss of Cat5p/Coq7p indirectly influences induction
of gluconeogenesis, and the defect can be completely suppressed by the
addition of Q6.
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Localization of Coq7p/Cat5 to the Mitochondria--
To determine
the localization of Coq7p/Cat5p in yeast, we used a polyclonal antibody
against Coq7p/Cat5p. As shown in Fig. 3,
the specific immunodetection of the protein was possible exclusively in
crude mitochondrial fractions of wild-type cells grown under nonfermentable conditions. Since Coq7p/Cat5p was not detectable in
glucose-repressed cells (Fig. 3), we assume that most of the protein
was synthesized during the transition from fermentative to respiratory
growth. As judged by the mobility in SDS-PAGE, no deviation from the
predicted molecular mass (31 kDa) was obtained. Further subcellular
localization was performed by employing a fusion of the COQ7
gene and the sequence coding for an epitope peptide from the
hemagglutinin viral protein. Western analysis of yeast subcellular
fractions indicates that the Coq7p-HA fusion protein cofractionates
with the mitochondria (Fig. 4). The lack of mitochondrial contamination in the cytosol was verified with the
mitochondrial marker F1-ATPase (41). The crude
mitochondrial fractions depicted in Figs. 3 and 4 contained some
contaminating organelles, detected with antibodies to Sec62p
(endoplasmic reticulum, Ref. 42), Kex2p (Golgi, Ref. 43), and ALPp
(vacuole, Ref. 44) (Fig. 5). When the
mitochondria were further purified over a Nycodenz gradient (33), the
abundance of all of the contaminating proteins dropped dramatically or
disappeared altogether (Fig. 5), indicating that Coq7p cofractionates
with the mitochondria.
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Localization of Coq7p/Cat5p to the Inner Mitochondrial
Membrane--
To determine the submitochondrial localization of Coq7p,
purified yeast mitochondria were subjected to various treatments which
break apart the mitochondria and allow the isolation of proteins from
different compartments (45). The outer membrane was disrupted through
osmotic swelling, thus releasing the soluble proteins of the
intermembrane space, but leaving the inner membrane intact. Western
analysis verified that Coq7p was absent from the intermembrane space
fraction as compared with the marker cytochrome b2 (data not shown). Peripherally bound and
soluble proteins were extracted from the resulting mitoplasts and
disrupted membranes and were treated with either alkaline carbonate or
urea. Both the alkaline carbonate and the urea treatments extract both
matrix and peripherally bound membrane proteins which remain in the
supernatant following high speed centrifugation (46, 47). As shown in Fig. 6, Coq7p fractionated in a manner
similar to cytochrome c1, an integral inner
membrane protein (48). Conversely, two soluble matrix proteins, Mas2
and Hsp60 (49, 50), and peripheral inner membrane proteins such as
F1-ATPase were released into the supernatant (Fig. 6,
B-D). OM45, an outer membrane protein with one transmembrane domain (51), was also extracted from the pellet fraction (Fig. 6F). In contrast, Coq7p remained solely in the pellet with
both extraction protocols (Fig. 6A), colocalizing with
cytochrome c1 (Fig. 6E). Therefore,
Coq7p is an inner mitochondrial membrane protein.
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DISCUSSION |
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This study provides evidence for the direct involvement of
Coq7p/Cat5p in Q biosynthesis. As shown in Fig. 2 the growth defect of
a coq7/cat5 and a coq3 mutant under
nonfermentable conditions can be restored by external feeding with 15 µM Q6. Moreover, addition of Q6
also restores the ability to activate gluconeogenic
(phosphoenolpyruvate carboxykinase and isocitrate lyase) enzymes during
the transition from glucose to ethanol metabolism (Table III). Such
gluconeogenic gene activation is absent in other mutants affecting Q
biosynthesis (coq3) as well as in a broad range of
respiratory deficient yeast strains (atp2
, cox7-7,
mtf2-30, cox15-44, and cox10-60) as seen by a
lack of derepression of gluconeogenic promoters (Table II). This
indicates that glucose derepression is dependent on intact respiratory
metabolism. Furthermore, our results indicate that while the glucose
derepression system influences Q biosynthesis, this regulatory system
is not essential for production of Q. It is known that high levels of
glucose repress yeast Q biosynthesis (11, 52), and COQ3 and
COQ7/CAT5 mRNA levels (8, 53). The expression of a
COQ7/CAT5-lacZ fusion gene is repressed 5-6-fold by glucose (7). Moreover, Coq7p/Cat5p is not detectable in glucose-repressed cells and is mainly synthesized after the transfer of
cells to nonfermentable growth conditions (YPE) (Fig. 4). Many nuclear-encoded mitochondrial proteins are regulated by carbon source,
although there can be significant variation depending on the strain
used (54). While the cat5/coq7 mutant fails to produce Q,
other cat mutants (cat1, cat3, and
cat8) with defects in glucose derepression produce Q (Fig. 1).
Thus neither the pleiotropic Cat1p/Cat3p (Snf1p/Snf4p) protein kinase
involved in glucose derepression nor the transcriptional activator of
gluconeogenic genes, Cat8p (20, 23), are essential for Q biosynthesis.
This finding supports the idea that the inability to produce Q is a
very specific defect (11) and cannot be caused by a lack of glucose
derepression in the examined mutant strains.
Western analysis of cell fractions indicates that both Coq7p and the
fusion protein Coq7p-HA cofractionate with mitochondria (Figs. 3 and
4). The lack of contaminating organelles in the pure mitochondrial
preparations provides compelling evidence for a mitochondrial location
of Coq7p (Fig. 5). Two other yeast polypeptides (Coq3p and Coq5p)
required for the respective O-methylation and C-methylation
steps of Q biosynthesis are also located in the mitochondria (24, 55,
56). The amino terminus of each of these latter polypeptides has a
typical mitochondrial leader sequence, including the presence of
several basic residues, an absence of acidic residues, and a sequence
consistent with the tendency to form amphipathic -helices (57). A
3-amino acid consensus motif common to mitochondiral matrix proteins
(58) is present in the amino termini of Coq2p (59), Coq3p (55), Coq5p
(24, 56), Coq4p, Coq6p, and
Coq8p.2 The amino terminus of
Coq7p contains neither a typical leader sequence nor the matrix motifs
and even has two acidic residues in its N-terminal region. However,
unlike the matrix proteins Coq3p and Coq5p, Coq7p is instead located in
the inner mitochondrial membrane, and cofractionates with cytochrome
c1, an integral inner membrane protein (Fig. 6).
An absence of such targeting motifs is not uncommon to inner
mitochondrial membrane proteins. There is a class of such inner
membrane proteins which have no cleavable targeting sequence, and
likely contain internal targeting sequences (60) most of which have yet
to be characterized (61).
The amino acid sequence of yeast Coq7p does contain a region (residues 154-175) predicted to be in an [alpha[-helical conformation (Fig. 7A), with the ability to insert into the membrane as determined by the MOMENT program (62, 63). The amphipathic nature of this predicted membrane helix in other systems leads to membrane protein association via charged pairs (64, 65). The orientation of the NH2- and COOH-terminal regions has yet to be experimentally determined. However, the abundance of positively charged residues NH2-terminal to the potential membrane insertion element of Coq7p indicates a matrix localization of this portion of the protein, as determined by the program PSORT (66). Interestingly, this program predicts Coq3p and Coq5p to be mitochondrial matrix proteins, a prediction that has been confirmed experimentally (24, 55, 56).
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Similar potential membrane insertion regions are also present in the C. elegans, rat, mouse, and human Coq7 homologs (Fig. 7B). These homologs have a high degree of identity throughout the entire protein; in the region between amino acids 92 and 272 of the yeast sequence, yeast and C. elegans are 41% identical, yeast and rat are 47% identical, and C. elegans and rat are 57% identical. Since C. elegans clk-1 (6) and the rat COQ7 (12) both complemented yeast coq7/cat5 mutants, it is evident that these proteins share the same function and location. The location of Coq7/Cat5 is consistent with its proposed role in aiding the conversion of HHB to Q (8); however, its specific function in the production of Q is not known. It is interesting to note that HHB is the predominant intermediate found in each of the yeast Q mutants coq3-coq8 (11). One possibility is that Coq7p serves to anchor a multisubunit complex composed of one or more of the Coq proteins to the inner membrane, thus facilitating their ability to act on the lipophilic Q intermediates.
Given the functional conservation of yeast, rat, and C. elegans Coq7p/Cat5p/Clk-1p, yeast provide a suitable model to
unravel the action of this protein in aging and delayed development.
Although results shown here identify COQ7 as a gene involved
in Q biosynthesis, we have not ruled out the possibility of an unknown
secondary function responsible for the C. elegans clk-1
mutant phenotype. It seems unlikely that the characterized
clk-1 mutations that effect increased longevity in C. elegans result in a complete loss of Q and respiratory function,
since the recovery of these mutant alleles represented a rare event
(4). In yeast, respiratory defective mutants arise at a high frequency
due to the large number of nuclear genes required to produce
respiratory competent mitochondria (10). In addition, yeast
mutants (lacking mitochondrial respiration) are
reported to have shorter life spans (67). Instead, it seems more likely
that the mutations in clk-1 and the resulting effects on
life span and development in C. elegans may relate to
changes in the amount of Q. In this event, the abundance of Q may
influence the extent to which oxidants are generated by mitochondrial
respiration (68, 69). Many in vitro studies implicate
ubisemiquinone (Q
) as the major site of electron leakage (70,
71). Accordingly, a decrease in Q in clk-1 mutants could
decrease respiratory electron transfer and perhaps the generation of
superoxide, hydrogen peroxide, and hydroxyl radicals that have been
proposed to contribute to cellular aging (72, 73). In this oxidative
stress theory of aging, mitochondria are considered to be both the main
source and the target of oxygen-derived free radicals (72). However, the sites of superoxide and hydrogen peroxide generation in
vivo is still an open question. The in vitro studies
employ conditions that enhance the propensity of radical production.
For example, drugs such as antimycin which enhance superoxide
production, modify the interaction of Q
with proteins, alter
the stability of Q
, and elicit superoxide production at sites
which in vivo may play a very minor role (74, 75). Hence the
influence of Q levels on both the pro- and antioxidant activities of
the mitochondrial respiratory chain remains to be determined (76-78).
Reduced Q (QH2) is capable of acting as a lipid-soluble
antioxidant, scavenging radicals both directly, in a manner similar to
that of vitamin E, and indirectly, by regenerating vitamin E (79-81).
Q has been shown to be a functional antioxidant in yeast under
conditions promoting lipid peroxidation (82). Q in the plasma
membrane also plays a role in extracellular ascorbate stabilization
(83). In view of this potential balance of antioxidant and pro-oxidant activities of Q, it will be very important to determine the effect of
the clk-1 mutations on levels and intracellular distribution of Q, cell cycle length, and life extension in the yeast model.
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ACKNOWLEDGEMENTS |
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We thank all those who generously donated antibodies: Dr. John Colicelli, Dr. Michael Yaffe, Dr. David Meyer, Dr. Greg Payne, Dr. Martin Horst, and Dr. Alexander Tzagoloff. We thank Dr. Peter Koetter and Niels Bojunga for providing yeast strains and Christian Wanner for preparation of Cat5p antibodies. We thank Dr. James Bowie for help with the structural analysis of Coq7p. We also thank the members of the Clarke and Entian laboratories for helpful suggestions and support.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM45952 (to C. F. C), United States Public Health Service National Research Service Award GM07185 (to T. J.), Deutsche Forschungsgemeinschaft, and a UCLA Center on Aging Pilot Research Grant, funded jointly by Dr. and Mrs. Ivan Mensh and the Retirement Research Foundation.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.
§ Contributed equally to the results of this work.
Supported by a grant of the Fonds der Chemischen Industrie.
Present address: Instituto de Biologia Molecular y Celular de Plantas,
Universidad Politecnica, CSIC, Camino de Vera 46022, Valencia, Spain.
** Supported by a grant from the Ministry of Education and Science of Spain. Present address: Instituto de Agroquimica y Technologia de Alimentos CSIC, Apartado de correos 73-46100 Burjassot, Valencia, Spain.
Present address: Dept. of Anesthesiology, University of Alabama
at Birmingham, Birmingham, AL 35233.
¶¶ Present address: School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
|| To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Ave., Los Angeles, CA 90095-1569. Tel.: 310-825-0771; Fax: 310-206-5213; E-mail: cathy{at}ewald.mbi.ucla.edu.
1 The abbreviations used are: Q, ubiquinone or coenzyme Q; QH2, reduced ubiquinone or ubiquinol; HHB, 3-hexaprenyl-4-hydroxybenzoate; Coq7p, the polypeptide encoded by COQ7; Coq7-1p, the polypeptide encoded by the point mutant allele of the yeast COQ7 gene; Cat5p, the polypeptide encoded by CAT5; Q6, ubiquinone containing six isoprene units; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
2 A. Y. Hsu, P. T. Lee, and C. F. Clarke, unpublished data.
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REFERENCES |
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