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Journal of Bacteriology, December 2000, p. 6933-6939, Vol. 182, No. 24
Department of Applied Bioscience and
Biotechnology, Faculty of Life and Environmental Science, Shimane
University, Matsue 690-8504, Japan
Received 13 June 2000/Accepted 26 September 2000
Ubiquinone is an essential component of the electron transfer
system in both prokaryotes and eukaryotes and is synthesized from
chorismate and polyprenyl diphosphate by eight steps.
p-Hydroxybenzoate (PHB) polyprenyl diphosphate
transferase catalyzes the condensation of PHB and polyprenyl
diphosphate in ubiquinone biosynthesis. We isolated the gene
(designated ppt1) encoding PHB polyprenyl diphosphate
transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene. This strain could not
grow on minimal medium supplemented with glucose. Expression of
COQ2 from Saccharomyces cerevisiae in the
defective S. pombe strain restored growth and enabled the
cells to produce ubiquinone-10, indicating that COQ2 and
ppt1 are functional homologs. The
ppt1-deficient strain required supplementation with
antioxidants, such as cysteine, glutathione, and Ubiquinone is known to be an
electron transporter in the respiratory chain in prokaryotes and
eukaryotes. It varies among organisms in the length of its isoprenoid
side chain. For example, Saccharomyces cerevisiae uses
ubiquinone-6 (UQ-6), Escherichia coli uses UQ-8, and
Schizosaccharomyces pombe uses UQ-10 (9, 16, 37).
It has been shown that the type of ubiquinone in organisms is
determined by the polyprenyl diphosphate synthase enzyme, which
catalyzes the condensation reaction of isopentenyl diphosphate with
allylic diphosphate to give a defined length of the isoprenoid
(22, 26). When polyprenyl diphosphate synthase genes
from other sources were expressed in S. cerevisiae and
E. coli, the ubiquinone generated was of the same type as
that expressed in the donor organism (22-26). By this
method, we successfully produced various ubiquinone species (UQ-5 to
UQ-10) in the S. cerevisiae COQ1 mutant (22),
which in turn indicates that p-hydroxybenzoate (PHB)
polyprenyl diphosphate transferase, which catalyzes the condensation reaction between the isoprenoid side chain and PHB, has a
broad substrate specificity. This is supported by consistent observations showing that purified PHB polyprenyl diphosphate transferases from Pseudomonas putida (12, 40) and
E. coli (17) have fairly wide substrate
specificities in terms of polyprenols. In contrast, PHB
geranyltransferase, which is responsible for the synthesis of shikonin,
is highly specific, as it uses only geranyl diphosphate as a
substrate (21). Studying the PHB polyprenyl diphosphate transferases from different sources may enhance our understanding of this type of enzyme.
Ubiquinone appears to play roles in addition to acting as a component
of the electron transfer system. One such role is that of an
antioxidant, as indicated by a number of studies (1, 5, 6, 7,
8, 14). A strain of S. cerevisiae unable to produce
ubiquinone is sensitive to lipid peroxide, suggesting that ubiquinone
protects against oxidants (5). Similarly, an S. pombe strain which does not produce ubiquinone because of a deficiency of decaprenyl diphosphate synthase is sensitive to H2O2 and requires an antioxidant to grow on
glucose-containing medium (37). Antioxidant roles of
ubiquinone in E. coli also have been reported recently
(18, 36). Furthermore, physiological concentrations of
ubiquinone act as antioxidants on human low-density lipoprotein
(1, 7). Another role of ubiquinone is that it can accept
electrons from sources other than the respiratory chain. Recently, it
was elegantly shown that ubiquinone (or menaquinone) will accept
electrons generated by the formation of protein disulfide in E. coli (3). Sulfide-ubiquinone oxidoreductase,
previously thought to occur mainly in photobiosynthetic bacteria as a
component in energy metabolism, has been shown to be present in
S. pombe and other eukaryotic organisms (42).
This suggests that there may be a link between sulfide metabolism and
ubiquinone in eukaryotes.
To increase our knowledge of the ubiquinone biosynthetic pathway and
the various functions of ubiquinone, we have characterized in this
study a strain of S. pombe that cannot produce
ubiquinone because of a defect in its PHB polyprenyl
transferase gene. We show clearly that ubiquinone can act both as
an antioxidant and as an acceptor of electrons from sulfide.
Materials.
Restriction enzymes and other DNA-modifying
enzymes were purchased from Takara Shuzo Co. Ltd. and New England
Biolabs, Inc.
Strains, plasmids, and media.
E. coli strains DH10B
and DH5 DNA manipulations.
Cloning, restriction enzyme analysis, and
preparation of plasmid DNAs were performed essentially as described
previously (32). PCR was done according to the procedure
described before (31). DNA sequences were determined by the
dideoxynucleotide chain termination method (33) using an
ABI377 DNA sequencer. To clone the ppt1 gene, the following
four primers were designed. Two oligonucleotides, 5'-TGAATTCGATGATAATTAAGCCTATAGCGT-3' (creates an
EcoRI site) and 5'-TCCAAGACTGCAGTAGAACGTTTAAGAATC-3',
were used to amplify the ppt1 gene. The amplified
fragment was then cloned into pT7Blue-T to yield pSP5. The two
additional oligonucleotides
5'-TGATGAACCACATTTACTTGATTTAGTCGA-3' and
5'-TCGAGCTCTTCTGACACCTCAACCTTTAAA-3' were used to amplify the 4.5-kb fragment containing the ppt1 gene and
the surrounding region. The amplified fragment was then cloned
into pT7Blue-T to yield pSP7. To make pSP11, pSP7 was digested with
SnaBI and ligated with the ura4 cassette derived
from pHSG398-ura4 (39). The 1.8-kb
SnaBI fragment containing ppt1 was cloned into
the SmaI site of pREP1 to yield pREP1-PPT1. The
SacI-BamHI fragment containing COQ2
(38) was cloned into pREP1 to yield pREP1-COQ2. Putative
mitochondrial transit sequences of ppt1 were amplified by
PCR using the oligonucleotides 5'-AGGTCGACAGATTAGCATGTAAATAG-3' (sense primer; creates a SalI site) and
5'-ATGGATCCGGGGGTTACAGAGTTTGA-3' (antisense primer; creates
a BamHI site) or 5'-TAGGATCCTTCAGCGTAGTATTGCCA-3' (antisense primer; creates a BamHI site). The PCR
products were cloned into the SalI and BamHI
sites of pREP1-GFPS65A (39), which contains the GFPS65A gene
(20) in pREP1, to yield pGFP-TP45 and pGFP-TP68.
Gene disruption.
The one-step gene disruption technique was
performed according to the procedure of Rothstein (30).
Plasmid pSP11 was linearized by appropriate restriction enzymes, and
the linearized plasmid was used to transform SP870 and SP826 to uracil
prototrophy. Southern hybridization was performed as described before
(32).
Ubiquinone extraction and measurement.
Ubiquinone was
extracted as previously described (37, 41). S. pombe cells were grown in a PMA-based medium (20 ml) until the
mid-log phase. After harvesting, the cells were lysed with 3 mg of
Novozyme, and ubiquinone was extracted with 3 ml of hexane-acetone (1:1, vol/vol), followed by evaporation of the organic solution to
dryness. Samples were then redissolved in 1 ml of chloroform-methanol (1:1, vol/vol) and the solution was washed with 0.5 ml of 0.7% NaCl.
After evaporation to dryness, the residue was taken up in 30 µl of
chloroform-methanol (2:1, vol/vol) and analyzed by normal-phase thin-layer chromatography on a Kiesel gel 60 F254 plate (Merck) with
benzene-acetone (93:7, vol/vol). A UQ-10 standard (Kaneka) was also
applied. The UV-visualized band containing ubiquinone was collected
from the thin-layer chromatography plate and extracted with
chloroform-methanol (1:1, vol/vol). The solution was evaporated to
dryness and the residue was redissolved in ethanol. The purified ubiquinone was further analyzed by high-pressure liquid chromatography using ethanol as a solvent (41).
Measurement of sulfide.
Hydrogen sulfide was detected by
production of PbS from lead acetate. A quantitative determination of
sulfide was performed by the methylene blue method as previously
described (28). Briefly, S. pombe cells were
grown in YEA medium (50 ml) until the late log phase. The cells were
collected and disrupted by glass beads, and cell extracts were
resuspended in 0.1 ml of 0.1% dimethylphenylenediamine (in 5.5 N HCl)
and 0.1 ml of 23 mM FeCl3 (in 1.2 N HCl). The samples were
incubated at 37°C for 5 min, after which the absorbance at 670 nm was
determined using a blank consisting of the reagents alone.
Staining of mitochondria and fluorescence microscopy.
Mitochondria were stained by the mitochondrion-specific dye MitoTracker
Green FM (Molecular Probes, Inc.). Cells were suspended in 10 mM HEPES,
pH 7.4, containing 5% glucose, and MitoTracker Green FM was added to
yield a final concentration of 100 nM. After standing for 15 min at
room temperature, cells were visualized by fluorescence microscopy at
490 nm. Fluorescence microscopy was carried out with a Zeiss Axioskop
microscope at a magnification of ×1,000. GFPS65A fluorescence was
observed by illumination at 485 nm. Images were captured by a Hamamatsu
C5985 CCD camera.
Cloning of the ppt1 gene and construction of
strains with a defective ppt1 gene.
We found a
putative gene for PHB polyprenyl diphosphate transferase in the
S. pombe genomic DNA sequence determined by the Sanger
Center. This gene (SPAC56F8.04c) shows high sequence similarity with
the COQ2 gene from S. cerevisiae and was
designated ppt1 (for PHB polyprenyl diphosphate
transferase). ppt1 and putative PHB polyprenyl
diphosphate transferases from other species could also be found in the
National Center for Biotechnology Information database (Fig.
1). Of these genes, only ubiA
from E. coli and COQ2 from S. cerevisiae have been functionally characterized (2, 16, 17,
35, 38).
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Phenotypes of Fission Yeast Defective in Ubiquinone Production
Due to Disruption of the Gene for p-Hydroxybenzoate
Polyprenyl Diphosphate Transferase
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-tocopherol, to
grow on minimal medium. This suggests that ubiquinone can act as an
antioxidant, a premise supported by our observation that the
ppt1-deficient strain is sensitive to
H2O2 and Cu2+. Interestingly, we
also found that the ppt1-deficient strain produced a
significant amount of H2S, which suggests that oxidation of
sulfide by ubiquinone may be an important pathway for sulfur metabolism
in S. pombe. Ppt1-green fluorescent protein fusion proteins
localized to the mitochondria, indicating that ubiquinone biosynthesis
occurs in the mitochondria in S. pombe. Thus, analysis of
the phenotypes of S. pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
were used for the general construction of plasmids
(32). Plasmids pBluescript II KS+/
, pT7Blue-T (Novagen),
pREP1 (15), and pREP1-GFPS65A (39) were used as vectors. The S. pombe homothallic haploid wild-type strain
SP870 (h90 leu1-32 ade6-M210 ura4-D18)
(13) and the diploid strains SP826 (h+
leu1-32 ade6-M210 ura4-D18/h+ leu1-32 ade6-M216
ura4-D18) (13) and TP4-1D/TP4-5A (h+
leu1-32 ura4-D18 his2 ade6-M216/h
leu1-32 ura4-D18
ade6-M210) (37) were used to produce
ppt1::ura4 strains by homologous
recombination. KS10 (h+ leu1-32 ade6-M216 ura4-D18
dps::ura4) was previously described (37). JV5 (h ura4-294 leu1-32
hmt2::URA3+) was obtained from
D. W. Ow (42). Yeast cells were grown in YE (0.5%
yeast extract, 3% glucose) or PM minimal medium (111 mM glucose, 93.5 mM NH4Cl, 15.5 mM Na2HPO, 14.7 mM potassium
hydrogen phthalate, 5.2 mM MgCl2 · 6H2O,
13.4 mM KCl, 0.28 mM Na2SO4, 0.1 mM
CaCl2 · 2H2O, 81.2 µM nicotinic acid,
55.5 µM myo-inositol, 40.8 µM biotin, 4.2 µM calcium
pantothenate, 8.1 µM boric acid, 2.37 µM MnSO4,
1.39 µM ZnSO4 · 7H2O, 0.74 µM
FeCl3 · 6H2O, 0.25 µM
MoO4 · 2H2O, 0.6 µM KI, 0.16 µM
CuSO4 · 5H2O, 4.76 µM citric acid)
with appropriate supplements as described by Moreno et al. (19). YEA and PMA contain 75 µg of adenine per ml in YE
and PM, respectively. The concentration of supplemented amino acids was
100 µg/ml. Yeast transformation was performed according to the method
described by Rose et al. (29).
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (86K):
[in a new window]
FIG. 1.
Comparison of the amino acid sequences of
PHB-polyprenyl diphosphate transferases. EC, E. coli
(accession no. X66619); SY, Synechocystis sp. strain PCC6803
(D64006); SC, S. cerevisiae (M81698); SP, S. pombe (Z69728); CE, Caenorhabditis elegans (U13876);
Dm, Drosophila melanogaster (AE003678). A putative substrate
recognition sequence is indicated by the underline. Conserved amino
acids of at least three in six sequences are highlighted.
ppt1, was allowed to make spores. Germinated haploid
cells were plated in replicates on plates containing YEA and PMA-Leu. While all cells grew well on YEA medium some grew only very slowly on
the PMA-Leu plate, and these were examined for ubiquinone synthesis. As
none synthesized ubiquinone (Fig. 3),
these strains were considered to potentially have a disruption in
ppt1. One such haploid strain, designated NU609, was
used for further experiments. Transformation of TP4-1D/TP4-5A
similarly generated a strain with a putative disruption in
ppt1 that was designation TP4-1D/TP4-5Appt1.
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Verification of ppt1 disruption by Southern
hybridization analysis.
Genomic DNAs from SP826,
SP826ppt1, NU609, and TP4-1D/TP4-5Appt1
were subjected to Southern hybridization analysis to confirm the
disruption of ppt1 by ura4. The genomic DNAs were
first digested with EcoRV and run on an agarose gel. The
ura4 cassette and the ppt1 gene were used as
probes. In lanes containing SP826ppt1 and
TP4-1D/TP4-5Appt1 DNAs, 1.5- and 4.5-kb bands
appeared with both probes (Fig. 2B and Fig.
4, lanes 2, 3, 6, and 7), because SP826ppt1 and TP4-1D/TP4-5Appt1 contain
both the complete ppt1 gene and the
ura4-disrupted ppt1 gene. When the
ura4 cassette was used as a probe, no band appeared with DNA
from SP826 (Fig. 4, lane 1), but 1.5- and 4.5-kb bands appeared with
the DNAs from SP826ppt1 and
TP4-1D/TP4-5Appt1 strains (Fig. 4, lanes 2 and 3, respectively) as well as with NU609 DNA (Fig. 4, lane 4). When the
ppt1 fragment was used as a probe, four bands of 1.5, 2.0, 4.5, and 6.0 kb appeared with SP826ppt1 and
TP4-1D/TP4-5Appt1 DNAs (Fig. 4, lanes 6 and 7), and three
bands of 1.5, 2.0, and 4.5 kb appeared with NU609 DNA (Fig. 4, lane 8).
Thus, the ppt1 gene is properly disrupted in
SP826ppt1, TP4-1D/TP4-5Appt1, and NU609.
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Complementation of ppt1 disruption-containing cells with COQ2. In the disruption of the ppt1 gene in NU609 by homologous recombination, it is possible that the upstream and downstream deletion of ppt1 could have damaged other genes. To eliminate this possibility, the plasmid pREP1-PPT1, which includes only the ppt1 gene, was used in a complementation assay. We also constructed pREP1-COQ2, in which only the COQ2 region is expressed under the control of the strong promoter nmt1, to test the functional conservation between Coq2 and Ppt1. Thus, NU609 harboring either or both of the vectors pREP1-PPT1 and pREP1-COQ2 were plated on PM-based medium and growth was observed. A few days later, NU609 harboring only the pREP1 vector formed only a very tiny colony, while NU609 harboring pREP1-PPT1 and pREP1-COQ2 grew as well as the wild-type strain. Thus, only the ppt1 function was abolished in NU609. That pREP1-PPT1 is as competent as pREP1-COQ2 in correcting the poor growth of the ppt1-defective strain indicates that ppt1 is a functional homologue of COQ2. When we extracted ubiquinone from each strain, UQ-10 was detected in the wild-type strain, in NU609 harboring pREP1-PPT1, and in NU609 harboring pREP1-COQ2, but not in NU609 alone (Fig. 3). That COQ2 complements the ppt1 disruptant and allows the production of UQ-10 in S. pombe is consistent with the idea that PHB polyprenyl diphosphate transferase has a broad substrate specificity.
Phenotypes of the NU609 ppt1 disruptant.
It was
previously reported that KS10 (dps::ura4),
a strain of S. pombe with a disruption in the dps
(decaprenyl diphosphate synthase) gene, is unable to produce ubiquinone
and has some notable phenotypes, including H2O2
and Cu2+ sensitivity and a requirement of cysteine or
glutathione for growth on minimal medium (37). Thus, NU609
was tested for these phenotypes. NU609 (ppt1) was first
grown on PM-based medium with and without supplementation with 200 µg
of cysteine per ml or 200 µg of glutathione per ml. The addition of
cysteine or glutathione effectively caused a recovery of NU609 growth
similar to that of the dps disruptant KS10 when it was
treated similarly (data not shown). NU609 was next tested for growth on
PM-based liquid medium supplemented with -tocopherol, a well-known
lipid antioxidant. NU609 cells did not grow on the minimal medium, but
interestingly, NU609 cells grew well when 1 mM -tocopherol was added
(data not shown). This suggests that ubiquinone may act as an
antioxidant in S. pombe. If this is so, it follows that the
ppt1-deficient strain might be susceptible to oxygen radical
producers, and we duly noted that NU609 growth is severely inhibited by
the presence of 2.5 mM H2O2 (Fig.
5A) or 0.5 mM Cu2+ (Fig. 5B).
The oxidants at these concentrations did not, however, affect the
growth of wild-type cells (Fig. 5). These results suggest strongly that
ubiquinone can serve as an antioxidant in normal fission yeast cells.
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, 
, 
, and 
) and NU609
(
, 
, 
, and 
)
strains were pregrown in YEA liquid medium until saturation and then
placed in 40-fold dilutions in fresh YEA medium with 2.5 mM
H2O2 (A) (Production of hydrogen sulfide in S. pombe strains
unable to produce ubiquinone.
We found that when the S. pombe strains with disruptions in ppt1 or
dps were cultivated, they smelled unpleasant. This was found
to be due to their production of H2S when we tested for the
formation of PbS by the chemical reaction of H2S with lead acetate (data not shown). Strains deficient in either ppt1
or dps could produce H2S, but the wild-type
cells could not. Since HMT2 catalyzes sulfide oxidation by concomitant
reduction of ubiquinone in S. pombe (42), we
tested for H2S production in hmt2 mutants, but
H2S could not be detected using this method. The production of H2S was also observed in NU609 cells grown on liquid
minimal medium supplemented with -tocopherol, indicating that the
antioxidant function of -tocopherol could not overcome the
production of H2S. We measured the amount of acid-labile
sulfide present in the cells and found that while JV5
(hmt2) produced a 2.5-fold-larger amount of
S2 than wild-type cells (82.1 and 33.7 nmol/106 cells, respectively), KS10 (dps) and
NU609 (ppt1) produced 9-fold-larger amounts of
S2 than the hmt2 strain (758.1 and
718.6 nmol/109 cells, respectively). This surprisingly high
level of S2 production presumably leads to the production
of H2S in the ppt1- and dps-deficient
strains. This unexpected phenotype suggests that ubiquinone may be
important in sulfide oxidation in S. pombe.
Mitochondrial localization of Ppt1.
Since ubiquinone
biosynthetic enzymes are localized to the mitochondria of S. cerevisiae (4, 11, 27), it has been suggested that
ubiquinone biosynthesis occurs in the mitochondria. To assess the
case for homologous enzymes from S. pombe, the localization of S. pombe Ppt1 was examined by Ppt1-green
fluorescent protein (GFP) fusions. Thus, genes expressing putative Ppt1
mitochondrial transit peptides of either 45 (pGFP-TP45) or 68 (pGFP-TP68) amino acids fused with GFP were constructed. pGFP-TP45 and
pGFP-TP68 were used to transform the S. pombe wild-type
strain, and Leu+ transformants were selected. When selected
transformants were examined by fluorescence microscopy, accumulation of
the fusion proteins in the mitochondria was observed (Fig.
6). The transformants were simultaneously
stained with MitoTracker Green FM, which stains mitochondria. The dye
stained the cells in exactly the same pattern produced by fluorescing
of the Ppt1-GFP fusions, indicating that Ppt1 localizes to the
mitochondria.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we examined the ppt1 gene, which encodes a 358-amino-acid protein with high homology to E. coli UbiA and S. cerevisiae Coq2 (34 and 48% identity, respectively). In S. cerevisiae, Coq2 acts to transfer six isoprenoid units to PHB to produce UQ-6. If, however, the COQ2 gene is expressed in an E. coli ubiA mutant, the cells produce UQ-8 (38). Similarly, as shown in this study, expression of the COQ2 gene in an S. pombe ppt1 disruptant resulted in production of UQ-10. Thus, COQ2 can transfer both octaprenyl diphosphate and decaprenyl diphosphate to PHB. S. cerevisiae can also generate various ubiquinone species (UQ-5 to UQ-10) when polyprenyl diphosphate synthases from other species are expressed in the COQ1 mutant (22). Those observations all indicate that PHB polyprenyl diphosphate transferases can act with a broad range of different polyprenyl diphosphate substrates.
Sequence alignment of the various PHB polyprenyl transferases suggests a putative substrate binding site constituted by an aspartic acid-rich motif (NDXXDXXXD) (35). This motif is well conserved in homolog proteins from Providencia stuartii, Neisseria meningitidis, Pasteurella haemolytica, and Arabidopsis thaliana as well as in the proteins listed in Fig. 1. However, the assumption that this motif is the substrate binding site is based merely on its similarity with the substrate recognition site (DDXXD) in polyprenyl diphosphate synthases (23, 24). The exact substrate recognition sequence in PHB polyprenyl diphosphate transferases remains to be determined.
While eight COQ genes (COQ1 to COQ8) are known to be involved in ubiquinone biosynthesis in S. cerevisiae (2, 4, 10, 11, 27), the ppt1 gene was only the second gene found to be involved in S. pombe biosynthesis of ubiquinone. When we subsequently examined the database from the S. pombe genome project for more genes, we found several COQ homologs. Besides dps (COQ1 homolog) and ppt1 (COQ2 homolog), there are also COQ3, COQ4, COQ5, COQ6, and COQ7 homologs in the S. pombe genome, with amino acid sequence identities of 40, 42, 54, 37, and 51%, respectively (the sequence of COQ8 is not public). Thus, the entire gene set known to be involved in S. cerevisiae ubiquinone biosynthesis is also preserved in S. pombe. However, the enzymatic activities of Coq4 and Coq8 have, as yet, not been determined, and it is also not clear if all eight S. cerevisiae COQs are necessary and sufficient for the biosynthesis of ubiquinone.
Coq1 (our unpublished observations), Coq3 (27), Coq5 (4), and Coq7 (11) have all been localized to the mitochondria of S. cerevisiae, indicating that ubiquinone biosynthesis occurs in mitochondria. When we examined Ppt1 localization in this study, we found that it also localized to the mitochondria, suggesting that biosynthesis of ubiquinone in S. pombe also occurs in the mitochondria.
S. pombe strains whose ppt1 gene had been
disrupted had several interesting phenotypes. First, while the
ppt1 disruption strain did not grow well on PMA-glucose,
growth was greatly improved by the presence of cysteine or glutathione.
The addition of the lipid antioxidant -tocopherol also
improved growth. A requirement for cysteine, glutathione, or
-tocopherol for growth on minimal medium is interesting and is
consistent with the concept that ubiquinone acts as an antioxidant.
Supporting this idea further is that the ppt1-deficient
strain is sensitive to active oxygen-producing reagents, such as
H2O2 and Cu2+. These phenotypes of
ppt1-deficient S. pombe are essentially equivalent to those observed for the dps-deficient S. pombe (37), confirming that these phenotypes arise as a
consequence of not being able to produce ubiquinone. A role for
ubiquinone as an antioxidant has also been reported for E. coli, S. cerevisiae, and mammalian cells (1,
7, 14, 18, 36). In E. coli, a ubiquinoneless mutant is
more susceptible to H2O2 and Cu2+
(36). In S. cerevisiae, strains unable to produce
ubiquinone are more susceptible to lipid peroxide and show lower
stabilities of extracellular ascorbate (34). In mammalian
cells, ubiquinone works synergistically with -tocopherol to reduce
lipid peroxide or low-density lipoprotein (1, 7). It is
deduced that ubiquinone in S. pombe also has the role of
suppressing lipid peroxidation of the membrane, although more direct
evidence will be necessary to prove this point.
This study also detected an additional, and very interesting, phenotype of fission yeast strains unable to produce ubiquinone. The ppt1 and dps mutants both produced large amounts of H2S. This observation could not be explained by ordinary metabolic pathways. However, the recent finding that sulfide-ubiquinone reductase exists in S. pombe (42) suggested to us that there may be a metabolic link between ubiquinone and H2S production. We speculate that in the absence of ubiquinone in the cell, sulfide-ubiquinone reductase cannot function and thus the cell accumulates H2S. Since sulfide-ubiquinone reductase is not present in S. cerevisiae, mutants of S. cerevisiae that are unable to produce ubiquinone do not produce H2S (our unpublished observation). Interestingly, humans as well as some other higher eukaryotes possess sulfide-ubiquinone reductases similar to the one found in photosynthetic bacteria (42). HMT2 catalyzes sulfide oxidation by concomitant reduction of ubiquinone in S. pombe. That the hmt2 mutant does not release H2S in equivalent quantities as strains unable to produce ubiquinone is perhaps due to the gradual oxidization of sulfide by ubiquinone that occurs despite the absence of sulfide-ubiquinone reductase.
All the observed phenotypes of S. pombe strains that are unable to produce ubiquinone serve to emphasize the fact that ubiquinone does not function solely as a component of the electron transfer system, as is generally believed. Ubiquinone appears to also be important in the oxidative stress response and the sulfide oxidation pathways, at least in S. pombe. The former role seems to be common in eukaryotes, while the latter role may occur in the majority of eukaryotes that have sulfide-ubiquinone reductases. Further investigation into the importance of the alternative functions of ubiquinone in other species can be carried out by the construction of ubiquinone-deficient organisms.
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ACKNOWLEDGMENTS |
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This study was supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan.
JV5 was kindly provided by D. W. Ow.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan. Phone: 81-852-32-6587. Fax: 81-852-32-6092. E-mail: kawamuka{at}life.shimane-u.ac.jp.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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M. Tomasetti,
M. Battino,
G. Curatola,
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Journal of Bacteriology, December 2000, p. 6933-6939, Vol. 182, No. 24
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