19F nuclear magnetic resonance analysis of 5-fluorouracil metabolism in wild-type and 5-fluorouracil-resistant Nectria haematococca

A mutant (furA3) was isolated from the S1 wild-type strain of Nectria haematococca on the basis of its resistance to 5-fluorouracil (5FU). This mutant has greatly reduced activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme catalyzing the synthesis of UMP from uracil. The metabolism of 5FU was examined in both strains by using 19F nuclear magnetic resonance spectroscopy. In the S1 strain, 5FU appears to be metabolized by two pathways operating simultaneously: (i) conversion to fluoronucleotides and (ii) degradation into alpha-fluoro-beta-alanine. The furA3 mutant shows metabolic changes consistent with a uracil phosphoribosyltransferase lesion, since it takes up 5FU and forms a small amount of alpha-fluoro-beta-alanine but does not synthesize fluoronucleotides. Since pigment synthesis is strongly enhanced by 5FU in the S1 wild-type strain but not in the furA3 mutant, these results support the hypothesis that 5FU stimulation of secondary metabolism in N. haematococca is not mediated by the drug itself but involves a phosphorylated anabolite.     

19F nuclear magnetic resonance analysis of 5-fluorouracil metabolism in wild-type and 5-fluorouracil-resistant Nectria haematococca.

A mutant (furA3) was isolated from the S1 wild-type strain of Nectria haematococca on the basis of its resistance to 5-fluorouracil (5FU). This mutant has greatly reduced activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme catalyzing the synthesis of UMP from uracil. The metabolism of 5FU was examined in both strains by using 19F nuclear magnetic resonance spectroscopy. In the S1 strain, 5FU appears to be metabolized by two pathways operating simultaneously: (i) conversion to fluoronucleotides and (ii) degradation into alpha-fluoro-beta-alanine. The furA3 mutant shows metabolic changes consistent with a uracil phosphoribosyltransferase lesion, since it takes up 5FU and forms a small amount of alpha-fluoro-beta-alanine but does not synthesize fluoronucleotides. Since pigment synthesis is strongly enhanced by 5FU in the S1 wild-type strain but not in the furA3 mutant, these results support the hypothesis that 5FU stimulation of secondary metabolism in N. haematococca is not mediated by the drug itself but involves a phosphorylated anabolite.     

Ubiquinone biosynthesis in Saccharomyces cerevisiae. Isolation and sequence of COQ3, the 3,4-dihydroxy-5-hexaprenylbenzoate methyltransferase gene

Ubiquinone (or coenzyme Q) is a lipid component of the respiratory chain in the inner mitochondrial membrane, in which it functions in electron transport. Recent reports show that ubiquinone and ubiquinone biosynthetic enzymes are present in both mitochondrial and nonmitochondrial membranes of cells (Kalen, A., Appelkvist, E.-L., Chojnacki, T., and Dallner, G. (1990) J. Biol. Chem. 265, 1158-1164) although the functions that ubiquinone may play outside of the mitochondrion are not understood. To study coenzyme Q synthesis and function we cloned the 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase gene by functional complementation of a yeast coenzyme Q mutant strain, defective in the COQ3 gene (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225). This gene restores both coenzyme Q synthesis in the mutant strain and the ability to grow on media containing glycerol, a nonfermentable substrate. A one-step in situ gene replacement with the cloned DHHB methyltransferase DNA directs integration to the yeast COQ3 locus on chromosome XV of Saccharomyces cerevisiae, establishing that the COQ3 locus encodes the DHHB methyltransferase structural gene. The predicted amino acid sequence of the yeast DHHB methyltransferase contains a methyltransferase consensus sequence and shows a 40% identity with an open reading frame of Escherichia coli, the gyrA5' hypothetical protein. This open reading frame is adjacent to the gyrA gene and close to the mapped location of the ubiG gene at 48 min on the E. coli chromosome. These results suggest that the E. coli gyrA5' open reading frame encodes a methyltransferase and may correspond to the ubiG gene, which is required for ubiquinone biosynthesis.     

An Escherichia coli mutant resistant to phleomycin, bleomycin, and heat inactivation is defective in ubiquinone synthesis.

A mutant of Escherichia coli, selected for resistance to the antibiotic and antitumor agent phleomycin, has been characterized, and the phleomycin resistance determinant has been identified. The mutant is equally resistant to bleomycins. The resistance to phleomycin is strongly dependent on the nature of the C-terminal amine of the drug, with the greatest resistance being shown to phleomycins and bleomycins with the most basic terminal amines. The mutation also confers resistance to the lethal effects of heating at 52 degrees C. Other characteristics of the phleomycin-resistant strain include a slow growth rate, an inability to grow on succinate as the sole carbon source (Suc- phenotype), cross resistance to aminoglycoside antibiotics, and a slight sensitivity to hydrogen peroxide, methyl methanesulfonate, and gamma-irradiation. Some of these characteristics, together with mapping data, suggested that the phleomycin resistance and Suc- determinant probably lies within the ubiF gene coding for an enzyme effecting a step in the biosynthesis of ubiquinone. The phenotypes of known mutants defective in this and other steps of the ubiquinone pathway were found to be closely similar to those of the original phleomycin-resistant strain.     

The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli.

The Escherichia coli ispB gene encoding octaprenyl diphosphate synthase is responsible for the synthesis of the side chain of isoprenoid quinones. We tried to construct an E. coli ispB-disrupted mutant but could not isolate the chromosomal ispB disrupted mutant unless the ispB gene or its homolog was supplied on a plasmid. The chromosomal ispB disruptants that harbored plasmids carrying the ispB homologs from Haemophilus influenzae and Synechocystis sp. strain PCC6803 produced mainly ubiquinone 7 and ubiquinone 9, respectively. Our results indicate that the function of the ispB gene is essential for normal growth and that this function can be substituted for by homologs of the ispB gene from other organisms that produce distinct forms of ubiquinone.     

Chemotherapy agents, response rates and mechanisms of resistance in the therapy of the colorectal carcinoma

Colorectal carcinoma is one of the most common cancers in Hungary, responsible for about 5000 deaths each year. In the first line treatment the most commonly used drugs are 5-fluorouracil, oxaliplatin and irinotecan. The most frequently used drug is 5-fluorouracil, which has no effect in 90% of the cases. In combination with leukovorin or with 5-ethyl-2'-deoxyuridin fluorouracil has an increased effect. The main mechanisms of the resistance against 5-fluorouracil are due to the overexpression of dihydropyrimidine dehydrogenase, MRP8, thymidylate synthase, and NFkB p65. Oxaliplatin forms reactive platinum complexes, which are believed to inhibit DNA synthesis by forming interstrand and intrastrand cross-linking of DNA molecules. The oxaliplatin-5-fluorouracil-leucovorin combination was the first to reach more than 20 months median survival. The main mechanisms of resistance are decreased accumulation, increased detoxification and increased DNA repair. Irinotecan inhibits the topoisomerase I enzyme, resulting in the inhibition of the repair of DNA breaks occurring during DNA synthesis. With sequential 5-fluorouracil, oxaliplatin, irinotecan combination 26 months median survival was reached. Mechanisms resulting in resistance are decreased accumulation, increased enzymatic detoxification, alterations of ABC transporters, DNA repair system, apoptotic pathways and topoisomerase I. Survival can be elongated using biological therapy (cetuximab, bevacizumab). In the near future biological therapy is expected to spread.     

Selectable mutant phenotypes of the extremely thermophilic archaebacterium Sulfolobus acidocaldarius.

As a first step toward developing the genetic potential of extremely thermophilic archaebacteria, mutant strains of Sulfolobus acidocaldarius were selected by plating cells directly on solid medium containing one of several growth inhibitors. Three spontaneous resistance phenotypes were observed (5-fluorouracil resistance, novobiocin resistance, and L-ethionine resistance), each at a different average frequency. Characterization of representative strains showed each of the three mutant phenotypes to provide a potentially useful genetic marker.     

De novo synthesis and desaturation of fatty acids at the mitochondrial acyl-carrier protein, a subunit of NADH:ubiquinone oxidoreductase in Neurospora crassa.

We have cultivated the cel mutant of Neurospora crassa defective in cytosolic fatty acid synthesis with [2-14C]malonate and found radioactivity covalently attached to the mitochondrial acyl-carrier protein (ACP), a subunit of the respiratory chain NADH:ubiquinone oxidoreductase. We purified the ACP by reverse-phase HPLC: the bound acyl groups were trans-esterified to methylesters and analyzed by gas chromatography. The saturated C6 to C18 fatty acids and oleic acid were detected. De novo synthesis and desaturation of fatty acids at the ACP subunit of NADH:ubiquinone oxidoreductase and use of the products of this mitochondrial synthetic pathway for cardiolipin synthesis is discussed.     

Production of isoprenoid compounds in the facultative methylotroph Protomonas extorquens

Screening of a strain which contained a large amount of ubiquinone Q-10 and a variety of isoprenoid compounds using different culture conditions and mutations was carried out.Protomonas extorquens TK 0045, which was found to contain carotenoid pigments, Hop-22(29)-ene, and Hopan-22-ol, was selected on the basis of cell yield and the content of ubiquinone Q-10. The contents of ubiquinone and sterols increased as the age of the culture increased, and reached a maximum level during the stationary phase.The contents of ubiquinone, sterols and carotenoid pigments, and ubiquinone homologs produced by P. extorquens TK 0045 were varied using mutagenesis. Mutants that had increased or decreased contents of carotenoid pigments were obtained with a high frequency. Most mutants had varying contents of other isoprenoid compounds. The ubiquinone homologs obtained by mutagenesis varied with a high frequency, and mutants which possessed increased levels of ubiquinone Q-9, Q-11 or Q-12 were isolated. However, the major ubiquinone component in these mutants was Q-10 the same as that in the wild strain. The production of ubiquinone was increased considerablyby repeated mutagenesis, with the content of ubiquinone produced by the third generation mutant (strains HB-5) being approximately 3.3 mg·g dry cell⁻¹ (2.5 times that of the wild strain). The acquisition of mutants exhibiting altered synthesis of carotenoid pigments would be useful for increasing the content of ubiquinone Q-10 in bacterial cells.     

Molecular mechanism of maternal rescue in the clk-1 mutants of Caenorhabditis elegans

The clk-1 mutants of Caenorhabditis elegans display an average slowing down of physiological rates, including those of development, various behaviors, and aging. clk-1 encodes a hydroxylase involved in the biosynthesis of the redox-active lipid ubiquinone (co-enzyme Q), and in clk-1 mutants, ubiquinone is replaced by its biosynthetic precursor demethoxyubiquinone. Surprisingly, homozygous clk-1 mutants display a wild-type phenotype when issued from a heterozygous mother. Here, we show that this maternal effect is the result of the persistence of small amounts of maternally derived CLK-1 protein and that maternal CLK-1 is sufficient for the synthesis of considerable amounts of ubiquinone during development. However, gradual depletion of CLK-1 and ubiquinone, and expression of the mutant phenotype, can be produced experimentally by developmental arrest. We also show that the very long lifespan observed in daf-2 clk-1 double mutants is not abolished by the maternal effect. This suggests that, like developmental arrest, the increased lifespan conferred by daf-2 allows for depletion of maternal CLK-1, resulting in the expression of the synergism between clk-1 and daf-2. Thus, increased adult longevity can be uncoupled from the early mutant phenotypes, indicating that it is possible to obtain an increased adult lifespan from the late inactivation of processes required for normal development and reproduction.     

Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis.

Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms. The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cremoris MG1363 by complementation of an Escherichia coli mutant. The gene was sequenced, and the putative amino acid sequence was deduced. The promoter was mapped by both primer extension and analysis of beta-galactosidase expressed from strains carrying fusion between upp promoter fragments and the lacLM gene. The results showed that the upp gene was expressed from its own promoter. After in vitro construction of an internal deletion, a upp mutant was constructed by a double-crossover event. This implicated the utilization of a plasmid with a thermosensitive origin of replication and a new and easy way to screen for double crossover events in both gram-positive and gram-negative bacterial strains. The phenotype of the uracil phosphoribosyltransferase-deficient strain was established. Surprisingly, the upp strain is resistant only to very low concentrations of 5-fluorouracil. Secondary mutants in thymidine phosphorylase and thymidine kinase were isolated by selection for resistance to high concentrations of 5-fluorouracil.     

The oxidation of external NADH by an intermembrane electron transfer in mitochondria from the ubiquinone-deficient mutant E3-24 of Saccharomyces cerevisiae

Cells of the E3-24 mutant of the strain D273-10B of Saccharomyces cerevisiae, grown in a fermentable substrate not showing catabolite repression of respiration (2% galactose), are able to respire, in spite of their ubiquinone deficiency in mitochondrial membranes. Mitochondria isolated from these mutant cells oxidize exogenous NADH through a pathway insensitive to antimycin A but inhibited by cyanide. Addition of methanolic solutions of ubiquinone homologs stimulates the oxidation rate and restores antimycin A sensitivity in both isolated mitochondria and whole cells. Mersalyl preincubation of isolated mitochondria inhibits both NADH oxidation and NADH-cytochrome c oxido-reductase activity (assayed in the presence of cyanide) with the same pattern. Electrons resulting from the oxidation of exogenous NADH reduce both cytochrome b5 and endogenous cytochrome c. The increase in ionic strength stimulates NADH oxidation, which is also coupled to the ATP synthesis with an ATP/O ratio similar to that obtained with ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) as substrate. The effect of cyanide on these activities and on NADH-induced endogenous cytochrome c reduction is also comparable. These results support the existence in vivo and in isolated mitochondria of a energy-conserving pathway for the oxidation of cytoplasmatic NADH not related to the dehydrogenases of the inner membrane, the ubiquinone, and the b-c1 complex, but involving a cytochrome c shuttle between the NADH-cytochrome c reductase of the outer membrane and cytochrome oxidase in the inner membrane.     

Cloning and functional expression of AtCOQ3, the Arabidopsis homologue of the yeast COQ3 gene, encoding a methyltransferase from plant mitochondria involved in ubiquinone biosynthesis

A mutant of Saccharomyces cerevisiae deleted for the COQ3 gene was constructed. COQ3 encodes a 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase that catalyses the fourth step in the biosynthesis of ubiquinone from p-hydroxybenzoic acid. A full length cDNA encoding a homologue of DHHB-methyltransferase was cloned from an Arabidopsis thaliana cDNA library by functional complementation of a yeast coq3 deletion mutant. The Arabidopsis thaliana cDNA (AtCOQ3) was able to restore the respiration ability and ubiquinone synthesis of the mutant. The product of the 1372 bp cDNA contained 322 amino acids and had a molecular mass of 35 360 Da. The predicted amino acid sequence contained all consensus regions for S-adenosyl methionine methyltransferases and presented 26% identity with Saccharomyces cerevisiae DHHB-methyltransferase and 38% identity with the rat protein, as well as with a bacterial (Escherichia coli and Salmonella typhimurium) methyltransferase encoded by the UBIG gene. Southern analysis showed that the Arabidopsis thaliana enzyme was encoded by a single nuclear gene. The NH₂-terminal part of the cDNA product contained features consistent with a putative mitochondrial transit sequence. The cDNA in Escherichia coli was overexpressed and antibodies were raised against the recombinant protein. Western blot analysis of Arabidopsis thaliana and pea protein extracts indicated that the AtCOQ3 gene product is localized within mitochondrial membranes. This result suggests that at least this step of ubiquinone synthesis takes place in mitochondria.     

The function of ubiquinone in Escherichia coli

1. The function of ubiquinone in Escherichia coli was studied by using whole cells and membrane preparations of normal E. coli and of a mutant lacking ubiquinone. 2. The mutant lacking ubiquinone, strain AN59 (Ubi(-)), when grown under aerobic conditions, gave an anaerobic type of growth yield and produced large quantities of lactic acid, indicating that ubiquinone plays a vital role in electron transport. 3. NADH and lactate oxidase activities in membranes from strain AN59 (Ubi(-)) were greatly impaired and activity was restored by the addition of ubiquinone (Q-1). 4. Comparison of the percentage reduction of flavin, cytochrome b(1) and cytochrome a(2) in the aerobic steady state in membranes from the normal strain (AN62) and strain AN59 (Ubi(-)) and the effect of respiratory inhibitors on these percentages in membranes from strain AN62 suggest that ubiquinone functions at more than one site in the electron-transport chain. 5. Membranes from strain AN62, in the absence of substrate, showed an electron-spin-resonance signal attributed to ubisemiquinone. The amount of reduced ubiquinone (50%) found after rapid solvent extraction is consistent with the existence of ubiquinone in membranes as a stabilized ubisemiquinone. 6. The effects of piericidin A on membranes from strain AN62 suggest that this inhibitor acts at the ubiquinone sites: thus inhibition of electron transport is reversed by ubiquinone (Q-1); the aerobic steady-state oxidation-reduction levels of flavins and cytochrome b(1) in the presence of the inhibitor are raised to values approximating those found in the membranes of strain AN59 (Ubi(-)); the inhibitor rapidly eliminates the electron-spin-resonance signal attributed to ubisemiquinone and allows slow oxidation of endogenous ubiquinol in the absence of substrate and prevents reduction of ubiquinone in the presence of substrate. It is concluded that piericidin A separates ubiquinone from the remainder of the electron-transport chain. 7. A scheme is proposed in which ubisemiquinone, complexed to an electron carrier, functions in at least two positions in the electron-transport sequence.     

Metabolism of ubiquinone in relation to thyroxine status

1. Under conditions of thyrotoxicosis induced by feeding rats with iodinated casein, ubiquinone concentration was found to increase in the liver by increased synthesis and by partly decreased catabolism leading to its accumulation. The increased ubiquinone was found primarily in the mitochondrial and supernatant fractions. 2. Supplementing the diet with thyroxine, at less than toxic doses, also increased the synthesis and the concentration of ubiquinone in the liver. 3. In the condition of hypothyroidism obtained by feeding rats with thiouracil the concentration and the synthesis of ubiquinone in the liver showed a small decrease. 4. Synthesis of ubiquinone in liver slices was partially inhibited by addition of thyroxine in vitro. Therefore the activation effect on ubiquinone synthesis of excess of thyroxine in the intact animals appears to be by an indirect mechanism.     

Molecular Mechanisms of Resistance to 5-Fluorocytosine in Laboratory Mutants of <Emphasis Type="Italic">Candida glabrata</Emphasis>

Resistance to 5-fluorocytosine (5-FC) has been poorly investigated in the yeast Candida glabrata. This study was conducted on laboratory mutants obtained by exposure of a wild-type isolate to 5-FC. Based on their susceptibility to 5-fluorouracil (5-FU), two of these mutants were selected for further analysis of the molecular mechanisms of 5-FC resistance. One mutant, resistant to both compounds, exhibited a missense mutation in the gene coding the cytosine deaminase and a decrease in the expression level of the gene coding the uridine monophosphate pyrophosphorylase. The other mutant that showed a reduced susceptibility to 5-FC and 5-FU exhibited an overexpression of the genes coding the thymidylate synthase and a cytosine permease, associated with a missense mutation in the last gene. Thus, beside mutations in the FUR1 gene which represent the most common cause of resistance to 5-FC, other mechanisms may also occur in C. glabrata.     

Interrelationships of Ubiquinone and Sterol Syntheses in Cultured Cells of Neural Origin

Abstract: Ubiquinone synthesis has been studied in cultured C-6 glial and neuroblastoma cells by utilizing an inhibitor, 3-β-(2-diethylaminoethoxy) androst-5-en-17-one hydrochloride (U18666A), of cholesterol biosynthesis. Exposure of C-6 glial cells to nanomolar quantities of U18666A caused a marked inhibition of total sterol synthesis from [¹⁴C]acetate or [³H]mevalonate within minutes. A 95% inhibition was apparent after a 3-h exposure to 200 ng/ml of U18666A. These observations, together with studies of the incorporation of radioactivity from the two precursors into cholesterol, desmosterol, lanosterol, and squalene, indicated that although the most sensitive site to inhibition by U18666A is desmosterol reduction to cholesterol, a major site of inhibition is demonstrable at a more proximal site, perhaps squalene synthetase. As a consequence of the latter inhibition, exposure of C-6 glial cells to U18666A caused a marked stimulation of incorporation of [¹⁴C]acetate or [³H]mevalonate into ubiquinone. Over a wide range of U18666A concentrations, the increase in ubiquinone synthesis was accompanied by an approximately similar decrease in total sterol synthesis. Whereas in the absence of U18666A only approximately 7% of the radioactivity incorporated from [³H]mevalonate into isoprenoid compounds was found in ubiquinone, in the presence of the drug approximately 90% of incorporated radioactivity was found in ubiquinone. The reciprocal effects of U18666A on ubiquinone and sterol syntheses were apparent also in the neuronal cells. The data thus demonstrate a tight relationship between ubiquinone and sterol biosyntheses in cultured cells of neural origin. In such cells ubiquinone synthesis is exquisitely sensitive to the availability of isoprenoid precursors derived from the cholesterol biosynthetic pathway.     

Genetic and Physiological Aspects of Resistance to 5-Fluoropyrimidines in Saccharomyces cerevisiae

Mutants resistant to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine were selected in yeast, and the mechanisms of their resistance were investigated. The investigated mutations map in seven different loci. (i) A mutation at the locus FUI 1 gives specifically resistance to 5-fluorouridine. (ii) Two loci are involved in a specific 5-fluorocytosine resistance: a mutation at locus FCY 1 produces a loss of cytosine deaminase activity; a mutation at locus FCY 2 results in the loss of the activity of a cytosine-specific permease. (iii) A mutation at the locus FUR 4 gives a simultaneous resistance to 5-fluorouracil and to 5-fluorouridine by loss in the activity of the uracil-specific permease. (iv) We found three types of mutants in the locus FUR 1. One is dominant and weakly resistant to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine. The two others are recessive and are unable to catalyze one of the steps involved in uracil transformation into uridine 5'-monophosphate; this block-age explains their strong resistance to 5-fluorouracil and 5-fluorocytosine. Of these two mutants, one is resistant to 5-fluorouridine and the other is not. (v) Mutations at locus FUR 2 give resistance to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine. These mutations are dominant and lead to a loss in the feedback regulation of the aspartic transcarbamylase activity by uridine triphosphate. (vi) The mutants FUR 3 are resistant to 5-fluorocytosine and 5-fluorouridine. They are dominant and physiologically related to the mutants of the locus FUR 1 but their mechanism of resistance is not understood.     

Inhibition of Light-Dependent Development of Chloroplasts by 5-Fluorouracil

The uracil analogue, 5-fluorouracil, inhibited the developmentof chloroplasts in Euglena gracilis, strain Z. Chlorophyll synthesiswas inhibited when dark-grown cells were illuminated in thepresence of 5-fluorouracil, but only if the 5-fluorouracil waspresent during the lag phase of chlorophyll synthesis. Ribonucleaseshowed a similar inhibition. Equimolar concentrations of uracilreleased inhibition by 5-fluorouracil, but if cells were incubatedin the light with 5-fluorouracil before addition of uracil,the ability of uracil to effect rapid reversal of 5-fluorouracilinhibition was decreased. In contrast, prior incubation with5-fluorouracil in the dark did not affect reversibility by uracil.The synthesis of a chloroplast-localized protein, cytochromec (552, Euglena), was also inhibited by 5-fluorouracil, whereasthe light-stimulated synthesis of a number of cytoplasmic enzymeswas enhanced. The results suggest that addition of 5-fluorouracilat the beginning of the illumination period preferentially interfereswith the synthesis of chloroplast protein compared with thesynthesis of cytoplasmic protein by inhibiting the formationof a ribosomal system, presumably localized in the chloroplast,that functions in the synthesis of chloroplast protein. Thedata also suggest that in uninhibited cells, the formation ofthis ribosomal system was largely completed within the first10 to 14 h of illumination and before the main period of synthesisof chloroplastproteins.     

Pleiotropic phenotypes of fission yeast defective in ubiquinone-10 production. A study from the abc1Sp (coq8Sp) mutant

We previously constructed two Schizosaccahromyces pombe ubiquinone-10 (or Coenzyme Q10) less mutants, which are either defective for decaprenyl diphosphate synthase or p-hydroxybenzoate polyprenyl diphosphate transferase. To further confirm the roles of ubiquinone in S. pombe, we examined the phenotype of the abc1Sp (coq8Sp) mutant, which is highly speculated to be defective in ubiquinone biosynthesis. We show here that the abc1Sp defective strain did not produce UQ-10 and could not grow on minimal medium. The abc1Sp-deficient strain required supplementation with antioxidants such as cysteine or glutathione to grow on minimal medium. In support of the antioxidant function of ubiquinone, the abc1Sp-deficient strain is sensitive to H2O2 and Cu2+. In addition, expression of the stress inducible ctt1 gene was much induced in the ubiquinone less mutant than wild type. Interestingly, we also found that the abc1-deficient strain as well as other ubiquinone less mutants 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. Thus, analysis of the phenotypes of S. pombe ubiquinone less mutants clearly demonstrate that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.