Enhanced sensitivity of ubiquinone-deficient mutants of Saccharomyces cerevisiae to products of autoxidized polyunsaturated fatty acids.

Thai, Quang Tung; Schultz, Jeffery R.; Clarke, Catherine F.

doi:10.1073/pnas.93.15.7534

Cited by 152 publications

(115 citation statements)

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“…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).…”

Section: ؉mentioning

confidence: 99%

Phenotypes of Fission Yeast Defective in Ubiquinone Production Due to Disruption of the Gene for p -Hydroxybenzoate Polyprenyl Diphosphate Transferase

et al. 2000

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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 ␣-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 H 2 O 2 and Cu 2؉. Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H 2 S, 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. 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)(23)(24)(25)(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 g...

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Section: ؉mentioning

confidence: 99%

Phenotypes of Fission Yeast Defective in Ubiquinone Production Due to Disruption of the Gene for p -Hydroxybenzoate Polyprenyl Diphosphate Transferase

et al. 2000

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“…Because PUFA treatment has previously been shown to cause accumulation of lipid peroxides in yeast (Do et al, 1996) and the cyanobacterium Synechococcus sp. PCC 7002 (Sakamoto et al, 1998), the level of total peroxides in the growth media of wild-type and slr1736 mutant strains during different treatments were measured using the ferrous oxidation-xylenol orange (FOX) assay (Griffiths et al, 2000;Sattler et al, 2004) and correlated with growth rates.…”

Section: Pufa Treatments Increase Peroxides In the Growth Mediamentioning

confidence: 99%

“…To test this hypothesis, a variety of chemicals were used to induce lipid peroxidation in the wild type and the tocopherol-deficient mutants. PUFAs, which are known to generate LOOH and LOO by autoxidation reactions in the presence of oxygen (Porter, 1986), have been used to induce lipid peroxidation in yeast (Do et al, 1996) and cyanobacteria (Sakamoto et al, 1998 ), hereafter referred to as 18:2 and 18:3, respectively, were applied in combination with HL stress to the wild type and the slr1736 and slr1737 mutants. In the presence of 10 mM 18:2/HL, growth of the slr1736 mutant ceased after 20 h, whereas the wild type and the slr1737 mutant were able to grow as well as untreated controls (Fig.…”

Section: Sensitivity Of Tocopherol-deficient Mutants To Compounds Thamentioning

confidence: 99%

Tocopherols Protect Synechocystis sp. Strain PCC 6803 from Lipid Peroxidation

et al. 2005

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Tocopherols (vitamin E) are lipid-soluble antioxidants synthesized only by photosynthetic eukaryotes and some cyanobacteria, and have been assumed to play important roles in protecting photosynthetic membranes from oxidative stress. To test this hypothesis, tocopherol-deficient mutants of Synechocystis sp. strain PCC 6803 (slr1736 and slr1737 mutants) were challenged with a series of reactive oxygen species-generating and lipid peroxidation-inducing chemicals in combination with high-light (HL) intensity stress. The tocopherol-deficient mutants and wild type were indistinguishable in their growth responses to HL in the presence and absence of superoxide and singlet oxygen-generating chemicals. However, the mutants showed enhanced sensitivity to linoleic or linolenic acid treatments in combination with HL, consistent with tocopherols playing a crucial role in protecting Synechocystis sp. strain PCC 6803 cells from lipid peroxidation. The tocopherol-deficient mutants were also more susceptible to HL treatment in the presence of sublethal levels of norflurazon, an inhibitor of carotenoid synthesis, suggesting carotenoids and tocopherols functionally interact or have complementary or overlapping roles in protecting Synechocystis sp. strain PCC 6803 from lipid peroxidation and HL stress.Oxygenic photosynthetic organisms continuously produce oxygen in the presence of light, and as such cellular damage from various reactive oxygen species (ROS), including singlet oxygen (2 ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (OH ), is a constant threat. Photosynthetic organisms have therefore evolved extensive detoxifying and protective mechanisms, which both limit the production of and potential damage by ROS. Examples include superoxide dismutase (SOD), which reduces O 2 2 to H 2 O 2 ; ascorbate peroxidase, which reduces H 2 O 2 to H 2 O; and nonphotochemical quenching that quenches singlet state chlorophylls ( 1 Chl*) and harmlessly dissipates excessive excitation energy as heat, thereby reducing 1 O 2 production (Asada, 1999;Muller et al., 2001). ROS, such as OH , can trigger a lipid peroxidation chain reaction by abstracting an allylic hydrogen from polyunsaturated fatty acid (PUFA)-containing lipids producing lipid radicals that are converted to lipid peroxyl radicals (LOO ) upon O 2 addition. LOO can subsequently attack another PUFA generating a second LOO and propagating a chain reaction of lipid peroxidation that perturbs membrane structure and function (Porter, 1986). Given the susceptibility of PUFAs to ROS damage, it seems counterintuitive that the PUFA-enriched thylakoid membranes would house the photosynthetic machinery, a potential ROS generator. In contrast to the well-studied mechanisms of water-soluble ROS detoxification in photosynthetic organisms (Asada, 1999), the mechanisms preventing or limiting oxidative damage in photosynthetic membranes are less well understood. Several peroxiredoxins have been implicated in reducing lipid hydroperoxides (LOOH) to the less toxic lipid hydroxides (LOH) in ...

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“…Yeast cells normally do not produce or require PUFAs, but are able to take them up and incorporate them into membrane phospholipids [45][46][47]. Although wild-type yeast cells are quite tolerant of PUFAs, coq mutant yeast lacking Q, an endogenously produced antioxidant lipid, are exquisitely sensitive to PUFA treatment [27,48]. The site-specific replacement of the 11,11-bis-allylic H atoms of Lin with D rescues the hypersensitivity of the yeast Q-less mutants to PUFA treatment (Fig.…”

Section: Discussionmentioning

confidence: 99%

Small amounts of isotope-reinforced polyunsaturated fatty acids suppress lipid autoxidation

Shchepinov

Hill

Lamberson

et al. 2012

Free Radical Biology and Medicine

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Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D 2 -Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress.Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA: natural H-PUFA are protected from lipid autoxidationmediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of non-deuterated PUFAs in yeast.

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Enhanced sensitivity of ubiquinone-deficient mutants of Saccharomyces cerevisiae to products of autoxidized polyunsaturated fatty acids.

Cited by 152 publications

References 53 publications

Phenotypes of Fission Yeast Defective in Ubiquinone Production Due to Disruption of the Gene for p -Hydroxybenzoate Polyprenyl Diphosphate Transferase

Phenotypes of Fission Yeast Defective in Ubiquinone Production Due to Disruption of the Gene for p -Hydroxybenzoate Polyprenyl Diphosphate Transferase

Tocopherols Protect Synechocystis sp. Strain PCC 6803 from Lipid Peroxidation

Small amounts of isotope-reinforced polyunsaturated fatty acids suppress lipid autoxidation

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