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

Abstract

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...

Ubiquinone Synthesis in Mitochondrial and Microsomal Subcellular Fractions of Pneumocystis spp.: Differential Sensitivities to Atovaquone

“…High concentrations of CoQ can be found in cell surface membranes (13), and CoQ was shown to serve as an electron and proton carrier in cell surface membranes of mammalian cells (5,55,61,70). CoQ also serves as an important endogenous antioxidant (25,47,59,70) and plays a role in development and longevity (20,40,52,54,71) in addition to several other functions now known to involve this ubiquitous molecule (70). Except for its role in cellular respiration, very little is known about the functions of CoQ in Pneumocystis spp.…”

Expression of NAD(P)H:Quinone Oxidoreductase 1 in HeLa Cells

“…Also, ubiquinol prevents adriamycininduced generation of H 2 O 2 in isolated hepatocytes (10,11), and this protection is abolished by the NQO1 inhibitor dicumarol (10). Finally, strains of Schizosaccharomyces pombe defective in ubiquinone biosynthesis display enhanced sensitivity to H 2 O 2 (50,51) and enrichment of hepatocytes with ubiquinol protects cells against H 2 O 2 (52). In sum, these data support the possibility that NQO1 may contribute to protect against H 2 O 2 via the generation and maintenance of hydroquinones.…”

Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis

“…Eco_ppsA, phosphoenolpyruvate synthase; Eco_aroF FBR , feedback-inhibitionresistant (FBR) 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase; Eco_ubiC, chorismate lyase; Spo_aro2, chorismate synthase; Spo_aro7, chorismate mutase; Sce_thmgr1, truncated 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase 1; Sce_mvk FBR , FBR mevalonate kinase; Spo_erg8, phosphomevalonate kinase; Spo_mvd1, diphosphomevalonate decarboxylase; dps1-dlp1, decaprenyl diphosphate synthase; ppt1, PHB-decaprenyl diphosphate transferase. CoQ 10 biosynthesis 2,19,24,26) ; thus, it was a promising organism to use with this approach.…”

“…For example, CoQ acts as an electron donor during disulfide bond formation in E. coli, 1) and CoQ reduction is coupled to sulfide oxidation in Schizosaccharomyces pombe and other organisms. 2) CoQ is also required for de novo synthesis of UMP in eukaryotes. 3,4) Living organisms possess different species of CoQ, and these are classified according to the length of the isoprenoid side chain.…”