Ubiquinone (coenzyme Q) functions as an electron transporter in aerobic respiration and oxidative phosphorylation in the respiratory chain [1]. In addition, many reports suggest that ubiquinone also functions as a lipid-soluble antioxidant in cellular biomembranes, scavenging reactive oxygen species [2][3][4][5]. Indeed, several studies using yeast strains that do not produce ubiquinone suggest that an in vitro function of ubiquinone is to protect against oxidants [6,7]. Another phenotype of such ubiquinone-deficient fission yeast is that they generate high levels of hydrogen sulfide [8][9][10]. As Schizosaccharomyces pombe and other eukaryotes are known to carry sulfide-ubiquinone reductase, an enzyme that oxidizes sulfide via ubiquinone [11], it has been suggested that ubiquinone is linked to sulfide metabolism in many organisms. In addition, it was The isoprenoid chain of ubiquinone (Q) is determined by trans-polyprenyl diphosphate synthase in micro-organisms and presumably in mammals. Because mice and humans produce Q 9 and Q 10 , they are expected to possess solanesyl and decaprenyl diphosphate synthases as the determining enzyme for a type of ubiquinone. Here we show that murine and human solanesyl and decaprenyl diphosphate synthases are heterotetramers composed of newly characterized hDPS1 (mSPS1) and hDLP1 (mDLP1), which have been identified as orthologs of Schizosaccharomyces pombe Dps1 and Dlp1, respectively. Whereas hDPS1 or mSPS1 can complement the S. pombe dps1 disruptant, neither hDLP1 nor mDLP1 could complement the S. pombe dLp1 disruptant. Thus, only hDPS1 and mSPS1 are functional orthologs of SpDps1. Escherichia coli was engineered to express murine and human SpDps1 and ⁄ or SpDlp1 homologs and their ubiquinone types were determined. Whereas transformants expressing a single component produced only Q 8 of E. coli origin, double transformants expressing mSPS1 and mDLP1 or hDPS1 and hDLP1 produced Q 9 or Q 10 , respectively, and an in vitro activity of solanesyl or decaprenyl diphosphate synthase was verified. The complex size of the human and murine long-chain transprenyl diphosphate synthases, as estimated by gel-filtration chromatography, indicates that they consist of heterotetramers. Expression in E. coli of heterologous combinations, namely, mSPS1 and hDLP1 or hDPS1 and mDLP1, generated both Q 9 and Q 10 , indicating both components are involved in determining the ubiquinone side chain. Thus, we identified the components of the enzymes that determine the side chain of ubiquinone in mammals and they resembles the S. pombe, but not plant or Saccharomyces cerevisiae, type of enzyme.Abbreviations
The analysis of the structure and function of long chainproducing polyprenyl diphosphate synthase, which synthesizes the side chain of ubiquinone, has largely focused on the prokaryotic enzymes, and little is known about the eukaryotic counterparts. Here we show that decaprenyl diphosphate synthase from Schizosaccharomyces pombe is comprised of a novel protein named Dlp1 acting in partnership with Dps1. Dps1 is highly homologous to other prenyl diphosphate synthases but Dlp1 shares only weak homology with Dps1. We showed that the two proteins must be present simultaneously in Escherichia coli transformants before ubiquinone-10, which is produced by S. pombe but not by E. coli, is generated. Furthermore, the two proteins were shown to form a heterotetrameric complex. This is unlike the prokaryotic counterparts, which are homodimers. The deletion mutant of dlp1 lacked the enzymatic activity of decaprenyl diphosphate synthase, did not produce ubiquinone-10 and had the typical ubiquinone-deficient S. pombe phenotypes, namely hypersensitivity to hydrogen peroxide, the need for antioxidants for growth on minimal medium and an elevated production of H 2 S. Both the dps1 (formerly dps) and dlp1 mutants could generate ubiquinone when they were transformed with a bacterial decaprenyl diphosphate synthase, which functions in its host as a homodimer. This indicates that both dps1 and dlp1 are required for the S. pombe enzymatic activity. Thus, decaprenyl diphosphate from a eukaryotic origin has a heterotetrameric structure that is not found in prokaryotes.
The Sendai virus pi strain (SeVpi) isolated from cells persistently infected with SeV shows mainly two phenotypes: (1) temperature sensitivity and (2) an ability of establishing persistent infection (steady state). Three amino acid substitutions are found in the Lpi protein and are located at aa 1088, 1618, and 1664. Recombinant SeV(Lpi) (rSeV(Lpi)) having all these substitutions is temperature sensitive and is capable of establishing persistent infection (steady state). rSeVs carrying the fragment containing L1618V show both phenotypes. rSeV(L1618V), in which leucine at aa 1618 is replaced with valine, has the ability of establishing persistent infection, but is not a temperature-sensitive mutant, indicating that the ability of a virus to establish persistent infection can be separated from temperature sensitivity. The amino acid change at 1618(L-->V) coexisting with aa 1169 threonine is required for acquirement of a temperature-sensitive phenotype. Three amino acid substitutions are also found in the Ppi protein, but rSeV(Ppi) does not show these phenotypes.
We prepared the chimeric recombinant Sendai virus [rSeV(Ppi)] by replacing the P gene of the Z strain with that of pi strain for analyzing the function of Ppi, Vpi and Cpi proteins. Intriguingly, HA production by rSeV(Ppi) is significantly lower at 38 degrees C than at 32 degrees C, showing that virus growth of rSeV(Ppi) is slightly suppressed at 38 degrees C. However, the main phenotypes of SeVpi, a marked temperature sensitivity as viral replication and an ability of establishing persistent infection, are not explained by the Ppi, Vpi and Cpi proteins. The V and C proteins form inclusion bodies in L929 cells infected with rSeV(Ppi) and incubated at 38 degrees C. L929 cells infected with rSeV(Ppi) and L929 cells stably expressing the Cpi protein show resistance to interferon-beta at 32 and 38 degrees C, indicating that the Cpi protein per se is not temperature-sensitive to inhibition of IFN signaling. The complete genome sequences of Sendai virus (SeV) pi and parent Nagoya strains were determined. Fifty nucleic acid substitutions are found in the genome sequence of SeV pi strain in comparison with Nagoya strain. There are three nucleic acid substitutions in the leader sequence, while the trailer, intergenic, gene-end and gene-start sequences of both strains are completely identical. Deletions and insertions of nucleotide are not found. There are 32 amino acid substitutions in Sendai virus pi strain. The specific amino acid substitutions unique to the SeVpi are 18. Information about the complete genome sequences of SeVpi strain is important to totally understand the persistent infection and lower pathogenicity of SeV.
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