Makoto Kawamukai

Characterization of a Fission Yeast SUMO-1 Homologue, Pmt3p, Required for Multiple Nuclear Events, Including the Control of Telomere Length and Chromosome Segregation

ABSTRACT Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of thepmt3 + gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. Thepmt3Δ cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found thatpmt3 + is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase- and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.

Biosynthesis, bioproduction and novel roles of ubiquinone

Ubiquinone (coenzyme Q) is a well-known component of the electron transfer system in living organisms. It is known that ubiquinone transfers electrons from Complex I (or Complex II) to Complex III in the respiratory chain. However, recent evidence indicates that an involvement in respiration is not the sole role of ubiquinone, and various novel roles have been elucidated. A role as a lipid soluble antioxidant is now widely accepted. The relationship between lifespan and ubiquinone has attracted much interest based on the study of a Caenorhabditis elegans clk-1 mutant. The connection between disulfide bond formation and ubiquinone (or menaquinone) in Escherichia coli has been well studied. The production of hydrogen sulfide in a ubiquinone-deficient fission yeast is an interesting phenotype recently observed. These are some examples of the novel roles of ubiquinone and this review summarizes the recent findings relating to the biosynthesis, bioproduction and novel roles of ubiquinone.

Polyprenyl diphosphate synthase essentially defines the length of the side chain of ubiquinone

Ubiquinone, known as a component of the electron transfer system in many organisms, has a different length of the isoprenoid side chain depending on the species, e.g., Escherichia coli, Saccharomyces cerevisiae and humans have 8, 6, and 10 isoprene units in the side chain, respectively. No direct evidence has yet shown what factors define the length of the side chain of ubiquinone. Here we proved that the polyprenyl diphosphate that was available in cells determined the length of the side chain of ubiquinone. E. coli octaprenyl diphosphate synthase (IspB) was expressed with the mitochondrial import signal in S. cerevisiae. Such cells produced ubiquinone-8 in addition to the originally existing ubiquinone-6. When IspB was expressed in a S. cerevisiae COQ1 defective strain. IspB complemented the defect of the growth on the non-fermentable carbon source. Those cells had the activity of octaprenyl diphosphate synthase and produced only ubiquinone-8. These results opened the possibility of producing the type of ubiquinone that we need in S. cerevisiae simply by expressing the corresponding polyprenyl diphosphate synthase.

PAD1 and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae

The volatile phenols, to which Saccharomyces cerevisiae converts from phenylacrylic acids including ferulic acid, p-coumaric acid, and cinnamic acid, generate off-flavors in alcoholic beverages such as beer and wine. Using gene disruptants, transformants and cell-free extracts of these strains, we have verified that the adjacent PAD1 (phenylacrylic acid decarboxylase, YDR538W) and FDC1 (ferulic acid decarboxylase, YDR539W) genes are essential for the decarboxylation of phenylacrylic acids in S. cerevisiae. Pad1p and Fdc1p are homologous with UbiX and UbiD, respectively, in the ubiquinone synthetic pathway of Escherichia coli. However, ubiquinone was detected quantitatively in all of the yeast single-deletion mutants, Delta pad1, Delta fdc1, and double-deletion mutant, Delta pad1 Delta fdc1.

Geranylgeranyl pyrophosphate synthase encoded by the newly isolated gene GGPS6 from Arabidopsis thaliana is localized in mitochondria

We have cloned a new geranylgeranyl pyrophosphate (GGPP) synthase gene, designated GGPS6/, from Arabidopsis thaliana genomic DNA. Nucleotide sequence analysis revealed that the GGPS6 gene contains an open reading frame coding for a protein of 343 amino acid residues with a calculated molecular mass of 37 507 Da. Also, the gene is not interrupted by an intron. The predicted amino acid sequence of the GGPS6 gene shows significant homology (34.0–57.7%) with other GGPP synthases from Arabidopsis. The GGPS6 protein contains a N-terminal signal peptide which is thought to function as an organelle targeting sequence. In fact, the GGPS6-GFP fusion protein was found to be localized exclusively in mitochondria when expressed in tobacco BY-2 cells. In vitro analysis of the enzyme activity as well as genetic complementation analysis with Erwinia uredovora crt gene cluster expressed in Escherichia coli showed that the GGPS6 gene most certainly encodes a GGPP synthase catalyzing the conversion of farnesyl pyrophosphate to GGPP.

Characterization of solanesyl and decaprenyl diphosphate synthases in mice and humans

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 Q9 and Q10, 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 Q8 of E. coli origin, double transformants expressing mSPS1 and mDLP1 or hDPS1 and hDLP1 produced Q9 or Q10, respectively, and an in vitro activity of solanesyl or decaprenyl diphosphate synthase was verified. The complex size of the human and murine long‐chain trans‐prenyl 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 Q9 and Q10, 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.

Biosynthesis and bioproduction of coenzyme Q10 by yeasts and other organisms.

CoQ (coenzyme Q), an isoprenylated benzoquinone, is a well‐known component of the electron‐transfer system in eukaryotes. The main role of CoQ is to transfer electrons from NADH dehydrogenase and succinate dehydrogenase to CoQ:cytochrome c reductase in the respiratory chain. However, recent evidence indicates that an involvement in respiration is not the only role of CoQ. The second apparent role of CoQ is its anti‐oxidation property, and other novel roles for CoQ, such as in disulfide‐bond formation, sulfide oxidation and pyrimidine metabolism, have been reported. CoQ10, having ten isoprene units in the isoprenoid side chain, has been used as a medicine and is now commercially popular as a food supplement. Two yeast species, namely the budding yeast Saccharomyces cerevisiae, which produces CoQ6, and the fission yeast Schizosaccharomyces pombe, which produces CoQ10, are the main subjects of the present minireview because they have greatly contributed to our basic knowledge of CoQ biosynthesis among eukaryotes. The biosynthetic pathway that converts p‐hydroxybenzoate into CoQ consists of eight steps in yeasts. The five enzymes involved in the biosynthetic pathway have been identified in both yeasts, yet the functions of three proteins were still not known. Analyses of the biosynthetic pathway in yeasts also contribute to the understanding of human genetic diseases related to CoQ deficiency. In the present minireview I focus on the biochemical and commercial aspects of CoQ in yeasts and in other organisms for comparison.

Development of R4 gateway binary vectors (R4pGWB) enabling high-throughput promoter swapping for plant research

We developed a new series of Gateway binary vectors, R4pGWBs, that are plant transformation vectors designed for one-step construction of chimeric genes between any promoter and any cDNA. The structure of R4pGWBs is almost the same as the promoterless type of improved pGWBs (ImpGWBs), except that the attR1 site is replaced with attR4, which enables tripartite recombination of these vectors with promoter- and cDNA-entry clones. While ImpGWBs are suitable for promoter analysis and constitutive expression of cDNAs in higher plants, R4pGWBs have a great advantage in expressing a cDNA under the regulation of desired promoters.

Biological significance of the side chain length of ubiquinone in Saccharomyces cerevisiae

Ubiquinone (UQ), an important component of the electron transfer system, is constituted of a quinone structure and a side chain isoprenoid. The side chain length of UQ differs between microorganisms, and this difference has been used for taxonomic study. In this study, we have addressed the importance of the length of the side chain of UQ for cells, and examined the effect of chain length by producing UQs with isoprenoid chain lengths between 5 and 10 in Saccharomyces cerevisiae. To make the different UQ species, different types of prenyl diphosphate synthases were expressed in a S. cerevisiae COQ1 mutant defective for hexaprenyl diphosphate synthesis. As a result, we found that the original species of UQ (in this case UQ‐6) had maximum functionality. However, we found that other species of UQ could replace UQ‐6. Thus a broad spectrum of different UQ species are biologically functional in yeast cells, although cells seem to display a preference for their own particular type of UQ.

Coenzyme Q10 supplementation rescues renal disease in Pdss2kd/kd mice with mutations in prenyl diphosphate synthase subunit 2.

Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q (Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days of age and thus precedes the onset of proteinuria and kidney disease by several weeks. The presence of the kd (V117M) mutation in PDSS2 does not prevent its association with PDSS1. However, heterologous expression of the kd mutant form of PDSS2 together with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2kd/kd mouse. Dietary supplementation with Q10 provides a dramatic rescue of both proteinuria and interstitial nephritis in the Pdss2kd/kd mutant mice. The results presented suggest that Q may be acting as a potent lipid-soluble antioxidant, rather than by boosting kidney mitochondrial respiration. Such Q10 supplementation may have profound and beneficial effects in treatment of certain forms of focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2kd/kd mouse.