Fission yeast decaprenyl diphosphate synthase consists of Dps1 and the newly characterized Dlp1 protein in a novel heterotetrameric structure.

The analysis of the structure and function of long chain-producing 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 H2S. 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.     

Genetic evidence for a multi-subunit complex in coenzyme Q biosynthesis in yeast and the role of the Coq1 hexaprenyl diphosphate synthase

Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants designated coq1-coq8. Here we provide genetic evidence that several of the Coq polypeptides interact with one another. Deletions in any of the COQ genes affect the steady-state expression of Coq3p, Coq4p, and Coq6p. Antibodies that recognize Coq1p, a hexaprenyl diphosphate synthase, were generated and used to determine that Coq1p is peripherally associated with the inner membrane on the matrix side. Yeast Deltacoq1 mutants harboring diverse Coq1 orthologs from prokaryotic species produce distinct sizes of polyprenyl diphosphate and hence distinct isoforms of Q including Q(7), Q(8), Q(9), or Q(10) (Okada, K., Kainou, T., Matsuda, H., and Kawamukai, M. (1998) FEBS Lett. 431, 241-244). We find that steady-state levels of Coq3p, Coq4p, and Coq6p are rescued in some cases to near wild-type levels by the presence of these diverse Coq1 orthologs in the Deltacoq1 mutant. These data suggest that the lipid product of Coq1p or a Q-intermediate derived from polyprenyl diphosphate is involved in stabilizing the Coq3, Coq4, and Coq6 polypeptides.     

Homologous mRNA 3'' end formation in fission and budding yeast.

Sequences resembling polyadenylation signals of higher eukaryotes are present downstream of the Schizosaccharomyces pombe ura4+ and cdc10+ coding regions and function in HeLa cells. However, these and other mammalian polyadenylation signals are inactive in S. pombe. Instead, we find that polyadenylation signals of the CYC1 gene of budding yeast Saccharomyces cerevisiae function accurately and efficiently in fission yeast. Furthermore, a 38 bp deletion which renders this RNA processing signal non-functional in S. cerevisiae has the equivalent effect in S. pombe. We demonstrate that synthetic pre-mRNAs encoding polyadenylation sites of S. pombe genes are accurately cleaved and polyadenylated in whole cell extracts of S. cerevisiae. Finally, as is the case in S. cerevisiae, DNA sequences encoding regions proximal to the S. pombe mRNA 3' ends are found to be extremely AT rich; however, no general sequence motif can be found. We conclude that although fission yeast has many genetic features in common with higher eukaryotes, mRNA 3' end formation is significantly different and appears to be formed by an RNA processing mechanism homologous to that of budding yeast. Since fission and budding yeast are evolutionarily divergent, this lower eukaryotic mechanism of mRNA 3' end formation may be generally conserved.     

Geranylgeranyl diphosphate synthase in fission yeast is a heteromer of farnesyl diphosphate synthase (FPS), Fps1, and an FPS-like protein, Spo9, essential for sporulation

Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1(+), but not spo9(+), suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1(+) nor spo9(+) expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Delta cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Delta cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.     

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.     

Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells

Cytokinesis is a temporally and spatially regulated process through which the cellular constituents of the mother cell are partitioned into two daughter cells, permitting an increase in cell number. When cytokinesis occurs in a polarized cell it can create daughters with distinct fates. In eukaryotes, cytokinesis is carried out by the coordinated action of a cortical actomyosin contractile ring and targeted membrane deposition. Recent use of model organisms with facile genetics and improved light-microscopy methods has led to the identification and functional characterization of many proteins involved in cytokinesis. To date, this analysis indicates that some of the basic components involved in cytokinesis are conserved from yeast to humans, although their organization into functional machinery that drives cytokinesis and the associated regulatory mechanisms bear species-specific features. Here, we briefly review the current status of knowledge of cytokinesis in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and animal cells, in an attempt to highlight both the common and the unique features. Although these organisms diverged from a common ancestor about a billion years ago, there are eukaryotes that are far more divergent. To evaluate the overall evolutionary conservation of cytokinesis, it will be necessary to include representatives of these divergent branches. Nevertheless, the three species discussed here provide substantial mechanistic diversity.     

Analysis of the splicing machinery in fission yeast: a comparison with budding yeast and mammals

Based on genetic and bioinformatic analysis, 80 proteins from the newly sequenced Schizosaccharomyces pombe genome appear to be splicing factors. The fission yeast splicing factors were compared to those of Homo sapiens and Saccharomyces cerevisiae in order to determine the extent of conservation or divergence that has occurred over the billion years of evolution that separate these organisms. Our results indicate that many of the factors present in all three organisms have been well conserved throughout evolution. It is calculated that 38% of the fission yeast splicing factors are more similar to the human proteins than to the budding yeast proteins (>10% more similar or similar over a greater region). Many of the factors in this category are required for recognition of the 3′ splice site. Ten fission yeast splicing factors, including putative regulatory factors, have human homologs, but no apparent budding yeast homologs based on sequence data alone. Many of the budding yeast factors that are absent in fission yeast are associated with the U1 and U4/U6.U5 snRNP. Collectively the data presented in this survey indicate that of the two yeasts, S.pombe contains a splicing machinery more closely reflecting the archetype of a spliceosome.     

An inducible expression vector for both fission and budding yeast

We have developed a vector system for inducible gene expression in both fission yeast (Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae). The autonomously replicating expression vector contains multiple glucocorticoid response elements, rendering a linked promoter inducible 20-70-fold by glucocorticoid hormones in the presence of the mammalian glucocorticoid receptor. A polylinker with several unique cloning sites allows insertion of cDNAs of interest. Glucocorticoids are gratuitous signalling molecules in yeast, exerting little or no effect on the expression of genes other than those fused to the regulated promoter.     

Connected cavity structure enables prenyl elongation across the dimer interface in mutated geranylfarnesyl diphosphate synthase from Methanosarcina mazei

The mechanism for product chain-length determination of geranylfarnesyl diphosphate synthase from the methanogenic archaeon Methanosarcina mazei was investigated by constructing mutants based on structural information. Among the mutants, in which each of the bulky residues that constitute the bottom of the product-accommodating cavity was replaced with alanine, those having mutations on an α-helix existing at the subunit interface yielded longer products. In particular, replacement of isoleucine 112 on the α-helix greatly elongated the product chain-length, probably by connecting the reaction cavities of two subunits across the dimer interface.     

Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4

The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis. Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks. Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses. Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all. However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well. Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point. For most substrates the leading strand template is cleaved. The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template.     

De novo growth zone formation from fission yeast spheroplasts

Eukaryotic cells often form polarized growth zones in response to internal or external cues. To understand the establishment of growth zones with specific dimensions we used fission yeast, which grows as a rod-shaped cell of near-constant width from growth zones located at the cell tips. Removing the cell wall creates a round spheroplast with a disorganized cytoskeleton and depolarized growth proteins. As spheroplasts recover, new growth zones form that resemble normal growing cell tips in shape and width, and polarized growth resumes. Regulators of the GTPase Cdc42, which control width in exponentially growing cells, also control spheroplast growth zone width. During recovery the Cdc42 scaffold Scd2 forms a polarized patch in the rounded spheroplast, demonstrating that a growth zone protein can organize independent of cell shape. Rga4, a Cdc42 GTPase activating protein (GAP) that is excluded from cell tips, is initially distributed throughout the spheroplast membrane, but is excluded from the growth zone after a stable patch of Scd2 forms. These results provide evidence that growth zones with normal width and protein localization can form de novo through sequential organization of cellular domains, and that the size of these growth zones is genetically controlled, independent of preexisting cell shape.     

Dielectrophoretic properties of yeast cells dividing by budding and by transversal fission

The dielectrophoretic behaviour of yeast cells dividing by budding or by transversal fission was analyzed. The results obtained show that the dielectrophoretic yield is a linear function of alternating voltage, cell concentration and the square root of the time of collection in all the species assayed. Dependence of the rate of collection on the frequency of the voltage applied (between 0.2 and 5 MHz) was also found. This behaviour is similar in the three microorganisms studied. The scale factor correlating the frequency spectrum for Saccharomyces cerevisiae and Saccharomycopsis lipolytica is proportional to cell size. However, these results can not be extended to Schizosaccharomyces pombe. A relationship between the dielectrophoretic yield and the age of the culture and the consumption of glucose has been established for the three yeast strains. Dielectrophoresis also permits the differentiation between viable and non-viable cells.     

Segregation of the nucleolus during mitosis in budding and fission yeast.

The segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schizosaccharomyces pombe by indirect immunofluorescence using antibodies directed to highly conserved anti-nucleolus antigens. In mitotic S. pombe cells, the nucleolus appears to trail the bulk of the DNA. In wild-type cells of S. cerevisiae, the nucleolus segregates alongside the bulk of the genomic DNA. Based on its distance from the centromere, we would expect the rDNA in both organisms to segregate behind the majority of the genomic DNA, if telomeric regions trail centromeric regions as in other eukaryotes. We therefore suggest that in S. cerevisiae the nucleolus is attached to other parts of the nucleus which enable it to segregate along with the bulk of the DNA. The segregation of the nucleolus in topoisomerase mutants and nuclear division mutants of S. cerevisiae was also investigated. In cdc14 mutants which arrest at late anaphase, the vast majority of the DNA is separated, but the nucleolar antigens remain extended between the mother and daughter cells. Thus, the CDC14 gene of S. cerevisiae appears to be important for the separation of the nucleolus at mitosis.     

The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene

Mutations in the fission yeast cut1+, cut2+, and cut10+ genes uncouple normally coordinated mitotic events and deregulate, rather than arrest, mitosis. DNA synthesis continues, making polyploid nuclei with several spindles. Multiple, aberrant spindle pole bodies (SPBs) are produced in cut1 mutant cells. The cut1+ and cut2+ genes are cloned by transformation. High gene dosage of cut1+ also complements cut2 and cut10 mutants. The cut2+ gene, however, complements only cut2. The 210 kd cut1+ gene product contains putative ATP binding and helical coil regions followed by a COOH-terminal domain homologous to the S. cerevisiae gene ESP1. Mutations in the ESP1 gene also result in many SPBs. The cut1+ product is shown by anti-cut1 antibody to be a rare component of the insoluble nuclear fraction. It may play a key role in coupling chromosome disjunction with other cell cycle events and is potentially a component, regulator, or motor for the SPB and/or kinetochores.     

The (1,3)beta-D-glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)beta-d-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1(+) shut-off and bgs1Delta cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)beta-d-glucan and primary septum formation. bgs1(+) shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)beta-d-glucan and partial primary septa. Conversely, both structures are absent in bgs1Delta cells, where there is no Bgs1p. The septum analysis of bgs1(+)-repressed cells indicates that linear (1,3)beta-d-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)beta-d-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die.     

Proteomics analysis identifies new components of the fission and budding yeast anaphase-promoting complexes

The anaphase-promoting complex (APC) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. Components of the APC have been identified through genetic screens in both Schizosaccharomyces pombe and Saccharomyces cerevisiae as well as through biochemical purification coupled with mass spectrometric protein identification. With these approaches, 11 subunits of the core S. cerevisiae APC have been identified. Here, we have applied a tandem affinity purification approach coupled with direct analysis of the purified complexes by mass spectrometry (DALPC) to reveal additional subunits of both the S. pombe and S. cerevisiae APCs. Our data increase the total number of identified APC subunits to 13 in both yeasts and indicate that previous approaches were biased against the identification of small subunits. These results underscore the power of direct analysis of protein complexes by mass spectrometry and set the foundation for further functional and structural studies of the APC.     

Mrc1 protects uncapped budding yeast telomeres from exonuclease EXO1

Mrc1 (Mediator of Replication Checkpoint 1) is a component of the DNA replication fork machinery and is necessary for checkpoint activation after replication stress. In this study, we addressed the role of Mrc1 at uncapped telomeres. Our experiments show that Mrc1 contributes to the vitality of both cdc13-1 and yku70Delta telomere capping mutants. Cells with telomere capping defects containing MRC1 or mrc1(AQ), a checkpoint defective allele, exhibit similar growth, suggesting growth defects of cdc13-1 mrc1Delta are not due to checkpoint defects. This is in accordance with Mrc1-independent Rad53 activation after telomere uncapping. Poor growth of cdc13-1 mutants in the absence of Mrc1 is a result of enhanced single stranded DNA accumulation at uncapped telomeres. Consistent with this, deletion of EXO1, encoding a nuclease that contributes to single stranded DNA accumulation after telomere uncapping, improves growth of cdc13-1 mrc1Delta strains and decreases ssDNA production. Our observations show that Mrc1, a core component of the replication fork, plays an important role in telomere capping, protecting from nucleases and checkpoint pathways.     

Mitochondrial fission proteins Fis1 and Mdv1, but not Dnm1, play a role in maintenance of heteroplasmy in budding yeast

In budding yeast, the mitochondrial DNA (mtDNA) replication pathway involving the homologous DNA pairing protein Mhr1 promotes mitochondrial allele segregation. Mitochondrial fusion facilitates the recombination-mediated replication pathway; however, the role of fission remains largely unknown. By monitoring mitochondrial allele segregation during zygotic division, we found that the absence of fission proteins Fis1 or Mdv1, but not Dnm1, resulted in increased initial homoplasmy levels and decreased mtDNA copy number. However, decreases in mtDNA copy number alone were not sufficient for rapid establishment of homoplasmy, suggesting that inhibiting the activities of certain fission proteins promotes homoplasmy by reducing the number of mtDNA segregation units.     

Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli.

Dehydrodolichyl diphosphate synthase (DDPPs) catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize long-chain dehydrodolichyl diphosphate, which serves as a precursor of glycosyl carrier in glycoprotein biosynthesis in eukaryotes. To perform kinetic and structural studies of DDPPs, we have expressed yeast DDPPs using Escherichia coli as the host cell. Thioredoxin and His tag were utilized to increase the solubility of the recombinant protein and facilitate its purification using Ni-nitrilotriacetic acid (NTA) column. The protein was overexpressed in E. coli but mostly existed in pellet in the absence of detergent. The low quantity of soluble DDPPs was purified using Ni-NTA, Mono Q anion-exchange, and size-column chromatographies. The protein in the pellet was solubilized with 7 M urea and purified using Ni-NTA under denaturing condition. The protein refolding was achieved via the stepwise dialysis to remove the denaturant in the presence of 6 mM beta-mercaptoethanol. Detergent n-octyl-beta-d-glucopyranoside and Triton X-100 increased the solubility of the DDPPs so that refolding can be performed at higher protein concentration. Alternatively, on-column refolding was carried out in a single step to obtain the active protein in large quantities. beta-Mercaptoethanol and Triton were both required in this quick refolding process. The kinetic studies indicated that the soluble and refolded DDPPs have comparable activities (k(cat) = 2 x 10(-4) s(-1)). Unlike its bacterial homologue, undecaprenyl diphosphate synthase, yeast DDPPs activity was not enhanced by Triton.