Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle

The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.     

A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-α-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A–Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-α-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism.     

Metabolism of phospholipids in the yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is an important model organism for the study of fundamental questions in eukaryotic cell and molecular biology. A plethora of cellular processes are membrane associated and/or dependent on the proper functioning of cellular membranes. Phospholipids are not only the basic building blocks of cellular membranes; they also serve as precursors to numerous signaling molecules. In this review, we describe the biosynthetic pathways leading to major S. pombe phospholipids, how these pathways are regulated, and what is known about degradation and turnover of fission yeast phospholipids. This review also addresses the synthesis, regulation and the role of water-soluble phospholipid precursors. The last chapter of the review is devoted to the use of S. pombe for the biotechnological production of value-added lipid molecules.     

Fission yeast translation initiation factor 3 subunit eIF3h is not essential for global translation initiation,but deletion of eif3h+ affects spore formation

The fission yeast Schizosaccharomyces pombe homologue of the p40/eIF3h subunit of mammalian translation initiation factor eIF3 has been characterized in this study. We show that this protein physically associates with the 40S ribosomal particles as a constituent of the multimeric eIF3 protein complex, which consists of all five known eIF3 core subunits (eIF3a, eIF3b, eIF3c, eIF3g and eIF3i) as well as the five non‐core subunits (eIF3d, eIF3e, eIF3f, eIF3h and eIF3m) that constitute an eIF3 holocomplex in fission yeast. However, affinity purification of eIF3 from fission yeast cells expressing TAP‐tagged eIF3h suggests the presence of distinct forms of eIF3 that differ in their composition of the non‐core subunits. Further characterization of eIF3h shows that strains lacking eif3h⁺ (eif3hΔ) are viable and show no gross defects, either in vegetative growth or in the rate of in vivo protein synthesis. Polysome profile analysis shows no apparent defects in translation initiation. Furthermore, deletion of eif3h⁺ does not affect the ability of the other eIF3 subunits to remain associated with one another in a tight protein complex similar to the situation in wild‐type cells. Additionally, we show that human eIF3h can functionally substitute fission yeast eIF3h in complementing in vivo a genetic deletion of eif3h⁺. Interestingly, mutant eif3hΔ cells show several prominent phenotypic properties. They are hypersensitive to caffeine and highly defective in meiosis, producing either no spores or incomplete tetrads with a very high frequency. The implications of these results in relation to the functions of eIF3h in Sz. pombe are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature‐sensitive fission yeast strains, rrs1‐D14/22G and rrs1‐L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1‐84 (D22/30G) and rrs1‐124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two‐hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts. Copyright © 2015 John Wiley & Sons, Ltd.     

Properties and Heterologous Expression of the Glucose Transporter GHT1 from Schizosaccharomyces pombe

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS-5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62·5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS-5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported d -glucose with substrate specificity similar to that in Sz. pombe wild-type cells. Moreover, the cells of the two transformed yeast strains accumulated 2-deoxy-d -glucose, a non-metabolizable d -glucose analogue, with an efficiency similar to Sz. pombe wild-type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an Δμdependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy-dependent uptake of glucose. The sequence of GHT1 was deposited at EMBL, Outstation EBI, Accession Number X91218. ©1997 John Wiley & Sons, Ltd.     

Discovery of genes involved in mitosis,cell division,cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains

The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the Sz. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini‐chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of non‐essential gene deletions will add to the toolkits available for the study of biological processes in Sz. pombe. Copyright © 2016 John Wiley & Sons, Ltd.     

A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe

Double‐strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double‐strand break into the genome by regulating the expression of the site‐specific HO endonuclease with a galactose inducible promoter. Variations of the HO site‐specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (~1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I‐PpoI is tightly regulated with a tetracycline‐inducible promoter. We show that induction of the I‐PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break‐adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well‐conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double‐stranded DNA breaks. Copyright © 2012 John Wiley & Sons, Ltd.     

An estradiol‐inducible promoter enables fast,graduated control of gene expression in fission yeast

The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose–response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β‐estradiol‐regulated function of the human oestrogen receptor, for use in S. pombe. We show that this promoter system, termed Z₃EV, turns on quickly, can reach a maximal induction of 20‐fold, and exhibits a linear dose response over its entire induction range, with few off‐target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β‐estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast. Copyright © 2017 John Wiley & Sons, Ltd.     

Induction of a heat shock-type response in fission yeast following nitrogen starvation

When cells of the fission yeast, Schizosaccharomyces pombe, are incubated in medium devoid of a nitrogen source, they accelerate into cell division and differentially synthesize two polypeptides at 46 and 27 kD (named p46 and p27) after a delay of about an hour. The synthesis of p46 and p27 is transient. These proteins have no obvious cell cycle connection since they are also evident in nitrogen-starved (but not accelerated) cells of the temperature-sensitive mutant of S. pombe, wee 1·50h^?. We infer from this that p46 and p27 are synthesized as a direct result of nutritional stress. The possibility that p46 and p27 represent examples of general environmental stress proteins was investigated by comparing nitrogen starvation with the heat-shock response in S. pombe. Heat-shock analysis of cells revealed the existence of two proteins of similar Mr to p46 and p27. In addition, nitrogen-starved cells acquired thermotolerance in a manner similar to heat-shocking cells. We suggest that nitrogen starvation in fission yeast induces a subset of the total array of heat-shock proteins.     

Isolation and quantitation of M-factor,a diffusible mating factor from the fission yeast Schizosaccharomyces pombe

Although there have been several reports demonstrating the existence of mating factors in the fission yeast Schizosaccharomyces pombe it has not been possible to isolate these factors as cell-free preparations. Such an ability is the first requirement towards a molecular characterization of these factors and here I report the successful isolation of a mating factor from S. pombe. This factor, termed M-factor, is released by cells of the cellular mating type M (Minus) and induces mating-specific changes in P-type cells. A reliable and accurate assay for the quantitation of the M-factor, based upon changes in cell volume following exposure to the factor, is also described.     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

Isolation of a Schizosaccharomyces pombe Gene Which in High Copy Confers Resistance to the Nucleoside Analogue 5-Azacytidine

Treatment of Schizosaccharomyces pombe with the C5 DNA methyltransferase (C5Mtase) inhibitor 5-azacytidine (5-azaC) has previously been shown to induce G2 checkpoint-dependent cell cycle arrest. S. pombe strains defective in both the checkpoint control pathways and in DNA repair processes are sensitive to 5-azaC. Here we describe the isolation of azr1as a multi-copy suppressor of the 5-azaC sensitivity of G2 checkpoint and DNA repair-deficient strains. azr1⁺ encodes a putative 25 kDa protein with limited homology to a Saccharomyces cerevisiae open reading frame of unknown function. The azr1⁺ gene is not essential and the null mutant shows no alteration in either DNA repair or checkpoint properties. We also report the sequence of the putative fission yeast cytidine deaminase gene, designated pcd1⁺, which lies immediately adjacent to azr1⁺ but which plays only a moderate role in suppression of 5-azaC sensitivity. These data have been deposited with EMBL nucleotide sequence database, Accession Number X98329. © 1997 John Wiley & Sons, Ltd.     

Quantitative analysis of human ras localization and function in the fission yeast Schizosaccharomyces pombe

Ras signalling is central to fundamental and diverse cellular processes. In higher eukaryotes ras signalling is highly complex, involving multiple isoforms, regulatory proteins and effectors. As a consequence, the study of ras activity in mammalian systems presents a number of technical challenges. The model organism Schizosaccharomyces pombe has previously proved a key system for the study of human signalling components and provides an ideal model for the study of ras, as it contains just one ras protein (Ras1p), which is non‐essential and controls a number of downstream processes. Here we present data demonstrating the quantitative analysis of three distinct Ras1‐related signalling outputs, utilizing the three most abundant human ras isoforms, H‐Ras, N‐Ras and K‐Ras4B, in Sz. pombe. Further, we have characterized the localization of these three human ras isoforms in Sz. pombe, utilizing quantitative image analysis techniques. These data indicate that all three human ras isoforms are functional in fission yeast, displaying differing localization patterns which correlate strongly with function in the regulation of pheromone response and cell shape. These data demonstrate that such yeast strains could provide powerful tools for the investigation of ras biology, and potentially in the development of cancer therapies. Copyright © 2013 John Wiley & Sons, Ltd.     

Use of a fluoride channel as a new selection marker for fission yeast plasmids and application to fast genome editing with CRISPR/Cas9

Fission yeast is a powerful model organism that has provided insights into important cellular processes thanks to the ease of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in Schizosaccharomyces pombe. In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular,functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed ‘feed-forward’ regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

A fission yeast gene encoding a protein that preferentially associates with curved DNA

We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic curved DNA sequence, by means of a DNA-binding gel shift assay in the presence of an excess amount of a non-curved DNA sequence as a competitor. We identified such a protein in S. pombe. The protein, thus purified, has an apparent molecular weight of 42 000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was suggested that this protein (42 K-protein) recognizes and binds to a curved DNA structure in a given nucleotide sequence, although it also binds to a non-curved DNA sequence with lower affinity. As its putative coding sequence, a 1·9-kilobase genomic DNA from S. pombe was cloned and sequenced. Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381-amino-acid protein with a calculated molecular mass, 41 597. This protein appears to be located in the nucleus. The predicted protein sequence revealed that the 42 K-protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.     

Characterization of an active GST-human Cdc2 fusion protein kinase expressed in the fission yeast Schizosaccharomyces pombe: A new approach to the study of cell cycle control proteins

Characterization of cdk (c yclin d ependent k inases) substrates and studies of their regulation require purified enzymatic complexes of cdc2-related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S-transferase (GST). The GST-human Cdc2p fusion protein was active in vivo since it rescued a temperature-sensitive allele of cdc2. The fusion protein was purified using a one-step chromatography procedure with glutathione–Sepharose and exhibited a catalytic activity in vitro. Yeast cyclin B and suc1 were found in association with GST-Cdc2. A 17-fold stimulation of GST-Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S. pombe cellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.     

Yeast sequencing reports. Molecular cloning and sequencing of the hcs gene,which encodes 3-hydroxy-3-methylglutaryl coenzyme a synthase of Schizosaccharomyces pombe

We have cloned and sequenced the hcs gene, which is thought to encode a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase consisting of 447 amino acids, from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence of the hcs product of S. pombe has homology with the HMG-CoA synthase of rat (47·8%), chicken (49·2%), hamster (47·1%) and human cells (46·9%). One of the hcs genes was replaced with a marker gene in the diploid cell. No viable hcs-disrupted haploid was isolated after tetrad dissection, suggesting that the hcs gene is essential for growth. However the hcs-defective mutant could be grown on a medium containing 5 mg/ml mevalonate. These results strongly support that the hcs gene encodes HMG-CoA synthase and S. pombe contains a single copy of the hcs gene. The sequence of the hcs gene has been entered into the public data libraries under Accession Number U32187.     

The natural diversity and ecology of fission yeast

While the fission yeast is a powerful model of eukaryote biology, there have been few studies of quantitative genetics, phenotypic or genetic diversity. Here I survey the small collection of fission yeast diversity research. I discuss what we can infer about the ecology and origins of Schizosaccharomyces pombe from microbiology field studies and the few strains that have been collected.