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Sample records for fission yeast amp-activated

  1. Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress.

    PubMed

    Toyama, Erin Quan; Herzig, Sébastien; Courchet, Julien; Lewis, Tommy L; Losón, Oliver C; Hellberg, Kristina; Young, Nathan P; Chen, Hsiuchen; Polleux, Franck; Chan, David C; Shaw, Reuben J

    2016-01-15

    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA-linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission. PMID:26816379

  2. Fission yeast septation

    PubMed Central

    Cortés, Juan C. G.; Ramos, Mariona; Osumi, Masako; Pérez, Pilar; Ribas, Juan Carlos

    2016-01-01

    ABSTRACT In animal cells cytokinesis relies on the contraction of an actomyosin ring that pulls the plasma membrane to create a cleavage furrow, whose ingression finally divides the mother cell into two daughter cells. Fungal cells are surrounded by a tough and flexible structure called cell wall, which is considered to be the functional equivalent of the extracellular matrix in animal cells. Therefore, in addition to cleavage furrow ingression, fungal cytokinesis also requires the centripetal formation of a septum wall structure that develops between the dividing cells, whose genesis must be strictly coordinated with both the actomyosin ring closure and plasma membrane ingression. Here we briefly review what is known about the septum structure and composition in the fission yeast Schizosaccharomyces pombe, the recent progress about the relationship between septum biosynthesis and actomyosin ring constriction, and the importance of the septum and ring in the steady progression of the cleavage furrow. PMID:27574536

  3. Fission yeast septation.

    PubMed

    Cortés, Juan C G; Ramos, Mariona; Osumi, Masako; Pérez, Pilar; Ribas, Juan Carlos

    2016-01-01

    In animal cells cytokinesis relies on the contraction of an actomyosin ring that pulls the plasma membrane to create a cleavage furrow, whose ingression finally divides the mother cell into two daughter cells. Fungal cells are surrounded by a tough and flexible structure called cell wall, which is considered to be the functional equivalent of the extracellular matrix in animal cells. Therefore, in addition to cleavage furrow ingression, fungal cytokinesis also requires the centripetal formation of a septum wall structure that develops between the dividing cells, whose genesis must be strictly coordinated with both the actomyosin ring closure and plasma membrane ingression. Here we briefly review what is known about the septum structure and composition in the fission yeast Schizosaccharomyces pombe, the recent progress about the relationship between septum biosynthesis and actomyosin ring constriction, and the importance of the septum and ring in the steady progression of the cleavage furrow. PMID:27574536

  4. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  5. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-01

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase. PMID:24815694

  6. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress

    PubMed Central

    Courchet, Julien; Lewis, Tommy L.; Losón, Oliver C.; Hellberg, Kristina; Young, Nathan P.; Chen, Hsiuchen; Polleux, Franck; Chan, David C.; Shaw, Reuben J.

    2016-01-01

    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA–linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)–activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission. PMID:26816379

  7. Overview of fission yeast septation.

    PubMed

    Pérez, Pilar; Cortés, Juan C G; Martín-García, Rebeca; Ribas, Juan C

    2016-09-01

    Cytokinesis is the final process of the vegetative cycle, which divides a cell into two independent daughter cells once mitosis is completed. In fungi, as in animal cells, cytokinesis requires the formation of a cleavage furrow originated by constriction of an actomyosin ring which is connected to the plasma membrane and causes its invagination. Additionally, because fungal cells have a polysaccharide cell wall outside the plasma membrane, cytokinesis requires the formation of a septum coincident with the membrane ingression. Fission yeast Schizosaccharomyces pombe is a unicellular, rod-shaped fungus that has become a popular model organism for the study of actomyosin ring formation and constriction during cell division. Here we review the current knowledge of the septation and separation processes in this fungus, as well as recent advances in understanding the functional interaction between the transmembrane enzymes that build the septum and the actomyosin ring proteins. PMID:27155541

  8. Molecular control of fission yeast cytokinesis.

    PubMed

    Rincon, Sergio A; Paoletti, Anne

    2016-05-01

    Cytokinesis gives rise to two independent daughter cells at the end of the cell division cycle. The fission yeast Schizosaccharomyces pombe has emerged as one of the most powerful systems to understand how cytokinesis is controlled molecularly. Like in most eukaryotes, fission yeast cytokinesis depends on an acto-myosin based contractile ring that assembles at the division site under the control of spatial cues that integrate information on cell geometry and the position of the mitotic apparatus. Cytokinetic events are also tightly coordinated with nuclear division by the cell cycle machinery. These spatial and temporal regulations ensure an equal cleavage of the cytoplasm and an accurate segregation of the genetic material in daughter cells. Although this model system has specificities, the basic mechanisms of contractile ring assembly and function deciphered in fission yeast are highly valuable to understand how cytokinesis is controlled in other organisms that rely on a contractile ring for cell division. PMID:26806637

  9. Fission Yeast Cell Cycle Synchronization Methods.

    PubMed

    Tormos-Pérez, Marta; Pérez-Hidalgo, Livia; Moreno, Sergio

    2016-01-01

    Fission yeast cells can be synchronized by cell cycle arrest and release or by size selection. Cell cycle arrest synchronization is based on the block and release of temperature-sensitive cell cycle mutants or treatment with drugs. The most widely used approaches are cdc10-129 for G1; hydroxyurea (HU) for early S-phase; cdc25-22 for G2, and nda3-KM311 for mitosis. Cells can also be synchronized by size selection using centrifugal elutriation or a lactose gradient. Here we describe the methods most commonly used to synchronize fission yeast cells. PMID:26519320

  10. Elutriation for Cell Cycle Synchronization in Fission Yeast.

    PubMed

    Kume, Kazunori

    2016-01-01

    Cell synchronization is a powerful technique for studying the eukaryotic cell cycle events precisely. The fission yeast is a rod-shaped cell whose growth is coordinated with the cell cycle. Monitoring the cellular growth of fission yeast is a relatively simple way to measure the cell cycle stage of a cell. Here, we describe a detailed method of unperturbed cell synchronization, named centrifugal elutriation, for fission yeast. PMID:26254921

  11. Microscopy of Fission Yeast Sexual Lifecycle.

    PubMed

    Vjestica, Aleksandar; Merlini, Laura; Dudin, Omaya; Bendezu, Felipe O; Martin, Sophie G

    2016-01-01

    The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores. PMID:27022830

  12. FYPO: the fission yeast phenotype ontology

    PubMed Central

    Harris, Midori A.; Lock, Antonia; Bähler, Jürg; Oliver, Stephen G.; Wood, Valerie

    2013-01-01

    Motivation: To provide consistent computable descriptions of phenotype data, PomBase is developing a formal ontology of phenotypes observed in fission yeast. Results: The fission yeast phenotype ontology (FYPO) is a modular ontology that uses several existing ontologies from the open biological and biomedical ontologies (OBO) collection as building blocks, including the phenotypic quality ontology PATO, the Gene Ontology and Chemical Entities of Biological Interest. Modular ontology development facilitates partially automated effective organization of detailed phenotype descriptions with complex relationships to each other and to underlying biological phenomena. As a result, FYPO supports sophisticated querying, computational analysis and comparison between different experiments and even between species. Availability: FYPO releases are available from the Subversion repository at the PomBase SourceForge project page (https://sourceforge.net/p/pombase/code/HEAD/tree/phenotype_ontology/). The current version of FYPO is also available on the OBO Foundry Web site (http://obofoundry.org/). Contact: mah79@cam.ac.uk or vw253@cam.ac.uk PMID:23658422

  13. Snf1/AMP-activated protein kinase activates Arf3p to promote invasive yeast growth via a non-canonical GEF domain

    PubMed Central

    Hsu, Jia-Wei; Chen, Kuan-Jung; Lee, Fang-Jen S.

    2015-01-01

    Active GTP-bound Arf GTPases promote eukaryotic cell membrane trafficking and cytoskeletal remodelling. Arf activation is accelerated by guanine nucleotide-exchange factors (GEFs) using the critical catalytic glutamate in all known Sec7 domain sequences. Yeast Arf3p, a homologue of mammalian Arf6, is required for yeast invasive responses to glucose depletion. Here we identify Snf1p as a GEF that activates Arf3p when energy is limited. SNF1 is the yeast homologue of AMP-activated protein kinase (AMPK), which is a key regulator of cellular energy homeostasis. As activation of Arf3p does not depend on the Snf1p kinase domain, assay of regulatory domain fragments yield evidence that the C-terminal hydrophobic α-helix core of Snf1p is a non-canonical GEF for Arf3p activation. Thus, our study reveals a novel mechanism for regulating cellular responses to energy deprivation, in particular invasive cell growth, through direct Arf activation by Snf1/AMPK. PMID:26198097

  14. Fission yeast Schizosaccharomyces pombe in continuous culture

    SciTech Connect

    Vrana, D.

    1983-08-01

    The fission yeast Schizosaccharomyces pombe was cultivated in a chemostat at dilution rates of D = 0.03, 0.05, 0.10, and 0.20/h. After steady state has been reached, the amount of dry matter, number of cells, concentration of residual sugar, yield coefficient (Y), and some morphological properties of the cells were estimated. Curves reflecting the dry mass, number of cells, and cell mean volume show a changing coordination between the growth rate and the rate of cell division, with respect of D. In addition, it could be concluded that in dividing cells the cell septum is localized asymmetrically; two nonidentical cells differing both in length and volume result. The degree of asymmetry is a function of the dilution rate. (25 Refs.)

  15. Mechanics of cell division in fission yeast

    NASA Astrophysics Data System (ADS)

    Chang, Fred

    2012-02-01

    Cytokinesis is the stage of cell division in which a cell divides into two. A paradigm of cytokinesis in animal cells is that the actomyosin contractile ring provides the primary force to squeeze the cell into two. In the fission yeast Schizosaccharomyces pombe, cytokinesis also requires a actomyosin ring, which has been generally assumed to provide the force for cleavage. However, in contrast to animal cells, yeast cells assemble a cell wall septum concomitant with ring contraction and possess large (MPa) internal turgor pressure. Here, we show that the inward force generated by the division apparatus opposes turgor pressure; a decrease in effective turgor pressure leads to an increase in cleavage rate. We show that the ring cannot be the primary force generator. Scaling arguments indicate that the contractile ring can only provide a tiny fraction of the mechanical stress required to overcome turgor. Further, we show that cleavage can occur even in the absence of the contractile ring. Instead of the contractile ring, scaling arguments and modeling suggest that the large forces for cytokinesis are produced by the assembly of cell wall polymers in the growing septum.

  16. Dielectric modelling of cell division for budding and fission yeast

    NASA Astrophysics Data System (ADS)

    Asami, Koji; Sekine, Katsuhisa

    2007-02-01

    The frequency dependence of complex permittivity or the dielectric spectrum of a system including a cell in cell division has been simulated by a numerical technique based on the three-dimensional finite difference method. Two different types of cell division characteristic of budding and fission yeast were examined. The yeast cells are both regarded as a body of rotation, and thus have anisotropic polarization, i.e. the effective permittivity of the cell depends on the orientation of the cell to the direction of an applied electric field. In the perpendicular orientation, where the rotational axis of the cell is perpendicular to the electric field direction, the dielectric spectra for both yeast cells included one dielectric relaxation and its intensity depended on the cell volume. In the parallel orientation, on the other hand, two dielectric relaxations appeared with bud growth for budding yeast and with septum formation for fission yeast. The low-frequency relaxation was shifted to a lower frequency region by narrowing the neck between the bud and the mother cell for budding yeast and by increasing the degree of septum formation for fission yeast. After cell separation, the low-frequency relaxation disappeared. The simulations well interpreted the oscillation of the relative permittivity of culture broth found for synchronous cell growth of budding yeast.

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

    PubMed Central

    Käufer, Norbert F.; Potashkin, Judith

    2000-01-01

    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. PMID:10931913

  18. Modeling the Control of DNA Replication in Fission Yeast

    NASA Astrophysics Data System (ADS)

    Novak, Bela; Tyson, John J.

    1997-08-01

    A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses (``endoreplication'') or initiation of mitosis before DNA is fully replicated (``mitotic catastrophe''). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of ``Start'' control in Schizosaccharomyces pombe. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1- (size control at Start), cdc13Δ and rum1OP (endoreplication), and wee1- rum1Δ (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.

  19. Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae.

    PubMed

    Niki, Hironori

    2014-03-01

    The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast. PMID:24375690

  20. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe

    PubMed Central

    Benko, Zsigmond; Elder, Robert T.; Li, Ge; Liang, Dong; Zhao, Richard Y.

    2016-01-01

    Background HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. Results A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. Conclusions This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings. PMID:26982200

  1. PomBase 2015: updates to the fission yeast database

    PubMed Central

    McDowall, Mark D.; Harris, Midori A.; Lock, Antonia; Rutherford, Kim; Staines, Daniel M.; Bähler, Jürg; Kersey, Paul J.; Oliver, Stephen G.; Wood, Valerie

    2015-01-01

    PomBase (http://www.pombase.org) is the model organism database for the fission yeast Schizosaccharomyces pombe. PomBase provides a central hub for the fission yeast community, supporting both exploratory and hypothesis-driven research. It provides users easy access to data ranging from the sequence level, to molecular and phenotypic annotations, through to the display of genome-wide high-throughput studies. Recent improvements to the site extend annotation specificity, improve usability and allow for monthly data updates. Both in-house curators and community researchers provide manually curated data to PomBase. The genome browser provides access to published high-throughput data sets and the genomes of three additional Schizosaccharomyces species (Schizosaccharomyces cryophilus, Schizosaccharomyces japonicus and Schizosaccharomyces octosporus). PMID:25361970

  2. The price of independence: cell separation in fission yeast.

    PubMed

    Martín-García, Rebeca; Santos, Beatriz

    2016-04-01

    The ultimate goal of cell division is to give rise to two viable independent daughter cells. A tight spatial and temporal regulation between chromosome segregation and cytokinesis ensures the viability of the daughter cells. Schizosaccharomyces pombe, commonly known as fission yeast, has become a leading model organism for studying essential and conserved mechanisms of the eukaryotic cell division process. Like many other eukaryotic cells it divides by binary fission and the cleavage furrow undergoes ingression due to the contraction of an actomyosin ring. In contrast to mammalian cells, yeasts as cell-walled organisms, also need to form a division septum made of cell wall material to complete the process of cytokinesis. The division septum is deposited behind the constricting ring and it will constitute the new ends of the daughter cells. Cell separation also involves cell wall degradation and this process should be precisely regulated to avoid cell lysis. In this review, we will give a brief overview of the whole cytokinesis process in fission yeast, from the positioning and assembly of the contractile ring to the final step of cell separation, and the problems generated when these processes are not precise. PMID:26931605

  3. Fission Yeast Scp3 Potentially Maintains Microtubule Orientation through Bundling

    PubMed Central

    Ozaki, Kanako; Chikashige, Yuji; Hiraoka, Yasushi; Matsumoto, Tomohiro

    2015-01-01

    Microtubules play important roles in organelle transport, the maintenance of cell polarity and chromosome segregation and generally form bundles during these processes. The fission yeast gene scp3+ was identified as a multicopy suppressor of the cps3-81 mutant, which is hypersensitive to isopropyl N-3-chlorophenylcarbamate (CIPC), a poison that induces abnormal multipolar spindle formation in higher eukaryotes. In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe. Microscopic observation revealed that treatment with CIPC, cps3-81 mutation and scp3+ gene deletion disturbed the orientation of microtubules in interphase cells. Overexpression of scp3+ suppressed the abnormal orientation of microtubules by promoting bundling. Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle. A strain lacking the ase1+ gene was more sensitive to CIPC, with the drug affecting the integrity of the mitotic spindle, indicating that CIPC has a mitotic target that has a role redundant with Ase1. These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast. PMID:25767875

  4. Drug Synergy Drives Conserved Pathways to Increase Fission Yeast Lifespan

    PubMed Central

    Huang, Xinhe; Leggas, Markos; Dickson, Robert C.

    2015-01-01

    Aging occurs over time with gradual and progressive loss of physiological function. Strategies to reduce the rate of functional loss and mitigate the subsequent onset of deadly age-related diseases are being sought. We demonstrated previously that a combination of rapamycin and myriocin reduces age-related functional loss in the Baker’s yeast Saccharomyces cerevisiae and produces a synergistic increase in lifespan. Here we show that the same drug combination also produces a synergistic increase in the lifespan of the fission yeast Schizosaccharomyces pombe and does so by controlling signal transduction pathways conserved across a wide evolutionary time span ranging from yeasts to mammals. Pathways include the target of rapamycin complex 1 (TORC1) protein kinase, the protein kinase A (PKA) and a stress response pathway, which in fission yeasts contains the Sty1 protein kinase, an ortholog of the mammalian p38 MAP kinase, a type of Stress Activated Protein Kinase (SAPK). These results along with previous studies in S. cerevisiae support the premise that the combination of rapamycin and myriocin enhances lifespan by regulating signaling pathways that couple nutrient and environmental conditions to cellular processes that fine-tune growth and stress protection in ways that foster long term survival. The molecular mechanisms for fine-tuning are probably species-specific, but since they are driven by conserved nutrient and stress sensing pathways, the drug combination may enhance survival in other organisms. PMID:25786258

  5. The Spontaneous Mutation Rate in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Farlow, Ashley; Long, Hongan; Arnoux, Stéphanie; Sung, Way; Doak, Thomas G; Nordborg, Magnus; Lynch, Michael

    2015-10-01

    The rate at which new mutations arise in the genome is a key factor in the evolution and adaptation of species. Here we describe the rate and spectrum of spontaneous mutations for the fission yeast Schizosaccharomyces pombe, a key model organism with many similarities to higher eukaryotes. We undertook an ∼1700-generation mutation accumulation (MA) experiment with a haploid S. pombe, generating 422 single-base substitutions and 119 insertion-deletion mutations (indels) across the 96 replicates. This equates to a base-substitution mutation rate of 2.00 × 10(-10) mutations per site per generation, similar to that reported for the distantly related budding yeast Saccharomyces cerevisiae. However, these two yeast species differ dramatically in their spectrum of base substitutions, the types of indels (S. pombe is more prone to insertions), and the pattern of selection required to counteract a strong AT-biased mutation rate. Overall, our results indicate that GC-biased gene conversion does not play a major role in shaping the nucleotide composition of the S. pombe genome and suggest that the mechanisms of DNA maintenance may have diverged significantly between fission and budding yeasts. Unexpectedly, CpG sites appear to be excessively liable to mutation in both species despite the likely absence of DNA methylation. PMID:26265703

  6. A Proteome-wide Fission Yeast Interactome Reveals Network Evolution Principles from Yeasts to Human.

    PubMed

    Vo, Tommy V; Das, Jishnu; Meyer, Michael J; Cordero, Nicolas A; Akturk, Nurten; Wei, Xiaomu; Fair, Benjamin J; Degatano, Andrew G; Fragoza, Robert; Liu, Lisa G; Matsuyama, Akihisa; Trickey, Michelle; Horibata, Sachi; Grimson, Andrew; Yamano, Hiroyuki; Yoshida, Minoru; Roth, Frederick P; Pleiss, Jeffrey A; Xia, Yu; Yu, Haiyuan

    2016-01-14

    Here, we present FissionNet, a proteome-wide binary protein interactome for S. pombe, comprising 2,278 high-quality interactions, of which ∼ 50% were previously not reported in any species. FissionNet unravels previously unreported interactions implicated in processes such as gene silencing and pre-mRNA splicing. We developed a rigorous network comparison framework that accounts for assay sensitivity and specificity, revealing extensive species-specific network rewiring between fission yeast, budding yeast, and human. Surprisingly, although genes are better conserved between the yeasts, S. pombe interactions are significantly better conserved in human than in S. cerevisiae. Our framework also reveals that different modes of gene duplication influence the extent to which paralogous proteins are functionally repurposed. Finally, cross-species interactome mapping demonstrates that coevolution of interacting proteins is remarkably prevalent, a result with important implications for studying human disease in model organisms. Overall, FissionNet is a valuable resource for understanding protein functions and their evolution. PMID:26771498

  7. Molecular Genetic Tools and Techniques in Fission Yeast.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    The molecular genetic tools used in fission yeast have generally been adapted from methods and approaches developed for use in the budding yeast, Saccharomyces cerevisiae Initially, the molecular genetics of Schizosaccharomyces pombe was developed to aid gene identification, but it is now applied extensively to the analysis of gene function and the manipulation of noncoding sequences that affect chromosome dynamics. Much current research using fission yeast thus relies on the basic processes of introducing DNA into the organism and the extraction of DNA for subsequent analysis. Targeted integration into specific genomic loci is often used to create site-specific mutants or changes to noncoding regulatory elements for subsequent phenotypic analysis. It is also regularly used to introduce additional sequences that generate tagged proteins or to create strains in which the levels of wild-type protein can be manipulated through transcriptional regulation and/or protein degradation. Here, we draw together a collection of core molecular genetic techniques that underpin much of modern research using S. pombe We summarize the most useful methods that are routinely used and provide guidance, learned from experience, for the successful application of these methods. PMID:27140925

  8. Mathematical model of the cell division cycle of fission yeast.

    PubMed

    Novak, Bela; Pataki, Zsuzsa; Ciliberto, Andrea; Tyson, John J.

    2001-03-01

    Much is known about the genes and proteins controlling the cell cycle of fission yeast. Can these molecular components be spun together into a consistent mechanism that accounts for the observed behavior of growth and division in fission yeast cells? To answer this question, we propose a mechanism for the control system, convert it into a set of 14 differential and algebraic equations, study these equations by numerical simulation and bifurcation theory, and compare our results to the physiology of wild-type and mutant cells. In wild-type cells, progress through the cell cycle (G1-->S-->G2-->M) is related to cyclic progression around a hysteresis loop, driven by cell growth and chromosome alignment on the metaphase plate. However, the control system operates much differently in double-mutant cells, wee1(-) cdc25Delta, which are defective in progress through the latter half of the cell cycle (G2 and M phases). These cells exhibit "quantized" cycles (interdivision times clustering around 90, 160, and 230 min). We show that these quantized cycles are associated with a supercritical Hopf bifurcation in the mechanism, when the wee1 and cdc25 genes are disabled. (c) 2001 American Institute of Physics. PMID:12779461

  9. Model of Exploratory Search for Mating Partners by Fission Yeast

    NASA Astrophysics Data System (ADS)

    Hurwitz, Daniel; Bendezu, Felipe; Martin, Sophie; Vavylonis, Dimitrios

    2014-03-01

    During conditions of nitrogen starvation, the model eukaryote S. pombe (fission yeast) undergoes sexual sporulation. Because fission yeast are non-motile, contact between opposite mating types during spore formation is accomplished by polarizing growth, via the Rho GTP-ase Cdc42, in each mating type towards the selected mate, a process known as shmooing. Recent findings showed that cells pick one of their neighboring compatible mates by randomizing the position of the Cdc42 complex about the cell membrane, such that the complex is stabilized near areas of high concentration of the opposite mating type pheromone. We developed Monte Carlo simulations to model partner finding in populations of mating cells and in small cell clusters. We assume that pheromones are secreted at the site of Cdc42 accumulation and that the Cdc42 dwell time increases in response to increasing pheromone concentration. We measured the number of cells that succeed in successful reciprocal pairing, the number of cells that were unable to find a partner, and the number of cells that picked a partner already engaged with another cell. For optimal cell pairing, we find the pheromone concentration decay length is around 1 micron, of order the cell size. We show that non-linear response of Cdc42 dwell time to pheromone concentration improves the number of successful pairs for a given spatial cell distribution. We discuss how these results compare to non-exploratory pairing mechanisms.

  10. Pom1 and cell size homeostasis in fission yeast

    PubMed Central

    Wood, Elizabeth; Nurse, Paul

    2013-01-01

    Cells sense their size and use this information to coordinate cell division with cell growth to maintain a constant cell size within a given population. A model has been proposed for cell size control in the rod-shaped cells of the fission yeast, Schizosaccharomyces pombe. This involves a protein localized to the cell ends, which inhibits mitotic activators in the middle of the cell in a cell size-dependent manner. This protein, Pom1, along with another tip-localized protein, Nif1, have been implicated as direct sensors of cell size controlling the onset of mitosis. Here we have investigated cell size variability and size homeostasis at the G2/M transition, focusing on the role of pom1 and nif1. Cells deleted for either of these 2 genes show wild-type size homeostasis both in size variability analyses and size homeostasis experiments. This indicates that these genes do not have a critical role as direct cell size sensors in the control mechanism. Cell size homeostasis also seems to be independent of Cdc2–Tyr15 phosphorylation, suggesting that the size sensing mechanism in fission yeast may act through an unidentified pathway regulating CDK activity by an unknown mechanism. PMID:24047646

  11. Inner Kinetochore Protein Interactions with Regional Centromeres of Fission Yeast

    PubMed Central

    Thakur, Jitendra; Talbert, Paul B.; Henikoff, Steven

    2015-01-01

    Centromeres of the fission yeast Schizosaccharomyces pombe lack the highly repetitive sequences that make most other "regional" centromeres refractory to analysis. To map fission yeast centromeres, we applied H4S47C-anchored cleavage mapping and native and cross-linked chromatin immunoprecipitation with paired-end sequencing. H3 nucleosomes are nearly absent from the central domain, which is occupied by centromere-specific H3 (cenH3 or CENP-A) nucleosomes with two H4s per particle that are mostly unpositioned and are more widely spaced than nucleosomes elsewhere. Inner kinetochore proteins CENP-A, CENP-C, CENP-T, CENP-I, and Scm3 are highly enriched throughout the central domain except at tRNA genes, with no evidence for preferred kinetochore assembly sites. These proteins are weakly enriched and less stably incorporated in H3-rich heterochromatin. CENP-A nucleosomes protect less DNA from nuclease digestion than H3 nucleosomes, while CENP-T protects a range of fragment sizes. Our results suggest that CENP-T particles occupy linkers between CENP-A nucleosomes and that classical regional centromeres differ from other centromeres by the absence of CENP-A nucleosome positioning. PMID:26275423

  12. Inner Kinetochore Protein Interactions with Regional Centromeres of Fission Yeast.

    PubMed

    Thakur, Jitendra; Talbert, Paul B; Henikoff, Steven

    2015-10-01

    Centromeres of the fission yeast Schizosaccharomyces pombe lack the highly repetitive sequences that make most other "regional" centromeres refractory to analysis. To map fission yeast centromeres, we applied H4S47C-anchored cleavage mapping and native and cross-linked chromatin immunoprecipitation with paired-end sequencing. H3 nucleosomes are nearly absent from the central domain, which is occupied by centromere-specific H3 (cenH3 or CENP-A) nucleosomes with two H4s per particle that are mostly unpositioned and are more widely spaced than nucleosomes elsewhere. Inner kinetochore proteins CENP-A, CENP-C, CENP-T, CENP-I, and Scm3 are highly enriched throughout the central domain except at tRNA genes, with no evidence for preferred kinetochore assembly sites. These proteins are weakly enriched and less stably incorporated in H3-rich heterochromatin. CENP-A nucleosomes protect less DNA from nuclease digestion than H3 nucleosomes, while CENP-T protects a range of fragment sizes. Our results suggest that CENP-T particles occupy linkers between CENP-A nucleosomes and that classical regional centromeres differ from other centromeres by the absence of CENP-A nucleosome positioning. PMID:26275423

  13. Mediator promotes CENP-a incorporation at fission yeast centromeres.

    PubMed

    Carlsten, Jonas O; Szilagyi, Zsolt; Liu, Beidong; Lopez, Marcela Davila; Szászi, Erzsébet; Djupedal, Ingela; Nyström, Thomas; Ekwall, Karl; Gustafsson, Claes M; Zhu, Xuefeng

    2012-10-01

    At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A(Cnp1), which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A(Cnp1) from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ cells appears to directly block CENP-A(Cnp1) incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function. PMID:22851695

  14. Kinetochore and heterochromatin domains of the fission yeast centromere.

    PubMed

    Pidoux, Alison L; Allshire, Robin C

    2004-01-01

    Fission yeast centromeres are composed of two distinctive chromatin domains. The central domain nucleosomes contain the histone H3-like protein CENP-A(Cnp1). In contrast, the flanking repeats are coated with silent chromatin in which Swi6 (HP1) binds histone H3 methylated on lysine 9 that is induced by the action of the RNA interference pathway on non-coding centromeric transcripts. The overall structure is similar to that of metazoan centromeres where the kinetochore is embedded in surrounding heterochromatin. Kinetochore specific proteins associate with the central domain and affect silencing in that region. The flanking heterochromatin is required to recruit cohesin and mediate tight physical cohesion between sister centromeres. The loss of silencing that accompanies defects in heterochromatin has been invaluable as a tool in the investigation of centromere function. Both the heterochromatin and kinetochore regions are required for the de novo assembly of a functional centromere on DNA constructs, suggesting that heterochromatin may provide an environment that promotes kinetochore assembly within the central domain. The process is clearly epigenetically regulated. Fission yeast kinetochores associate with 2-4 microtubules, and flanking heterochromatin may be required to promote the orientation of multiple microtubule binding sites on one kinetochore towards the same pole and thus prevent merotelic orientation. PMID:15289660

  15. Bidirectional motility of the fission yeast kinesin-5, Cut7

    SciTech Connect

    Edamatsu, Masaki

    2014-03-28

    Highlights: • Motile properties of Cut7 (fission yeast kinesin-5) were studied for the first time. • Half-length Cut7 moved toward plus-end direction of microtubule. • Full-length Cut7 moved toward minus-end direction of microtubule. • N- and C-terminal microtubule binding sites did not switch the motile direction. - Abstract: Kinesin-5 is a homotetrameric motor with its motor domain at the N-terminus. Kinesin-5 crosslinks microtubules and functions in separating spindle poles during mitosis. In this study, the motile properties of Cut7, fission yeast kinesin-5, were examined for the first time. In in vitro motility assays, full-length Cut7 moved toward minus-end of microtubules, but the N-terminal half of Cut7 moved toward the opposite direction. Furthermore, additional truncated constructs lacking the N-terminal or C-terminal regions, but still contained the motor domain, did not switch the motile direction. These indicated that Cut7 was a bidirectional motor, and microtubule binding regions at the N-terminus and C-terminus were not involved in its directionality.

  16. Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis.

    PubMed

    Chatterjee, Gautam; Sankaranarayanan, Sundar Ram; Guin, Krishnendu; Thattikota, Yogitha; Padmanabhan, Sreedevi; Siddharthan, Rahul; Sanyal, Kaustuv

    2016-02-01

    The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species--Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast. PMID:26845548

  17. Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

    PubMed Central

    Chatterjee, Gautam; Sankaranarayanan, Sundar Ram; Guin, Krishnendu; Thattikota, Yogitha; Padmanabhan, Sreedevi; Siddharthan, Rahul; Sanyal, Kaustuv

    2016-01-01

    The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast. PMID:26845548

  18. Nucleation and spreading of a heterochromatic domain in fission yeast

    PubMed Central

    Obersriebnig, Michaela J.; Pallesen, Emil M. H.; Sneppen, Kim; Trusina, Ala; Thon, Geneviève

    2016-01-01

    Outstanding questions in the chromatin field bear on how large heterochromatin domains are formed in space and time. Positive feedback, where histone-modifying enzymes are attracted to chromosomal regions displaying the modification they catalyse, is believed to drive the formation of these domains; however, few quantitative studies are available to assess this hypothesis. Here we quantified the de novo establishment of a naturally occurring ∼20-kb heterochromatin domain in fission yeast through single-cell analyses, measuring the kinetics of heterochromatin nucleation in a region targeted by RNAi and its subsequent expansion. We found that nucleation of heterochromatin is stochastic and can take from one to ten cell generations. Further silencing of the full region takes another one to ten generations. Quantitative modelling of the observed kinetics emphasizes the importance of local feedback, where a nucleosome-bound enzyme modifies adjacent nucleosomes, combined with a feedback where recruited enzymes can act at a distance. PMID:27167753

  19. Cell-Cycle Analyses Using Thymidine Analogues in Fission Yeast

    PubMed Central

    Anda, Silje; Boye, Erik; Grallert, Beata

    2014-01-01

    Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2′-deoxyuridine (EdU) and 5-Chloro-2′-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2′-deoxyuridine (BrdU). Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry. PMID:24551125

  20. Cell-based screens and phenomics with fission yeast.

    PubMed

    Rallis, Charalampos; Bähler, Jürg

    2016-01-01

    Next-generation sequencing approaches have considerably advanced our understanding of genome function and regulation. However, the knowledge of gene function and complex cellular processes remains a challenge and bottleneck in biological research. Phenomics is a rapidly emerging area, which seeks to rigorously characterize all phenotypes associated with genes or gene variants. Such high-throughput phenotyping under different conditions can be a potent approach toward gene function. The fission yeast Schizosaccharomyces pombe (S. pombe) is a proven eukaryotic model organism that is increasingly used for genomewide screens and phenomic assays. In this review, we highlight current large-scale, cell-based approaches used with S. pombe, including computational colony-growth measurements, genetic interaction screens, parallel profiling using barcodes, microscopy-based cell profiling, metabolomic methods and transposon mutagenesis. These diverse methods are starting to offer rich insights into the relationship between genotypes and phenotypes. PMID:26523839

  1. Nucleation and spreading of a heterochromatic domain in fission yeast.

    PubMed

    Obersriebnig, Michaela J; Pallesen, Emil M H; Sneppen, Kim; Trusina, Ala; Thon, Geneviève

    2016-01-01

    Outstanding questions in the chromatin field bear on how large heterochromatin domains are formed in space and time. Positive feedback, where histone-modifying enzymes are attracted to chromosomal regions displaying the modification they catalyse, is believed to drive the formation of these domains; however, few quantitative studies are available to assess this hypothesis. Here we quantified the de novo establishment of a naturally occurring ∼20-kb heterochromatin domain in fission yeast through single-cell analyses, measuring the kinetics of heterochromatin nucleation in a region targeted by RNAi and its subsequent expansion. We found that nucleation of heterochromatin is stochastic and can take from one to ten cell generations. Further silencing of the full region takes another one to ten generations. Quantitative modelling of the observed kinetics emphasizes the importance of local feedback, where a nucleosome-bound enzyme modifies adjacent nucleosomes, combined with a feedback where recruited enzymes can act at a distance. PMID:27167753

  2. Brownian dynamics simulation of fission yeast mitotic spindle formation

    NASA Astrophysics Data System (ADS)

    Edelmaier, Christopher

    2014-03-01

    The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies.

  3. Swi1Timeless Prevents Repeat Instability at Fission Yeast Telomeres.

    PubMed

    Gadaleta, Mariana C; Das, Mukund M; Tanizawa, Hideki; Chang, Ya-Ting; Noma, Ken-ichi; Nakamura, Toru M; Noguchi, Eishi

    2016-03-01

    Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1(Timeless), a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Δ mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of Swi1(Timeless) in regulation of telomere stability in cancer cells. PMID:26990647

  4. Swi1Timeless Prevents Repeat Instability at Fission Yeast Telomeres

    PubMed Central

    Gadaleta, Mariana C.; Das, Mukund M.; Tanizawa, Hideki; Chang, Ya-Ting; Noma, Ken-ichi; Nakamura, Toru M.; Noguchi, Eishi

    2016-01-01

    Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Δ mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of Swi1Timeless in regulation of telomere stability in cancer cells. PMID:26990647

  5. Role of turgor pressure in endocytosis in fission yeast

    PubMed Central

    Basu, Roshni; Munteanu, Emilia Laura; Chang, Fred

    2014-01-01

    Yeast and other walled cells possess high internal turgor pressure that allows them to grow and survive in the environment. This turgor pressure, however, may oppose the invagination of the plasma membrane needed for endocytosis. Here we study the effects of turgor pressure on endocytosis in the fission yeast Schizosaccharomyces pombe by time-lapse imaging of individual endocytic sites. Decreasing effective turgor pressure by addition of sorbitol to the media significantly accelerates early steps in the endocytic process before actin assembly and membrane ingression but does not affect the velocity or depth of ingression of the endocytic pit in wild-type cells. Sorbitol also rescues endocytic ingression defects of certain endocytic mutants and of cells treated with a low dose of the actin inhibitor latrunculin A. Endocytosis proceeds after removal of the cell wall, suggesting that the cell wall does not contribute mechanically to this process. These studies suggest that endocytosis is governed by a mechanical balance between local actin-dependent inward forces and opposing forces from high internal turgor pressure on the plasma membrane. PMID:24403609

  6. De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae) ▿

    PubMed Central

    Hansen, Esben H.; Møller, Birger Lindberg; Kock, Gertrud R.; Bünner, Camilla M.; Kristensen, Charlotte; Jensen, Ole R.; Okkels, Finn T.; Olsen, Carl E.; Motawia, Mohammed S.; Hansen, Jørgen

    2009-01-01

    Vanillin is one of the world's most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker's yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin β-d-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity. PMID:19286778

  7. Incompatibility with Formin Cdc12p Prevents Human Profilin from Substituting for Fission Yeast Profilin: Insights from Crystal Structures of Fission Yeast Proflin

    SciTech Connect

    Ezezika, O.; Younger, N; Lu, J; Kaiser, D; Corbin, Z; Nolen, B; Kovar, D; Pollard, T

    2009-01-01

    Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-l-proline, but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider 'wings' that interact with actin.

  8. Sterol-Rich Membrane Domains Define Fission Yeast Cell Polarity.

    PubMed

    Makushok, Tatyana; Alves, Paulo; Huisman, Stephen Michiel; Kijowski, Adam Rafal; Brunner, Damian

    2016-05-19

    Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization. PMID:27180904

  9. Fission yeast kinesin-8 controls chromosome congression independently of oscillations

    PubMed Central

    Mary, Hadrien; Fouchard, Jonathan; Gay, Guillaume; Reyes, Céline; Gauthier, Tiphaine; Gruget, Clémence; Pécréaux, Jacques; Tournier, Sylvie; Gachet, Yannick

    2015-01-01

    ABSTRACT In higher eukaryotes, efficient chromosome congression relies, among other players, on the activity of chromokinesins. Here, we provide a quantitative analysis of kinetochore oscillations and positioning in Schizosaccharomyces pombe, a model organism lacking chromokinesins. In wild-type cells, chromosomes align during prophase and, while oscillating, maintain this alignment throughout metaphase. Chromosome oscillations are dispensable both for kinetochore congression and stable kinetochore alignment during metaphase. In higher eukaryotes, kinesin-8 family members control chromosome congression by regulating their oscillations. By contrast, here, we demonstrate that fission yeast kinesin-8 controls chromosome congression by an alternative mechanism. We propose that kinesin-8 aligns chromosomes by controlling pulling forces in a length-dependent manner. A coarse-grained model of chromosome segregation implemented with a length-dependent process that controls the force at kinetochores is necessary and sufficient to mimic kinetochore alignment, and prevents the appearance of lagging chromosomes. Taken together, these data illustrate how the local action of a motor protein at kinetochores provides spatial cues within the spindle to align chromosomes and to prevent aneuploidy. PMID:26359299

  10. Fission yeast RNA triphosphatase reads an Spt5 CTD code.

    PubMed

    Doamekpor, Selom K; Schwer, Beate; Sanchez, Ana M; Shuman, Stewart; Lima, Christopher D

    2015-01-01

    mRNA capping enzymes are directed to nascent RNA polymerase II (Pol2) transcripts via interactions with the carboxy-terminal domains (CTDs) of Pol2 and transcription elongation factor Spt5. Fission yeast RNA triphosphatase binds to the Spt5 CTD, comprising a tandem repeat of nonapeptide motif TPAWNSGSK. Here we report the crystal structure of a Pct1·Spt5-CTD complex, which revealed two CTD docking sites on the Pct1 homodimer that engage TPAWN segments of the motif. Each Spt5 CTD interface, composed of elements from both subunits of the homodimer, is dominated by van der Waals contacts from Pct1 to the tryptophan of the CTD. The bound CTD adopts a distinctive conformation in which the peptide backbone makes a tight U-turn so that the proline stacks over the tryptophan. We show that Pct1 binding to Spt5 CTD is antagonized by threonine phosphorylation. Our results fortify an emerging concept of an "Spt5 CTD code" in which (i) the Spt5 CTD is structurally plastic and can adopt different conformations that are templated by particular cellular Spt5 CTD receptor proteins; and (ii) threonine phosphorylation of the Spt5 CTD repeat inscribes a binary on-off switch that is read by diverse CTD receptors, each in its own distinctive manner. PMID:25414009

  11. High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification.

    PubMed

    Nie, Minghua; Vashisht, Ajay A; Wohlschlegel, James A; Boddy, Michael N

    2015-01-01

    Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO "cloud" phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function. PMID:26404184

  12. High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification

    PubMed Central

    Nie, Minghua; Vashisht, Ajay A.; Wohlschlegel, James A.; Boddy, Michael N.

    2015-01-01

    Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO “cloud” phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function. PMID:26404184

  13. Ectopic Centromere Nucleation by CENP-A in Fission Yeast

    PubMed Central

    Gonzalez, Marlyn; He, Haijin; Dong, Qianhua; Sun, Siyu; Li, Fei

    2014-01-01

    The centromere is a specific chromosomal locus that organizes the assembly of the kinetochore. It plays a fundamental role in accurate chromosome segregation. In most eukaryotic organisms, each chromosome contains a single centromere the position and function of which are epigenetically specified. Occasionally, centromeres form at ectopic loci, which can be detrimental to the cell. However, the mechanisms that protect the cell against ectopic centromeres (neocentromeres) remain poorly understood. Centromere protein-A (CENP-A), a centromere-specific histone 3 (H3) variant, is found in all centromeres and is indispensable for centromere function. Here we report that the overexpression of CENP-ACnp1 in fission yeast results in the assembly of CENP-ACnp1 at noncentromeric chromatin during mitosis and meiosis. The noncentromeric CENP-A preferentially assembles near heterochromatin and is capable of recruiting kinetochore components. Consistent with this, cells overexpressing CENP-ACnp1 exhibit severe chromosome missegregation and spindle microtubule disorganization. In addition, pulse induction of CENP-ACnp1 overexpression reveals that ectopic CENP-A chromatin can persist for multiple generations. Intriguingly, ectopic assembly of CENP-Acnp1 is suppressed by overexpression of histone H3 or H4. Finally, we demonstrate that deletion of the N-terminal domain of CENP-Acnp1 results in an increase in the number of ectopic CENP-A sites and provide evidence that the N-terminal domain of CENP-A prevents CENP-A assembly at ectopic loci via the ubiquitin-dependent proteolysis. These studies expand our current understanding of how noncentromeric chromatin is protected from mistakenly assembling CENP-A. PMID:25298518

  14. Ectopic centromere nucleation by CENP--a in fission yeast.

    PubMed

    Gonzalez, Marlyn; He, Haijin; Dong, Qianhua; Sun, Siyu; Li, Fei

    2014-12-01

    The centromere is a specific chromosomal locus that organizes the assembly of the kinetochore. It plays a fundamental role in accurate chromosome segregation. In most eukaryotic organisms, each chromosome contains a single centromere the position and function of which are epigenetically specified. Occasionally, centromeres form at ectopic loci, which can be detrimental to the cell. However, the mechanisms that protect the cell against ectopic centromeres (neocentromeres) remain poorly understood. Centromere protein-A (CENP-A), a centromere-specific histone 3 (H3) variant, is found in all centromeres and is indispensable for centromere function. Here we report that the overexpression of CENP-A(Cnp1) in fission yeast results in the assembly of CENP-A(Cnp1) at noncentromeric chromatin during mitosis and meiosis. The noncentromeric CENP-A preferentially assembles near heterochromatin and is capable of recruiting kinetochore components. Consistent with this, cells overexpressing CENP-A(Cnp1) exhibit severe chromosome missegregation and spindle microtubule disorganization. In addition, pulse induction of CENP-A(Cnp1) overexpression reveals that ectopic CENP-A chromatin can persist for multiple generations. Intriguingly, ectopic assembly of CENP-A(cnp1) is suppressed by overexpression of histone H3 or H4. Finally, we demonstrate that deletion of the N-terminal domain of CENP-A(cnp1) results in an increase in the number of ectopic CENP-A sites and provide evidence that the N-terminal domain of CENP-A prevents CENP-A assembly at ectopic loci via the ubiquitin-dependent proteolysis. These studies expand our current understanding of how noncentromeric chromatin is protected from mistakenly assembling CENP-A. PMID:25298518

  15. Advancing our understanding of functional genome organisation through studies in the fission yeast

    PubMed Central

    Olsson, Ida

    2010-01-01

    Significant progress has been made in understanding the functional organisation of the cell nucleus. Still many questions remain to be answered about the relationship between the spatial organisation of the nucleus and the regulation of the genome function. There are many conflicting data in the field making it very difficult to merge published results on mammalian cells into one model on subnuclear chromatin organisation. The fission yeast, Schizosaccharomyces pombe, over the last decades has emerged as a valuable model organism in understanding basic biological mechanisms, especially the cell cycle and chromosome biology. In this review we describe and compare the nuclear organisation in mammalian and fission yeast cells. We believe that fission yeast is a good tool to resolve at least some of the contradictions and unanswered questions concerning functional nuclear architecture, since S. pombe has chromosomes structurally similar to that of human. S. pombe also has the advantage over higher eukaryotes in that the genome can easily be manipulated via homologous recombination making it possible to integrate the tools needed for visualisation of chromosomes using live-cell microscopy. Classical genetic experiments can be used to elucidate what factors are involved in a certain mechanism. The knowledge we have gained during the last few years indicates similarities between the genome organisation in fission yeast and mammalian cells. We therefore propose the use of fission yeast for further advancement of our understanding of functional nuclear organisation. PMID:21113595

  16. Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation.

    PubMed Central

    Tang, Zhaohua; Käufer, Norbert F; Lin, Ren-Jang

    2002-01-01

    The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1-Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast. PMID:12186627

  17. Observation of magnetic field-induced contraction of fission yeast cells using optical projection microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Xi; Beckwith, Andrew; Miller, John; Wood, Lowell

    2004-12-01

    The charges in live cells interact with or produce electric fields, which results in enormous dielectric responses, flexoelectricity, and related phenomena. Here we report on a contraction of Schizosaccharomyces pombe (fission yeast) cells induced by magnetic fields, as observed using a phase-sensitive projection imaging technique. Unlike electric fields, magnetic fields only act on moving charges. The observed behavior is therefore quite remarkable, and may result from a contractile Lorentz force acting on diamagnetic screening currents. This would indicate extremely high intracellular charge mobilities. Besides, we observed a large electro-optic response from fission yeast cells.

  18. Observation of magnetic field-induced contraction of fission yeast cells using optical projection microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Xi; Beckwith, A. W.

    2005-03-01

    The charges in live cells interact with or produce electric fields, which results in enormous dielectric responses, flexoelectricity, and related phenomena. Here we report on a contraction of Schizosaccharomyces pombe (fission yeast) cells induced by magnetic fields, as observed using a phase-sensitive projection imaging technique. Unlike electric fields, magnetic fields only act on moving charges. The observed behavior is therefore quite remarkable, and may result from a contractile Lorentz force acting on diamagnetic screening currents. This would indicate extremely high intracellular charge mobilities. Besides, we observed a large electro-optic response from fission yeast cells.

  19. Comparative 3D Genome Structure Analysis of the Fission and the Budding Yeast

    PubMed Central

    Gong, Ke; Tjong, Harianto; Zhou, Xianghong Jasmine; Alber, Frank

    2015-01-01

    We studied the 3D structural organization of the fission yeast genome, which emerges from the tethering of heterochromatic regions in otherwise randomly configured chromosomes represented as flexible polymer chains in an nuclear environment. This model is sufficient to explain in a statistical manner many experimentally determined distinctive features of the fission yeast genome, including chromatin interaction patterns from Hi-C experiments and the co-locations of functionally related and co-expressed genes, such as genes expressed by Pol-III. Our findings demonstrate that some previously described structure-function correlations can be explained as a consequence of random chromatin collisions driven by a few geometric constraints (mainly due to centromere-SPB and telomere-NE tethering) combined with the specific gene locations in the chromosome sequence. We also performed a comparative analysis between the fission and budding yeast genome structures, for which we previously detected a similar organizing principle. However, due to the different chromosome sizes and numbers, substantial differences are observed in the 3D structural genome organization between the two species, most notably in the nuclear locations of orthologous genes, and the extent of nuclear territories for genes and chromosomes. However, despite those differences, remarkably, functional similarities are maintained, which is evident when comparing spatial clustering of functionally related genes in both yeasts. Functionally related genes show a similar spatial clustering behavior in both yeasts, even though their nuclear locations are largely different between the yeast species. PMID:25799503

  20. Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

    PubMed Central

    Sun, Ling-Ling; Shen, En-Zhi; Yang, Bing; Dong, Meng-Qiu; He, Wan-Zhong; Du, Li-Lin

    2013-01-01

    Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy. PMID:23950735

  1. Meiotic chromosome mobility in fission yeast is resistant to environmental stress.

    PubMed

    Illner, Doris; Lorenz, Alexander; Scherthan, Harry

    2016-01-01

    The formation of healthy gametes requires pairing of homologous chromosomes (homologs) as a prerequisite for their correct segregation during meiosis. Initially, homolog alignment is promoted by meiotic chromosome movements feeding into intimate homolog pairing by homologous recombination and/or synaptonemal complex formation. Meiotic chromosome movements in the fission yeast, Schizosaccharomyces pombe, depend on astral microtubule dynamics that drag the nucleus through the zygote; known as horsetail movement. The response of microtubule-led meiotic chromosome movements to environmental stresses such as ionizing irradiation (IR) and associated reactive oxygen species (ROS) is not known. Here, we show that, in contrast to budding yeast, the horsetail movement is largely radiation-resistant, which is likely mediated by a potent antioxidant defense. IR exposure of sporulating S. pombe cells induced misrepair and irreparable DNA double strand breaks causing chromosome fragmentation, missegregation and gamete death. Comparing radiation outcome in fission and budding yeast, and studying meiosis with poisoned microtubules indicates that the increased gamete death after IR is innate to fission yeast. Inhibition of meiotic chromosome mobility in the face of IR failed to influence the course of DSB repair, indicating that paralysis of meiotic chromosome mobility in a genotoxic environment is not a universal response among species. PMID:27074839

  2. Meiotic chromosome mobility in fission yeast is resistant to environmental stress

    PubMed Central

    Illner, Doris; Lorenz, Alexander; Scherthan, Harry

    2016-01-01

    The formation of healthy gametes requires pairing of homologous chromosomes (homologs) as a prerequisite for their correct segregation during meiosis. Initially, homolog alignment is promoted by meiotic chromosome movements feeding into intimate homolog pairing by homologous recombination and/or synaptonemal complex formation. Meiotic chromosome movements in the fission yeast, Schizosaccharomyces pombe, depend on astral microtubule dynamics that drag the nucleus through the zygote; known as horsetail movement. The response of microtubule-led meiotic chromosome movements to environmental stresses such as ionizing irradiation (IR) and associated reactive oxygen species (ROS) is not known. Here, we show that, in contrast to budding yeast, the horsetail movement is largely radiation-resistant, which is likely mediated by a potent antioxidant defense. IR exposure of sporulating S. pombe cells induced misrepair and irreparable DNA double strand breaks causing chromosome fragmentation, missegregation and gamete death. Comparing radiation outcome in fission and budding yeast, and studying meiosis with poisoned microtubules indicates that the increased gamete death after IR is innate to fission yeast. Inhibition of meiotic chromosome mobility in the face of IR failed to influence the course of DSB repair, indicating that paralysis of meiotic chromosome mobility in a genotoxic environment is not a universal response among species. PMID:27074839

  3. An IF-FISH Approach for Covisualization of Gene Loci and Nuclear Architecture in Fission Yeast.

    PubMed

    Kim, K-D; Iwasaki, O; Noma, K

    2016-01-01

    Recent genomic studies have revealed that chromosomal structures are formed by a hierarchy of organizing processes ranging from gene associations, including interactions among enhancers and promoters, to topologically associating domain formations. Gene associations identified by these studies can be characterized by microscopic analyses. Fission yeast is a model organism, in which gene associations have been broadly mapped across the genome, although many of those associations have not been further examined by cell biological approaches. To address the technically challenging process of the visualization of associating gene loci in the fission yeast nuclei, we provide, in detail, an IF-FISH procedure that allows for covisualizing both gene loci and nuclear structural markers such as the nuclear membrane and nucleolus. PMID:27423862

  4. The domain structure of centromeres is conserved from fission yeast to humans.

    PubMed

    Kniola, B; O'Toole, E; McIntosh, J R; Mellone, B; Allshire, R; Mengarelli, S; Hultenby, K; Ekwall, K

    2001-09-01

    The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes. PMID:11553715

  5. Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast.

    PubMed

    Osaka, F; Saeki, M; Katayama, S; Aida, N; Toh-E, A; Kominami, K; Toda, T; Suzuki, T; Chiba, T; Tanaka, K; Kato, S

    2000-07-01

    A ubiquitin-like modifier, NEDD8, is covalently attached to cullin-family proteins, but its physiological role is poorly understood. Here we report that the NEDD8-modifying pathway is essential for cell viability and function of Pcu1 (cullin-1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin-ligase was completely modified by NEDD8. Pcu1(K713R) defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1(K713R) or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin-dependent kinase inhibitor Rum1, which is a substrate of the SCF complex. Based on these findings, we propose that covalent modification of cullin-1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast. PMID:10880460

  6. Chromosome and mitotic spindle dynamics in fission yeast kinesin-8 mutants

    NASA Astrophysics Data System (ADS)

    Crapo, Ammon M.; Gergley, Zachary R.; McIntosh, J. Richard; Betterton, M. D.

    2014-03-01

    Fission yeast proteins Klp5p and Klp6p are plus-end directed motors of the kinesin-8 family which promote microtubule (MT) depolymerization and also affect chromosome segregation, but the mechanism of these activities is not well understood. Using live-cell time-lapse fluorescence microscopy of fission yeast wild-type (WT) and klp5/6 mutant strains, we quantify and compare the dynamics of kinetochore motion and mitotic spindle length in 3D. In WT cells, the spindle, once formed, remains a consistent size and chromosomes are correctly organized and segregated. In kinesin-8 mutants, spindles undergo large length fluctuations of several microns. Kinetochore motions are also highly fluctuating, with kinetochores frequently moving away from the spindle rather than toward it. We observe transient pushing of chromosomes away from the spindle by as much as 10 microns in distance.

  7. A stochastic model of kinetochore–microtubule attachment accurately describes fission yeast chromosome segregation

    PubMed Central

    Gay, Guillaume; Courtheoux, Thibault; Reyes, Céline

    2012-01-01

    In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs. The model accurately reproduces the timing of correct chromosome biorientation and segregation seen in fission yeast. Prevention of attachment defects requires both appropriate kinetochore orientation and an Aurora B–like activity. The model also reproduces abnormal chromosome segregation behavior (caused by, for example, inhibition of Aurora B). It predicts that, in metaphase, merotelic attachment is prevented by a kinetochore orientation effect and corrected by an Aurora B–like activity, whereas in anaphase, it is corrected through unbalanced forces applied to the kinetochore. These unbalanced forces are sufficient to prevent aneuploidy. PMID:22412019

  8. Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles

    PubMed Central

    Převorovský, Martin; Oravcová, Martina; Tvarůžková, Jarmila; Zach, Róbert; Folk, Petr; Půta, František; Bähler, Jürg

    2015-01-01

    Background Cbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped. Methodology/Principal Findings Using a combination of transcriptome profiling under various conditions and genome-wide analysis of CSL-DNA interactions, we identify genes regulated directly and indirectly by CSL proteins in fission yeast. We show that the expression of stress-response genes and genes that are expressed periodically during the cell cycle is deregulated upon genetic manipulation of cbf11 and/or cbf12. Accordingly, the coordination of mitosis and cytokinesis is perturbed in cells with genetically manipulated CSL protein levels, together with other specific defects in cell-cycle progression. Cbf11 activity is nutrient-dependent and Δcbf11-associated defects are mitigated by inactivation of the protein kinase A (Pka1) and stress-activated MAP kinase (Sty1p38) pathways. Furthermore, Cbf11 directly regulates a set of lipid metabolism genes and Δcbf11 cells feature a stark decrease in the number of storage lipid droplets. Conclusions/Significance Our results provide a framework for a more detailed understanding of the role of CSL proteins in the regulation of cell-cycle progression in fission yeast. PMID:26366556

  9. Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast.

    PubMed

    Sun, Xiaoying; Hirai, Go; Ueki, Masashi; Hirota, Hiroshi; Wang, Qianqian; Hongo, Yayoi; Nakamura, Takemichi; Hitora, Yuki; Takahashi, Hidekazu; Sodeoka, Mikiko; Osada, Hiroyuki; Hamamoto, Makiko; Yoshida, Minoru; Yashiroda, Yoko

    2016-01-01

    Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4(+) and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast. PMID:26892493

  10. Fission yeast mitochondria are distributed by dynamic microtubules in a motor-independent manner

    PubMed Central

    Li, Tianpeng; Zheng, Fan; Cheung, Martin; Wang, Fengsong; Fu, Chuanhai

    2015-01-01

    The cytoskeleton plays a critical role in regulating mitochondria distribution. Similar to axonal mitochondria, the fission yeast mitochondria are distributed by the microtubule cytoskeleton, but this is regulated by a motor-independent mechanism depending on the microtubule associated protein mmb1p as the absence of mmb1p causes mitochondria aggregation. In this study, using a series of chimeric proteins to control the subcellular localization and motility of mitochondria, we show that a chimeric molecule containing a microtubule binding domain and the mitochondria outer membrane protein tom22p can restore the normal interconnected mitochondria network in mmb1-deletion (mmb1∆) cells. In contrast, increasing the motility of mitochondria by using a chimeric molecule containing a kinesin motor domain and tom22p cannot rescue mitochondria aggregation defects in mmb1∆ cells. Intriguingly a chimeric molecule carrying an actin binding domain and tom22p results in mitochondria associated with actin filaments at the actomyosin ring during mitosis, leading to cytokinesis defects. These findings suggest that the passive motor-independent microtubule-based mechanism is the major contributor to mitochondria distribution in wild type fission yeast cells. Hence, we establish that attachment to microtubules, but not kinesin-dependent movement and the actin cytoskeleton, is required and crucial for proper mitochondria distribution in fission yeast. PMID:26046468

  11. Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast

    PubMed Central

    Sun, Xiaoying; Hirai, Go; Ueki, Masashi; Hirota, Hiroshi; Wang, Qianqian; Hongo, Yayoi; Nakamura, Takemichi; Hitora, Yuki; Takahashi, Hidekazu; Sodeoka, Mikiko; Osada, Hiroyuki; Hamamoto, Makiko; Yoshida, Minoru; Yashiroda, Yoko

    2016-01-01

    Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4+ and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast. PMID:26892493

  12. Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin.

    PubMed

    Pidoux, Alison L; Choi, Eun Shik; Abbott, Johanna K R; Liu, Xingkun; Kagansky, Alexander; Castillo, Araceli G; Hamilton, Georgina L; Richardson, William; Rappsilber, Juri; He, Xiangwei; Allshire, Robin C

    2009-02-13

    The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin. PMID:19217404

  13. Fission Yeast Scm3: A CENP-A Receptor Required for Integrity of Subkinetochore Chromatin

    PubMed Central

    Pidoux, Alison L.; Choi, Eun Shik; Abbott, Johanna K.R.; Liu, Xingkun; Kagansky, Alexander; Castillo, Araceli G.; Hamilton, Georgina L.; Richardson, William; Rappsilber, Juri; He, Xiangwei; Allshire, Robin C.

    2009-01-01

    Summary The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3Sp, the ortholog of budding yeast Scm3Sc. Scm3Sp depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3Sp coaffinity purifies with CENP-ACnp1 and associates with CENP-ACnp1 in vitro, yet localizes independently of intact CENP-ACnp1 chromatin and is differentially released from chromatin. While Scm3Sc has been proposed to form a unique hexameric nucleosome with CENP-ACse4 and histone H4 at budding yeast point centromeres, we favor a model in which Scm3Sp acts as a CENP-ACnp1 receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-ACnp1 from the Sim3 escort and mediate assembly of CENP-ACnp1 into subkinetochore chromatin. PMID:19217404

  14. Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe.

    PubMed

    Rostron, Kerry A; Rolph, Carole E; Lawrence, Clare L

    2015-07-01

    Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321-328, 2012), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses. PMID:25948336

  15. Histone H3K36 trimethylation is essential for multiple silencing mechanisms in fission yeast.

    PubMed

    Suzuki, Shota; Kato, Hiroaki; Suzuki, Yutaka; Chikashige, Yuji; Hiraoka, Yasushi; Kimura, Hiroshi; Nagao, Koji; Obuse, Chikashi; Takahata, Shinya; Murakami, Yota

    2016-05-19

    In budding yeast, Set2 catalyzes di- and trimethylation of H3K36 (H3K36me2 and H3K36me3) via an interaction between its Set2-Rpb1 interaction (SRI) domain and C-terminal repeats of RNA polymerase II (Pol2) phosphorylated at Ser2 and Ser5 (CTD-S2,5-P). H3K36me2 is sufficient for recruitment of the Rpd3S histone deacetylase complex to repress cryptic transcription from transcribed regions. In fission yeast, Set2 is also responsible for H3K36 methylation, which represses a subset of RNAs including heterochromatic and subtelomeric RNAs, at least in part via recruitment of Clr6 complex II, a homolog of Rpd3S. Here, we show that CTD-S2P-dependent interaction of fission yeast Set2 with Pol2 via the SRI domain is required for formation of H3K36me3, but not H3K36me2. H3K36me3 silenced heterochromatic and subtelomeric transcripts mainly through post-transcriptional and transcriptional mechanisms, respectively, whereas H3K36me2 was not enough for silencing. Clr6 complex II appeared not to be responsible for heterochromatic silencing by H3K36me3. Our results demonstrate that H3K36 methylation has multiple outputs in fission yeast; these findings provide insights into the distinct roles of H3K36 methylation in metazoans, which have different enzymes for synthesis of H3K36me1/2 and H3K36me3. PMID:26792892

  16. Phenotypes and fed-batch fermentation of ubiquinone-overproducing fission yeast using ppt1 gene.

    PubMed

    Zhang, Dawei; Shrestha, Binaya; Niu, Weining; Tian, Pingfang; Tan, Tianwei

    2007-01-30

    Ubiquinone (UQ), a component of the electron transfer system in many organisms, has been widely used for pharmaceuticals and cosmetics. In this study, we cloned and overexpressed the full-length ppt1 (MTppt1) gene, which encodes p-hydroxybenzoate:polyprenyltransferase and ERppt1 gene, which was modified to be localized on endoplasmic reticulum in fission yeast. The yeast MTppt1 and ERppt1 transgenic lines showed about 3.7 and 5.1 times increment in UQ content and the recombinant yeasts with a higher UQ level are more resistant to H(2)O(2), Cu(2+) and NaCl, and interestingly their growth was also faster than the wild type at lower temperature. For large-scale cultivation, the direct feedback control of glucose using an on-line ethanol concentration monitor for ubiquinone production of yeast ERppt1 by high-cell-density fermentation was investigated and the fermentation parameters (e.g., dissolved oxygen, pH, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiration quotient) were also discussed. After 90 h cultures, the yeast dry cell weight reached 57 gl(-1) and the ubiquinone yield reached 23 mgl(-1). In addition, plasmid stability was maintained at high level throughout the fermentation. PMID:17069919

  17. Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

    SciTech Connect

    Kita, Ayako; Higa, Mari; Doi, Akira; Satoh, Ryosuke; Sugiura, Reiko

    2015-02-13

    Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2{sup +} gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis with multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2{sup +} gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking.

  18. Mapping epigenetic mutations in fission yeast using whole-genome next-generation sequencing

    PubMed Central

    Irvine, Danielle V.; Goto, Derek B.; Vaughn, Matthew W.; Nakaseko, Yukinobu; McCombie, W. Richard; Yanagida, Mitsuhiro; Martienssen, Rob

    2009-01-01

    Fission yeast is an important model for epigenetic studies due to the ease with which genetic mutants can be isolated. However, it can be difficult to complement epigenetic phenotypes with genomic libraries in order to identify the genes responsible. This is because epigenetic phenotypes are typically unstable, and can prohibit complementation if silencing cannot be reestablished. Here we have resequenced the fission yeast genome following mutagenesis to readily identify a novel mutant involved in heterochromatic silencing. Candidate genes were identified as functional single base changes linked to the mutation, which were then reconstituted in a wild-type strain to recapitulate the mutant phenotype. By this procedure we identified a weak allele of ubc4, which encodes an essential E2 ubiquitin ligase, as responsible for the swi*603 mutant phenotype. In combination with a large collection of mutants and suppressor plasmids, next-generation genomic resequencing promises to dramatically enhance the power of yeast genetics, permitting the isolation of subtle alleles of essential genes, alleles with quantitative effects, and enhancers and suppressors of heterochromatic silencing. PMID:19423874

  19. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  20. Bulk Segregant Analysis Reveals the Genetic Basis of a Natural Trait Variation in Fission Yeast

    PubMed Central

    Hu, Wen; Suo, Fang; Du, Li-Lin

    2015-01-01

    Although the fission yeast Schizosaccharomyces pombe is a well-established model organism, studies of natural trait variations in this species remain limited. To assess the feasibility of segregant-pool-based mapping of phenotype-causing genes in natural strains of fission yeast, we investigated the cause of a maltose utilization defect (Mal-) of the S. pombe strain CBS5557 (originally known as Schizosaccharomyces malidevorans). Analyzing the genome sequence of CBS5557 revealed 955 nonconservative missense substitutions, and 61 potential loss-of-function variants including 47 frameshift indels, 13 early stop codons, and 1 splice site mutation. As a side benefit, our analysis confirmed 146 sequence errors in the reference genome and improved annotations of 27 genes. We applied bulk segregant analysis to map the causal locus of the Mal- phenotype. Through sequencing the segregant pools derived from a cross between CBS5557 and the laboratory strain, we located the locus to within a 2.23-Mb chromosome I inversion found in most S. pombe isolates including CBS5557. To map genes within the inversion region that occupies 18% of the genome, we created a laboratory strain containing the same inversion. Analyzing segregants from a cross between CBS5557 and the inversion-containing laboratory strain narrowed down the locus to a 200-kb interval and led us to identify agl1, which suffers a 5-bp deletion in CBS5557, as the causal gene. Interestingly, loss of agl1 through a 34-kb deletion underlies the Mal- phenotype of another S. pombe strain CGMCC2.1628. This work adapts and validates the bulk segregant analysis method for uncovering trait-gene relationship in natural fission yeast strains. PMID:26615217

  1. The fission yeast MTREC and EJC orthologs ensure the maturation of meiotic transcripts during meiosis.

    PubMed

    Marayati, Bahjat Fadi; Hoskins, Victoria; Boger, Robert W; Tucker, James F; Fishman, Emily S; Bray, Andrew S; Zhang, Ke

    2016-09-01

    Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3'-5' exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes. PMID:27365210

  2. The fission yeast MTREC and EJC orthologs ensure the maturation of meiotic transcripts during meiosis

    PubMed Central

    Marayati, Bahjat Fadi; Hoskins, Victoria; Boger, Robert W.; Tucker, James F.; Fishman, Emily S.; Bray, Andrew S.; Zhang, Ke

    2016-01-01

    Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3′–5′ exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe. In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes. PMID:27365210

  3. Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1.

    PubMed

    Schwer, Beate; Ghosh, Agnidipta; Sanchez, Ana M; Lima, Christopher D; Shuman, Stewart

    2015-06-01

    Protein phosphatases regulate mRNA synthesis and processing by remodeling the carboxy-terminal domain (CTD) of RNA polymerase II (Pol2) to dynamically inscribe a Pol2 CTD code. Fission yeast Fcp1 (SpFcp1) is an essential 723-amino acid CTD phosphatase that preferentially hydrolyzes Ser2-PO4 of the YS(2)PTSPS repeat. The SpFcp1 catalytic domain (aa 140-580) is composed of a DxDxT acyl-phosphatase module (FCPH) and a BRCT module. Here we conducted a genetic analysis of SpFcp1, which shows that (i) phosphatase catalytic activity is required for vegetative growth of fission yeast; (ii) the flanking amino-terminal domain (aa 1-139) and its putative metal-binding motif C(99)H(101)Cys(109)C(112) are essential; (iii) the carboxy-terminal domain (aa 581-723) is dispensable; (iv) a structurally disordered internal segment of the FCPH domain (aa 330-393) is dispensable; (v) lethal SpFcp1 mutations R271A and R299A are rescued by shortening the Pol2 CTD repeat array; and (vi) CTD Ser2-PO4 is not the only essential target of SpFcp1 in vivo. Recent studies highlight a second CTD code involving threonine phosphorylation of a repeat motif in transcription elongation factor Spt5. We find that Fcp1 can dephosphorylate Thr1-PO4 of the fission yeast Spt5 CTD nonamer repeat T(1)PAWNSGSK. We identify Arg271 as a governor of Pol2 versus Spt5 CTD substrate preference. Our findings implicate Fcp1 as a versatile sculptor of both the Pol2 and Spt5 CTD codes. Finally, we report a new 1.45 Å crystal structure of SpFcp1 with Mg(2+) and AlF3 that mimics an associative phosphorane transition state of the enzyme-aspartyl-phosphate hydrolysis reaction. PMID:25883047

  4. Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1

    PubMed Central

    Schwer, Beate; Ghosh, Agnidipta; Sanchez, Ana M.; Lima, Christopher D.; Shuman, Stewart

    2015-01-01

    Protein phosphatases regulate mRNA synthesis and processing by remodeling the carboxy-terminal domain (CTD) of RNA polymerase II (Pol2) to dynamically inscribe a Pol2 CTD code. Fission yeast Fcp1 (SpFcp1) is an essential 723-amino acid CTD phosphatase that preferentially hydrolyzes Ser2-PO4 of the YS2PTSPS repeat. The SpFcp1 catalytic domain (aa 140–580) is composed of a DxDxT acyl-phosphatase module (FCPH) and a BRCT module. Here we conducted a genetic analysis of SpFcp1, which shows that (i) phosphatase catalytic activity is required for vegetative growth of fission yeast; (ii) the flanking amino-terminal domain (aa 1–139) and its putative metal-binding motif C99H101Cys109C112 are essential; (iii) the carboxy-terminal domain (aa 581–723) is dispensable; (iv) a structurally disordered internal segment of the FCPH domain (aa 330–393) is dispensable; (v) lethal SpFcp1 mutations R271A and R299A are rescued by shortening the Pol2 CTD repeat array; and (vi) CTD Ser2-PO4 is not the only essential target of SpFcp1 in vivo. Recent studies highlight a second CTD code involving threonine phosphorylation of a repeat motif in transcription elongation factor Spt5. We find that Fcp1 can dephosphorylate Thr1-PO4 of the fission yeast Spt5 CTD nonamer repeat T1PAWNSGSK. We identify Arg271 as a governor of Pol2 versus Spt5 CTD substrate preference. Our findings implicate Fcp1 as a versatile sculptor of both the Pol2 and Spt5 CTD codes. Finally, we report a new 1.45 Å crystal structure of SpFcp1 with Mg2+ and AlF3 that mimics an associative phosphorane transition state of the enzyme-aspartyl-phosphate hydrolysis reaction. PMID:25883047

  5. Phosphorylation of Pex11p does not regulate peroxisomal fission in the yeast Hansenula polymorpha

    PubMed Central

    Thomas, Ann S.; Krikken, Arjen M.; van der Klei, Ida J.; Williams, Chris P.

    2015-01-01

    Pex11p plays a crucial role in peroxisomal fission. Studies in Saccharomyces cerevisiae and Pichia pastoris indicated that Pex11p is activated by phosphorylation, which results in enhanced peroxisome proliferation. In S. cerevisiae but not in P. pastoris, Pex11p phosphorylation was shown to regulate the protein’s trafficking to peroxisomes. However, phosphorylation of PpPex11p was proposed to influence its interaction with Fis1p, another component of the organellar fission machinery. Here, we have examined the role of Pex11p phosphorylation in the yeast Hansenula polymorpha. Employing mass spectrometry, we demonstrate that HpPex11p is also phosphorylated on a Serine residue present at a similar position to that of ScPex11p and PpPex11p. Furthermore, through the use of mutants designed to mimic both phosphorylated and unphosphorylated forms of HpPex11p, we have investigated the role of this post-translational modification. Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p. Also, no effect on peroxisome inheritance was observed. Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p. PMID:26099236

  6. Replication stress in early S phase generates apparent micronuclei and chromosome rearrangement in fission yeast

    PubMed Central

    Sabatinos, Sarah A.; Ranatunga, Nimna S.; Yuan, Ji-Ping; Green, Marc D.; Forsburg, Susan L.

    2015-01-01

    DNA replication stress causes genome mutations, rearrangements, and chromosome missegregation, which are implicated in cancer. We analyze a fission yeast mutant that is unable to complete S phase due to a defective subunit of the MCM helicase. Despite underreplicated and damaged DNA, these cells evade the G2 damage checkpoint to form ultrafine bridges, fragmented centromeres, and uneven chromosome segregations that resembles micronuclei. These micronuclei retain DNA damage markers and frequently rejoin with the parent nucleus. Surviving cells show an increased rate of mutation and chromosome rearrangement. This first report of micronucleus-like segregation in a yeast replication mutant establishes underreplication as an important factor contributing to checkpoint escape, abnormal chromosome segregation, and chromosome instability. PMID:26246602

  7. CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast.

    PubMed

    Coffman, Valerie C; Wu, Pengcheng; Parthun, Mark R; Wu, Jian-Qiu

    2011-11-14

    The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ~40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1-DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models. PMID:22084306

  8. Rga6 is a fission yeast Rho GAP involved in Cdc42 regulation of polarized growth

    PubMed Central

    Revilla-Guarinos, M. T.; Martín-García, Rebeca; Villar-Tajadura, M. Antonia; Estravís, Miguel; Coll, Pedro M.; Pérez, Pilar

    2016-01-01

    Active Cdc42 is essential for the establishment of polarized growth. This GTPase is negatively regulated by the GTPase-activating proteins (GAPs), which are important for the spatial specificity of Cdc42 function. Rga4 is the only GAP described as negative regulator of fission yeast Cdc42. We report here that Rga6, another fission yeast Cdc42 GAP, shares some functions with Rga4. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking Rga6 and Rga4 simultaneously are rounded. In these cells, active Cdc42 is observed all around the membrane. These additive effects indicate that both GAPs collaborate in the spatial regulation of active Cdc42. Rga6 localizes to the plasma membrane, forming clusters different from those formed by Rga4. A polybasic region at the Rga6 C-terminus is responsible for its membrane localization. Rga6-GFP fluorescence decreases considerably at the growing tips, and this decrease is dependent on the actin cables. Of note, in the absence of Rga6, the amplitude of active Cdc42 oscillations at the tips decreases, and less GTP-Cdc42 accumulates at the new end of the cells. We propose that Rga6 collaborates with Rga4 to spatially restrict active Cdc42 at the cell tips and maintain cell dimensions. PMID:26960792

  9. Nickel resistance in fission yeast associated with the magnesium transport system.

    PubMed

    Sarikaya, Aysegul Topal; Akman, Gokhan; Temizkan, Guler

    2006-02-01

    We isolated and characterized a nickel (Ni2+)-resistant mutant (GA1) of Schizosaccharomyces pombe. This mutant strain displayed resistance to both Ni2+ and Zn2+, but not to Cd2+, Co2+, and Cu2+. The growth rate of GA1 increased proportionally with increasing Mg2+ concentrations until 50 mM Mg2+. The GA1 mutation phenotype suggests a defect in Mg2+ uptake. Sequence analysis of the GA1 open reading frame (ORF) O13779, which is homologous to the prokaryotic and eukaryotic CorA Mg2+ transport systems, revealed a point mutation at codon 153 (ccc to acc) resulting in a Pro153Thr substitution in the N-terminus of the CorA domain. Our results provide novel genetic information about Ni2+ resistance in fission yeast. Specifically, that reducing Mg2+ influx through the CorA Mg2+ transport membrane protein confers Ni2+ resistance in S. pombe. We also report that Ni2+ ion detoxification of the fission yeast is related to histidine metabolism and pH. PMID:16444015

  10. Mmi1 RNA surveillance machinery directs RNAi complex RITS to specific meiotic genes in fission yeast

    PubMed Central

    Hiriart, Edwige; Vavasseur, Aurélia; Touat-Todeschini, Leila; Yamashita, Akira; Gilquin, Benoit; Lambert, Emeline; Perot, Jonathan; Shichino, Yuichi; Nazaret, Nicolas; Boyault, Cyril; Lachuer, Joel; Perazza, Daniel; Yamamoto, Masayuki; Verdel, André

    2012-01-01

    RNA interference (RNAi) silences gene expression by acting both at the transcriptional and post-transcriptional levels in a broad range of eukaryotes. In the fission yeast Schizosaccharomyces pombe the RNA-Induced Transcriptional Silencing (RITS) RNAi complex mediates heterochromatin formation at non-coding and repetitive DNA. However, the targeting and role of RITS at other genomic regions, including protein-coding genes, remain unknown. Here we show that RITS localizes to specific meiotic genes and mRNAs. Remarkably, RITS is guided to these meiotic targets by the RNA-binding protein Mmi1 and its associated RNA surveillance machinery that together degrade selective meiotic mRNAs during vegetative growth. Upon sexual differentiation, RITS localization to the meiotic genes and mRNAs is lost. Large-scale identification of Mmi1 RNA targets reveals that RITS subunit Chp1 associates with the vast majority of them. In addition, loss of RNAi affects the effective repression of sexual differentiation mediated by the Mmi1 RNA surveillance machinery. These findings uncover a new mechanism for recruiting RNAi to specific meiotic genes and suggest that RNAi participates in the control of sexual differentiation in fission yeast. PMID:22522705

  11. The structure of cell wall alpha-glucan from fission yeast.

    PubMed

    Grün, Christian H; Hochstenbach, Frans; Humbel, Bruno M; Verkleij, Arie J; Sietsma, J Hans; Klis, Frans M; Kamerling, Johannis P; Vliegenthart, Johannes F G

    2005-03-01

    Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis. PMID:15470229

  12. Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Kakui, Yasutaka; Sunaga, Tomonari; Arai, Kunio; Dodgson, James; Ji, Liang; Csikász-Nagy, Attila; Carazo-Salas, Rafael; Sato, Masamitsu

    2015-06-01

    Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a 'module', can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. PMID:26108218

  13. Dissecting Fission Yeast Shelterin Interactions via MICro-MS Links Disruption of Shelterin Bridge to Tumorigenesis.

    PubMed

    Liu, Jinqiang; Yu, Clinton; Hu, Xichan; Kim, Jin-Kwang; Bierma, Jan C; Jun, Hyun-Ik; Rychnovsky, Scott D; Huang, Lan; Qiao, Feng

    2015-09-29

    Shelterin, a six-member complex, protects telomeres from nucleolytic attack and regulates their elongation by telomerase. Here, we have developed a strategy, called MICro-MS (Mapping Interfaces via Crosslinking-Mass Spectrometry), that combines crosslinking-mass spectrometry and phylogenetic analysis to identify contact sites within the complex. This strategy allowed identification of separation-of-function mutants of fission yeast Ccq1, Poz1, and Pot1 that selectively disrupt their respective interactions with Tpz1. The various telomere dysregulation phenotypes observed in these mutants further emphasize the critical regulatory roles of Tpz1-centered shelterin interactions in telomere homeostasis. Furthermore, the conservation between fission yeast Tpz1-Pot1 and human TPP1-POT1 interactions led us to map a human melanoma-associated POT1 mutation (A532P) to the TPP1-POT1 interface. Diminished TPP1-POT1 interaction caused by hPOT1-A532P may enable unregulated telomere extension, which, in turn, helps cancer cells to achieve replicative immortality. Therefore, our study reveals a connection between shelterin connectivity and tumorigenicity. PMID:26365187

  14. The Actomyosin Ring Recruits Early Secretory Compartments to the Division Site in Fission Yeast

    PubMed Central

    Vjestica, Aleksandar; Tang, Xin-Zi

    2008-01-01

    The ultimate goal of cytokinesis is to establish a membrane barrier between daughter cells. The fission yeast Schizosaccharomyces pombe utilizes an actomyosin-based division ring that is thought to provide physical force for the plasma membrane invagination. Ring constriction occurs concomitantly with the assembly of a division septum that is eventually cleaved. Membrane trafficking events such as targeting of secretory vesicles to the division site require a functional actomyosin ring suggesting that it serves as a spatial landmark. However, the extent of polarization of the secretion apparatus to the division site is presently unknown. We performed a survey of dynamics of several fluorophore-tagged proteins that served as markers for various compartments of the secretory pathway. These included markers for the endoplasmic reticulum, the COPII sites, and the early and late Golgi. The secretion machinery exhibited a marked polarization to the division site. Specifically, we observed an enrichment of the transitional endoplasmic reticulum (tER) accompanied by Golgi cisternae biogenesis. These processes required actomyosin ring assembly and the function of the EFC-domain protein Cdc15p. Cdc15p overexpression was sufficient to induce tER polarization in interphase. Thus, fission yeast polarizes its entire secretory machinery to the cell division site by utilizing molecular cues provided by the actomyosin ring. PMID:18184749

  15. Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality.

    PubMed Central

    Takahashi, K; Yamada, H; Yanagida, M

    1994-01-01

    Precise chromosome transmission in cell division cycle is maintained by a number of genes. The attempt made in the present study was to isolate temperature-sensitive (ts) fission yeast mutants that display high loss rates of minichromosomes at permissive or semipermissive temperature (designated mis). By colony color assay of 539 ts strains that contain a minichromosome, we have identified 12 genetic loci (mis1-mis12) and determined their phenotypes at restrictive temperature. Seven of them are related to cell cycle block phenotype at restrictive temperature, three of them in mitosis. Unequal distribution of regular chromosomes in the daughters is extensive in mis6 and mis12. Cells become inviable after rounds of cell division due to missegregation. The phenotype of mis5 is DNA replication defect and hypersensitivity to UV ray and hydroxyurea. mis5+ encodes a novel member of the ubiquitous MCM family required for the onset of replication. The mis5+ gene is essential for viability and functionally distinct from other previously identified members in fission yeast, cdc21+, nda1+, and nda4+. The mis11 mutant phenotype was the cell division block with reduced cell size. Progression of the G1 and G2 phases is blocked in mis11. The cloned mis11+ gene is identical to prp2+, which is essential for RNA splicing and similar to a mammalian splicing factor U2AF65. Images PMID:7865880

  16. Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast.

    PubMed

    Bendezú, Felipe O; Martin, Sophie G

    2011-01-01

    Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂)-dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP₂-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types. PMID:21148300

  17. Epigenetic Inheritance of Transcriptional Silencing and Switching Competence in Fission Yeast

    PubMed Central

    Thon, G.; Friis, T.

    1997-01-01

    Epigenetic events allow the inheritance of phenotypic changes that are not caused by an alteration in DNA sequence. Here we characterize an epigenetic phenomenon occuring in the mating-type region of fission yeast. Cells of fission yeast switch between the P and M mating-type by interconverting their expressed mating-type cassette between two allelic forms, mat1-P and mat1-M. The switch results from gene conversions of mat1 by two silent cassettes, mat2-P and mat3-M, which are linked to each other and to mat1. GREWAL and KLAR observed that the ability to both switch mat1 and repress transcription near mat2-P and mat3-M was maintained epigenetically in a strain with an 8-kb deletion between mat2 and mat3. Using a strain very similar to theirs, we determined that interconversions between the switching-and silencing-proficient state and the switching and silencing-deficient state occurred less frequently than once per 1000 cell divisions. Although transcriptional silencing was alleviated by the 8-kb deletion, it was not abolished. We performed a mutant search and obtained a class of trans-acting mutations that displayed a strong cumulative effect with the 8-kb deletion. These mutations allow to assess the extent to which silencing is affected by the deletion and provide new insights on the redundancy of the silencing mechanism. PMID:9055078

  18. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl

    2014-02-07

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

  19. Splicing factor Spf30 assists exosome-mediated gene silencing in fission yeast.

    PubMed

    Bernard, Pascal; Drogat, Julie; Dheur, Sonia; Genier, Sylvie; Javerzat, Jean-Paul

    2010-03-01

    Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways. PMID:20028739

  20. Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

    PubMed Central

    Kakui, Yasutaka; Sunaga, Tomonari; Arai, Kunio; Dodgson, James; Ji, Liang; Csikász-Nagy, Attila; Carazo-Salas, Rafael; Sato, Masamitsu

    2015-01-01

    Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. PMID:26108218

  1. Divergence of a conserved elongation factor and transcription regulation in budding and fission yeast.

    PubMed

    Booth, Gregory T; Wang, Isabel X; Cheung, Vivian G; Lis, John T

    2016-06-01

    Complex regulation of gene expression in mammals has evolved from simpler eukaryotic systems, yet the mechanistic features of this evolution remain elusive. Here, we compared the transcriptional landscapes of the distantly related budding and fission yeast. We adapted the Precision Run-On sequencing (PRO-seq) approach to map the positions of RNA polymerase active sites genome-wide in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Additionally, we mapped preferred sites of transcription initiation in each organism using PRO-cap. Unexpectedly, we identify a pause in early elongation, specific to S. pombe, that requires the conserved elongation factor subunit Spt4 and resembles promoter-proximal pausing in metazoans. PRO-seq profiles in strains lacking Spt4 reveal globally elevated levels of transcribing RNA Polymerase II (Pol II) within genes in both species. Messenger RNA abundance, however, does not reflect the increases in Pol II density, indicating a global reduction in elongation rate. Together, our results provide the first base-pair resolution map of transcription elongation in S. pombe and identify divergent roles for Spt4 in controlling elongation in budding and fission yeast. PMID:27197211

  2. Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

    SciTech Connect

    Kjaerulff, Soren

    2005-10-28

    In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae {alpha}-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The {alpha}-factor signal did not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.

  3. Valproic Acid Affects Membrane Trafficking and Cell-Wall Integrity in Fission Yeast

    PubMed Central

    Miyatake, Makoto; Kuno, Takayoshi; Kita, Ayako; Katsura, Kosaku; Takegawa, Kaoru; Uno, Satoshi; Nabata, Toshiya; Sugiura, Reiko

    2007-01-01

    Valproic acid (VPA) is widely used to treat epilepsy and manic-depressive illness. Although VPA has been reported to exert a variety of biochemical effects, the exact mechanisms underlying its therapeutic effects remain elusive. To gain further insights into the molecular mechanisms of VPA action, a genetic screen for fission yeast mutants that show hypersensitivity to VPA was performed. One of the genes that we identified was vps45+, which encodes a member of the Sec1/Munc18 family that is implicated in membrane trafficking. Notably, several mutations affecting membrane trafficking also resulted in hypersensitivity to VPA. These include ypt3+ and ryh1+, both encoding a Rab family protein, and apm1+, encoding the μ1 subunit of the adaptor protein complex AP-1. More importantly, VPA caused vacuolar fragmentation and inhibited the glycosylation and the secretion of acid phosphatase in wild-type cells, suggesting that VPA affects membrane trafficking. Interestingly, the cell-wall-damaging agents such as micafungin or the inhibition of calcineurin dramatically enhanced the sensitivity of wild-type cells to VPA. Consistently, VPA treatment of wild-type cells enhanced their sensitivity to the cell-wall-digesting enzymes. Altogether, our results suggest that VPA affects membrane trafficking, which leads to the enhanced sensitivity to cell-wall damage in fission yeast. PMID:17287531

  4. Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

    PubMed Central

    Stark, Benjamin C.; Sladewski, Thomas E.; Pollard, Luther W.

    2010-01-01

    Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system. PMID:20110347

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

    PubMed

    Brennwald, P; Wise, J A

    1994-02-01

    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-alpha-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-alpha-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. PMID:8203158

  6. Convergent domestication of pogo-like transposases into centromere-binding proteins in fission yeast and mammals

    PubMed Central

    Casola, Claudio; Hucks, Donald; Feschotte, Cédric

    2007-01-01

    The mammalian centromeric protein CENP-B shares significant sequence similarity with three proteins in fission yeast (Abp1, Cbh1 and Cbh2) that also bind centromeres and have essential function for chromosome segregation and centromeric heterochromatin formation. Each of these proteins displays extensive sequence similarity with pogo-like transposases, which have been previously identified in the genomes of various insects and vertebrates, in the protozoan Entamoeba and in plants. Based on this distribution, it has been proposed that the mammalian and fission yeast centromeric proteins are derived from ‘domesticated’ pogo-like transposons. Here we took advantage of the vast amount of sequence information that has become recently available for a wide range of fungal and animal species to investigate the origin of the mammalian CENP-B and yeast CENP-B-like genes. A highly conserved ortholog of CENP-B was detected in 31 species of mammals, including opossum and platypus, but was absent from all non-mammalian species represented in the databases. Similarly, no ortholog of the fission yeast centromeric proteins was identified in any of the various fungal genomes currently available. In contrast, we discovered a plethora of novel pogo-like transposons in diverse invertebrates and vertebrates and in several filamentous fungi. Phylogenetic analysis revealed that the mammalian and fission yeast CENP-B proteins fall into two distinct monophyletic clades, each of which includes a different set of pogo-like transposons. These results are most parsimoniously explained by independent domestication events of pogo-like transposases into centromeric proteins in the mammalian and fission yeast lineages, a case of ‘convergent domestication’. These findings highlight the propensity of transposases to give rise to new host proteins and the potential of transposons as sources of genetic innovation. PMID:17940212

  7. Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach

    SciTech Connect

    Han, Sangjo; Lee, Minho; Chang, Hyeshik; Nam, Miyoung; Park, Han-Oh; Kwak, Youn-Sig; Ha, Hye-jeong; Kim, Dongsup; Hwang, Sung-Ook; Hoe, Kwang-Lae; Kim, Dong-Uk

    2013-07-12

    Highlights: •The first compendium of chemical-genetic profiles form fission yeast was generated. •The first HTS of drug mode-of-action in fission yeast was performed. •The first comparative chemical genetic analysis between two yeasts was conducted. -- Abstract: Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at (http://pombe.kaist.ac.kr/compendium)

  8. Substrate preference of citrus naringenin rhamnosyltransferases and their application to flavonoid glycoside production in fission yeast.

    PubMed

    Ohashi, Takao; Hasegawa, Yuka; Misaki, Ryo; Fujiyama, Kazuhito

    2016-01-01

    Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo (Citrus maxima) and orange (Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 (AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7-O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3-O-glucoside in addition to naringenin-7-O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7-O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1,2RhaT or the Cs1,6RhaT genes as a whole-cell-biocatalyst. PMID:26433966

  9. The Arabidopsis CDC25 induces a short cell length when overexpressed in fission yeast: evidence for cell cycle function.

    PubMed

    Sorrell, D A; Chrimes, D; Dickinson, J R; Rogers, H J; Francis, D

    2005-02-01

    The putative mitotic inducer gene, Arath;CDC25 cloned in Arabidopsis thaliana, was screened for cell cycle function by overexpressing it in Schizosaccharomyces pombe (fission yeast). The expression pattern of Arath;CDC25 was also examined in different tissues of A. thaliana. Fission yeast was transformed with plasmids pREP1 and pREP81 with the Arath;CDC25 gene under the control of the thiamine-repressible nmt promoter. Using reverse transcription-polymerase chain reaction (RT-PCR), the expression of Arath;CDC25 was examined in seedlings, flower buds, mature leaves and stems of A. thaliana; actin (ACT2) was used as a control. In three independent transformants of fission yeast, cultured in the absence of thiamine (T), pREP1::Arath;CDC25 induced a highly significant reduction in mitotic cell length compared with wild type, pREP::Arath;CDC25 +T, and empty vector (pREP1 +/- T). The extent of cell shortening was greater using the stronger pREP1 compared with the weaker pREP81. However, Arath;CDC25 was expressed at low levels in all tissues examined. The data indicate that Arath;CDC25 can function as a mitotic accelerator in fission yeast. However, unlike other plant cell cycle genes, expression of Arath;CDC25 was not enhanced in rapidly dividing compared with non-proliferative Arabidopsis tissues. PMID:15720653

  10. Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast

    SciTech Connect

    Umehara, Takashi; Otta, Yumi; Tsuganezawa, Keiko; Matsumoto, Takehisa; Tanaka, Akiko; Horikoshi, Masami; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2005-11-01

    The histone chaperone cia1 from fission yeast has been overexpressed in E. coli, purified and crystallized using the vapour-diffusion method. In fission yeast, cia1{sup +} is an essential gene that encodes a histone chaperone, a homologue of human CIA (CCG1-interacting factor A) and budding yeast Asf1p (anti-silencing function-1), which both facilitate nucleosome assembly by interacting with the core histones H3/H4. The conserved domain (residues 1–161) of the cia1{sup +}-encoded protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The protein was crystallized in the monoclinic space group C2, with unit-cell parameters a = 79.16, b = 40.53, c = 69.79 Å, β = 115.93° and one molecule per asymmetric unit. The crystal diffracted to beyond 2.10 Å resolution using synchrotron radiation.

  11. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel; Busenlehner, Laura; Marcus, Stevan

    2010-08-20

    Research highlights: {yields} Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. {yields} The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. {yields} Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. {yields} PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  12. Plasticity of fission yeast CENP-A chromatin driven by relative levels of histone H3 and H4.

    PubMed

    Castillo, Araceli G; Mellone, Barbara G; Partridge, Janet F; Richardson, William; Hamilton, Georgina L; Allshire, Robin C; Pidoux, Alison L

    2007-07-01

    The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-A(Cnp1) chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-A(Cnp1) can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-A(Cnp1) chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-A(Cnp1) associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-A(Cnp1) for assembly into central domain chromatin, resulting in less CENP-A(Cnp1) and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-A(Cnp1) influence the extent of DNA at centromeres that is packaged in CENP-A(Cnp1) chromatin and the composition of this chromatin. Thus, CENP-A(Cnp1) chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-A(Cnp1) and other core histones. PMID:17677001

  13. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

  14. Highly condensed chromatins are formed adjacent to subtelomeric and decondensed silent chromatin in fission yeast

    PubMed Central

    Matsuda, Atsushi; Chikashige, Yuji; Ding, Da-Qiao; Ohtsuki, Chizuru; Mori, Chie; Asakawa, Haruhiko; Kimura, Hiroshi; Haraguchi, Tokuko; Hiraoka, Yasushi

    2015-01-01

    It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or ‘knobs'. Knob regions span ∼50 kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe. PMID:26205977

  15. DNA replication components as regulators of epigenetic inheritance--lesson from fission yeast centromere.

    PubMed

    He, Haijin; Gonzalez, Marlyn; Zhang, Fan; Li, Fei

    2014-06-01

    Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome. PMID:24691906

  16. Identification of noncoding transcripts from within CENP-A chromatin at fission yeast centromeres.

    PubMed

    Choi, Eun Shik; Strålfors, Annelie; Castillo, Araceli G; Durand-Dubief, Mickaël; Ekwall, Karl; Allshire, Robin C

    2011-07-01

    The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1). PMID:21531710

  17. Identification of Noncoding Transcripts from within CENP-A Chromatin at Fission Yeast Centromeres*

    PubMed Central

    Choi, Eun Shik; Strålfors, Annelie; Castillo, Araceli G.; Durand-Dubief, Mickaël; Ekwall, Karl; Allshire, Robin C.

    2011-01-01

    The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-ACnp1 chromatin establishment, but the underlying features governing where CENP-ACnp1 chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-ACnp1 associates with gene promoters where histone H3 is depleted by the activity of the Hrp1Chd1 chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-ACnp1 chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-ACnp1. PMID:21531710

  18. Fitness Profiling Links Topoisomerase II Regulation of Centromeric Integrity to Doxorubicin Resistance in Fission Yeast

    PubMed Central

    Nguyen, Thi Thuy Trang; Lim, Julia Sze Lynn; Tang, Richard Ming Yi; Zhang, Louxin; Chen, Ee Sin

    2015-01-01

    Doxorubicin, a chemotherapeutic agent, inhibits the religation step of topoisomerase II (Top2). However, the downstream ramifications of this action are unknown. Here we performed epistasis analyses of top2 with 63 genes representing doxorubicin resistance (DXR) genes in fission yeast and revealed a subset that synergistically collaborate with Top2 to confer DXR. Our findings show that the chromatin-regulating RSC and SAGA complexes act with Top2 in a cluster that is functionally distinct from the Ino80 complex. In various DXR mutants, doxorubicin hypersensitivity was unexpectedly suppressed by a concomitant top2 mutation. Several DXR proteins showed centromeric localization, and their disruption resulted in centromeric defects and chromosome missegregation. An additional top2 mutation could restore centromeric chromatin integrity, suggesting a counterbalance between Top2 and these DXR factors in conferring doxorubicin resistance. Overall, this molecular basis for mitotic catastrophe associated with doxorubicin treatment will help to facilitate drug combinatorial usage in doxorubicin-related chemotherapeutic regimens. PMID:25669599

  19. Uncleavable Nup98–Nup96 is functional in the fission yeast Schizosaccharomyces pombe

    PubMed Central

    Asakawa, Haruhiko; Mori, Chie; Ohtsuki, Chizuru; Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2015-01-01

    Essential nucleoporins Nup98 and Nup96 are coded by a single open reading frame, and produced by autopeptidase cleavage. The autocleavage site of Nup98–Nup96 is highly conserved in a wide range of organisms. To understand the importance of autocleavage, we examined a mutant that produces the Nup98–Nup96 joint molecule as a sole protein product of the nup189+ gene in the fission yeast Schizosaccharomyces pombe. Cells expressing only the joint molecule were found to be viable. This result indicates that autocleavage of Nup98–Nup96 is dispensable for cell growth, at least under normal culture conditions in S. pombe. PMID:26137436

  20. Fission yeast retrotransposon Tf1 integration is targeted to 5′ ends of open reading frames

    PubMed Central

    Behrens, Ralf; Hayles, Jacky; Nurse, Paul

    2000-01-01

    Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor. We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe. By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome. Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100–420 bp upstream of the translation start site. Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed. PMID:11095681

  1. The fission yeast heterochromatin protein Rik1 is required for telomere clustering during meiosis

    PubMed Central

    Tuzon, Creighton T.; Borgstrom, Britta; Weilguny, Dietmar; Egel, Richard; Cooper, Julia Promisel; Nielsen, Olaf

    2004-01-01

    Telomeres share the ability to silence nearby transcription with heterochromatin, but the requirement of heterochromatin proteins for most telomere functions is unknown. The fission yeast Rik1 protein is required for heterochromatin formation at centromeres and the mating-type locus, as it recruits the Clr4 histone methyltransferase, whose modification of histone H3 triggers binding by Swi6, a conserved protein involved in spreading of heterochromatin. Here, we demonstrate that Rik1 and Clr4, but not Swi6, are required along with the telomere protein Taz1 for crucial chromosome movements during meiosis. However, Rik1 is dispensable for the protective roles of telomeres in preventing chromosome end-fusion. Thus, a Swi6-independent heterochromatin function distinct from that at centromeres and the mating-type locus operates at telomeres during sexual differentiation. PMID:15197176

  2. Measurements and models of synchronous growth of fission yeast induced by temperature oscillations. [Schizosaccharomyces pombe

    SciTech Connect

    Agar, D.W.; Bailey, J.E.

    1982-01-01

    Pulsing of temperature in a fermentor at intervals coincident with cell generation time was used to induce synchrony in a population of the fission yeast Schizosaccharomyces pombe. Measurements of culture protein, RNA, and DNA during synchronous growth confirm continuous synthesis of protein and RNA and discontinuous synthesis of DNA as previously reported. Flow microfluorometry of populations at different times during the synchrony cycle was used to monitor the changes in single-cell protein, RNA, and DNA frequency functions. These measurements illustrate very clearly the degree of synchrony and patterns of macromolecular synthesis and also confirm previous estimates of the cellular protein contents characteristic of dividing cells. Additional insights into single-cell kinetics and division controls are provided by two-parameter flow microfluorometry measurements and by mathematical modeling of population dynamics. Such data are necessary foundations for robust population balance models of microbial processes. (Refs. 31).

  3. Fission yeast IQGAP arranges actin filaments into the cytokinetic contractile ring.

    PubMed

    Takaine, Masak; Numata, Osamu; Nakano, Kentaro

    2009-10-21

    The contractile ring (CR) consists of bundled actin filaments and myosin II; however, the actin-bundling factor remains elusive. We show that the fission yeast Schizosaccharomyces pombe IQGAP Rng2 is involved in the generation of CR F-actin and required for its arrangement into a ring. An N-terminal fragment of Rng2 is necessary for the function of Rng2 and is localized to CR F-actin. In vitro the fragment promotes actin polymerization and forms linear arrays of F-actin, which are resistant to the depolymerization induced by the actin-depolymerizing factor Adf1. Our findings indicate that Rng2 is involved in the generation of CR F-actin and simultaneously bundles the filaments and regulates its dynamics by counteracting the effects of Adf1, thus enabling the reconstruction of CR F-actin bundles, which provides an insight into the physical properties of the building blocks that comprise the CR. PMID:19713940

  4. Genome-Wide Screening for Genes Associated with FK506 Sensitivity in Fission Yeast

    PubMed Central

    Ma, Yan; Jiang, Weijuan; Liu, Qingbin; Ryuko, Sayomi; Kuno, Takayoshi

    2011-01-01

    We have been studying calcineurin signal transduction pathway in fission yeast Schizosaccharomyces pombe (S. pombe) by developing a genetic screen for mutants that show hypersensitivity to the immunosuppressive calcineurin inhibitor FK506 (tacrolimus). In the present study, to identify nonessential genes that are functionally related to the calcineurin signaling pathway, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 72 deletion strains to be FK506 sensitive. These 72 genes are classified into nine functional groups to include membrane trafficking (16 genes), signal transduction (10 genes), ubiquitination (8 genes), chromatin remodeling (6 genes), cytokinesis (4 genes), ribosomal protein (3 genes), RNA binding protein (3 genes), and a variety of other known functions (17 genes) or still unknown functions (5 genes) in the biological system. In our previous screening of FK506-sensitive mutants we isolated several membrane-trafficking mutants showing defective cell wall integrity. Here, we further examined the vacuolar fusion, the v-SNARE synaptobrevin Syb1 localization, and the sensitivity to the β-glucan synthase inhibitor micafungin in these 72 FK506-sensitive strains. Results showed that 25 deletion strains exhibited abnormal vacuole fusion, 19 deletion strains exhibited Syb1 mislocalization, and 14 deletion strains exhibited both abnormal vacuole fusion and Syb1 mislocalization, while 42 deletion strains showed both normal vacuole fusion and Syb1 localization. Likewise, 16 deletion strains showed sensitivity to micafungin. Altogether, our present study indicates that calcineurin mediates a plethora of physiological processes in fission yeast, and that calcineurin is extensively involved in cross-talk between signaling pathways. PMID:21850271

  5. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    SciTech Connect

    Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji; Habu, Toshiyuki; Hiraoka, Yasushi; Maki, Takahisa; Hayashi, Ikuko; Obuse, Chikashi; Matsumoto, Tomohiro

    2012-02-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

  6. Mga2 Transcription Factor Regulates an Oxygen-responsive Lipid Homeostasis Pathway in Fission Yeast.

    PubMed

    Burr, Risa; Stewart, Emerson V; Shao, Wei; Zhao, Shan; Hannibal-Bach, Hans Kristian; Ejsing, Christer S; Espenshade, Peter J

    2016-06-01

    Eukaryotic lipid synthesis is oxygen-dependent with cholesterol synthesis requiring 11 oxygen molecules and fatty acid desaturation requiring 1 oxygen molecule per double bond. Accordingly, organisms evaluate oxygen availability to control lipid homeostasis. The sterol regulatory element-binding protein (SREBP) transcription factors regulate lipid homeostasis. In mammals, SREBP-2 controls cholesterol biosynthesis, whereas SREBP-1 controls triacylglycerol and glycerophospholipid biosynthesis. In the fission yeast Schizosaccharomyces pombe, the SREBP-2 homolog Sre1 regulates sterol homeostasis in response to changing sterol and oxygen levels. However, notably missing is an SREBP-1 analog that regulates triacylglycerol and glycerophospholipid homeostasis in response to low oxygen. Consistent with this, studies have shown that the Sre1 transcription factor regulates only a fraction of all genes up-regulated under low oxygen. To identify new regulators of low oxygen adaptation, we screened the S. pombe nonessential haploid deletion collection and identified 27 gene deletions sensitive to both low oxygen and cobalt chloride, a hypoxia mimetic. One of these genes, mga2, is a putative transcriptional activator. In the absence of mga2, fission yeast exhibited growth defects under both normoxia and low oxygen conditions. Mga2 transcriptional targets were enriched for lipid metabolism genes, and mga2Δ cells showed disrupted triacylglycerol and glycerophospholipid homeostasis, most notably with an increase in fatty acid saturation. Indeed, addition of exogenous oleic acid to mga2Δ cells rescued the observed growth defects. Together, these results establish Mga2 as a transcriptional regulator of triacylglycerol and glycerophospholipid homeostasis in S. pombe, analogous to mammalian SREBP-1. PMID:27053105

  7. A Genetic and Pharmacological Analysis of Isoprenoid Pathway by LC-MS/MS in Fission Yeast

    PubMed Central

    Takami, Tomonori; Fang, Yue; Zhou, Xin; Jaiseng, Wurentuya; Ma, Yan; Kuno, Takayoshi

    2012-01-01

    Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level. PMID:23145048

  8. Role of the α-Glucanase Agn1p in Fission-Yeast Cell Separation

    PubMed Central

    Dekker, Nick; Speijer, Dave; Grün, Christian H.; van den Berg, Marlene; de Haan, Annett; Hochstenbach, Frans

    2004-01-01

    Cell division in the fission yeast Schizosaccharomyces pombe yields two equal-sized daughter cells. Medial fission is achieved by deposition of a primary septum flanked by two secondary septa within the dividing cell. During the final step of cell division, cell separation, the primary septum is hydrolyzed by an endo-(1,3)-β-glucanase, Eng1p. We reasoned that the cell wall material surrounding the septum, referred to here as the septum edging, also must be hydrolyzed before full separation of the daughter cells can occur. Because the septum edging contains (1,3)-α-glucan, we investigated the cellular functions of the putative (1,3)-α-glucanases Agn1p and Agn2p. Whereas agn2 deletion results in a defect in endolysis of the ascus wall, deletion of agn1 leads to clumped cells that remained attached to each other by septum-edging material. Purified Agn1p hydrolyzes (1,3)-α-glucan predominantly into pentasaccharides, indicating an endo-catalytic mode of hydrolysis. Furthermore, we show that the transcription factors Sep1p and Ace2p regulate both eng1 and agn1 expression in a cell cycle-dependent manner. We propose that Agn1p acts in concert with Eng1p to achieve efficient cell separation, thereby exposing the secondary septa as the new ends of the daughter cells. PMID:15194814

  9. Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast.

    PubMed

    Riveline, Daniel; Thiagarajan, Raghavan; Lehn, Jean-Marie; Carlier, Marie-France

    2014-01-01

    Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research. PMID:25664996

  10. Cdc42 explores the cell periphery for mate selection in fission yeast.

    PubMed

    Bendezú, Felipe O; Martin, Sophie G

    2013-01-01

    How cells polarize in response to external cues is a fundamental biological problem. For mating, yeast cells orient growth toward the source of a pheromone gradient produced by cells of the opposite mating type. Polarized growth depends on the small GTPase Cdc42, a central eukaryotic polarity regulator that controls signaling, cytoskeleton polarization, and vesicle trafficking. However, the mechanisms of polarity establishment and mate selection in complex cellular environments are poorly understood. Here we show that, in fission yeast, low-level pheromone signaling promotes a novel polarization state, where active Cdc42, its GEF Scd1, and scaffold Scd2 form colocalizing dynamic zones that sample the periphery of the cell. Two direct Cdc42 effectors--actin cables marked by myosin V Myo52 and the exocyst complex labeled by Sec6 and Sec8--also dynamically colocalize with active Cdc42. However, these cells do not grow due to a block in the exocytosis of cell wall synthases Bgs1 and Bgs4. High-level pheromone stabilizes active Cdc42 zones and promotes cell wall synthase exocytosis and polarized growth. However, in the absence of prior low-level pheromone signaling, exploration fails, and cells polarize growth at cell poles by default. Consequently, these cells show altered partner choice, mating preferentially with sister rather than nonsister cells. Thus, Cdc42 exploration serves to orient growth for partner selection. This process may also promote genetic diversification. PMID:23200991

  11. Virtual Nuclear Envelope Breakdown and Its Regulators in Fission Yeast Meiosis.

    PubMed

    Asakawa, Haruhiko; Yang, Hui-Ju; Hiraoka, Yasushi; Haraguchi, Tokuko

    2016-01-01

    Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called "open mitosis." In contrast, many fungi undergo a process termed "closed mitosis" in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis (anaphase of the second meiosis; called "anaphase II") when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This "virtual" nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis. PMID:26870731

  12. Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast

    PubMed Central

    Riveline, Daniel; Thiagarajan, Raghavan; Lehn, Jean-Marie; Carlier, Marie-France

    2014-01-01

    Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research. PMID:25664996

  13. Virtual Nuclear Envelope Breakdown and Its Regulators in Fission Yeast Meiosis

    PubMed Central

    Asakawa, Haruhiko; Yang, Hui-Ju; Hiraoka, Yasushi; Haraguchi, Tokuko

    2016-01-01

    Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called “open mitosis.” In contrast, many fungi undergo a process termed “closed mitosis” in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis (anaphase of the second meiosis; called “anaphase II”) when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This “virtual” nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis. PMID:26870731

  14. TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast.

    PubMed

    Rallis, Charalampos; Codlin, Sandra; Bähler, Jürg

    2013-08-01

    Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond. PMID:23551936

  15. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    SciTech Connect

    Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki; Shinohara, Akira; Maekawa, Shohei; Miyamoto, Masaaki

    2013-11-29

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

  16. Rab-family GTPase regulates TOR complex 2 signaling in fission yeast

    PubMed Central

    Tatebe, Hisashi; Morigasaki, Susumu; Murayama, Shinichi; Zeng, Cui Tracy; Shiozaki, Kazuhiro

    2010-01-01

    Summary Background From yeast to human, TOR (Target Of Rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators. Results We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2–Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyper-phosphorylation. The loss of functional Ryh1, TORC2 or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe and therefore, Rab6 may be a potential activator of TORC2 in mammals. Conclusions In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2–Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe. PMID:21035342

  17. Cytokinesis-Based Constraints on Polarized Cell Growth in Fission Yeast

    PubMed Central

    Bohnert, K. Adam; Gould, Kathleen L.

    2012-01-01

    The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state. PMID:23093943

  18. Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth

    SciTech Connect

    Takahashi, Hidekazu; Shirai, Atsuko; Matsuyama, Akihisa; Yoshida, Minoru

    2011-03-04

    Research highlights: {yields} Fission yeast manganese superoxide dismutase (MnSOD) is acetylated. {yields} The mitochondrial targeting sequence (MTS) is required for the acetylation of MnSOD. {yields} The MTS is not crucial for MnSOD activity, but is important for respiratory growth. {yields} Posttranslational regulation of MnSOD differs between budding and fission yeast. -- Abstract: Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

  19. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

    PubMed Central

    Rallis, Charalampos; López-Maury, Luis; Georgescu, Teodora; Pancaldi, Vera; Bähler, Jürg

    2014-01-01

    Summary Target of rapamycin complex 1 (TORC1), which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not. PMID:24463365

  20. Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

    PubMed Central

    Ortiz, D F; Kreppel, L; Speiser, D M; Scheel, G; McDonald, G; Ow, D W

    1992-01-01

    In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants. Images PMID:1396551

  1. Wall mechanics and exocytosis define the shape of growth domains in fission yeast

    NASA Astrophysics Data System (ADS)

    Abenza, Juan F.; Couturier, Etienne; Dodgson, James; Dickmann, Johanna; Chessel, Anatole; Dumais, Jacques; Salas, Rafael E. Carazo

    2015-10-01

    The amazing structural variety of cells is matched only by their functional diversity, and reflects the complex interplay between biochemical and mechanical regulation. How both regulatory layers generate specifically shaped cellular domains is not fully understood. Here, we report how cell growth domains are shaped in fission yeast. Based on quantitative analysis of cell wall expansion and elasticity, we develop a model for how mechanics and cell wall assembly interact and use it to look for factors underpinning growth domain morphogenesis. Surprisingly, we find that neither the global cell shape regulators Cdc42-Scd1-Scd2 nor the major cell wall synthesis regulators Bgs1-Bgs4-Rgf1 are reliable predictors of growth domain geometry. Instead, their geometry can be defined by cell wall mechanics and the cortical localization pattern of the exocytic factors Sec6-Syb1-Exo70. Forceful re-directioning of exocytic vesicle fusion to broader cortical areas induces proportional shape changes to growth domains, demonstrating that both features are causally linked.

  2. The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis

    PubMed Central

    Zhou, Zhou; Munteanu, Emilia Laura; He, Jun; Ursell, Tristan; Bathe, Mark; Huang, Kerwyn Casey; Chang, Fred

    2015-01-01

    The functions of the actin-myosin–based contractile ring in cytokinesis remain to be elucidated. Recent findings show that in the fission yeast Schizosaccharomyces pombe, cleavage furrow ingression is driven by polymerization of cell wall fibers outside the plasma membrane, not by the contractile ring. Here we show that one function of the ring is to spatially coordinate septum cell wall assembly. We develop an improved method for live-cell imaging of the division apparatus by orienting the rod-shaped cells vertically using microfabricated wells. We observe that the septum hole and ring are circular and centered in wild-type cells and that in the absence of a functional ring, the septum continues to ingress but in a disorganized and asymmetric manner. By manipulating the cleavage furrow into different shapes, we show that the ring promotes local septum growth in a curvature-dependent manner, allowing even a misshapen septum to grow into a more regular shape. This curvature-dependent growth suggests a model in which contractile forces of the ring shape the septum cell wall by stimulating the cell wall machinery in a mechanosensitive manner. Mechanical regulation of the cell wall assembly may have general relevance to the morphogenesis of walled cells. PMID:25355954

  3. Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function.

    PubMed Central

    Pidoux, A L; LeDizet, M; Cande, W Z

    1996-01-01

    We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle. Images PMID:8898367

  4. The fission yeast CENP-B protein Abp1 prevents pervasive transcription of repetitive DNA elements.

    PubMed

    Daulny, Anne; Mejía-Ramírez, Eva; Reina, Oscar; Rosado-Lugo, Jesus; Aguilar-Arnal, Lorena; Auer, Herbert; Zaratiegui, Mikel; Azorin, Fernando

    2016-10-01

    It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites. PMID:27345571

  5. Cellular economy in fission yeast cells continuously cultured with limited nitrogen resources

    PubMed Central

    Chikashige, Yuji; Arakawa, Shin'ichi; Leibnitz, Kenji; Tsutsumi, Chihiro; Mori, Chie; Osakada, Hiroko; Murata, Masayuki; Haraguchi, Tokuko; Hiraoka, Yasushi

    2015-01-01

    In ribosome biogenesis, a large fraction of ribosomes is used for producing ribosomal proteins themselves. Here, we applied simulation and experimentation to determine what fraction of ribosomes should be allocated for the synthesis of ribosomal proteins to optimize cellular economy for growth. We define the “r-fraction” as the fraction of mRNA of the ribosomal protein genes out of the total mRNA, and we simulated the effect of the r-fraction on the number of ribosomes. We then empirically measured the amount of protein and RNA in fission yeast cells cultured with high and low nitrogen sources. In the cells cultured with a low nitrogen source, the r-fraction decreased from 0.46 to 0.42 with a 40% reduction of rRNA, but the reduction of the total protein was smaller at 30%. These results indicate that the r-fraction is internally controlled to optimize the efficiency of protein synthesis at a limited cellular cost. PMID:26486373

  6. The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

    PubMed Central

    Fankhauser, C; Simanis, V

    1994-01-01

    Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation. Images PMID:8039497

  7. Wall mechanics and exocytosis define the shape of growth domains in fission yeast

    PubMed Central

    Abenza, Juan F.; Couturier, Etienne; Dodgson, James; Dickmann, Johanna; Chessel, Anatole; Dumais, Jacques; Salas, Rafael E. Carazo

    2015-01-01

    The amazing structural variety of cells is matched only by their functional diversity, and reflects the complex interplay between biochemical and mechanical regulation. How both regulatory layers generate specifically shaped cellular domains is not fully understood. Here, we report how cell growth domains are shaped in fission yeast. Based on quantitative analysis of cell wall expansion and elasticity, we develop a model for how mechanics and cell wall assembly interact and use it to look for factors underpinning growth domain morphogenesis. Surprisingly, we find that neither the global cell shape regulators Cdc42-Scd1-Scd2 nor the major cell wall synthesis regulators Bgs1-Bgs4-Rgf1 are reliable predictors of growth domain geometry. Instead, their geometry can be defined by cell wall mechanics and the cortical localization pattern of the exocytic factors Sec6-Syb1-Exo70. Forceful re-directioning of exocytic vesicle fusion to broader cortical areas induces proportional shape changes to growth domains, demonstrating that both features are causally linked. PMID:26455310

  8. Characterization of a UV endonuclease gene from the fission yeast Schizosaccharomyces pombe and its bacterial homolog.

    PubMed Central

    Takao, M; Yonemasu, R; Yamamoto, K; Yasui, A

    1996-01-01

    From the fission yeast Schizosaccharomyces pombe, a cDNA fragment was isolated, which confers UV resistance on repair deficient Escherichia coli host cells. The cloned cDNA encodes a protein of 68,815 Da, which has a 36.6% identity of amino acid sequence with the previously identified 74 kDa UV endonuclease of the filamentous fungus Neurospora crassa. Analysis of several truncated gene constructs shows that only the C-terminal two thirds region, which has 54% identity of amino acid sequence with the C-terminal region of the Neurospora homolog, is necessary for complementing activity of UV-sensitivity in the E. coli host cells. Purified recombinant protein from E. coli host cells incises both UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts at the sites immediately 5' to the DNA damage in the same fashion as the Neurospora protein. Furthermore, a bacterial homologous sequence was isolated from Bacillus subtilis and shows a similar complementing activity of UV sensitivity in E. coli host cells, indicating a wide distribution of this alternative excision repair mechanism in life. PMID:8614629

  9. Wall mechanics and exocytosis define the shape of growth domains in fission yeast.

    PubMed

    Abenza, Juan F; Couturier, Etienne; Dodgson, James; Dickmann, Johanna; Chessel, Anatole; Dumais, Jacques; Carazo Salas, Rafael E

    2015-01-01

    The amazing structural variety of cells is matched only by their functional diversity, and reflects the complex interplay between biochemical and mechanical regulation. How both regulatory layers generate specifically shaped cellular domains is not fully understood. Here, we report how cell growth domains are shaped in fission yeast. Based on quantitative analysis of cell wall expansion and elasticity, we develop a model for how mechanics and cell wall assembly interact and use it to look for factors underpinning growth domain morphogenesis. Surprisingly, we find that neither the global cell shape regulators Cdc42-Scd1-Scd2 nor the major cell wall synthesis regulators Bgs1-Bgs4-Rgf1 are reliable predictors of growth domain geometry. Instead, their geometry can be defined by cell wall mechanics and the cortical localization pattern of the exocytic factors Sec6-Syb1-Exo70. Forceful re-directioning of exocytic vesicle fusion to broader cortical areas induces proportional shape changes to growth domains, demonstrating that both features are causally linked. PMID:26455310

  10. A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression.

    PubMed

    Kühl, Inge; Dujeancourt, Laurent; Gaisne, Mauricette; Herbert, Christopher J; Bonnefoy, Nathalie

    2011-10-01

    Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast Schizosaccharomyces pombe and identified, in addition to the mitochondrial RNA polymerase, eight proteins all of which localized to the mitochondria, and showed some association with the membrane. The absence of all but one of these PPR proteins leads to a respiratory deficiency and modified patterns of steady state mt-mRNAs or newly synthesized mitochondrial proteins. Some cause a general defect, whereas others affect specific mitochondrial RNAs, either coding or non-coding: cox1, cox2, cox3, 15S rRNA, atp9 or atp6, sometimes leading to secondary defects. Interestingly, the two possible homologs of LRPPRC, ppr4 and ppr5, play opposite roles in the expression of the cox1 mt-mRNA, ppr4 being the first mRNA-specific translational activator identified in S. pombe, whereas ppr5 appears to be a general negative regulator of mitochondrial translation. PMID:21727087

  11. Spatial control of calcineurin in response to heat shock in fission yeast.

    PubMed

    Higa, Mari; Kita, Ayako; Hagihara, Kanako; Kitai, Yuki; Doi, Akira; Nagasoko, Rie; Satoh, Ryosuke; Sugiura, Reiko

    2015-02-01

    In fission yeast, Ppb1, the Ca2+/calmodulin-dependent protein phosphatase calcineurin regulates multiple biological processes, such as cytokinesis, Ca2+-homeostasis, membrane trafficking and cell wall integrity. Calcineurin dephosphorylates the Prz1 transcription factor, leading to its nuclear translocation and gene expression under the control of CDRE (calcineurin-dependent response element). Although the calcineurin-mediated spatial control of downstream transcription factors has been intensively studied in many organisms, less is known about the spatial regulation of calcineurin on stresses. Here, we show that heat shock stimulates calcineurin-dependent nuclear translocation of Prz1 and CDRE-dependent gene expression. Notably, calcineurin exhibited a dramatic change in subcellular localization, translocating from diffuse cytoplasmic to dot-like structures on heat shock. The calcineurin dots colocalized with Dcp2 or Pabp, the constituent of P-bodies or stress granules, respectively, thus suggesting that calcineurin is a component of RNA granules under heat shock. Importantly, the calcineurin inhibitor FK506 markedly inhibited the accumulation of calcineurin granules, whereas the constitutively active calcineurin strongly accumulated in the granules on heat shock, suggesting that phosphatase activity is important for calcineurin localization. Notably, the depletion of calcineurin induced a rapid appearance of Nrd1- and Pabp-positive RNA granules. The possible roles of calcineurin in response to heat shock will be discussed. PMID:25529221

  12. Multiple regulation of Rad51-mediated homologous recombination by fission yeast Fbh1.

    PubMed

    Tsutsui, Yasuhiro; Kurokawa, Yumiko; Ito, Kentaro; Siddique, Md Shahjahan P; Kawano, Yumiko; Yamao, Fumiaki; Iwasaki, Hiroshi

    2014-08-01

    Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated. PMID:25165823

  13. Coordination of DNA damage tolerance mechanisms with cell cycle progression in fission yeast

    PubMed Central

    Callegari, A. John; Kelly, Thomas J.

    2016-01-01

    ABSTRACT DNA damage tolerance (DDT) mechanisms allow cells to synthesize a new DNA strand when the template is damaged. Many mutations resulting from DNA damage in eukaryotes are generated during DDT when cells use the mutagenic translesion polymerases, Rev1 and Polζ, rather than mechanisms with higher fidelity. The coordination among DDT mechanisms is not well understood. We used live-cell imaging to study the function of DDT mechanisms throughout the cell cycle of the fission yeast Schizosaccharomyces pombe. We report that checkpoint-dependent mitotic delay provides a cellular mechanism to ensure the completion of high fidelity DDT, largely by homology-directed repair (HDR). DDT by mutagenic polymerases is suppressed during the checkpoint delay by a mechanism dependent on Rad51 recombinase. When cells pass the G2/M checkpoint and can no longer delay mitosis, they completely lose the capacity for HDR and simultaneously exhibit a requirement for Rev1 and Polζ. Thus, DDT is coordinated with the checkpoint response so that the activity of mutagenic polymerases is confined to a vulnerable period of the cell cycle when checkpoint delay and HDR are not possible. PMID:26652183

  14. Centromeric motion facilitates the mobility of interphase genomic regions in fission yeast

    PubMed Central

    Kim, Kyoung-Dong; Tanizawa, Hideki; Iwasaki, Osamu; Corcoran, Christopher J.; Capizzi, Joseph R.; Hayden, James E.; Noma, Ken-ichi

    2013-01-01

    Summary Dispersed genetic elements, such as retrotransposons and Pol-III-transcribed genes, including tRNA and 5S rRNA, cluster and associate with centromeres in fission yeast through the function of condensin. However, the dynamics of these condensin-mediated genomic associations remains unknown. We have examined the 3D motions of genomic loci including the centromere, telomere, rDNA repeat locus, and the loci carrying Pol-III-transcribed genes or long-terminal repeat (LTR) retrotransposons in live cells at as short as 1.5-second intervals. Treatment with carbendazim (CBZ), a microtubule-destabilizing agent, not only prevents centromeric motion, but also reduces the mobility of the other genomic loci during interphase. Further analyses demonstrate that condensin-mediated associations between centromeres and the genomic loci are clonal, infrequent and transient. However, when associated, centromeres and the genomic loci migrate together in a coordinated fashion. In addition, a condensin mutation that disrupts associations between centromeres and the genomic loci results in a concomitant decrease in the mobility of the loci. Our study suggests that highly mobile centromeres pulled by microtubules in cytoplasm serve as ‘genome mobility elements’ by facilitating physical relocations of associating genomic regions. PMID:23986481

  15. Nup132 modulates meiotic spindle attachment in fission yeast by regulating kinetochore assembly

    PubMed Central

    Yang, Hui-Ju; Asakawa, Haruhiko; Haraguchi, Tokuko

    2015-01-01

    During meiosis, the kinetochore undergoes substantial reorganization to establish monopolar spindle attachment. In the fission yeast Schizosaccharomyces pombe, the KNL1–Spc7-Mis12-Nuf2 (KMN) complex, which constitutes the outer kinetochore, is disassembled during meiotic prophase and is reassembled before meiosis I. Here, we show that the nucleoporin Nup132 is required for timely assembly of the KMN proteins: In the absence of Nup132, Mis12 and Spc7 are precociously assembled at the centromeres during meiotic prophase. In contrast, Nuf2 shows timely dissociation and reappearance at the meiotic centromeres. We further demonstrate that depletion of Nup132 activates the spindle assembly checkpoint in meiosis I, possibly because of the increased incidence of erroneous spindle attachment at sister chromatids. These results suggest that precocious assembly of the kinetochores leads to the meiosis I defects observed in the nup132-disrupted mutant. Thus, we propose that Nup132 plays an important role in establishing monopolar spindle attachment at meiosis I through outer kinetochore reorganization at meiotic prophase. PMID:26483559

  16. Chromosomes rein back the spindle pole body during horsetail movement in fission yeast meiosis.

    PubMed

    Chikashige, Yuji; Yamane, Miho; Okamasa, Kasumi; Mori, Chie; Fukuta, Noriko; Matsuda, Atsushi; Haraguchi, Tokuko; Hiraoka, Yasushi

    2014-01-01

    In meiosis, pairing and recombination of homologous chromosomes are crucial for the correct segregation of chromosomes, and substantial movements of chromosomes are required to achieve homolog pairing. During this process, it is known that telomeres cluster to form a bouquet arrangement of chromosomes. The fission yeast Schizosaccharomyces pombe provides a striking example of bouquet formation, after which the entire nucleus oscillates between the cell poles (these oscillations are generally called horsetail nuclear movements) while the telomeres remain clustered to the spindle pole body (SPB; a centrosome-equivalent structure in fungi) at the leading edge of the moving nucleus. S. pombe mutants defective in telomere clustering frequently form aberrant spindles, such as monopolar or nonpolar spindles, leading to missegregation of the chromosomes at the subsequent meiotic divisions. Here we demonstrate that such defects in meiotic spindle formation caused by loss of meiotic telomere clustering are rescued when nuclear movement is prevented. On the other hand, stopping nuclear movement does not rescue defects in telomere clustering, nor chromosome missgregation even in cells that have formed a bipolar spindle. These results suggest that movement of the SPB without attachment of telomeres leads to the formation of aberrant spindles, but that recovering bipolar spindles is not sufficient for rescue of chromosome missegregation in mutants lacking telomere clustering. PMID:24954111

  17. Glucosidase II β Subunit Modulates N-Glycan Trimming in Fission Yeasts and Mammals

    PubMed Central

    Stigliano, Ivan D.; Caramelo, Julio J.; Labriola, Carlos A.; Parodi, Armando J.

    2009-01-01

    Glucosidase II (GII) plays a key role in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is responsible for the sequential removal of the two innermost glucose residues from the glycan (Glc3Man9GlcNAc2) transferred to Asn residues in proteins. GII participates in the calnexin/calreticulin cycle; it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimer whose α subunit (GIIα) bears the glycosyl hydrolase active site, whereas its β subunit (GIIβ) role is controversial and has been reported to be involved in GIIα ER retention and folding. Here, we report that in the absence of GIIβ, the catalytic subunit GIIα of the fission yeast Schizosaccharomyces pombe (an organism displaying a glycoprotein folding quality control mechanism similar to that occurring in mammalian cells) folds to an active conformation able to hydrolyze p-nitrophenyl α-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc2Man9GlcNAc2 (G2M9) and Glc1Man9GlcNAc2 (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIβ and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIβ modulates G2M9 and G1M9 trimming. PMID:19605557

  18. Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Senoo, Takanori; Yamanaka, Mayumi; Nakamura, Atori; Terashita, Tomoki; Kawano, Shinji; Ikeda, Shogo

    2016-08-01

    Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10kb) in mtDNA were quantitatively amplified using 0.1ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging. PMID:27236021

  19. Sim4: a novel fission yeast kinetochore protein required for centromeric silencing and chromosome segregation.

    PubMed

    Pidoux, Alison L; Richardson, William; Allshire, Robin C

    2003-04-28

    Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore. PMID:12719471

  20. The DASH complex and Klp5/Klp6 kinesin coordinate bipolar chromosome attachment in fission yeast.

    PubMed

    Sanchez-Perez, Isabel; Renwick, Steven J; Crawley, Karen; Karig, Inga; Buck, Vicky; Meadows, John C; Franco-Sanchez, Alejandro; Fleig, Ursula; Toda, Takashi; Millar, Jonathan B A

    2005-08-17

    We identified a truncated allele of dam1 as a multicopy suppressor of the sensitivity of cdc13-117 (cyclin B) and mal3-1 (EB-1) cells to thiabendazole, a microtubule poison. We find that Dam1 binds to the plus end of spindle microtubules and kinetochores as cells enter mitosis and this is dependent on other components of the fission yeast DASH complex, including Ask1, Duo1, Spc34 and Dad1. By contrast, Dad1 remains bound to kinetochores throughout the cell cycle and its association is dependent on the Mis6 and Mal2, but not Mis12, Nuf2 or Cnp1, kinetochore proteins. In cells lacking Dam1, or other components of the DASH complex, anaphase is delayed due to activation of the spindle assembly checkpoint and lagging sister chromatids are frequently observed and occasionally sister chromatid pairs segregate to the same spindle pole. We find that the mitotic centromere-associated Klp5/Klp6 kinesin complex is essential in cells lacking components of the DASH complex. Cells lacking both Dam1 and Klp5 undergo a first cell cycle arrest in mitosis due to a failure to establish bipolar chromosome attachment. PMID:16079915

  1. Diverse roles of HP1 proteins in heterochromatin assembly and functions in fission yeast.

    PubMed

    Fischer, Tamás; Cui, Bowen; Dhakshnamoorthy, Jothy; Zhou, Ming; Rubin, Chanan; Zofall, Martin; Veenstra, Timothy D; Grewal, Shiv I S

    2009-06-01

    Conserved chromosomal HP1 proteins capable of binding to histone H3 methylated at lysine 9 are believed to provide a dynamic platform for the recruitment and/or spreading of various regulatory proteins involved in diverse chromosomal processes. The fission yeast Schizosaccharomyces pombe HP1 family members Chp2 and Swi6 are important for heterochromatin assembly and transcriptional silencing, but their precise roles are not fully understood. Here, we show that Swi6 and Chp2 associate with histone deacetylase (HDAC) protein complexes containing class I HDAC Clr6 and class II HDAC Clr3 (a component of Snf2/HDAC repressor complex), which are critical for transcriptional silencing of centromeric repeats targeted by the heterochromatin machinery. Mapping of RNA polymerase (Pol) II distribution in single and double mutant backgrounds revealed that Swi6 and Chp2 proteins and their associated HDAC complexes have overlapping functions in limiting Pol II occupancy across pericentromeric heterochromatin domains. The purified Swi6 fraction also contains factors involved in various chromosomal processes such as chromatin remodeling and DNA replication. Also, Swi6 copurifies with Mis4 protein, a cohesin loading factor essential for sister chromatid cohesion, and with centromere-specific histone H3 variant CENP-A, which is incorporated into chromatin in a heterochromatin-dependent manner. These analyses suggest that among other functions, HP1 proteins associate with chromatin-modifying factors that in turn cooperate to assemble repressive chromatin; thus, precluding accessibility of underlying DNA sequences to transcriptional machinery. PMID:19443688

  2. Biphasic incorporation of centromeric histone CENP-A in fission yeast.

    PubMed

    Takayama, Yuko; Sato, Hiroshi; Saitoh, Shigeaki; Ogiyama, Yuki; Masuda, Fumie; Takahashi, Kohta

    2008-02-01

    CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In Delta ams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in Delta ams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in Delta ams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged. PMID:18077559

  3. Shape and Size of the Fission Yeast Nucleus are governed by Equilibrium Mechanics

    NASA Astrophysics Data System (ADS)

    Lim, Gerald; Huber, Greg; Miller, Jonathan; Sazer, Shelley

    2006-03-01

    Nuclear morphogenesis in the asexual reproduction of Schizosaccharomyces pombe (fission yeast) consists of two stages: (i) volume-doubling growth, in which a round nucleus inflates uniformly, and (ii) division, in which the nucleus undergoes shape changes from round to oblong to peanut to dumbbell before it resolves into two smaller, round daughter nuclei, driven by the formation and elongation of a microtubule-based spindle within the nucleus. The combined volume of the daughter nuclei immediately after division is the same as the volume of the single nucleus at the onset of division. Consequently, the nuclear envelope (NE) area must increase by 26% during division. We are developing a model in order to determine the mechanics governing these shape and size changes. It is based on current knowledge of the nuclear structure, insight from normal and abnormal nuclei, and concepts from the mechanics governing lipid-bilayer membranes. We predict that (a) the NE prefers to be flat, (b) the NE is under tension, (c) the nucleus has an internal pressure, (d) nuclear growth is governed by the Law of Laplace, and (e) some abnormal nuclei behave like vesicles with encapsulated microtubules.

  4. Stress-dependent regulation of the gene encoding thioredoxin reductase from the fission yeast.

    PubMed

    Hong, Sung-Min; Lim, Hye-Won; Kim, Il-Han; Kim, Kanghwa; Park, Eun-Hee; Lim, Chang-Jin

    2004-05-15

    The unique putative gene for thioredoxin reductase (TrxR) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined DNA sequence carries 3125 bp, and encodes the plausible 322 amino acid sequence of TrxR with a molecular mass of 34,618 Da. The S. pombe cells harboring the cloned TrxR gene contain increased TrxR activity, and shows higher survivals on solid media with mercuric chloride or aluminum chloride. The 1526 bp upstream region was fused into promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid. The synthesis of beta-galactosidase from the fusion plasmid pYUTR10 was enhanced by menadione, mercuric chloride, hydrogen peroxide, aluminium chloride and sodium selenite. Menadione significantly enhanced the TrxR mRNA level in the S. pombe cells, which was detected by RT-PCR. Induction of the S. pombe TrxR gene by menadione and mercuric chloride occurs through the mediation of the transcription factor Pap1. These results suggest that the S. pombe TrxR gene is one of the stress response-related genes. PMID:15135546

  5. Requirement of fission yeast Cid14 in polyadenylation of rRNAs.

    PubMed

    Win, Thein Z; Draper, Simon; Read, Rebecca L; Pearce, James; Norbury, Chris J; Wang, Shao-Win

    2006-03-01

    Polyadenylation in eukaryotes is conventionally associated with increased nuclear export, translation, and stability of mRNAs. In contrast, recent studies suggest that the Trf4 and Trf5 proteins, members of a widespread family of noncanonical poly(A) polymerases, share an essential function in Saccharomyces cerevisiae that involves polyadenylation of nuclear RNAs as part of a pathway of exosome-mediated RNA turnover. Substrates for this pathway include aberrantly modified tRNAs and precursors of snoRNAs and rRNAs. Here we show that Cid14 is a Trf4/5 functional homolog in the distantly related fission yeast Schizosaccharomyces pombe. Unlike trf4 trf5 double mutants, cells lacking Cid14 are viable, though they suffer an increased frequency of chromosome missegregation. The Cid14 protein is constitutively nucleolar and is required for normal nucleolar structure. A minor population of polyadenylated rRNAs was identified. These RNAs accumulated in an exosome mutant, and their presence was largely dependent on Cid14, in line with a role for Cid14 in rRNA degradation. Surprisingly, both fully processed 25S rRNA and rRNA processing intermediates appear to be channeled into this pathway. Our data suggest that additional substrates may include the mRNAs of genes involved in meiotic regulation. Polyadenylation-assisted nuclear RNA turnover is therefore likely to be a common eukaryotic mechanism affecting diverse biological processes. PMID:16478992

  6. Fission yeast switches mating type by a replication-recombination coupled process.

    PubMed

    Arcangioli, B; de Lahondès, R

    2000-03-15

    Fission yeast exhibits a homothallic life cycle, in which the mating type of the cell mitotically alternates in a highly regulated fashion. Pedigree analysis of dividing cells has shown that only one of the two sister cells switches mating type. It was shown recently that a site- and strand-specific DNA modification at the mat1 locus precedes mating-type switching. By tracking the fate of mat1 DNA throughout the cell cycle with a PCR assay, we identified a novel DNA intermediate of mating-type switching in S-phase. The time and rate of appearance and disappearance of this DNA intermediate are consistent with a model in which mating-type switching occurs through a replication-recombination coupled pathway. Such a process provides experimental evidence in support of a copy choice recombination model in Schizosaccharomyces pombe mating-type switching and is reminiscent of the sister chromatid recombination used to complete replication in the presence of certain types of DNA damage. PMID:10716938

  7. Dynamic transition of transcription and chromatin landscape during fission yeast adaptation to glucose starvation.

    PubMed

    Oda, Arisa; Takemata, Naomichi; Hirata, Yoshito; Miyoshi, Tomoichiro; Suzuki, Yutaka; Sugano, Sumio; Ohta, Kunihiro

    2015-05-01

    Shortage of glucose, the primary energy source for all organisms, is one of the most critical stresses influencing cell viability. Glucose starvation promptly induces changes in mRNA and noncoding RNA (ncRNA) transcription. We previously reported that glucose starvation induces long ncRNA (lncRNA) transcription in the 5' segment of a fission yeast gluconeogenesis gene (fbp1+), which leads to stepwise chromatin alteration around the fbp1+ promoter and to subsequent robust gene activation. Here, we analyzed genomewide transcription by strand-specific RNA sequencing, together with chromatin landscape by immunoprecipitation sequencing (ChIP-seq). Clustering analysis showed that distinct mRNAs and ncRNAs are induced at the early, middle and later stages of cellular response to glucose starvation. The starvation-induced transcription depends substantially on the stress-responsive transcription factor Atf1. Using a new computer program that examines dynamic changes in expression patterns, we identified ncRNAs with similar behavior to the fbp1+ lncRNA. We confirmed that there are continuous lncRNAs associated with local reduction of histone density. Overlapping with the regions for transcription of these lncRNAs, antisense RNAs are antagonistically transcribed under glucose-rich conditions. These results suggest that Atf1-dependent integrated networks of mRNA and lncRNA govern drastic changes in cell physiology in response to glucose starvation. PMID:25728061

  8. Unique properties of cd-binding peptides induced in fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    Hayashi, Y.; Nakagawa, C.W.; Murasugi, A.

    1986-03-01

    Metallothioneins, a class of low molecular weight cysteine-rich proteins that bind heavy metal ions, have been found in various eucaryotic organisms. When fission yeasts are grown in the presence of high concentration of CdCl/sub 2/, large amounts of Cd-binding peptides (Cd-BP1 and Cd-BP2) are synthesized. While Cd-BP2 shows similarities to mammalian Cd-thioneins in UV and CD spectra, Cd-BP1has a characteristic shoulder at 265 nm in the UV absorption spectrum and shows two marked Cotton bands at 257 nm (negative) and 275 nm (positive). These characteristics of Cd-BP1 are not found in the other Cd-thioneins. The UV and CD spectra differences between reconstituted and native Cd-BP1 suggest the requirement for some additional molecular architecture including another peptide-Cd/sup 2 +/ interaction. Induction of cadystin synthesis is almost exclusive for Cd, but an exception is a small amount of cadystin also induced by the higher concentration of CuCl/sub 2/ (2.5 mM). The UV spectrum of the natural Cu-cadystin complex was similar to that of Cd-BP1. On the basis of these findings the models for Cd-BP1 and Cd-BP2 are proposed.

  9. Endocytosis is essential for dynamic translocation of a syntaxin 1 orthologue during fission yeast meiosis

    PubMed Central

    Kashiwazaki, Jun; Yamasaki, Yuriko; Itadani, Akiko; Teraguchi, Erika; Maeda, Yukari; Shimoda, Chikashi; Nakamura, Taro

    2011-01-01

    Syntaxin is a component of the target soluble N-ethylmaleimide–sensitive factor attachment protein receptor complex, which is responsible for fusion of membrane vesicles at the target membrane. The fission yeast syntaxin 1 orthologue Psy1 is essential for both vegetative growth and spore formation. During meiosis, Psy1 disappears from the plasma membrane (PM) and dramatically relocalizes on the nascent forespore membrane, which becomes the PM of the spore. Here we report the molecular details and biological significance of Psy1 relocalization. We find that, immediately after meiosis I, Psy1 is selectively internalized by endocytosis. In addition, a meiosis-specific signal induced by the transcription factor Mei4 seems to trigger this internalization. The internalization of many PM proteins is facilitated coincident with the initiation of meiosis, whereas Pma1, a P-type ATPase, persists on the PM even during the progression of meiosis II. Ergosterol on the PM is also important for the internalization of PM proteins in general during meiosis. We consider that during meiosis in Schizosaccharomyces pombe cells, the characteristics of endocytosis change, thereby facilitating internalization of Psy1 and accomplishing sporulation. PMID:21832151

  10. Isolation and Characterization of High-Osmolarity-Sensitive Mutants of Fission Yeast

    PubMed Central

    Aiba, Hirofumi; Kawaura, Ryosuke; Yamamoto, Eiji; Yamada, Hisami; Takegawa, Kaoru; Mizuno, Takeshi

    1998-01-01

    For the fission yeast Schizosaccharomyces pombe, adaptation to high-osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase. The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (gpd1+) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant. We previously characterized a set of high-osmolarity-sensitive S. pombe mutants, including wis1, sty1, and gpd1. In this study, we attempted to further isolate novel osmolarity-sensitive mutants. For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types. They were classified into three distinct types genetically and, thus, were designated hos1, hos2, and hos3 (high osmolarity sensitive) mutants. One of them, the hos1 mutant, was characterized in detail. The hos1 mutant was demonstrated to have a mutational lesion in the known ryh1+ gene, which encodes a small GTP-binding protein. Disruption of the ryh1+ gene results not only in osmosensitivity but also in temperature sensitivity for growth. It was also found that the Δryh1 mutant is severely sterile. These results are discussed with special reference to the osmoadaptation of S. pombe. PMID:9748434

  11. Identification and transcription control of fission yeast genes repressed by an ammonium starvation growth arrest.

    PubMed

    Bonnet, C; Perret, E; Dumont, X; Picard, A; Caput, D; Lenaers, G

    2000-01-15

    In fission yeast Schizosaccharomyces pombe, ammonium starvation induces a growth arrest, a cell cycle exit in G(1) and a further switch to meiosis. This process is regulated by the cAMP-dependent protein kinase and the Wis1-dependent MAP kinase cascade, and downstream transcription factors. In order to understand how cells adapt their genetic programme to the switch from mitotic cycling to starvation, a differential transcript analysis comparing mRNA from exponentially growing and ammonium-starved cells was performed. Genes repressed by this stimulus mainly concern cell growth, i.e. protein synthesis and global metabolism. Comparison of the expression of two of them, the ribosomal proteins Rps6 and TCTP, in many different growing conditions, evidenced a strong correlation, suggesting that their transcriptions are coordinately regulated. Nevertheless, by repeating the ammonium starvation on strains constitutively activated for the PKA pathway (Deltacgs1), or unable to activate the Wis1-dependent MAP kinase pathway (Deltawis1), or with both characteristics (Deltacgs1+Deltawis1), the transcriptional inhibition was found to be governed either by the PKA pathway, or by the Wis1 pathway, or by both. These results suggest that during the switch from exponential growth to ammonium starvation, cell homeostasis is maintained by downregulating the transcription of the most expressed genes by a PKA and a Wis1-dependent process. Accession Nos for the S30 and L14 ribosomal protein cDNA sequences are AJ2731 and AJ2732, respectively. PMID:10620772

  12. Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast.

    PubMed

    Hipp, Katharina; Schäfer, Benjamin; Kepp, Gabi; Jeske, Holger

    2016-01-01

    The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses. PMID:27399762

  13. Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast

    PubMed Central

    Hipp, Katharina; Schäfer, Benjamin; Kepp, Gabi; Jeske, Holger

    2016-01-01

    The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses. PMID:27399762

  14. Fission yeast mtr1p regulates interphase microtubule cortical dwell-time

    PubMed Central

    Carlier-Grynkorn, Frédérique; Ji, Liang; Fraisier, Vincent; Lombard, Berangère; Dingli, Florent; Loew, Damarys; Paoletti, Anne; Ronot, Xavier; Tran, Phong T.

    2014-01-01

    ABSTRACT The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1+, a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1Δ) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3Δ results in short microtubules, but can be partially rescued by mtr1Δ, as the double mutant mal3Δ mtr1Δ exhibits longer microtubules than mal3Δ single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1Δ. The double-mutant mal3Δ rps1801Δ also exhibits longer microtubules than mal3Δ single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics. PMID:24928430

  15. Fission yeast mtr1p regulates interphase microtubule cortical dwell-time.

    PubMed

    Carlier-Grynkorn, Frédérique; Ji, Liang; Fraisier, Vincent; Lombard, Berangère; Dingli, Florent; Loew, Damarys; Paoletti, Anne; Ronot, Xavier; Tran, Phong T

    2014-01-01

    The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1(+), a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1Δ) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3Δ results in short microtubules, but can be partially rescued by mtr1Δ, as the double mutant mal3Δ mtr1Δ exhibits longer microtubules than mal3Δ single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1Δ. The double-mutant mal3Δ rps1801Δ also exhibits longer microtubules than mal3Δ single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics. PMID:24928430

  16. Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Meyers, Alex; Del Rio, Zuania P; Beaver, Rachael A; Morris, Ryan M; Weiskittel, Taylor M; Alshibli, Amany K; Mannik, Jaana; Morrell-Falvey, Jennifer; Dalhaimer, Paul

    2016-06-01

    Eukaryotic cells store cholesterol/sterol esters (SEs) and triacylglycerols (TAGs) in lipid droplets, which form from the contiguous endoplasmic reticulum (ER) network. However, it is not known if droplets preferentially form from certain regions of the ER over others. Here, we used fission yeast Schizosaccharomyces pombe cells where the nuclear and cortical/peripheral ER domains are distinguishable by light microscopy to show that SE-enriched lipid droplets form away from the nucleus at the cell tips, whereas TAG-enriched lipid droplets form around the nucleus. Sterols localize to the regions of the cells where droplets enriched in SEs are observed. TAG droplet formation around the nucleus appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which coverts DAG into phosphatidylcholine and phosphatidylethanolamine localized exclusively to the nuclear ER. Also, Dgk1p, which converts DAG into phosphatidic acid localized strongly to the nuclear ER over the cortical/peripheral ER. We also show that TAG more readily translocates from the ER to lipid droplets than do SEs. The results augment the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis point in the cell. PMID:26990381

  17. Global effects on gene expression in fission yeast by silencing and RNA interference machineries.

    PubMed

    Hansen, Klavs R; Burns, Gavin; Mata, Juan; Volpe, Thomas A; Martienssen, Robert A; Bähler, Jürg; Thon, Geneviève

    2005-01-01

    Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing. PMID:15632061

  18. Regulation of actin assembly by microtubules in fission yeast cell polarity.

    PubMed

    Chang, Fred; Feierbach, Becket; Martin, Sophie

    2005-01-01

    It has been speculated that microtubule plus ends function to regulate the actin cytoskeleton in processes such as cytokinesis, cell polarization and cell migration. In the fission yeast Schizosaccharomyces pombe, interphase microtubules regulate cell polarity through proteins such as tea1p, a kelch repeat protein, and for3p, a formin that nucleates actin cable assembly at cell tips. Here, we review recent progress on understanding tea1p regulation and function. Microtubules may govern the localization of tea1p by transporting it on the plus ends of microtubules and depositing it directly onto the cell tip when the microtubule catastrophes. The interaction of tea1p with the CLIP170 protein tip1p is responsible for its localization at growing microtubule plus ends. Tea1p may regulate cell polarity by associating with large 'polarisome' complexes that include for3p. For3p is present at both cell tips, but is not on the microtubules. Tea1p is needed to localize the formin to establish polarized cell growth at cell tips that have not grown previously. These studies begin to elucidate a molecular pathway for how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth. PMID:16355535

  19. Cdc18/CDC6 activates the Rad3-dependent checkpoint in the fission yeast.

    PubMed

    Fersht, Naomi; Hermand, Damien; Hayles, Jacqueline; Nurse, Paul

    2007-01-01

    A screen for genes that can ectopically activate a Rad3-dependent checkpoint block over mitosis in fission yeast has identified the DNA replication initiation factor cdc18 (known as CDC6 in other organisms). Either a stabilized form of Cdc18, the Cdc18-T6A phosphorylation mutant, or overexpression of wild type Cdc18, activate the Rad3-dependent S-M checkpoint in the apparent absence of detectable replication structures and gross DNA damage. This cell cycle block relies on the Rad checkpoint pathway and requires Chk1 phosphorylation and activation. Unexpectedly, Cdc18-T6A induces changes in the mobility of Chromosome III, affecting the size of a restriction fragment containing rDNA repeats and producing aberrant nucleolar structures. Recombination events within the rDNA appear to contribute at least in part to the cell cycle delay. We propose that an elevated level of Cdc18 activates the Rad3-dependent checkpoint either directly or indirectly, and additionally causes expansion of the rDNA repeats on Chromosome III. PMID:17690116

  20. Novel interactions of fission yeast kinesin 8 revealed through in vivo expression of truncation alleles.

    PubMed

    West, Robert R; McIntosh, J Richard

    2008-08-01

    Fission yeast expresses two kinesin 8s, klp5+ and klp6+, which are important for diverse cellular functions: mitosis, meiosis, and the maintenance of normal cell morphology. During vegetative growth these motors display complex localization patterns, moving from the cytoplasm during interphase to the kinetochores in early mitosis, the interpolar spindle in anaphase B, and then back into the cytoplasm. We have expressed GFP-tagged alleles of domains from these motors, seeking the signals required for their localizations. The tail of Klp5p localized to the interphase nucleus, more specifically to telomeres. Addition of the neck re-directed this fragment to microtubules in the cytoplasm. Klp6-tail and the neck-tail domains of both motors localized at microtubule ends. Klp6-neck-tail localized to the spindle in early mitosis but to the pole-proximal ends of the spindle in anaphase B. The Klp5-motor and motor-neck localized to microtubules, often causing them to bundle. Over-expression of Klp6-motor or motor-neck resulted in shorter microtubules. These localization patterns were no different when constructs were expressed in strains lacking either or both of the endogenous, full-length proteins. Our results indicate that the localization signals for these kinesins are not derived from simple amino acid sequences but from complex interactions among multiple domains of each motor. PMID:18553361

  1. Identification of a novel type of spacer element required for imprinting in fission yeast.

    PubMed

    Sayrac, Suha; Vengrova, Sonya; Godfrey, Emma L; Dalgaard, Jacob Z

    2011-03-01

    Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during lagging-strand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers. PMID:21423720

  2. A role for DNA polymerase alpha in epigenetic control of transcriptional silencing in fission yeast.

    PubMed

    Nakayama Ji; Allshire, R C; Klar, A J; Grewal, S I

    2001-06-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase alpha (pol(alpha)) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that pol(alpha) mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polalpha and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Pol(alpha) in vitro. Moreover, silencing-defective mutant Pol(alpha) displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Pol(alpha), in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  3. Genome-wide Screens for Sensitivity to Ionizing Radiation Identify the Fission Yeast Nonhomologous End Joining Factor Xrc4

    PubMed Central

    Li, Jun; Yu, Yang; Suo, Fang; Sun, Ling-Ling; Zhao, Dan; Du, Li-Lin

    2014-01-01

    Nonhomologous end joining (NHEJ) is the main means for repairing DNA double-strand breaks (DSBs) in human cells. Molecular understanding of NHEJ has benefited from analyses in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In human cells, the DNA ligation reaction of the classical NHEJ pathway is carried out by a protein complex composed of DNA ligase IV (LigIV) and XRCC4. In S. cerevisiae, this reaction is catalyzed by a homologous complex composed of Dnl4 and Lif1. Intriguingly, no homolog of XRCC4 has been found in S. pombe, raising the possibility that such a factor may not always be required for classical NHEJ. Here, through screening the ionizing radiation (IR) sensitivity phenotype of a genome-wide fission yeast deletion collection in both the vegetative growth state and the spore state, we identify Xrc4, a highly divergent homolog of human XRCC4. Like other fission yeast NHEJ factors, Xrc4 is critically important for IR resistance of spores, in which no homologous recombination templates are available. Using both extrachromosomal and chromosomal DSB repair assays, we show that Xrc4 is essential for classical NHEJ. Exogenously expressed Xrc4 colocalizes with the LigIV homolog Lig4 at the chromatin region of the nucleus in a mutually dependent manner. Furthermore, like their human counterparts, Xrc4 and Lig4 interact with each other and this interaction requires the inter-BRCT linker and the second BRCT domain of Lig4. Our discovery of Xrc4 suggests that an XRCC4 family protein is universally required for classical NHEJ in eukaryotes. PMID:24847916

  4. Rhn1, a Nuclear Protein, Is Required for Suppression of Meiotic mRNAs in Mitotically Dividing Fission Yeast

    PubMed Central

    Hada, Kazumasa; Niwa, Ryusuke

    2012-01-01

    In the fission yeast Schizosaccharomyces pombe, many meiotic mRNAs are transcribed during mitosis and meiosis and selectively eliminated in mitotic cells. However, this pathway for mRNA decay, called the determinant of selective removal (DSR)-Mmi1 system, targets only some of the numerous meiotic mRNAs that are transcribed in mitotic cells. Here we describe Rhn1, a nuclear protein involved in meiotic mRNA suppression in vegetative fission yeast. Rhn1 is homologous to budding yeast Rtt103 and localizes to one or a few discrete nuclear dots in growing vegetative cells. Rhn1 colocalizes with a pre-mRNA 3′-end processing factor, Pcf11, and with the 5′–3′ exoribonuclease, Dhp1; moreover, Rhn1 coimmunoprecipitates with Pcf11. Loss of rhn1 results in elevated sensitivity to high temperature, to thiabendazole (TBZ), and to UV. Interestingly, meiotic mRNAs—including moa1+, mcp5+, and mug96+—accumulate in mitotic rhn1Δ cells. Accumulation of meiotic mRNAs also occurs in strains lacking Lsk1, a kinase that phosphorylates serine 2 (Ser-2) in the C-terminal domain (CTD) of RNA polymerase II (Pol II), and in strains lacking Sen1, an ATP-dependent 5′–3′ RNA/DNA helicase: notably, both Lsk1 and Sen1 have been implicated in termination of Pol II-dependent transcription. Furthermore, RNAi knockdown of cids-2, a Caenorhabditis elegans ortholog of rhn1+, leads to elevated expression of a germline-specific gene, pgl-1, in somatic cells. These results indicate that Rhn1 contributes to the suppression of meiotic mRNAs in vegetative fission yeast and that the mechanism by which Rhn1 downregulates germline-specific transcripts may be conserved in unicellular and multicellular organisms. PMID:22912768

  5. Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast.

    PubMed

    Chatfield-Reed, Kate; Vachon, Lianne; Kwon, Eun-Joo Gina; Chua, Gordon

    2016-04-01

    Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeastSchizosaccharomyces pombe Genome-wide studies of theCrz1and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2and tunicamycin treatment, as well as a∆pmr1genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of theSaccharomyces cerevisiaeorthologCrz1 These genes were functionally enriched forCrz1-conserved processes such as cell-wall biosynthesis. Overexpression ofprz1(+)increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of theO-mannosyltransferase encoding geneomh1(+) Loss ofomh1(+)abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss ofprz1(+)resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the∆prz1strain was abrogated by the loss ofgsf2(+)orcbf12(+) This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes betweenCrz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network. PMID:26896331

  6. Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast

    PubMed Central

    Chatfield-Reed, Kate; Vachon, Lianne; Kwon, Eun-Joo Gina; Chua, Gordon

    2016-01-01

    Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2 and tunicamycin treatment, as well as a ∆pmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell-wall biosynthesis. Overexpression of prz1+ increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the ∆prz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network. PMID:26896331

  7. AnGeLi: A Tool for the Analysis of Gene Lists from Fission Yeast.

    PubMed

    Bitton, Danny A; Schubert, Falk; Dey, Shoumit; Okoniewski, Michal; Smith, Graeme C; Khadayate, Sanjay; Pancaldi, Vera; Wood, Valerie; Bähler, Jürg

    2015-01-01

    Genome-wide assays and screens typically result in large lists of genes or proteins. Enrichments of functional or other biological properties within such lists can provide valuable insights and testable hypotheses. To systematically detect these enrichments can be challenging and time-consuming, because relevant data to compare against query gene lists are spread over many different sources. We have developed AnGeLi (Analysis of Gene Lists), an intuitive, integrated web-tool for comprehensive and customized interrogation of gene lists from the fission yeast, Schizosaccharomyces pombe. AnGeLi searches for significant enrichments among multiple qualitative and quantitative information sources, including gene and phenotype ontologies, genetic and protein interactions, numerous features of genes, transcripts, translation, and proteins such as copy numbers, chromosomal positions, genetic diversity, RNA polymerase II and ribosome occupancy, localization, conservation, half-lives, domains, and molecular weight among others, as well as diverse sets of genes that are co-regulated or lead to the same phenotypes when mutated. AnGeLi uses robust statistics which can be tailored to specific needs. It also provides the option to upload user-defined gene sets to compare against the query list. Through an integrated data submission form, AnGeLi encourages the community to contribute additional curated gene lists to further increase the usefulness of this resource and to get the most from the ever increasing large-scale experiments. AnGeLi offers a rigorous yet flexible statistical analysis platform for rich insights into functional enrichments and biological context for query gene lists, thus providing a powerful exploratory tool through which S. pombe researchers can uncover fresh perspectives and unexpected connections from genomic data. AnGeLi is freely available at: www.bahlerlab.info/AnGeLi. PMID:26635866

  8. Transcriptional and cellular responses to defective mitochondrial proteolysis in fission yeast.

    PubMed

    Guha, Suranjana; López-Maury, Luis; Shaw, Michael; Bähler, Jürg; Norbury, Chris J; Agashe, Vishwas R

    2011-04-29

    Lon and m-AAA are the principal, regulated proteases required for protein maturation and turnover in the mitochondrial matrix of diverse species. To understand their roles in fission yeast (Schizosaccharomyces pombe) mitochondria, we generated deletion strains lacking Lon and m-AAA, individually (Δlon1 and Δm-AAA) or together, Δlon1Δm-AAA (Δ/Δ). All three strains were viable but incapable of respiratory growth on a non-fermentable carbon source due to mitochondrial dysfunction. Confocal and electron microscopy revealed a decrease in membrane potential and ultrastructural changes in Δlon1, Δm-AAA and Δ/Δ mitochondria, consistent with a respiratory defect and aggregation of proteins in the mitochondrial matrix. To understand the global adaptations required for cell survival in the absence of Lon and m-AAA proteases, we compared genome-wide gene expression signatures of the deletion strains with the isogenic wild-type strain. Deletion of lon1 caused a distinctive transcriptional footprint of just 12 differentially expressed genes, 9 of which were up-regulated genes located on the proximal mitochondrial genome (mitochondrial DNA). In contrast, m-AAA deletion caused a much larger transcriptional response involving 268 almost exclusively nuclear genes. Genes ameliorating stress and iron assimilation were up-regulated, while diverse mitochondrial genes and other metabolic enzymes were down-regulated. The connection with iron dysregulation was further explored using biochemical, chemical and cellular assays. Although Δm-AAA and Δ/Δ contained more cellular iron than the wild-type strain, their transcriptomes strongly resembled a signature normally evoked by iron insufficiency or disrupted assembly of iron-sulfur clusters in mitochondria. Based on these findings, we posit that excess iron accumulation could contribute to the pathology of human neurodegenerative disorders arising from defects in m-AAA function. PMID:21354177

  9. Structural Requirements for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast*

    PubMed Central

    Cheung, Rocky; Espenshade, Peter J.

    2013-01-01

    Sterol regulatory element-binding proteins (SREBPs) are central regulators of cellular lipid synthesis and homeostasis. Mammalian SREBPs are proteolytically activated and liberated from the membrane by Golgi Site-1 and Site-2 proteases. Fission yeast SREBPs, Sre1 and Sre2, employ a different mechanism that genetically requires the Golgi Dsc E3 ligase complex for cleavage activation. Here, we established Sre2 as a model to define structural requirements for SREBP cleavage. We showed that Sre2 cleavage does not require the N-terminal basic helix-loop-helix zipper transcription factor domain, thus separating cleavage of Sre2 from its transcription factor function. From a mutagenesis screen of 94 C-terminal residues of Sre2, we isolated 15 residues required for cleavage and further identified a glycine-leucine sequence required for Sre2 cleavage. Importantly, the glycine-leucine sequence is located at a conserved distance before the first transmembrane segment of both Sre1 and Sre2 and cleavage occurs in between this sequence and the membrane. Bioinformatic analysis revealed a broad conservation of this novel glycine-leucine motif in SREBP homologs of ascomycete fungi, including the opportunistic human pathogen Aspergillus fumigatus where SREBP is required for virulence. Consistent with this, the sequence was also required for cleavage of the oxygen-responsive transcription factor Sre1 and adaptation to hypoxia, demonstrating functional conservation of this cleavage recognition motif. These cleavage mutants will aid identification of the fungal SREBP protease and facilitate functional dissection of the Dsc E3 ligase required for SREBP activation and fungal pathogenesis. PMID:23729666

  10. Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast

    PubMed Central

    Bitton, Danny A.; Atkinson, Sophie R.; Rallis, Charalampos; Smith, Graeme C.; Ellis, David A.; Chen, Yuan Y.C.; Malecki, Michal; Codlin, Sandra; Lemay, Jean-François; Cotobal, Cristina; Bachand, François; Marguerat, Samuel; Mata, Juan; Bähler, Jürg

    2015-01-01

    Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5′-3′ exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼0.24% in wild type and ∼1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance. PMID:25883323

  11. Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe.

    PubMed

    Yazawa, Hisashi; Ogiso, Masayo; Kumagai, Hiromichi; Uemura, Hiroshi

    2014-11-01

    We previously succeeded to obtain a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we identified plg7, encoding phospholipase A2, as a multicopy suppressor that restored the growth defect by removing RA from phospholipids and induced secretion of a part of the released free RA into culture media. In this study, we extended our analysis and examined the effect of triglyceride (TG) lipase overexpression on the tolerance to RA toxicity and RA productivity. S. pombe has three TG lipase genes, ptl1, ptl2, and ptl3, which have high protein sequence similarities to each other and to Saccharomyces cerevisiae counterparts TGL3, TGL4, and TGL5, but only ptl2 overexpression suppressed the growth defect induced by RA production, and the culture grown at 20 °C secreted free RA into media like plg7 overexpression. Suppression by ptl2 was independent of plg7, and a large amount of free RA was accumulated in the cells concomitant with the decrease in RA moieties in phospholipids. Furthermore, the suppression by ptl2 was attenuated by bromoenol lactone (BEL), a phospholipase A2 specific inhibitor, suggesting that Ptl2p may have phospholipase activity. Simultaneous overexpression of ptl2 and plg7 in the FAH12 integrant increased secretion and intracellular accumulation of RA 1.2- and 1.3-fold, respectively, compared to those with single overexpression of plg7 on day 10 at 20 °C. PMID:25109267

  12. Functional analysis of the human NRAMP family expressed in fission yeast.

    PubMed Central

    Tabuchi, M; Yoshida, T; Takegawa, K; Kishi, F

    1999-01-01

    The Bcg/Ity/Lsh locus in the mouse genome regulates macrophage activation for antimicrobial activity against intracellular pathogens, and the positional cloning of this locus identified the Nramp1 (natural resistance-associated macrophage protein) gene. Nramp2 was initially isolated as a homologue of Nramp1. Recently, the rat divalent metal transporter DMT1 was identified electrophysiologically, and was found to be an isoform of Nramp2, a mutation which was subsequently identified in rats suffering from hereditary iron-deficiency anaemia. Despite the 64% amino acid sequence identity of Nramp1 and Nramp2, no divalent metal transport activity has yet been detected from Nramp1, and the function of Nramp1 on the molecular level is still unclear. To investigate the divalent metal transport activity of NRAMP molecules, we constructed four chimeric NRAMP genes by swapping the domains of human NRAMP1 and NRAMP2 with each other. The functional characteristics of wild-type NRAMP1, NRAMP2 and their chimeras were determined by expression in the divalent metal transporter-disrupted strain of fission yeast, pdt1Delta, and we analysed the divalent metal transport activity by complementation of the EGTA- and pH-sensitive phenotype of pdt1Delta. Replacement of the N-terminal cytoplasmic domain of NRAMP2 with the NRAMP1 counterpart resulted in inactive chimeras, indicating that the functional difference between NRAMP1 and NRAMP2 is located in this region. However, results obtained with the reverse construct and other chimeras indicated that these regions are not solely responsible for the differences in EGTA- and pH-sensitivity of NRAMP1 and NRAMP2. These findings indicate that NRAMP1 itself cannot represent the divalent metal transport activity in S. pombe and the additional protein segments of the molecules located elsewhere in NRAMP1 are also functionally distinct from their NRAMP2 counterparts. PMID:10548553

  13. Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus.

    PubMed

    Aoki, Keita; Nakajima, Reiko; Furuya, Kanji; Niki, Hironori

    2010-12-01

    Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4(+) of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early-log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4(+) probe. The plasmid containing ade6(+) suppressed the auxotrophic growth of the ade6-domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. PMID:20737410

  14. Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

    PubMed Central

    2011-01-01

    Background Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Findings Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. Conclusions We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls. PMID:22088094

  15. Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent

    SciTech Connect

    Griffiths, Karen; Masuda, Hirohisa; Dhut, Susheela; Toda, Takashi

    2008-04-11

    The Dam1/DASH outer kinetochore complex is required for high-fidelity chromosome segregation in budding and fission yeast. Unlike budding yeast, the fission yeast complex is non-essential, however it promotes bipolar microtubule attachment in conjunction with microtubule-depolymerising kinesin-8 Klp5 and Klp6. Here, we screened for dam1 temperature sensitive mutants in a klp5 null background and identified dam1-A8 that contains two amino acid substitutions in the C-terminus (H126R and E149G). dam1-A8klp5 mutant cells display massive chromosome missegregation with lagging chromosomes and monopolar attachment of sister chromatids to one SPB (spindle pole body). Unexpectedly contrary to a deletion mutant that is hypersensitive to microtubule-destabilising drugs, dam1-A8 is resistant and furthermore the temperature sensitivity of dam1-A8klp5 is rescued by addition of these drugs. This indicates that the hyper-stabilised rigidity of kinetochore-spindle mal-attachments is the primary cause of lethality. Our result shows that fine-tuning of Dam1 activity is essential for chromosome bi-orientation.

  16. Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent.

    PubMed

    Griffiths, Karen; Masuda, Hirohisa; Dhut, Susheela; Toda, Takashi

    2008-04-11

    The Dam1/DASH outer kinetochore complex is required for high-fidelity chromosome segregation in budding and fission yeast. Unlike budding yeast, the fission yeast complex is non-essential, however it promotes bipolar microtubule attachment in conjunction with microtubule-depolymerising kinesin-8 Klp5 and Klp6. Here, we screened for dam1 temperature sensitive mutants in a klp5 null background and identified dam1-A8 that contains two amino acid substitutions in the C-terminus (H126R and E149G). dam1-A8klp5 mutant cells display massive chromosome missegregation with lagging chromosomes and monopolar attachment of sister chromatids to one SPB (spindle pole body). Unexpectedly contrary to a deletion mutant that is hypersensitive to microtubule-destabilising drugs, dam1-A8 is resistant and furthermore the temperature sensitivity of dam1-A8klp5 is rescued by addition of these drugs. This indicates that the hyper-stabilised rigidity of kinetochore-spindle mal-attachments is the primary cause of lethality. Our result shows that fine-tuning of Dam1 activity is essential for chromosome bi-orientation. PMID:18262494

  17. The effect of the cwf14 gene of fission yeast on cell wall integrity is associated with rho1.

    PubMed

    Kim, Dong-Uk; Maeng, Shinae; Lee, Hyemi; Nam, Miyoung; Lee, Sook-Jeong; Hoe, Kwang-Lae

    2016-02-01

    In all eukaryotic organisms, a wide range of morphologies are responsible for critical cellular function and development. In particular, the Rho GTPases, which are highly conserved from yeast to mammals, are key molecules in signaling pathways that control cell polarity processes and cell wall biosynthesis, which are fundamental aspects of morphogenesis. Therefore, using haploinsufficiency deletion mutants of the fission yeast Schizosaccharomyces pombe, we screened the slow-growing mutants and their morphogenesis, specifically focusing on regulation of their Rho GTPases. Based on this screening, we found that the cwf14 mutant of S. pombe exhibited the slow growth and abnormal phenotypes with an elongated cell shape and thicker cell wall when compared with wild-type cells. In particular, cells with the cwf14 deletion showed excessive Rho1 expression. However, the wildtype strain with ectopically expressed Rho1 did not exhibited any significant change in the level of cwf14, suggesting that cwf14 may act on the upstream of Rho1. Furthermore, the cells with a cwf14 deletion also have increased sensitivity to β-glucanase, a cell wall-digesting enzyme, which is also seen in Rho1-overexpressing cells. Overall, our results suggest that the cwf14 plays a key role in fission yeast morphogenesis and cell wall biosynthesis and/or degradation possibly via the regulation of Rho1 expression. PMID:26832665

  18. Mitochondrial dynamics in model organisms: what yeasts, worms and flies have taught us about fusion and fission of mitochondria.

    PubMed

    Westermann, Benedikt

    2010-08-01

    Mitochondrial fusion and fission are important for a great variety of cellular functions, including energy metabolism, development, aging and cell death. Many of the core components mediating mitochondrial dynamics in human cells have been first identified and mechanistically analyzed in model organisms, such as Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. In particular, the functions of FZO/mitofusin and Mgm1/EAT-3/OPA1 in fusion and Dnm1/DRP1 in fission have been remarkably well conserved in yeasts, worms, flies and mammals. On the other hand, mechanisms to coordinate and regulate the activity of these molecular machines appear to be more diverse in different organisms. Here, I will discuss how S. cerevisiae, C. elegans and Drosophila have contributed to our current understanding of the cellular machineries mediating the dynamic behaviour of mitochondria. PMID:20006727

  19. Single site suppressors of a fission yeast temperature-sensitive mutant in cdc48 identified by whole genome sequencing.

    PubMed

    Marinova, Irina N; Engelbrecht, Jacob; Ewald, Adrian; Langholm, Lasse L; Holmberg, Christian; Kragelund, Birthe B; Gordon, Colin; Nielsen, Olaf; Hartmann-Petersen, Rasmus

    2015-01-01

    The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors. PMID:25658828

  20. Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells

    PubMed Central

    Recouvreux, Pierre; Sokolowski, Thomas R.; Grammoustianou, Aristea; ten Wolde, Pieter Rein; Dogterom, Marileen

    2016-01-01

    Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns. PMID:26831106

  1. Single Site Suppressors of a Fission Yeast Temperature-Sensitive Mutant in cdc48 Identified by Whole Genome Sequencing

    PubMed Central

    Marinova, Irina N.; Engelbrecht, Jacob; Ewald, Adrian; Langholm, Lasse L.; Holmberg, Christian; Kragelund, Birthe B.; Gordon, Colin; Nielsen, Olaf; Hartmann-Petersen, Rasmus

    2015-01-01

    The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors. PMID:25658828

  2. Chimera proteins with affinity for membranes and microtubule tips polarize in the membrane of fission yeast cells.

    PubMed

    Recouvreux, Pierre; Sokolowski, Thomas R; Grammoustianou, Aristea; Ten Wolde, Pieter Rein; Dogterom, Marileen

    2016-02-16

    Cell polarity refers to a functional spatial organization of proteins that is crucial for the control of essential cellular processes such as growth and division. To establish polarity, cells rely on elaborate regulation networks that control the distribution of proteins at the cell membrane. In fission yeast cells, a microtubule-dependent network has been identified that polarizes the distribution of signaling proteins that restricts growth to cell ends and targets the cytokinetic machinery to the middle of the cell. Although many molecular components have been shown to play a role in this network, it remains unknown which molecular functionalities are minimally required to establish a polarized protein distribution in this system. Here we show that a membrane-binding protein fragment, which distributes homogeneously in wild-type fission yeast cells, can be made to concentrate at cell ends by attaching it to a cytoplasmic microtubule end-binding protein. This concentration results in a polarized pattern of chimera proteins with a spatial extension that is very reminiscent of natural polarity patterns in fission yeast. However, chimera levels fluctuate in response to microtubule dynamics, and disruption of microtubules leads to disappearance of the pattern. Numerical simulations confirm that the combined functionality of membrane anchoring and microtubule tip affinity is in principle sufficient to create polarized patterns. Our chimera protein may thus represent a simple molecular functionality that is able to polarize the membrane, onto which additional layers of molecular complexity may be built to provide the temporal robustness that is typical of natural polarity patterns. PMID:26831106

  3. A microfluidic device for the hydrodynamic immobilisation of living fission yeast cells for super-resolution imaging☆

    PubMed Central

    Bell, Laurence; Seshia, Ashwin; Lando, David; Laue, Ernest; Palayret, Matthieu; Lee, Steven F.; Klenerman, David

    2014-01-01

    We describe a microfluidic device designed specifically for the reversible immobilisation of Schizosaccharomyces pombe (Fission Yeast) cells to facilitate live cell super-resolution microscopy. Photo-Activation Localisation Microscopy (PALM) is used to create detailed super-resolution images within living cells with a modal accuracy of >25 nm in the lateral dimensions. The novel flow design captures and holds cells in a well-defined array with minimal effect on the normal growth kinetics. Cells are held over several hours and can continue to grow and divide within the device during fluorescence imaging. PMID:25844024

  4. Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

    PubMed Central

    Lee, I-Ju; Wang, Ning; Hu, Wen; Schott, Kersey; Bähler, Jürg; Giddings, Thomas H.; Pringle, John R.; Du, Li-Lin; Wu, Jian-Qiu

    2014-01-01

    Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly. PMID:25031431

  5. Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast.

    PubMed

    Lee, I-Ju; Wang, Ning; Hu, Wen; Schott, Kersey; Bähler, Jürg; Giddings, Thomas H; Pringle, John R; Du, Li-Lin; Wu, Jian-Qiu

    2014-09-15

    Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly. PMID:25031431

  6. Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells.

    PubMed

    Appelgren, Henrik; Kniola, Barbara; Ekwall, Karl

    2003-10-01

    Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion

  7. Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast

    PubMed Central

    Hochstenbach, Frans; Klis, Frans M.; van den Ende, Herman; van Donselaar, Elly; Peters, Peter J.; Klausner, Richard D.

    1998-01-01

    The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1+, which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis. PMID:9689051

  8. A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.

    PubMed Central

    Osaka, F; Seino, H; Seno, T; Yamao, F

    1997-01-01

    A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis. PMID:9154838

  9. Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks.

    PubMed

    Willis, Nicholas A; Zhou, Chunshui; Elia, Andrew E H; Murray, Johanne M; Carr, Antony M; Elledge, Stephen J; Rhind, Nicholas

    2016-06-28

    The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase-specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes. PMID:27298342

  10. Mutation of histone H3 serine 86 disrupts GATA factor Ams2 expression and precise chromosome segregation in fission yeast

    PubMed Central

    Kiat Lim, Kim; Rui Ong, Terenze Yao; Rong Tan, Yue; Guorong Yang, Eugene; Ren, Bingbing; Shan Seah, Kwi; Yang, Zhe; Soo Tan, Tsu; Dymock, Brian W.; Sin Chen, Ee

    2015-01-01

    Eukaryotic genomes are packed into discrete units, referred to as nucleosomes, by organizing around scaffolding histone proteins. The interplay between these histones and the DNA can dynamically regulate the function of the chromosomal domain. Here, we interrogated the function of a pair of juxtaposing serine residues (S86 and S87) that reside within the histone fold of histone H3. We show that fission yeast cells expressing a mutant histone H3 disrupted at S86 and S87 (hht2-S86AS87A) exhibited unequal chromosome segregation, disrupted transcriptional silencing of centromeric chromatin, and reduced expression of Ams2, a GATA-factor that regulates localization of the centromere-specific histone H3 variant CENP-A. We found that overexpression of ams2+ could suppress the chromosome missegregation phenotype that arose in the hht2-S86AS87A mutant. We further demonstrate that centromeric localization of SpCENP-Acnp1-1 was significantly compromised in hht2-S86AS87A, suggesting synergism between histone H3 and the centromere-targeting domain of SpCENP-A. Taken together, our work presents evidence for an uncharacterized serine residue in fission yeast histone H3 that affects centromeric integrity via regulating the expression of the SpCENP-A-localizing Ams2 protein. [173/200 words] PMID:26369364

  11. Structure of the WD40 domain of SCAP from fission yeast reveals the molecular basis for SREBP recognition.

    PubMed

    Gong, Xin; Li, Jingxian; Shao, Wei; Wu, Jianping; Qian, Hongwu; Ren, Ruobing; Espenshade, Peter; Yan, Nieng

    2015-04-01

    The sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) are central players in the SREBP pathway, which control the cellular lipid homeostasis. SCAP binds to SREBP through their carboxyl (C) domains and escorts SREBP from the endoplasmic reticulum to the Golgi upon sterol depletion. A conserved pathway, with the homologues of SREBP and SCAP being Sre1 and Scp1, was identified in fission yeast Schizosaccharomyces pombe. Here we report the in vitro reconstitution of the complex between the C domains of Sre1 and Scp1 as well as the crystal structure of the WD40 domain of Scp1 at 2.1 Å resolution. The structure reveals an eight-bladed β-propeller that exhibits several distinctive features from a canonical WD40 repeat domain. Structural and biochemical characterization led to the identification of two Scp1 elements that are involved in Sre1 recognition, an Arg/Lys-enriched surface patch on the top face of the WD40 propeller and a 30-residue C-terminal tail. The structural and biochemical findings were corroborated by in vivo examinations. These studies serve as a framework for the mechanistic understanding and further functional characterization of the SREBP and SCAP proteins in fission yeast and higher organisms. PMID:25771684

  12. Structure of the WD40 domain of SCAP from fission yeast reveals the molecular basis for SREBP recognition

    PubMed Central

    Gong, Xin; Li, Jingxian; Shao, Wei; Wu, Jianping; Qian, Hongwu; Ren, Ruobing; Espenshade, Peter; Yan, Nieng

    2015-01-01

    The sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) are central players in the SREBP pathway, which control the cellular lipid homeostasis. SCAP binds to SREBP through their carboxyl (C) domains and escorts SREBP from the endoplasmic reticulum to the Golgi upon sterol depletion. A conserved pathway, with the homologues of SREBP and SCAP being Sre1 and Scp1, was identified in fission yeast Schizosaccharomyces pombe. Here we report the in vitro reconstitution of the complex between the C domains of Sre1 and Scp1 as well as the crystal structure of the WD40 domain of Scp1 at 2.1 Å resolution. The structure reveals an eight-bladed β-propeller that exhibits several distinctive features from a canonical WD40 repeat domain. Structural and biochemical characterization led to the identification of two Scp1 elements that are involved in Sre1 recognition, an Arg/Lys-enriched surface patch on the top face of the WD40 propeller and a 30-residue C-terminal tail. The structural and biochemical findings were corroborated by in vivo examinations. These studies serve as a framework for the mechanistic understanding and further functional characterization of the SREBP and SCAP proteins in fission yeast and higher organisms. PMID:25771684

  13. Kinesin-8 from Fission Yeast: A Heterodimeric, Plus-End–directed Motor that Can Couple Microtubule Depolymerization to Cargo Movement

    PubMed Central

    Grissom, Paula M.; Fiedler, Thomas; Grishchuk, Ekaterina L.; Nicastro, Daniela; West, Robert R.

    2009-01-01

    Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5+ and klp6+ genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end–directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A. PMID:19037096

  14. Analysis of Mcm2-7 chromatin binding during anaphase and in the transition to quiescence in fission yeast

    SciTech Connect

    Namdar, Mandana; Kearsey, Stephen E. . E-mail: stephen.kearsey@zoo.ox.ac.uk

    2006-10-15

    Mcm2-7 proteins are generally considered to function as a heterohexameric complex, providing helicase activity for the elongation step of DNA replication. These proteins are loaded onto replication origins in M-G1 phase in a process termed licensing or pre-replicative complex formation. It is likely that Mcm2-7 proteins are loaded onto chromatin simultaneously as a pre-formed hexamer although some studies suggest that subcomplexes are recruited sequentially. To analyze this process in fission yeast, we have compared the levels and chromatin binding of Mcm2-7 proteins during the fission yeast cell cycle. Mcm subunits are present at approximately 1 x 10{sup 4} molecules/cell and are bound with approximately equal stoichiometry on chromatin in G1/S phase cells. Using a single cell assay, we have correlated the timing of chromatin association of individual Mcm subunits with progression through mitosis. This showed that Mcm2, 4 and 7 associate with chromatin at about the same stage of anaphase, suggesting that licensing involves the simultaneous binding of these subunits. We also examined Mcm2-7 chromatin association when cells enter a G0-like quiescent state. Chromatin binding is lost in this transition in a process that does not require DNA replication or the selective degradation of specific subunits.

  15. Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast.

    PubMed

    Lando, David; Endesfelder, Ulrike; Berger, Harald; Subramanian, Lakxmi; Dunne, Paul D; McColl, James; Klenerman, David; Carr, Antony M; Sauer, Markus; Allshire, Robin C; Heilemann, Mike; Laue, Ernest D

    2012-07-01

    The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A(Cnp1) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A(Cnp1) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle. PMID:22870388

  16. A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

    PubMed

    Natsume, Toyoaki; Tsutsui, Yasuhiro; Sutani, Takashi; Dunleavy, Elaine M; Pidoux, Alison L; Iwasaki, Hiroshi; Shirahige, Katsuhiko; Allshire, Robin C; Yamao, Fumiaki

    2008-01-01

    Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication. PMID:18493607

  17. Mutation of histone H3 serine 86 disrupts GATA factor Ams2 expression and precise chromosome segregation in fission yeast.

    PubMed

    Lim, Kim Kiat; Ong, Terenze Yao Rui; Tan, Yue Rong; Yang, Eugene Guorong; Ren, Bingbing; Seah, Kwi Shan; Yang, Zhe; Tan, Tsu Soo; Dymock, Brian W; Chen, Ee Sin

    2015-01-01

    Eukaryotic genomes are packed into discrete units, referred to as nucleosomes, by organizing around scaffolding histone proteins. The interplay between these histones and the DNA can dynamically regulate the function of the chromosomal domain. Here, we interrogated the function of a pair of juxtaposing serine residues (S86 and S87) that reside within the histone fold of histone H3. We show that fission yeast cells expressing a mutant histone H3 disrupted at S86 and S87 (hht2-S86AS87A) exhibited unequal chromosome segregation, disrupted transcriptional silencing of centromeric chromatin, and reduced expression of Ams2, a GATA-factor that regulates localization of the centromere-specific histone H3 variant CENP-A. We found that overexpression of ams2(+) could suppress the chromosome missegregation phenotype that arose in the hht2-S86AS87A mutant. We further demonstrate that centromeric localization of SpCENP-A(cnp1-1) was significantly compromised in hht2-S86AS87A, suggesting synergism between histone H3 and the centromere-targeting domain of SpCENP-A. Taken together, our work presents evidence for an uncharacterized serine residue in fission yeast histone H3 that affects centromeric integrity via regulating the expression of the SpCENP-A-localizing Ams2 protein. [173/200 words]. PMID:26369364

  18. Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast

    PubMed Central

    Ard, Ryan; Tong, Pin; Allshire, Robin C.

    2014-01-01

    Most long non-coding RNAs (lncRNAs) encoded by eukaryotic genomes remain uncharacterized. Here we focus on a set of intergenic lncRNAs in fission yeast. Deleting one of these lncRNAs exhibited a clear phenotype: drug sensitivity. Detailed analyses of the affected locus revealed that transcription of the nc-tgp1 lncRNA regulates drug tolerance by repressing the adjacent phosphate-responsive permease gene transporter for glycerophosphodiester 1 (tgp1+). We demonstrate that the act of transcribing nc-tgp1 over the tgp1+ promoter increases nucleosome density, prevents transcription factor access and thus represses tgp1+ without the need for RNA interference or heterochromatin components. We therefore conclude that tgp1+ is regulated by transcriptional interference. Accordingly, decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation. Furthermore, nc-tgp1 loss induces tgp1+ even in repressive conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of the nc-tgp1 RNA. Thus, transcription of an lncRNA governs drug tolerance in fission yeast. PMID:25428589

  19. Anillin-related protein Mid1 regulates timely formation of the contractile ring in the fission yeast Schizosaccharomyces japonicus.

    PubMed

    Yasuda, Tsuyoshi; Takaine, Masak; Numata, Osamu; Nakano, Kentaro

    2016-06-01

    In the fission yeast Schizosaccharomyces pombe (Sp), Mid1/Dmf1 plays an important role in positioning the division site by inducing formation of the contractile ring (CR). Mid1, emanating from the nucleus located in the cell center, forms a dozen of nodes in the middle cell cortex ahead of mitosis, and actin filaments and myosin II accumulated at each node interact and assemble the CR in metaphase. Curiously, in another fission yeast S. japonicus (Sj), CR formation begins after nuclear segregation in late anaphase. Here, we investigated the role of S. japonicus Mid1 during mitosis to compare the molecular mechanisms that determine the cell division site in Schizosaccharomyces. Similar to Sp Mid1, Sj Mid1 often accumulated in the nucleus of interphase cells. Moreover, Sj Mid1 localized to cortical dots with myosin II in the future division site and formed a medial ring in mitotic cells. However, S. japonicus cells without Mid1 function still carried out symmetrical binary division. Therefore, the Mid1 dependency for positional control of the cell division site is possibly different between the two species. Meanwhile, we found that Sj Mid1 enhanced CR formation, in a manner possibly similar to that by Sp Mid1. PMID:27059155

  20. Cold-sensitive mutants of p34cdc2 that suppress a mitotic catastrophe phenotype in fission yeast.

    PubMed

    Ayscough, K; Hayles, J; MacNeill, S A; Nurse, P

    1992-04-01

    The p34cdc2 protein kinase plays a central role in the regulation of the eukaryotic cell cycle, being required both in late G1 for the commitment to S-phase and in late G2 for the initiation of mitosis. p34cdc2 also determines the precise timing of entry into mitosis in fission yeast, where a number of gene products that regulate p34cdc2 activity have been identified and characterised. To investigate further the mitotic role of p34cdc2 in this organism we have isolated new cold-sensitive p34cdc2 mutants. These are defective only in their G2 function and are extragenic suppressors of the lethal premature entry into mitosis brought about by mutating the mitotic inhibitor p107wee1 and overproducing the mitotic activator p80cdc25. One of the mutant proteins p34cdc2-E8 is only functional in the absence of p107wee1, and all the mutant strains have reduced histone H1 kinase activity in vitro. Each mutant allele has been cloned and sequenced, and the lesions responsible for the cold-sensitive phenotypes identified. All the mutations were found to map to regions that are conserved between the fission yeast p34cdc2 and functional homologues from higher eukaryotes. PMID:1316996

  1. Tts1, the fission yeast homologue of the TMEM33 family, functions in NE remodeling during mitosis

    PubMed Central

    Zhang, Dan; Oliferenko, Snezhana

    2014-01-01

    The fission yeast Schizosaccharomyces pombe undergoes “closed” mitosis in which the nuclear envelope (NE) stays intact throughout chromosome segregation. Here we show that Tts1, the fission yeast TMEM33 protein that was previously implicated in organizing the peripheral endoplasmic reticulum (ER), also functions in remodeling the NE during mitosis. Tts1 promotes insertion of spindle pole bodies (SPBs) in the NE at the onset of mitosis and modulates distribution of the nuclear pore complexes (NPCs) during mitotic NE expansion. Structural features that drive partitioning of Tts1 to the high-curvature ER domains are crucial for both aspects of its function. An amphipathic helix located at the C-terminus of Tts1 is important for ER shaping and modulating the mitotic NPC distribution. Of interest, the evolutionarily conserved residues at the luminal interface of the third transmembrane region function specifically in promoting SPB-NE insertion. Our data illuminate cellular requirements for remodeling the NE during “closed” nuclear division and provide insight into the structure and functions of the eukaryotic TMEM33 family. PMID:25103238

  2. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.

    PubMed

    Millar, J B; Buck, V; Wilkinson, M G

    1995-09-01

    Simultaneous inactivation of pyp1 and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pyp1 and pyp2 and reside in two complementation groups were isolated, sty1 and sty2. sty1- and sty2- mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the sty1 gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cervisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Sty1 and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Sty1 in vitro. We find that wis1-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Sty1 in vivo on prolonged exposure to osmotic stress. We conclude that Pyp1 and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast. PMID:7657164

  3. MADS Box Transcription Factor Mbx2/Pvg4 Regulates Invasive Growth and Flocculation by Inducing gsf2+ Expression in Fission Yeast

    PubMed Central

    Matsuzawa, Tomohiko; Yoritsune, Ken-ichi

    2012-01-01

    The fission yeast Schizosaccharomyces pombe exhibits invasive growth and nonsexual flocculation in response to nitrogen limitation. Gsf2, a flocculin of fission yeast, is required not only for nonsexual flocculation but also for invasive growth through the recognition of galactose residues on cell surface glycoconjugates. We found that pyruvylation negatively regulates nonsexual flocculation by capping the galactose residues of N-linked galactomannan. We investigated whether pyruvylation also regulates invasive growth. The pvg4+ gene originally was isolated as a multicopy suppressor of a pvg4 mutant defective in the pyruvylation of N-linked oligosaccharides. However, we did not detect a defect in cell surface pyruvylation in the pvg4/mbx2 deletion mutant, as assessed by alcian blue staining and a Q-Sepharose binding assay. Instead, the deletion prevented invasive growth under conditions of low nitrogen and high glucose, and it reduced the adhesion and flocculation of otherwise flocculent mutants by reducing gsf2+ expression. mbx2+-overexpressing strains exhibited nonsexual and calcium-dependent aggregation, which was inhibited in the presence of galactose but mediated by the induction of gsf2+. These findings indicate that Mbx2 mediates invasive growth and flocculation via the transcriptional activation of gsf2+ in fission yeast. In addition, we found that fission yeast Mbx2 induces the nonsexual flocculation of budding yeast by the activation of FLO1. PMID:22180499

  4. Profilin Binding to Poly-l-Proline and Actin Monomers along with Ability to Catalyze Actin Nucleotide Exchange Is Required for Viability of Fission Yeast

    PubMed Central

    Lu, Jia; Pollard, Thomas D.

    2001-01-01

    We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-l-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2–5% wild-type affinity for actin or poly-l-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-l-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast. PMID:11294914

  5. ATP-binding motifs play key roles in Krp1p, kinesin-related protein 1, function for bi-polar growth control in fission yeast

    SciTech Connect

    Rhee, Dong Keun; Cho, Bon A; Kim, Hyong Bai . E-mail: hbkim5212@hotmail.com

    2005-06-03

    Kinesin is a microtubule-based motor protein with various functions related to the cell growth and division. It has been reported that Krp1p, kinesin-related protein 1, which belongs to the kinesin heavy chain superfamily, localizes on microtubules and may play an important role in cytokinesis. However, the function of Krp1p has not been fully elucidated. In this study, we overexpressed an intact form and three different mutant forms of Krp1p in fission yeast constructed by site-directed mutagenesis in two ATP-binding motifs or by truncation of the leucine zipper-like motif (LZiP). We observed hyper-extended microtubules and the aberrant nuclear shape in Krp1p-overexpressed fission yeast. As a functional consequence, a point mutation of ATP-binding domain 1 (G89E) in Krp1p reversed the effect of Krp1p overexpression in fission yeast, whereas the specific mutation in ATP-binding domain 2 (G238E) resulted in the altered cell polarity. Additionally, truncation of the leucine zipper-like domain (LZiP) at the C-terminal of Krp1p showed a normal nuclear division. Taken together, we suggest that krp1p is involved in regulation of cell-polarized growth through ATP-binding motifs in fission yeast.

  6. The Fun30 Chromatin Remodeler Fft3 Controls Nuclear Organization and Chromatin Structure of Insulators and Subtelomeres in Fission Yeast

    PubMed Central

    Khorosjutina, Olga; Persson, Jenna; Smialowska, Agata; Javerzat, Jean-Paul; Ekwall, Karl

    2015-01-01

    In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope. PMID:25798942

  7. Inhibition of splicing and nuclear retention of pre-mRNA by spliceostatin A in fission yeast

    SciTech Connect

    Lo, Chor-Wai; Kaida, Daisuke; Nishimura, Shinichi; Matsuyama, Akihisa; Yashiroda, Yoko; Taoka, Hiroshi; Ishigami, Ken; Watanabe, Hidenori; Nakajima, Hidenori; Tani, Tokio; Horinouchi, Sueharu; Yoshida, Minoru

    2007-12-21

    Nuclear retention of pre-mRNAs is tightly regulated by several security mechanisms that prevent pre-mRNA export into the cytoplasm. Recently, spliceostatin A, a methylated derivative of a potent antitumor microbial metabolite FR901464, was found to cause pre-mRNA accumulation and translation in mammalian cells. Here we report that spliceostatin A also inhibits splicing and nuclear retention of pre-mRNA in a fission yeast strain that lacks the multidrug resistance protein Pmd1. As observed in mammalian cells, spliceostatin A is bound to components of the SF3b complex in the spliceosome. Furthermore, overexpression of nup211, a homolog of Saccharomyces cerevisiae MLP1, suppresses translation of pre-mRNAs accumulated by spliceostatin A. These results suggest that the SF3b complex has a conserved role in pre-mRNA retention, which is independent of the Mlp1 function.

  8. Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast.

    PubMed

    Takahashi, K; Chen, E S; Yanagida, M

    2000-06-23

    Mammalian kinetochores contain the centromere-specific histone H3 variant CENP-A, whose incorporation into limited chromosomal regions may be important for centromere function and chromosome segregation during mitosis. However, regulation of CENP-A localization and its role have not been clear. Here we report that the fission yeast homolog SpCENP-A is essential for establishing centromere chromatin associated with equal chromosome segregation. SpCENP-A binding to the nonrepetitious inner centromeres depended on Mis6, an essential centromere connector protein acting during G1-S phase of the cell cycle. Mis6 is likely required for recruiting SpCENP-A to form proper connection of sister centromeres. PMID:10864871

  9. Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast

    SciTech Connect

    Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko; Nakanishi, Makoto; Yoshida, Takashi; Aiba, Hirofumi; Murakami, Hiroshi

    2009-10-23

    The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2{sup +}. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint by regulating Mad2p.

  10. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

    SciTech Connect

    Iida, Tetsushi; Iida, Naoko; Tsutsui, Yasuhiro; Yamao, Fumiaki; Kobayashi, Takehiko

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer RNAi is linked to the cell cycle checkpoint in fission yeast. Black-Right-Pointing-Pointer Ptr1 co-purifies with Ago1. Black-Right-Pointing-Pointer The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. Black-Right-Pointing-Pointer ago1{sup +} and ptr1{sup +} regulate the cell cycle checkpoint via the same pathway. Black-Right-Pointing-Pointer Mutations in ago1{sup +} and ptr1{sup +} lead to the nuclear accumulation of poly(A){sup +} RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1{sup +}, the overexpression of ago1{sup +} alleviated the cell cycle defect in dcr1{Delta}. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1{sup +} is dependent on ptr1{sup +}. Nuclear accumulation of poly(A){sup +} RNAs was detected in mutants of ago1{sup +} and ptr1{sup +}, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  11. Two distinct pathways responsible for the loading of CENP-A to centromeres in the fission yeast cell cycle.

    PubMed

    Takahashi, Kohta; Takayama, Yuko; Masuda, Fumie; Kobayashi, Yasuyo; Saitoh, Shigeaki

    2005-03-29

    CENP-A is a centromere-specific histone H3 variant that is- essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast. PMID:15897182

  12. Factors That Promote H3 Chromatin Integrity during Transcription Prevent Promiscuous Deposition of CENP-ACnp1 in Fission Yeast

    PubMed Central

    Choi, Eun Shik; Strålfors, Annelie; Catania, Sandra; Castillo, Araceli G.; Svensson, J. Peter; Pidoux, Alison L.; Ekwall, Karl; Allshire, Robin C.

    2012-01-01

    Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-ACnp1 deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-ACnp1 incorporation at non-centromeric sites. FACT has little or no effect on CENP-ACnp1 assembly at endogenous centromeres where CENP-ACnp1 is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-ACnp1 at specific loci, including subtelomeric regions, where CENP-ACnp1 is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-ACnp1 chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-ACnp1 to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-ACnp1 chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification. PMID:23028377

  13. Structure of a Ca2+-Myristoyl Switch Protein That Controls Activation of a Phosphatidylinositol 4-Kinase in Fission Yeast*

    PubMed Central

    Lim, Sunghyuk; Strahl, Thomas; Thorner, Jeremy; Ames, James B.

    2011-01-01

    Neuronal calcium sensor (NCS) proteins transduce Ca2+ signals and are highly conserved from yeast to humans. We determined NMR structures of the NCS-1 homolog from fission yeast (Ncs1), which activates a phosphatidylinositol 4-kinase. Ncs1 contains an α-NH2-linked myristoyl group on a long N-terminal arm and four EF-hand motifs, three of which bind Ca2+, assembled into a compact structure. In Ca2+-free Ncs1, the N-terminal arm positions the fatty acyl chain inside a cavity near the C terminus. The C14 end of the myristate is surrounded by residues in the protein core, whereas its amide-linked (C1) end is flanked by residues at the protein surface. In Ca2+-bound Ncs1, the myristoyl group is extruded (Ca2+-myristoyl switch), exposing a prominent patch of hydrophobic residues that specifically contact phosphatidylinositol 4-kinase. The location of the buried myristate and structure of Ca2+-free Ncs1 are quite different from those in other NCS proteins. Thus, a unique remodeling of each NCS protein by its myristoyl group, and Ca2+-dependent unmasking of different residues, may explain how each family member recognizes distinct target proteins. PMID:21288895

  14. The AP-2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast.

    PubMed

    de León, Nagore; Hoya, Marta; Curto, M-Angeles; Moro, Sandra; Yanguas, Francisco; Doncel, Cristina; Valdivieso, M-Henar

    2016-05-01

    In metazoans the AP-2 complex has a well-defined role in clathrin-mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co- immunoprecipitation between the fission yeast AP-2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4-64 uptake. In an otherwise wild-type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP-2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP-2 in endocytosis has been conserved throughout evolution. PMID:26749213

  15. sud1+ targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast

    PubMed Central

    Jallepalli, Prasad V.; Tien, Deborah; Kelly, Thomas J.

    1998-01-01

    In the fission yeast Schizosaccharomyces pombe, S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein. Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication in vivo, we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast. In this paper we describe one such component, encoded by the sud1+ gene. sud1+ shares homology with the budding yeast CDC4 gene and is required to prevent spontaneous re-replication in fission yeast. Cells lacking sud1+ accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators. Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in Δsud1 cells, but is not the cause of the defect in Cdc18 proteolysis. Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18 in vivo. Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites. These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1. Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles. PMID:9653157

  16. The Ubiquitin Ligase Ubr11 Is Essential for Oligopeptide Utilization in the Fission Yeast Schizosaccharomyces pombe

    PubMed Central

    Nakase, Mai; Tohda, Hideki; Takegawa, Kaoru

    2012-01-01

    Uptake of extracellular oligopeptides in yeast is mediated mainly by specific transporters of the peptide transporter (PTR) and oligopeptide transporter (OPT) families. Here, we investigated the role of potential peptide transporters in the yeast Schizosaccharomyces pombe. Utilization of naturally occurring dipeptides required only Ptr2/SPBC13A2.04c and none of the other 3 OPT proteins (Isp4, Pgt1, and Opt3), whereas only Isp4 was indispensable for tetrapeptide utilization. Both Ptr2 and Isp4 localized to the cell surface, but under rich nutrient conditions Isp4 localized in the Golgi apparatus through the function of the ubiquitin ligase Pub1. Furthermore, the ubiquitin ligase Ubr11 played a significant role in oligopeptide utilization. The mRNA levels of both the ptr2 and isp4 genes were significantly reduced in ubr11Δ cells, and the dipeptide utilization defect in the ubr11Δ mutant was rescued by the forced expression of Ptr2. Consistent with its role in transcriptional regulation of peptide transporter genes, the Ubr11 protein was accumulated in the nucleus. Unlike the situation in Saccharomyces cerevisiae, the oligopeptide utilization defect in the S. pombe ubr11Δ mutant was not rescued by inactivation of the Tup11/12 transcriptional corepressors, suggesting that the requirement for the Ubr ubiquitin ligase in the upregulation of peptide transporter mRNA levels is conserved in both yeasts; however, the actual mechanism underlying the control appears to be different. We also found that the peptidomimetic proteasome inhibitor MG132 was still operative in a strain lacking all known PTR and OPT peptide transporters. Therefore, irrespective of its peptide-like structure, MG132 is carried into cells independently of the representative peptide transporters. PMID:22226946

  17. Role of the Small GTPase Rho3 in Golgi/Endosome Trafficking through Functional Interaction with Adaptin in Fission Yeast

    PubMed Central

    Kita, Ayako; Li, Cuifang; Yu, Yang; Umeda, Nanae; Doi, Akira; Yasuda, Mitsuko; Ishiwata, Shunji; Taga, Atsushi; Horiuchi, Yoshitaka; Sugiura, Reiko

    2011-01-01

    Background We had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Methodology/Principal Findings In the present study, we isolated rho3+, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl− sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl−, and valproic acid. Green fluorescent protein (GFP)-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner. Conclusions/Significance Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence of a direct link

  18. Sip1, an AP-1 Accessory Protein in Fission Yeast, Is Required for Localization of Rho3 GTPase

    PubMed Central

    Yu, Yang; Li, Cuifang; Kita, Ayako; Katayama, Yuta; Kubouchi, Koji; Udo, Masako; Imanaka, Yukako; Ueda, Shiho; Masuko, Takashi; Sugiura, Reiko

    2013-01-01

    Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3+ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi

  19. Sip1, an AP-1 accessory protein in fission yeast, is required for localization of Rho3 GTPase.

    PubMed

    Yu, Yang; Li, Cuifang; Kita, Ayako; Katayama, Yuta; Kubouchi, Koji; Udo, Masako; Imanaka, Yukako; Ueda, Shiho; Masuko, Takashi; Sugiura, Reiko

    2013-01-01

    Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3⁺ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of

  20. Arabidopsis Rho-Related GTPases: Differential Gene Expression in Pollen and Polar Localization in Fission Yeast1

    PubMed Central

    Li, Hai; Wu, Guang; Ware, Doreen; Davis, Keith R.; Yang, Zhenbiao

    1998-01-01

    The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction. Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases from plants). Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth. In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps, Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast. We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen. All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen. However, pollen-specific transcript accumulation for Rop1At is much higher than that for Rop3At and Rop5At. Furthermore, Rop1At is specifically expressed in anthers, whereas Rop3At and Rop5At are also expressed in vegetative tissues. In transgenic plants containing the Rop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes. We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen. PMID:9765526

  1. The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control.

    PubMed Central

    Kinoshita, N; Goebl, M; Yanagida, M

    1991-01-01

    The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis. Images PMID:1944266

  2. Characterization of the CTD phosphatase Fcp1 from fission yeast. Preferential dephosphorylation of serine 2 versus serine 5.

    PubMed

    Hausmann, Stéphane; Shuman, Stewart

    2002-06-14

    The C-terminal domain (CTD) of RNA polymerase II undergoes extensive phosphorylation and dephosphorylation at positions Ser2 and Ser5 during the transcription cycle. A single CTD phosphatase, Fcp1, has been identified in yeast and metazoans. Here we conducted a biochemical characterization of Fcp1 from the fission yeast Schizosaccharomyces pombe. The 723-amino acid Fcp1 protein was expressed at high levels in bacteria. Recombinant Fcp1 catalyzed the metal-dependent hydrolysis of para-nitrophenyl phosphate with a pH optimum of 5.5 (kcat = 2 s(-1); K(m) = 19 mm). Deletion analysis showed that 139- and 143-amino acid segments could be deleted from the N and C termini of Fcp1, respectively, without affecting phosphatase activity. A segment containing amino acids 487-580, deletion of which abolished activity, embraces a BRCT domain present in all known Fcp1 orthologs. Mutations of residues Asp170 and Asp172 abrogated Fcp1 phosphatase activity; the essential aspartates are located within a 170DXDXT172 motif that defines a superfamily of metal-dependent phosphotransferases. We exploited defined synthetic CTD phosphopeptide substrates to show for the first time that: (i) Fcp1 CTD phosphatase activity is not confined to native polymerase II and (ii) Fcp1 displays an inherent preference for a particular CTD phosphorylation array. Using equivalent concentrations (25 microm) of CTD peptides of identical amino acid sequence and phosphoserine content, which differed only in the positions of phosphoserine within the heptad, we found that Fcp1 was 10-fold more active in dephosphorylating Ser2-PO4 than Ser5-PO4. PMID:11934898

  3. Suppression of Sensitivity to Drugs and Antibiotics by High External Cation Concentrations in Fission Yeast

    PubMed Central

    Shabro, Aidin; Sunnerhagen, Per

    2015-01-01

    Background Potassium ion homeostasis plays an important role in regulating membrane potential and therefore resistance to cations, antibiotics and chemotherapeutic agents in Schizosaccharomyces pombe and other yeasts. However, the precise relationship between drug resistance in S. pombe and external potassium concentrations (particularly in its natural habitats) remains unclear. S. pombe can tolerate a wide range of external potassium concentrations which in turn affect plasma membrane polarization. We thus hypothesized that high external potassium concentrations suppress the sensitivity of this yeast to various drugs. Methods We have investigated the effect of external KCl concentrations on the sensitivity of S. pombe cells to a wide range of antibiotics, antimicrobial agents and chemotherapeutic drugs. We employed survival assays, immunoblotting and microscopy for these studies. Results We demonstrate that KCl, and to a lesser extent NaCl and RbCl can suppress the sensitivity of S. pombe to a wide range of antibiotics. Ammonium chloride and potassium hydrogen sulphate also suppressed drug sensitivity. This effect appears to depend in part on changes to membrane polarization and membrane transport proteins. Interestingly, we have found little relationship between the suppressive effect of KCl on sensitivity and the structure, polarity or solubility of the various compounds investigated. Conclusions High concentrations of external potassium and other cations suppress sensitivity to a wide range of drugs in S. pombe. Potassium-rich environments may thus provide S. pombe a competitive advantage in nature. Modulating potassium ion homeostasis may sensitize pathogenic fungi to antifungal agents. PMID:25793410

  4. Replication dynamics in fission and budding yeasts through DNA polymerase tracking.

    PubMed

    Vázquez, Enrique; Antequera, Francisco

    2015-10-01

    The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof-read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions. PMID:26293347

  5. Natural genetic variation impacts expression levels of coding, non-coding, and antisense transcripts in fission yeast

    PubMed Central

    Clément-Ziza, Mathieu; Marsellach, Francesc X; Codlin, Sandra; Papadakis, Manos A; Reinhardt, Susanne; Rodríguez-López, María; Martin, Stuart; Marguerat, Samuel; Schmidt, Alexander; Lee, Eunhye; Workman, Christopher T; Bähler, Jürg; Beyer, Andreas

    2014-01-01

    Our current understanding of how natural genetic variation affects gene expression beyond well-annotated coding genes is still limited. The use of deep sequencing technologies for the study of expression quantitative trait loci (eQTLs) has the potential to close this gap. Here, we generated the first recombinant strain library for fission yeast and conducted an RNA-seq-based QTL study of the coding, non-coding, and antisense transcriptomes. We show that the frequency of distal effects (trans-eQTLs) greatly exceeds the number of local effects (cis-eQTLs) and that non-coding RNAs are as likely to be affected by eQTLs as protein-coding RNAs. We identified a genetic variation of swc5 that modifies the levels of 871 RNAs, with effects on both sense and antisense transcription, and show that this effect most likely goes through a compromised deposition of the histone variant H2A.Z. The strains, methods, and datasets generated here provide a rich resource for future studies. PMID:25432776

  6. Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis.

    PubMed

    Wang, Ning; Lee, I-Ju; Rask, Galen; Wu, Jian-Qiu

    2016-04-01

    The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis. PMID:27082518

  7. Relationship between extracellular enzymes and cell growth during the cell cycle of the fission yeast Schizosaccharomyces pombe: acid phosphatase.

    PubMed Central

    Miyata, M; Miyata, H

    1978-01-01

    By using the intact cells of the fission yeast Schizosaccharomyces pombe, the activity of acid phosphatase (EC 3.1.3.2) was compared through the cell cycle with the growth in cell length as a measure of cell growth. The cells of a growing asynchronous culture increased exponentially in number and in total enzyme activity, but remained constant in average length and in specific activity, In a synchronous culture prepared by selection or by induction, the specific activity was periodic in parallel with the increase in average cell length. When hydroxyurea was added to an asynchronous or a synchronous culture by selection, both specific and total activity followed the same continuous pattern as the growth in cell length after the stoppage of cell division. When oversized cells produced by a hydroxyurea pulse treatment to the culture previously syndronized by selection were transferred to a poor medium, they divided synchronously but could hardly grow in the total cell length. In this experimental situation, the total enzyme activity also scarcely increased through three division cycles. These results suggested that the increase in acid phosphatase in dependent on cell elongation. PMID:711673

  8. The fission yeast NIMA kinase Fin1p is required for spindle function and nuclear envelope integrity.

    PubMed

    Krien, Michael J E; West, Robert R; John, Ulrik P; Koniaras, Kalli; McIntosh, J Richard; O'Connell, Matthew J

    2002-04-01

    NIMA kinases appear to be the least functionally conserved mitotic regulators, being implicated in chromosome condensation in fungi and in spindle function in metazoans. We demonstrate here that the fission yeast NIMA homologue, Fin1p, can induce profound chromosome condensation in the absence of the condensin and topoisomerase II, indicating that Fin1p-induced condensation differs from mitotic condensation. Fin1p expression is transcriptionally and post-translationally cell cycle-regulated, with Fin1p kinase activity maximal from the metaphase-anaphase transition to G(1). Fin1p is localized to the spindle pole body and fin1Delta cells are hypersensitive to anti-microtubule drugs, synthetically lethal with a number of spindle mutants and require the spindle checkpoint for viability. Moreover, fin1Delta cells show unusual and extensive elaborations of the nuclear envelope. These data support a role for Fin1p in spindle function and nuclear envelope transactions at or after the metaphase-anaphase transition that may be generally applicable to other NIMA-family members. PMID:11927555

  9. Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA

    PubMed Central

    Nakamura, Takahiro; Pluskal, Tomáš; Nakaseko, Yukinobu; Yanagida, Mitsuhiro

    2012-01-01

    Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA. PMID:23091701

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

    PubMed

    Moriyama, Daisuke; Hosono, Kouji; Fujii, Makoto; Washida, Motohisa; Nanba, Hirokazu; Kaino, Tomohiro; Kawamukai, Makoto

    2015-01-01

    Coenzyme Q10 (CoQ10) is essential for energy production and has become a popular supplement in recent years. In this study, CoQ10 productivity was improved in the fission yeast Schizosaccharomyces pombe. Ten CoQ biosynthetic genes were cloned and overexpressed in S. pombe. Strains expressing individual CoQ biosynthetic genes did not produce higher than a 10% increase in CoQ10 production. In addition, simultaneous expression of all ten coq genes did not result in yield improvements. Genes responsible for the biosynthesis of p-hydroxybenzoate and decaprenyl diphosphate, both of which are CoQ biosynthesis precursors, were also overexpressed. CoQ10 production was increased by overexpression of Eco_ubiC (encoding chorismate lyase), Eco_aroF(FBR) (encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase), or Sce_thmgr1 (encoding truncated HMG-CoA reductase). Furthermore, simultaneous expression of these precursor genes resulted in two fold increases in CoQ10 production. PMID:25647499