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  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  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. Sgf73, a subunit of SAGA complex, is required for the assembly of RITS complex in fission yeast

    PubMed Central

    Deng, Xiaolong; Zhou, Huan; Zhang, Guiping; Wang, Wenchao; Mao, Langyong; Zhou, Xing; Yu, Yao; Lu, Hong

    2015-01-01

    RNA interference (RNAi) is a widespread gene-silencing mechanism and is required for heterochromatin assembly in a variety of organisms. The RNA-induced transcriptional silencing complex (RITS), composed of Ago1, Tas3 and Chp1, is a key component of RNAi machinery in fission yeast that connects short interference RNA (siRNA) and heterochromatin formation. However, the process by which RITS is assembled is not well understood. Here, we identified Sgf73, a subunit of the SAGA co-transcriptional complex, is required for pericentromeric heterochromatin silencing and the generation of siRNA. This novel role of Sgf73 is independent of enzymatic activities or structural integrity of SAGA. Instead, Sgf73 is physically associated with Ago1 and Chp1. The interactions among the subunits of the RITS, including those between Tas3 and Chp1, between Chp1 and Ago1, between Ago1 and Tas3, were all impaired by the deletion of sgf73+. Consistently, the recruitment of Ago1 and Chp1 to the pericentromeric region was abolished in sgf73Δ cells. Our study unveils a moonlighting function of a SAGA subunit. It suggests Sgf73 is a novel factor that promotes assembly of RITS and RNAi-mediated heterochromatin formation. PMID:26443059

  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. 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

  2. Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring.

    PubMed

    Venkatram, Srinivas; Jennings, Jennifer L; Link, Andrew; Gould, Kathleen L

    2005-06-01

    Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis. PMID:15800064

  3. 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

  4. The RFTS Domain of Raf2 Is Required for Cul4 Interaction and Heterochromatin Integrity in Fission Yeast

    PubMed Central

    White, Sharon A.; Buscaino, Alessia; Sanchez-Pulido, Luis; Ponting, Chris P.; Nowicki, Matthew W.; Allshire, Robin C.

    2014-01-01

    Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 (equivalent to metazoan Suv3-9), a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-ACnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres. PMID:25090107

  5. TORC2 Is Required to Maintain Genome Stability during S Phase in Fission Yeast*

    PubMed Central

    Schonbrun, Miriam; Kolesnikov, Masha; Kupiec, Martin; Weisman, Ronit

    2013-01-01

    DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair, and reentry into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress but are insensitive to ionizing radiation. We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for the return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication restart following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development. PMID:23703609

  6. Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

    PubMed Central

    Curto, M.-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M.-Henar

    2014-01-01

    The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell–cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell–cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell–cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  7. Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

    PubMed

    Curto, M-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M-Henar

    2014-04-01

    The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  8. 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

  9. A proline-tyrosine nuclear localization signal (PY-NLS) is required for the nuclear import of fission yeast PAB2, but not of human PABPN1.

    PubMed

    Mallet, Pierre-Luc; Bachand, François

    2013-03-01

    Nuclear poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline-tyrosine nuclear localization signal (PY-NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin β2 (Kapβ2)-type receptors in the import of PY-NLS cargoes, we show that the fission yeast ortholog of human Kapβ2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N-terminal to the PY-core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub-optimal PY-NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY-NLS cargo. Although a sequence resembling a PY-NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY-NLS nor Kapβ2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY-NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1. PMID:23279110

  10. 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

  11. 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

  12. 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

  13. 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.

  14. A novel protein kinase gene ssp1+ is required for alteration of growth polarity and actin localization in fission yeast.

    PubMed Central

    Matsusaka, T; Hirata, D; Yanagida, M; Toda, T

    1995-01-01

    Temperature-sensitive suppressor mutants were isolated from two fission yeast mutants defective in cell shape control: ppe1, encoding a type 2A-like protein phosphatase, and sts5, one of 11 staurosporine-supersensitive mutants. Complementation tests showed that suppression was due to two chromosomal loci, ssp1 and ssp2. Cells of the ssp1 mutant grown at the restrictive temperature arrested uniformly with an elongated cell body and a 2C content of DNA. Interestingly, these mutant cells grew only in a monopolar manner. At a specific point in the G2 phase of the cell cycle, wild-type cells exhibit a drastic alteration in growth polarity, from mono- to bipolar. This change coincides with the distribution of cortical actin from one end of the cell to both ends. In the ssp1 mutant cells, cortical actin was localized only at one end, suggesting that the mutant fails to change growth polarity. Nucleotide sequence determination showed that ssp1+ encodes a novel protein kinase. Ectopic overexpression of ssp1+ resulted in an altered cell morphology and cortical actin was randomly dispersed within the cells. Immunocytological analysis revealed that the protein was primarily localized in the cytoplasm and that half of the protein existed in an insoluble fraction. These results show that the dynamics of actin-based growth polarity during the cell cycle are regulated, at least in part, by a novel set of protein kinases and phosphatases. Images PMID:7628434

  15. Condensin HEAT Subunits Required for DNA Repair, Kinetochore/Centromere Function and Ploidy Maintenance in Fission Yeast

    PubMed Central

    Xu, Xingya; Nakazawa, Norihiko; Yanagida, Mitsuhiro

    2015-01-01

    Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes) subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo) repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe) mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance. PMID:25764183

  16. Vgl1, a multi-KH domain protein, is a novel component of the fission yeast stress granules required for cell survival under thermal stress

    PubMed Central

    Wen, Wei-Ling; Stevenson, Abigail L.; Wang, Chun-Yu; Chen, Hsiang-Ju; Kearsey, Stephen E.; Norbury, Chris J.; Watt, Stephen; Bähler, Jürg; Wang, Shao-Win

    2010-01-01

    Multiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress. PMID:20547592

  17. Eng2 is a component of a dynamic protein complex required for endocytic uptake in fission yeast.

    PubMed

    Encinar del Dedo, Javier; Idrissi, Fatima-Zahra; Arnáiz-Pita, Yolanda; James, Michael; Dueñas-Santero, Encarnación; Orellana-Muñoz, Sara; del Rey, Francisco; Sirotkin, Vladimir; Geli, M Isabel; Vázquez de Aldana, Carlos R

    2014-10-01

    Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2-GFP dynamics did not match the pattern of other endocytic proteins. Eng2-GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3-YFP, a WASP-interacting protein, interacted with Eng2 by co-immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module. PMID:25040903

  18. 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

  19. Cleavage and polyadenylation factor, Rna14 is an essential protein required for the maintenance of genomic integrity in fission yeast Schizosaccharomyces pombe.

    PubMed

    Sonkar, Amit; Yadav, Sudhanshu; Ahmed, Shakil

    2016-02-01

    Faithful segregation of chromosomes is essential for the maintenance of genome integrity. In a genetic screen to identify genes related to checkpoint function, we have characterized the role of rna14, an essential gene in the maintenance of chromosome dynamics. We demonstrate that Rna14 localizes in the nucleus and in the absence of functional Rna14, the cells exhibit chromosomal segregation defects. The mutant allele of rna14 exhibits genetic interaction with key kinetochore components and spindle checkpoint proteins. Inactivation of rna14 leads to accumulation of Bub1-GFP foci, a protein required for spindle checkpoint activation that could be due to the defects in the attachment of mitotic spindle to the chromosome. Consistently, the double mutant of rna14-11 and bub1 knockout exhibits high degree of chromosome mis-segregation. At restrictive condition, the rna14-11 mutant cells exhibit defects in cell cycle progression with high level of septation. The orthologs of Rna14 in Saccharomyces cerevisiae (sc Rna14) and human (CstF3) contain similar domain architecture and are required for 3'-end processing of pre-mRNA. We have also demonstrated that the fission yeast Rna14 is required to prevent transcriptional read-through. These findings reveal the importance of transcription termination in the maintenance of genomic stability through the regulation of kinetochore function. PMID:26581324

  20. 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

  1. 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

  2. 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

  3. Fission yeast kinesin-8 Klp5 and Klp6 are interdependent for mitotic nuclear retention and required for proper microtubule dynamics.

    PubMed

    Unsworth, Amy; Masuda, Hirohisa; Dhut, Susheela; Toda, Takashi

    2008-12-01

    Fission yeast has two kinesin-8s, Klp5 and Klp6, which associate to form a heterocomplex. Here, we show that Klp5 and Klp6 are mutually dependent on each other for nuclear mitotic localization. During interphase, they are exported to the cytoplasm. In sharp contrast, during mitosis, Klp5 and Klp6 remain in the nucleus, which requires the existence of each counterpart. Canonical nuclear localization signal (NLS) is identified in the nonkinesin C-terminal regions. Intriguingly individual NLS mutants (NLSmut) exhibit loss-of-function phenotypes, suggesting that Klp5 and Klp6 enter the nucleus separately. Indeed, although neither Klp5-NLSmut nor Klp6-NLSmut enters the nucleus, wild-type Klp6 or Klp5, respectively, does so with different kinetics. In the absence of Klp5/6, microtubule catastrophe/rescue frequency and dynamicity are suppressed, whereas growth and shrinkage rates are least affected. Remarkably, chimera strains containing only the N-terminal Klp5 kinesin domains cannot disassemble interphase microtubules during mitosis, leading to the coexistence of cytoplasmic microtubules and nuclear spindles with massive chromosome missegregation. In this strain, a marked reduction of microtubule dynamism, even higher than in klp5/6 deletions, is evident. We propose that Klp5 and Klp6 play a vital role in promoting microtubule dynamics, which is essential for the spatiotemporal control of microtubule morphogenesis. PMID:18799626

  4. A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres.

    PubMed

    Dunleavy, Elaine M; Pidoux, Alison L; Monet, Marie; Bonilla, Carolina; Richardson, William; Hamilton, Georgina L; Ekwall, Karl; McLaughlin, Paul J; Allshire, Robin C

    2007-12-28

    A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin. PMID:18158900

  5. Swi6, a Gene Required for Mating-Type Switching, Prohibits Meiotic Recombination in the Mat2-Mat3 ``cold Spot'' of Fission Yeast

    PubMed Central

    Klar, AJS.; Bonaduce, M. J.

    1991-01-01

    Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 ``hot spot'' for transposition should be contrasted with the ``cold spot'' of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination. PMID:1783290

  6. A NASP (N1/N2)-Related Protein, Sim3, Binds CENP-A and Is Required for Its Deposition at Fission Yeast Centromeres

    PubMed Central

    Dunleavy, Elaine M.; Pidoux, Alison L.; Monet, Marie; Bonilla, Carolina; Richardson, William; Hamilton, Georgina L.; Ekwall, Karl; McLaughlin, Paul J.; Allshire, Robin C.

    2007-01-01

    Summary A defining feature of centromeres is the presence of the histone H3 variant CENP-ACnp1. It is not known how CENP-ACnp1 is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASPHuman and N1/N2Xenopus and aligns with Hif1S. cerevisiae, defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-ACnp1 and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-ACnp1 at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-ACnp1 to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-ACnp1 to chromatin assembly factors, allowing its incorporation into centromeric chromatin. PMID:18158900

  7. Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

    PubMed Central

    Masuda, Hirohisa; Mori, Risa; Yukawa, Masashi; Toda, Takashi

    2013-01-01

    γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1–6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1–3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not. Recently a novel conserved component, MOZART1, was identified in humans and plants, but its precise functions remain to be determined. In this paper, we characterize the fission yeast homologue Mzt1, showing that it is essential for cell viability. Mzt1 is present in approximately equal stoichiometry with Alp4/GCP2 and localizes to all the MTOCs, including the SPB and interphase and equatorial MTOCs. Temperature-sensitive mzt1 mutants display varying degrees of compromised microtubule organization, exhibiting multiple defects during both interphase and mitosis. Mzt1 is required for γ-TuC recruitment, but not sufficient to localize to the SPB, which depends on γ-TuC integrity. Intriguingly, the core γ-TuC assembles in the absence of Mzt1. Mzt1 therefore plays a unique role within the γ-TuC components in attachment of this complex to the major MTOC site. PMID:23885124

  8. Oxygen requirements of yeasts.

    PubMed Central

    Visser, W; Scheffers, W A; Batenburg-van der Vegte, W H; van Dijken, J P

    1990-01-01

    Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth. Images PMID:2082825

  9. 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

  10. 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.)

  11. The stress granule protein Vgl1 and poly(A)-binding protein Pab1 are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Morita, Takahiro; Satoh, Ryosuke; Umeda, Nanae; Kita, Ayako; Sugiura, Reiko

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Stress granules (SGs) as a mechanism of doxorubicin tolerance. Black-Right-Pointing-Pointer We characterize the role of stress granules in doxorubicin tolerance. Black-Right-Pointing-Pointer Deletion of components of SGs enhances doxorubicin sensitivity in fission yeast. Black-Right-Pointing-Pointer Doxorubicin promotes SG formation when combined with heat shock. Black-Right-Pointing-Pointer Doxorubicin regulates stress granule assembly independent of eIF2{alpha} phosphorylation. -- Abstract: Doxorubicin is an anthracycline antibiotic widely used for chemotherapy. Although doxorubicin is effective in the treatment of several cancers, including solid tumors and leukemias, the basis of its mechanism of action is not completely understood. Here, we describe the effects of doxorubicin and its relationship with stress granules formation in the fission yeast, Schizosaccharomyces pombe. We show that disruption of genes encoding the components of stress granules, including vgl1{sup +}, which encodes a multi-KH type RNA-binding protein, and pab1{sup +}, which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1{sup +} and pab1{sup +} genes did not confer sensitivity to other anti-cancer drugs such as cisplatin, 5-fluorouracil, and paclitaxel. We also showed that doxorubicin treatment promoted stress granule formation when combined with heat shock. Notably, doxorubicin treatment did not induce hyperphosphorylation of eIF2{alpha}, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2{alpha} phosphorylation. Our results demonstrate the usefulness of fission yeast for elucidating the molecular targets of doxorubicin toxicity and suggest a novel drug-resistance mechanism involving stress granule assembly.

  12. 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

  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. 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.

  15. Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

    2014-01-01

    The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

  16. Diverse fission yeast genes required for responding to oxidative and metal stress: Comparative analysis of glutathione-related and other defense gene deletions.

    PubMed

    Pluskal, Tomáš; Sajiki, Kenichi; Becker, Joanne; Takeda, Kojiro; Yanagida, Mitsuhiro

    2016-06-01

    Living organisms have evolved multiple sophisticated mechanisms to deal with reactive oxygen species. We constructed a collection of twelve single-gene deletion strains of the fission yeast Schizosaccharomyces pombe designed for the study of oxidative and heavy metal stress responses. This collection contains deletions of biosynthetic enzymes of glutathione (Δgcs1 and Δgsa1), phytochelatin (Δpcs2), ubiquinone (Δabc1) and ergothioneine (Δegt1), as well as catalase (Δctt1), thioredoxins (Δtrx1 and Δtrx2), Cu/Zn- and Mn- superoxide dismutases (SODs; Δsod1 and Δsod2), sulfiredoxin (Δsrx1) and sulfide-quinone oxidoreductase (Δhmt2). First, we employed metabolomic analysis to examine the mutants of the glutathione biosynthetic pathway. We found that ophthalmic acid was produced by the same enzymes as glutathione in S. pombe. The identical genetic background of the strains allowed us to assess the severity of the individual gene knockouts by treating the deletion strains with oxidative agents. Among other results, we found that glutathione deletion strains were not particularly sensitive to peroxide or superoxide, but highly sensitive to cadmium stress. Our results show the astonishing diversity in cellular adaptation mechanisms to various types of oxidative and metal stress and provide a useful tool for further research into stress responses. PMID:27005325

  17. 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

  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. 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

  20. 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

  1. The fission yeast ferric reductase gene frp1+ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human NADPH phagocyte oxidoreductase.

    PubMed Central

    Roman, D G; Dancis, A; Anderson, G J; Klausner, R D

    1993-01-01

    We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease. Images PMID:8321236

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. Some assembly required: yeast septins provide the instruction manual

    PubMed Central

    Versele, Matthias; Thorner, Jeremy

    2006-01-01

    Septins are a family of conserved proteins that form hetero-oligomeric complexes that assemble into filaments. The filaments can be organized into linear arrays, coils, rings and gauzes. They serve as membrane-associated scaffolds and as barriers to demarcate local compartments, especially for the establishment of the septation site for cytokinesis. Studies in budding and fission yeast have revealed many of the protein–protein interactions that govern the formation of multi-septin complexes. GTP binding and phosphorylation direct the polymerization of filaments that is required for septin-collar assembly in budding yeast, whereas a homolog of anillin instructs timely formation of the ring of septin filaments at the medial cortex in fission yeast. These insights should aid understanding of the organization and function of the diverse septin structures in animal cells. PMID:16009555

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. Mis17 Is a Regulatory Module of the Mis6-Mal2-Sim4 Centromere Complex That Is Required for the Recruitment of CenH3/CENP-A in Fission Yeast

    PubMed Central

    Shiroiwa, Yoshiharu; Hayashi, Takeshi; Fujita, Yohta; Villar-Briones, Alejandro; Ikai, Nobuyasu; Takeda, Kojiro; Ebe, Masahiro; Yanagida, Mitsuhiro

    2011-01-01

    Background The centromere is the chromosome domain on which the mitotic kinetochore forms for proper segregation. Deposition of the centromeric histone H3 (CenH3, CENP-A) is vital for the formation of centromere-specific chromatin. The Mis6-Mal2-Sim4 complex of the fission yeast S. pombe is required for the recruitment of CenH3 (Cnp1), but its function remains obscure. Methodology/Principal Findings Mass spectrometry was performed on the proteins precipitated with Mis6- and Mis17-FLAG. The results together with the previously identified Sim4- and Mal2-TAP precipitated proteins indicated that the complex contains 12 subunits, Mis6, Sim4, Mal2, Mis15, Mis17, Cnl2, Fta1-4, Fta6-7, nine of which have human centromeric protein (CENP) counterparts. Domain dissection indicated that the carboxy-half of Mis17 is functional, while its amino-half is regulatory. Overproduction of the amino-half caused strong negative dominance, which led to massive chromosome missegregation and hypersensitivity to the histone deacetylase inhibitor TSA. Mis17 was hyperphosphorylated and overproduction-induced negative dominance was abolished in six kinase-deletion mutants, ssp2 (AMPK), ppk9 (AMPK), ppk15 (Yak1), ppk30 (Ark1), wis4 (Ssk2), and lsk1 (P-TEFb). Conclusions Mis17 may be a regulatory module of the Mis6 complex. Negative dominance of the Mis17 fragment is exerted while the complex and CenH3 remain at the centromere, a result that differs from the mislocalization seen in the mis17-362 mutant. The known functions of the kinases suggest an unexpected link between Mis17 and control of the cortex actin, nutrition, and signal/transcription. Possible interpretations are discussed. PMID:21445296

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  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. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  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. Organic growth factor requirements of some yeasts.

    PubMed

    Madan, M; Gulati, N

    1980-01-01

    Some sporogenous yeasts (Brettanomyces bruxellensis, Debaryomyces hansenii, Hansenula ciferrii, Hansenula polymorpha, Pichia polymorpha, Saccharomycopsis guttulata, and Saccharomyces chevalieri), isolated from various fruits have been examined for their organic growth factor requisites. H. ciferrii was completely deficient in thiamine, biotin, inositol, riboflavin, niacin, and partially deficient in pantothenic acid. It required an external supply of 0.1-1.0 ppm thiamine, 0.01-0.1 ppm biotin, 10.0 ppm inositol, 0.10 ppm niacin and riboflavin for its optimum growth. H. polymorpha showed partial deficiency only in xanthine. P. polymorpha gave indications of partial deficiencies in thiamine and biotin. S. guttulata was completely deficient in biotin, and partially deficient in adenine sulphate. It required 0.01 ppm biotin for optimum growth. S chevalieri was completely deficient in pyridoxine and partially deficient in thiamine. It required 0.1 ppm pyridoxine for maximum growth. D. hansenii and B bruxellensis were auxoautotrophic for the various growth factors studied. PMID:7242379

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Characterization of genome-reduced fission yeast strains

    PubMed Central

    Sasaki, Mayumi; Kumagai, Hiromichi; Takegawa, Kaoru; Tohda, Hideki

    2013-01-01

    The Schizosaccharomyces pombe genome is one of the smallest among the free-living eukaryotes. We further reduced the S. pombe gene number by large-scale gene deletion to identify a minimal gene set required for growth under laboratory conditions. The genome-reduced strain has four deletion regions: 168.4 kb in the left arm of chromosome I, 155.4 kb in the right arm of chromosome I, 211.7 kb in the left arm of chromosome II and 121.6 kb in the right arm of chromosome II. The deletions corresponded to a loss of 223 genes of the original ∼5100. The quadruple-deletion strain, with a total deletion size of 657.3 kb, showed a decreased ability to uptake glucose and some amino acids in comparison with the parental strain. The strain also showed increased gene expression of the mating pheromone M-factor precursor and the nicotinamide adenine dinucleotide phosphate -specific glutamate dehydrogenase. There was also a 2.7-fold increase in the concentration of cellular adenosine triphosphate, and levels of the heterologous proteins, enhanced green fluorescent protein and secreted human growth hormone were increased by 1.7- and 1.8-fold, respectively. The transcriptome data from this study have been submitted to the Gene Expression Omnibus (GEO: http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE38620 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=vjkxjewuywgcovc&acc=GSE38620). PMID:23563150

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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)

  2. 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

  3. Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex

    PubMed Central

    Stewart, Emerson V.; Nwosu, Christine C.; Tong, Zongtian; Roguev, Assen; Cummins, Timothy D.; Kim, Dong-Uk; Hayles, Jacqueline; Park, Han-Oh; Hoe, Kwang-Lae; Powell, David W.; Krogan, Nevan J.; Espenshade, Peter J.

    2011-01-01

    SUMMARY Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here, we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1–4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2 conjugating enzyme Ubc4, the Dsc1 RING E3 ligase and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation. PMID:21504829

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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).

  13. 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

  14. 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.

  15. 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

  16. 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

  17. Study of Cyclin Proteolysis in Anaphase-Promoting Complex (APC) Mutant Cells Reveals the Requirement for APC Function in the Final Steps of the Fission Yeast Septation Initiation Network

    PubMed Central

    Chang, Louise; Morrell, Jennifer L.; Feoktistova, Anna; Gould, Kathleen L.

    2001-01-01

    Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombe mutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APC cut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cut mutants and the normal reorganization of septation initiation network components during anaphase. PMID:11533255

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. Fission yeast pak1+ encodes a protein kinase that interacts with Cdc42p and is involved in the control of cell polarity and mating.

    PubMed Central

    Ottilie, S; Miller, P J; Johnson, D I; Creasy, C L; Sells, M A; Bagrodia, S; Forsburg, S L; Chernoff, J

    1995-01-01

    A STE20/p65pak homolog was isolated from fission yeast by PCR. The pak1+ gene encodes a 72 kDa protein containing a putative p21-binding domain near its amino-terminus and a serine/threonine kinase domain near its carboxyl-terminus. The Pak1 protein autophosphorylates on serine residues and preferentially binds to activated Cdc42p both in vitro and in vivo. This binding is mediated through the p21 binding domain on Pak1p and the effector domain on Cdc42p. Overexpression of an inactive mutant form of pak1 gives rise to cells with markedly abnormal shape with mislocalized actin staining. Pak1 overexpression does not, however, suppress lethality associated with cdc42-null cells or the morphologic defeat caused by overexpression of mutant cdc42 alleles. Gene disruption of pak1+ establishes that, like cdc42+, pak1+ function is required for cell viability. In budding yeast, pak1+ expression restores mating function to STE20-null cells and, in fission yeast, overexpression of an inactive form of Pak inhibits mating. These results indicate that the Pak1 protein is likely to be an effector for Cdc42p or a related GTPase, and suggest that Pak1p is involved in the maintenance of cell polarity and in mating. Images PMID:8846783

  4. Nuclear data requirements for fission reactor neutronics calculations.

    SciTech Connect

    Finck, P.

    1998-06-29

    The paper discusses current European nuclear data measurement and evaluation requirements for fission reactor technology applications and problems involved in meeting the requirements. Reference is made to the NEA High Priority Nuclear Data Request List and to the production of the new JEFF-3 library of evaluated nuclear data. There are requirements for both differential (or basic) nuclear data measurements and for different types of integral measurement critical facility measurements and isotopic sample irradiation measurements. Cross-section adjustment procedures are being used to take into account the simpler types of integral measurement, and to define accuracy needs for evaluated nuclear data.

  5. 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.

  6. Cadmium-Induced Proteome Remodeling Regulated by Spc1/Sty1 and Zip1 in Fission Yeast

    PubMed Central

    Russell, Paul

    2012-01-01

    Stress-activated protein kinases and transcription factors are crucial for surviving exposure to cadmium and other environmental toxicants, but their effects on the proteome remain largely unexplored. In this study, isobaric tag for relative and absolute quantitation reveals that cadmium stress triggers rapid proteome remodeling in the fission yeast Schizosaccharomyces pombe. Spc1/Sty1, a mitogen/stress-activated protein kinase homologous to human p38 and Saccharomyces cerevisiae Hog1, controls many of these changes, including enzymes of the oxidative phase of the pentose phosphate pathway and trehalose metabolism. Genetic studies indicate that control of carbohydrate metabolism by Spc1 is required for cadmium tolerance. The bZIP transcription factor Zip1, which is functionally related to human Nrf2 and S. cerevisiae Met4, has a smaller effect on cadmium-induced proteome remodeling, but it is required for production of key proteins involved in sulfur metabolism, which are essential for cadmium resistance. These studies reveal how Spc1 and Zip1 independently reshape the proteome to modulate cellular defense mechanisms against the toxic effects of cadmium. PMID:22610605

  7. Drosophila Wee1 kinase rescues fission yeast from mitotic catastrophe and phosphorylates Drosophila Cdc2 in vitro.

    PubMed Central

    Campbell, S D; Sprenger, F; Edgar, B A; O'Farrell, P H

    1995-01-01

    Cdc2 kinase activity is required for triggering entry into mitosis in all known eukaryotes. Elaborate mechanisms have evolved for regulating Cdc2 activity so that mitosis occurs in a timely manner, when preparations for its execution are complete. In Schizosaccharomyces pombe, Wee1 and a related Mik1 kinase are Cdc2-inhibitory kinases that are required for preventing premature activation of the mitotic program. To identify Cdc2-inhibitory kinases in Drosophila, we screened for cDNA clones that rescue S. pombe wee1- mik1- mutants from lethal mitotic catastrophe. One of the genes identified in this screen, Drosophila wee1 (Dwee1), encodes a new Wee1 homologue. Dwee1 kinase is closely related to human and Xenopus Wee1 homologues, and can inhibit Cdc2 activity by phosphorylating a critical tyrosine residue. Dwee1 mRNA is maternally provided to embryos, and is zygotically expressed during the postblastoderm divisions of embryogenesis. Expression remains high in the proliferating cells of the central nervous system well after cells in the rest of the embryo have ceased dividing. The loss of zygotically expressed Dwee1 does not lead to mitotic catastrophe during postblastoderm cycles 14 to 16. This result may indicate that maternally provided Dwee1 is sufficient for regulating Cdc2 during embryogenesis, or it may reflect the presence of a redundant Cdc2 inhibitory kinase, as in fission yeast. Images PMID:8573790

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

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

    PubMed Central

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

    2016-01-01

    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

  19. Methionine sulphoxide reductases revisited: free methionine as a primary target of H₂O₂stress in auxotrophic fission yeast.

    PubMed

    García-Santamarina, Sarela; Boronat, Susanna; Ayté, José; Hidalgo, Elena

    2013-12-01

    Amino acid methionine can suffer reversible oxidation to sulphoxide and further irreversible over-oxidation to methionine sulphone. As part of the cellular antioxidant scavenging activities are the methionine sulphoxide reductases (Msrs), with a reported role in methionine sulphoxide reduction, both free and in proteins. Three families of Msrs have been described, but the fission yeast genome only includes one representative for two of these families: MsrA/Mxr1 and MsrB/Mxr2. We have investigated their role in methionine reduction and H2 O2 sensitivity. We show here that MsrA/Mxr1 is able to reduce free oxidized methionine. Cells lacking each one of the genes are not significantly sensitive to different types of oxidative stresses, neither display altered life span. However, only when deletion of msrA/mxr1 is combined with deletion of met6, which confers methionine auxotrophy, the survival upon H2 O2 stress decreases by 100-fold. In fact, cells lacking only Met6, and which therefore require addition of methionine to the growth media, are extremely sensitive to H2 O2 stress. These and other evidences suggest that oxidation of free methionine is a primary target of peroxide toxicity in cells devoid of methionine biosynthetic capacity, and that an important role of Msrs is to recycle this oxidized free amino acid. PMID:24118096

  20. The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast

    PubMed Central

    Mbogning, Jean; Nagy, Stephen; Pagé, Viviane; Schwer, Beate; Shuman, Stewart; Fisher, Robert P.; Tanny, Jason C.

    2013-01-01

    Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways. PMID:24385927

  1. 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

  2. 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

  3. Multi-step coordination of telomerase recruitment in fission yeast through two coupled telomere-telomerase interfaces

    PubMed Central

    Hu, Xichan; Liu, Jinqiang; Jun, Hyun-IK; Kim, Jin-Kwang; Qiao, Feng

    2016-01-01

    Tightly controlled recruitment of telomerase, a low-abundance enzyme, to telomeres is essential for regulated telomere synthesis. Recent studies in human cells revealed that a patch of amino acids in the shelterin component TPP1, called the TEL-patch, is essential for recruiting telomerase to telomeres. However, how TEL-patch—telomerase interaction integrates into the overall orchestration of telomerase regulation at telomeres is unclear. In fission yeast, Tel1ATM/Rad3ATR-mediated phosphorylation of shelterin component Ccq1 during late S phase is involved in telomerase recruitment through promoting the binding of Ccq1 to a telomerase accessory protein Est1. Here, we identify the TEL-patch in Tpz1TPP1, mutations of which lead to decreased telomeric association of telomerase, similar to the phosphorylation-defective Ccq1. Furthermore, we find that telomerase action at telomeres requires formation and resolution of an intermediate state, in which the cell cycle-dependent Ccq1-Est1 interaction is coupled to the TEL-patch—Trt1 interaction, to achieve temporally regulated telomerase elongation of telomeres. DOI: http://dx.doi.org/10.7554/eLife.15470.001 PMID:27253066

  4. Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16XPF and Inhibiting Dna2

    PubMed Central

    Zhang, Jia-Min; Liu, Xiao-Man; Ding, Yue-He; Xiong, Liang-Yao; Ren, Jing-Yi; Zhou, Zhi-Xiong; Wang, Hai-Tao; Zhang, Mei-Jun; Yu, Yang; Dong, Meng-Qiu; Du, Li-Lin

    2014-01-01

    Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3′ endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5′ endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases. PMID:25203555

  5. Sum1, a highly conserved WD-repeat protein, suppresses S-M checkpoint mutants and inhibits the osmotic stress cell cycle response in fission yeast.

    PubMed Central

    Humphrey, T; Enoch, T

    1998-01-01

    The S-M checkpoint ensures that entry into mitosis is dependent on completion of DNA replication. In the fission yeast Schizosaccharomyces pombe, the SM checkpoint mutant cdc2-3w is thought to be defective in receiving the checkpoint signal. To isolate genes that function in the checkpoint pathway, we screened an S. pombe cDNA library for genes that, when overexpressed, could suppress the checkpoint defect of cdc2-3w. Using this approach, we have identified a novel gene, sum1+ (suppressor of uncontrolled mitosis). sum1+ encodes a highly conserved WD-transducin repeat protein with striking sequence similarity to the human transforming growth factor (TGF)-beta-receptor interacting protein TRIP-1 and to the translation initiation factor 3 subunit eIF3-p39, encoded by the TIF34 gene in Saccharomyces cerevisiae. S. pombe sum1+ is an essential gene, required for normal cell growth and division. In addition to restoring checkpoint control, overexpression of sum1+ inhibits the normal cell cycle response to osmotic stress. Furthermore, we demonstrate that inactivation of the stress-activated MAP kinase pathway, required for cell cycle stress response, restores the S-M checkpoint in cdc2-3w cells. These results suggest that Suml interacts with the stress-activated MAP kinase pathway and raise the possibility that environmental conditions may influence the checkpoint response in fission yeast. PMID:9560390

  6. Oxygen requirements of yeasts. [Saccharomyces cerevisiae; Candida tropicalis

    SciTech Connect

    Visser, W.; Scheffers, W.A.; Batenburg-Van Der Vegte, W.H.; Van Dijken, J.P. )

    1990-12-01

    Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly ({mu}{sub max}, 0.03 and 0.05 h{sup {minus}1}, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.

  7. Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast.

    PubMed

    Yang, Hanna; Kwon, Chang Seob; Choi, Yoonjung; Lee, Daeyoup

    2016-08-01

    Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5' promoter and 3' termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast. PMID:27268234

  8. 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

  9. 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

  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. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  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. Sip1, a Conserved AP-1 Accessory Protein, Is Important for Golgi/Endosome Trafficking in Fission Yeast

    PubMed Central

    Yu, Yang; Kita, Ayako; Udo, Masako; Katayama, Yuta; Shintani, Mami; Park, Kwihwa; Hagihara, Kanako; Umeda, Nanae; Sugiura, Reiko

    2012-01-01

    We had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4+/sip1+, which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1-deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl− and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking. PMID:23028933

  19. Differential requirements for actin during yeast and mammalian endocytosis.

    PubMed

    Aghamohammadzadeh, Soheil; Ayscough, Kathryn R

    2009-08-01

    Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination. PMID:19597484

  20. 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

  1. 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

  2. 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

  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

    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

  5. 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

  6. Identification of genes encoding putative nucleoporins and transport factors in the fission yeast Schizosaccharomyces pombe: a deletion analysis.

    PubMed

    Chen, Xue Qin; Du, Xianming; Liu, Jianhua; Balasubramanian, Mohan K; Balasundaram, David

    2004-04-30

    In a systematic approach to study genes that are related to nucleocytoplasmic trafficking in the fission yeast Schizosaccharomyces pombe, the open reading frames (ORFs) of 26 putative nucleoporins and transport factors were deleted. Here we report the initial characterization of these deletion mutants. Of the 26 putative genes deleted, 14 were found to be essential for viability. Null mutations of essential genes resulted in failure to either complete one round or to sustain cell division. Four of the 14 essential genes, SPBC582.11c, SPBC17G9.04c, SPBC3B9.16c and SPCC162.08c, encode putative nucleoporins and a myosin-like protein with homologues NUP84, NUP85, NUP120 and MLP1, respectively, that are not required for viability in Saccharomyces cerevisiae, suggesting that their gene products perform critical functions in Sz. pombe. On the basis of combined drug sensitivity assays and genetic analysis we have identified five non-essential null mutants that were hypersensitive to the microtubule depolymerizing drug thiabendazole (TBZ) and exhibited a cut phenotype upon TBZ treatment, suggesting possible involvement in microtubule function. Three of the corresponding ORFs, SPCC18B5.07c, nup40 and SPAC1805.04, encode putative nucleoporins with low similarity to the S. cerevisiae nucleoporins NUP2p, NUP53p and NUP133p, respectively. Further genetic analysis revealed that one of the nucleoporin genes, nup40, and another gene, SPCC1322.06, encoding a putative importin-beta/Cse1p superfamily protein may have a spindle checkpoint function. PMID:15116432

  7. Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

    PubMed Central

    Tabuchi, M; Iwaihara, O; Ohtani, Y; Ohuchi, N; Sakurai, J; Morita, T; Iwahara, S; Takegawa, K

    1997-01-01

    PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal. PMID:9209031

  8. 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

  9. Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast

    PubMed Central

    G. Cortés, Juan C.; Pujol, Nuria; Sato, Mamiko; Pinar, Mario; Ramos, Mariona; Moreno, Belén; Osumi, Masako; Ribas, Juan Carlos; Pérez, Pilar

    2015-01-01

    In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast. PMID:26132084

  10. Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast.

    PubMed

    Cortés, Juan C G; Pujol, Nuria; Sato, Mamiko; Pinar, Mario; Ramos, Mariona; Moreno, Belén; Osumi, Masako; Ribas, Juan Carlos; Pérez, Pilar

    2015-07-01

    In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast. PMID:26132084

  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. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Rho4 interaction with exocyst and septins regulates cell separation in fission yeast.

    PubMed

    Pérez, Pilar; Portales, Elvira; Santos, Beatriz

    2015-05-01

    Rho GTPases are small proteins present in all eukaryotic cells, from yeast to mammals, with a function in actin organization and morphogenetic processes. Schizosaccharomyces pombe Rho4 is not essential but it displays a role during cell separation at high temperature. In fact, Rho4 is involved in the secretion of the hydrolytic enzymes that are required for cell septum degradation during this process. In rho4Δ cells, vesicles accumulate in the septum area and the glucanases Eng1 and Agn1 are not secreted to the culture medium. The localization of Eng1 and Agn1 depends on the exocyst and the septins. The exocyst is a conserved multiprotein complex important for the targeting and fusion of Golgi-derived vesicles with the plasma membrane. Septins are a family of GTP-binding proteins conserved in eukaryotes that function during cytokinesis. Here we show that Rho4 is required for the proper localization of the exocyst and septins at high temperature. Moreover, pull-down experiments demonstrate that Rho4 can interact with exocyst subunits, such as Sec8 and Exo70, and septin proteins, such as Spn3. We observe that Sec8 preferentially binds to activated GTP-Rho4, suggesting that Sec8 could be an effector of this GTPase. We propose that the interaction of Rho4 with the exocyst and septins confers a precise regulation for the secretion of glucanases at the appropriate place and time during the cell cycle. PMID:25724972

  20. PombeX: robust cell segmentation for fission yeast transillumination images.

    PubMed

    Peng, Jyh-Ying; Chen, Yen-Jen; Green, Marc D; Sabatinos, Sarah A; Forsburg, Susan L; Hsu, Chun-Nan

    2013-01-01

    Schizosaccharomyces pombe shares many genes and proteins with humans and is a good model for chromosome behavior and DNA dynamics, which can be analyzed by visualizing the behavior of fluorescently tagged proteins in vivo. Performing a genome-wide screen for changes in such proteins requires developing methods that automate analysis of a large amount of images, the first step of which requires robust segmentation of the cell. We developed a segmentation system, PombeX, that can segment cells from transmitted illumination images with focus gradient and varying contrast. Corrections for focus gradient are applied to the image to aid in accurate detection of cell membrane and cytoplasm pixels, which is used to generate initial contours for cells. Gradient vector flow snake evolution is used to obtain the final cell contours. Finally, a machine learning-based validation of cell contours removes most incorrect or spurious contours. Quantitative evaluations show overall good segmentation performance on a large set of images, regardless of differences in image quality, lighting condition, focus condition and phenotypic profile. Comparisons with recent related methods for yeast cells show that PombeX outperforms current methods, both in terms of segmentation accuracy and computational speed. PMID:24353754

  1. Factors that promote H3 chromatin integrity during transcription prevent promiscuous deposition of CENP-A(Cnp1) in fission yeast.

    PubMed

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

    2012-09-01

    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-A(Cnp1) deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites. FACT has little or no effect on CENP-A(Cnp1) assembly at endogenous centromeres where CENP-A(Cnp1) 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-A(Cnp1) at specific loci, including subtelomeric regions, where CENP-A(Cnp1) is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A(Cnp1) 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-A(Cnp1) 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-A(Cnp1) chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification. PMID:23028377

  2. 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.

  3. Its8, a fission yeast homolog of Mcd4 and Pig-n, is involved in GPI anchor synthesis and shares an essential function with calcineurin in cytokinesis.

    PubMed

    Yada, T; Sugiura, R; Kita, A; Itoh, Y; Lu, Y; Hong, Y; Kinoshita, T; Shuntoh, H; Kuno, T

    2001-04-27

    In fission yeast, calcineurin is required for cytokinesis and ion homeostasis; however, most of its physiological roles remain obscure. To identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature and isolated the mutant its8-1. its8(+) encodes a homolog of the budding yeast MCD4 and human Pig-n that are involved in glycosylphosphatidylinositol (GPI) anchor synthesis. Consistently, reduced inositol labeling of proteins suggested impaired GPI anchor synthesis in its8-1 mutants. The temperature upshift induced a further decrease in inositol labeling and caused dramatic increases in the frequency of septation in its8-1 mutants. BE49385A, an inhibitor of MCD4 and Pig-n, also increased the septation index of the wild-type cell. Osmotic stabilization suppressed these morphological defects, indicating that cell wall weakness caused by impaired GPI anchor synthesis resulted in abnormal cytokinesis. Furthermore, calcineurin-deleted cells exhibited hypersensitivity to BE49385A, and FK506 exacerbated the cytokinesis defects of the its8-1 mutant. Thus, calcineurin and Its8 may share an essential function in cytokinesis and cell viability through the regulation of cell wall integrity. PMID:11297516

  4. 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.

  5. 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

  6. The yeast nuclear import receptor is required for mitosis.

    PubMed Central

    Loeb, J D; Schlenstedt, G; Pellman, D; Kornitzer, D; Silver, P A; Fink, G R

    1995-01-01

    The nuclear import system is highly conserved among eukaryotes. Here we report the effects of a conditional mutation in SRP1, which encodes a Saccharomyces cerevisiae homolog of the vertebrate nuclear import receptor importin. Importin was isolated as a factor required for the initial targeting step of a nuclear import substrate to the nuclear envelope in a mammalian in vitro assay. We show that yeast Srp1 is similarly required for protein import. In addition, Srp1 is also required for the execution of mitosis: we demonstrate that cells containing a conditional mutation of SRP1 arrest with a G2/M phenotype in a manner analogous to classic cdc mutants. This defect may be due to the failure of the mutant to degrade the mitotic cyclin Clb2 and other proteins required for mitosis. The requirement of a nuclear import receptor for cell cycle-regulated proteolysis implies that import of cell cycle regulators into the nucleus is critical for cell cycle progression. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7644471

  7. Licensing of yeast centrosome duplication requires phosphoregulation of sfi1.

    PubMed

    Avena, Jennifer S; Burns, Shannon; Yu, Zulin; Ebmeier, Christopher C; Old, William M; Jaspersen, Sue L; Winey, Mark

    2014-10-01

    Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and is controlled in part by cyclin-dependent kinases (Cdks). Our study identifies Sfi1, a conserved component of centrosomes, as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. We found that reducing Cdk1 phosphorylation by changing Sfi1 phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated spindle pole bodies (SPBs, yeast centrosomes) and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. Our findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, we provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1. PMID:25340401

  8. Licensing of Yeast Centrosome Duplication Requires Phosphoregulation of Sfi1

    PubMed Central

    Avena, Jennifer S.; Burns, Shannon; Yu, Zulin; Ebmeier, Christopher C.; Old, William M.; Jaspersen, Sue L.; Winey, Mark

    2014-01-01

    Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and is controlled in part by cyclin-dependent kinases (Cdks). Our study identifies Sfi1, a conserved component of centrosomes, as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. We found that reducing Cdk1 phosphorylation by changing Sfi1 phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated spindle pole bodies (SPBs, yeast centrosomes) and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. Our findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, we provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1. PMID:25340401

  9. 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

  10. Real-Time Monitoring of Calcineurin Activity in Living Cells: Evidence for Two Distinct Ca2+-dependent Pathways in Fission Yeast

    PubMed Central

    Deng, Lu; Sugiura, Reiko; Takeuchi, Mai; Suzuki, Masahiro; Ebina, Hidemine; Takami, Tomonori; Koike, Atsushi; Iba, Shiori

    2006-01-01

    In fission yeast, calcineurin dephosphorylates and activates the Prz1 transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of prz1+ gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. Elevated extracellular CaCl2 caused an increase in calcineurin activity with an initial peak and then approached a sustained constant level in a concentration-dependent manner. In CaCl2-sensitive mutants such as Δpmc1, the response was markedly enhanced, reflecting its high intracellular Ca2+. Agents expected to induce Ca2+ influx showed distinct patterns of the CDRE-reporter activity, suggesting different mechanisms of calcineurin activation. Knockout of yam8+ or cch1+ encoding putative subunits of a Ca2+ channel abolished the activation of calcineurin upon exposure to various stimuli, including high extracellular NaCl and cell wall–damaging agents. However, knockout of yam8+ or cch1+ did not affect the activation of calcineurin upon stimulation by elevated extracellular Ca2+. The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca2+ influx, but it was not required for the stimulation by elevated extracellular Ca2+, suggesting two distinct pathways for calcineurin activation. PMID:16928959

  11. Role of the Rab GTP-Binding Protein Ypt3 in the Fission Yeast Exocytic Pathway and Its Connection to Calcineurin Function

    PubMed Central

    Cheng, Hong; Sugiura, Reiko; Wu, Wenlian; Fujita, Masaaki; Lu, Yabin; Sio, Susie O.; Kawai, Rena; Takegawa, Kaoru; Shuntoh, Hisato; Kuno, Takayoshi

    2002-01-01

    A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1+ leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes. PMID:12181359

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. A role for DNA polymerase α in epigenetic control of transcriptional silencing in fission yeast

    PubMed Central

    Nakayama, Jun-ichi; Allshire, Robin C.; Klar, Amar J.S.; Grewal, Shiv I.S.

    2001-01-01

    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 α (polα) 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 polα mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polα and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Polα in vitro. Moreover, silencing-defective mutant Polα displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Polα, in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  19. Intronic sequence elements impede exon ligation and trigger a discard pathway that yields functional telomerase RNA in fission yeast

    PubMed Central

    Kannan, Ram; Hartnett, Sean; Voelker, Rodger B.; Berglund, J. Andrew; Staley, Jonathan P.; Baumann, Peter

    2013-01-01

    The fission yeast telomerase RNA (TER1) precursor harbors an intron immediately downstream from its mature 3′ end. Unlike most introns, which are removed from precursor RNAs by the spliceosome in two sequential but tightly coupled transesterification reactions, TER1 only undergoes the first cleavage reaction during telomerase RNA maturation. The mechanism underlying spliceosome-mediated 3′ end processing has remained unclear. We now demonstrate that a strong branch site (BS), a long distance to the 3′ splice site (3′ SS), and a weak polypyrimidine (Py) tract act synergistically to attenuate the transition from the first to the second step of splicing. The observation that a strong BS antagonizes the second step of splicing in the context of TER1 suggests that the BS–U2 snRNA interaction is disrupted after the first step and thus much earlier than previously thought. The slow transition from first to second step triggers the Prp22 DExD/H-box helicase-dependent rejection of the cleaved products and Prp43-dependent “discard” of the splicing intermediates. Our findings explain how the spliceosome can function in 3′ end processing and provide new insights into the mechanism of splicing. PMID:23468430

  20. Two fission yeast B-type cyclins, cig2 and Cdc13, have different functions in mitosis.

    PubMed Central

    Bueno, A; Russell, P

    1993-01-01

    Cyclin B interacts with Cdc2 kinase to induce cell cycle events, particularly those of mitosis. The existence of cyclin B subtypes in several species has been known for some time, leading to speculation that key events of mitosis may be carried out by distinct functional classes of Cdc2/cyclin B. We report the discovery of cig2, a third B-type cyclin gene in Schizosaccharomyces pombe. Disruption of cig2 delays the onset of mitosis, to the degree that a cig2 null allele rescues mitotic catastrophe mutants, including those that are unable to carry out the inhibitory tyrosyl phosphorylation of Cdc2 kinase. Consistent with this, a cig2 null allele exhibits synthetic lethal interactions with cdc25ts and cdc2ts mutations. Mitotic phenotypes caused by disruption of cig2 are not reversed by increased production of Cdc13, the other fission yeast B-type cyclin that functions in mitosis. Likewise, a cdc13ts mutation is not rescued by increased gene dosage of cig2+. These data indicate that Cdc13 and Cig2 interact with Cdc2 to carry out different functions in mitosis. We suggest that some cyclin B subtypes found in other species, including humans, are also likely to have distinct, nonoverlapping functions in mitosis. Images PMID:8455610

  1. The Clr1 Locus Regulates the Expression of the Cryptic Mating-Type Loci of Fission Yeast

    PubMed Central

    Thon, G.; Klar, AJS.

    1992-01-01

    The mat2-P and mat3-M loci of fission yeast contain respectively the plus (P) and minus (M) mating-type information in a transcriptionally silent state. That information is transposed from the mat2 or mat3 donor locus via recombination into the expressed mating-type locus (mat1) resulting in switching of the cellular mating type. We have identified a gene, named clr1 (for cryptic loci regulator), whose mutations allow expression of the mat2 and mat3 loci. clr1 mutants undergo aberrant haploid meiosis, indicative of transcription of the silent genes. Production of mRNA from mat3 is detectable in clr1 mutants. Furthermore, the ura4 gene inserted near mat3, weakly expressed in wild-type cells, is derepressed in clr1 mutants. The clr1 mutations also permit meiotic recombination in the 15-kb mat2-mat3 interval, where recombination is normally inhibited. The clr1 locus is in the right arm of chromosome II. We suggest that clr1 regulates silencing of the mat2 and mat3 loci, and participates in establishing the ``cold spot'' for recombination by organizing the chromatin structure of the mating-type region. PMID:1644273

  2. Contributions of transcription and mRNA decay to gene expression dynamics of fission yeast in response to oxidative stress

    PubMed Central

    Marguerat, Samuel; Lawler, Katherine; Brazma, Alvis; Bähler, Jürg

    2014-01-01

    The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress. PMID:25007214

  3. Multiple layers of regulation influence cell integrity control by the PKC ortholog Pck2 in fission yeast.

    PubMed

    Madrid, Marisa; Jiménez, Rafael; Sánchez-Mir, Laura; Soto, Teresa; Franco, Alejandro; Vicente-Soler, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2015-01-15

    The fission yeast protein kinase C (PKC) ortholog Pck2 controls cell wall synthesis and is a major upstream activator of the cell integrity pathway (CIP) and its core component, the MAP kinase Pmk1 (also known as Spm1), in response to environmental stimuli. We show that in vivo phosphorylation of Pck2 at the conserved T842 activation loop during growth and in response to different stresses is mediated by the phosphoinositide-dependent kinase (PDK) ortholog Ksg1 and an autophosphorylation mechanism. However, T842 phosphorylation is not essential for Pmk1 activation, and putative phosphorylation at T846 might play an additional role in Pck2 catalytic activation and downstream signaling. These events, together with turn motif autophosphorylation at T984 and binding to small GTPases Rho1 and/or Rho2, stabilize Pck2 and render it competent to exert its biological functions. Remarkably, the target of rapamycin complex 2 (TORC2) does not participate in the catalytic activation of Pck2, but instead contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion. These results unveil a novel mechanism whereby TOR regulates PKC function at a translational level, and they add a new regulatory layer to MAPK signaling cascades. PMID:25416816

  4. 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

  5. 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

  6. Constriction model of actomyosin ring for cytokinesis by fission yeast using a two-state sliding filament mechanism

    NASA Astrophysics Data System (ADS)

    Jung, Yong-Woon; Mascagni, Michael

    2014-09-01

    We developed a model describing the structure and contractile mechanism of the actomyosin ring in fission yeast, Schizosaccharomyces pombe. The proposed ring includes actin, myosin, and α-actinin, and is organized into a structure similar to that of muscle sarcomeres. This structure justifies the use of the sliding-filament mechanism developed by Huxley and Hill, but it is probably less organized relative to that of muscle sarcomeres. Ring contraction tension was generated via the same fundamental mechanism used to generate muscle tension, but some physicochemical parameters were adjusted to be consistent with the proposed ring structure. Simulations allowed an estimate of ring constriction tension that reproduced the observed ring constriction velocity using a physiologically possible, self-consistent set of parameters. Proposed molecular-level properties responsible for the thousand-fold slower constriction velocity of the ring relative to that of muscle sarcomeres include fewer myosin molecules involved, a less organized contractile configuration, a low α-actinin concentration, and a high resistance membrane tension. Ring constriction velocity is demonstrated as an exponential function of time despite a near linear appearance. We proposed a hypothesis to explain why excess myosin heads inhibit constriction velocity rather than enhance it. The model revealed how myosin concentration and elastic resistance tension are balanced during cytokinesis in S. pombe.

  7. Structure and Biochemical Properties of Fission Yeast Arp2/3 Complex Lacking the Arp2 Subunit

    SciTech Connect

    Nolen, B.; Pollard, T

    2008-01-01

    Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2? Arp2/3 complex) from fission yeast. An x-ray crystal structure of the ?Arp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2? Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2? Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.

  8. The fission yeast pleckstrin homology domain protein Spo7 is essential for initiation of forespore membrane assembly and spore morphogenesis

    PubMed Central

    Nakamura-Kubo, Michiko; Hirata, Aiko; Shimoda, Chikashi; Nakamura, Taro

    2011-01-01

    Sporulation in fission yeast represents a unique mode of cell division in which a new cell is formed within the cytoplasm of a mother cell. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores. At prophase II, the spindle pole body (SPB) forms an outer plaque, from which formation of the FSM is initiated. Several components of the SPB play an indispensable role in SPB modification, and therefore in sporulation. In this paper, we report the identification of a novel SPB component, Spo7, which has a pleckstrin homology (PH) domain. We found that Spo7 was essential for initiation of FSM assembly, but not for SPB modification. Spo7 directly bound to Meu14, a component of the leading edge of the FSM, and was essential for proper localization of Meu14. The PH domain of Spo7 had affinity for phosphatidylinositol 3-phosphate (PI3P). spo7 mutants lacking the PH domain showed aberrant spore morphology, similar to that of meu14 and phosphatidylinositol 3-kinase (pik3) mutants. Our study suggests that Spo7 coordinates formation of the leading edge and initiation of FSM assembly, thereby accomplishing accurate formation of the FSM. PMID:21775631

  9. Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast.

    PubMed Central

    Jia, Z P; McCullough, N; Martel, R; Hemmingsen, S; Young, P G

    1992-01-01

    We have identified a new locus, sodium 2 (sod2) based on selection for increased LiCl tolerance in fission yeast, Schizosaccharomyces pombe. Tolerant strains have enhanced pH-dependent Na+ export capacity and sodium transport experiments suggest that the gene encodes an Na+/H+ antiport. The predicted sod2 gene product can be placed in the broad class of transporters which possess 12 hydrophobic transmembrane domains. The protein shows some sequence similarity to the human and bacterial Na+/H+ antiporters. Overexpression of sod2 increased Na+ export capacity and conferred sodium tolerance. Osmotolerance was not affected and sod2 cells were unaffected for growth in K+. In a sod2 disruption strain cells were incapable of exporting sodium. They were hypersensitive to Na+ or Li+ and could not grow under conditions that approximate pH7. The sod2 gene amplification could be selected stepwise and the degree of such amplification correlated with the level of Na+ or Li+ tolerance. Images PMID:1314171

  10. 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

  11. Regulation of the antioxidant system in cells of the fission yeast Schizosaccharomyces pombe after combined treatment with patulin and citrinin.

    PubMed

    Papp, Gábor; Máté, Gábor; Mike, Nóra; Gazdag, Zoltán; Pesti, Miklós

    2016-03-01

    The effects of combined treatment with patulin (PAT) and citrinin (CTN) on Schizosaccharomyces pombe cells were investigated in acute toxicity tests. In comparison with the controls the exposure of fission yeast cells (10(7) cells ml(-1)) to PAT + CTN (250 μM each) for 1 h at a survival rate of 66.6% significantly elevated the concentration of total reactive oxygen species (ROS) via increased levels of peroxides without affecting the concentrations of superoxides or the hydroxyl radical. This treatment induced a 3.08-fold increase in the specific concentration of glutathione and elevated specific activities of catalase and glutathione S-transferase, while at the same time the activity of glutathione reductase decreased. The pattern of the ROS was the same as that induced by CTN (Máté et al., 2014), while the presence of PAT in the PAT + CTN combination treatment modified the activities of the antioxidant system (Papp et al., 2012) in comparison with the individual PAT or CTN treatment, suggesting toxin-specific regulation of glutathione and the enzymes of the antioxidant system and the possibility that the transcription factor (pap1 and atf1) -regulated processes might be influenced directly by ROS. PMID:26752674

  12. Constriction model of actomyosin ring for cytokinesis by fission yeast using a two-state sliding filament mechanism

    SciTech Connect

    Jung, Yong-Woon; Mascagni, Michael

    2014-09-28

    We developed a model describing the structure and contractile mechanism of the actomyosin ring in fission yeast, Schizosaccharomyces pombe. The proposed ring includes actin, myosin, and α-actinin, and is organized into a structure similar to that of muscle sarcomeres. This structure justifies the use of the sliding-filament mechanism developed by Huxley and Hill, but it is probably less organized relative to that of muscle sarcomeres. Ring contraction tension was generated via the same fundamental mechanism used to generate muscle tension, but some physicochemical parameters were adjusted to be consistent with the proposed ring structure. Simulations allowed an estimate of ring constriction tension that reproduced the observed ring constriction velocity using a physiologically possible, self-consistent set of parameters. Proposed molecular-level properties responsible for the thousand-fold slower constriction velocity of the ring relative to that of muscle sarcomeres include fewer myosin molecules involved, a less organized contractile configuration, a low α-actinin concentration, and a high resistance membrane tension. Ring constriction velocity is demonstrated as an exponential function of time despite a near linear appearance. We proposed a hypothesis to explain why excess myosin heads inhibit constriction velocity rather than enhance it. The model revealed how myosin concentration and elastic resistance tension are balanced during cytokinesis in S. pombe.

  13. The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast*

    PubMed Central

    Muñoz, Sofía; Manjón, Elvira; Sánchez, Yolanda

    2014-01-01

    The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3+ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation. PMID:24947517

  14. Casein Kinase 1γ Ensures Monopolar Growth Polarity under Incomplete DNA Replication Downstream of Cds1 and Calcineurin in Fission Yeast

    PubMed Central

    Koyano, Takayuki; Konishi, Manabu; Martin, Sophie G.; Ohya, Yoshikazu; Hirata, Dai

    2015-01-01

    Cell polarity is essential for various cellular functions during both proliferative and developmental stages, and it displays dynamic alterations in response to intracellular and extracellular cues. However, the molecular mechanisms underlying spatiotemporal control of polarity transition are poorly understood. Here, we show that fission yeast Cki3 (a casein kinase 1γ homolog) is a critical regulator to ensure persistent monopolar growth during S phase. Unlike the wild type, cki3 mutant cells undergo bipolar growth when S phase is blocked, a condition known to delay transition from monopolar to bipolar growth (termed NETO [new end takeoff]). Consistent with this role, Cki3 kinase activity is substantially increased, and cells lose their viability in the absence of Cki3 upon an S-phase block. Cki3 acts downstream of the checkpoint kinase Cds1/Chk2 and calcineurin, and the latter physically interacts with Cki3. Autophosphorylation in the C terminus is inhibitory toward Cki3 kinase activity, and calcineurin is responsible for its dephosphorylation. Cki3 localizes to the plasma membrane, and this localization requires the palmitoyltransferase complex Erf2-Erf4. Membrane localization is needed not only for proper NETO timing but also for Cki3 kinase activity. We propose that Cki3 acts as a critical inhibitor of cell polarity transition under S-phase arrest. PMID:25691662

  15. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    SciTech Connect

    Kobayashi, Ayaho; Kanaba, Teppei; Satoh, Ryosuke; Fujiwara, Toshinobu; Ito, Yutaka; Sugiura, Reiko; Mishima, Masaki

    2013-07-19

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.

  16. lncRNA recruits RNAi and the exosome to dynamically regulate pho1 expression in response to phosphate levels in fission yeast

    PubMed Central

    Shah, Sneha; Wittmann, Sina; Kilchert, Cornelia; Vasiljeva, Lidia

    2014-01-01

    Numerous noncoding transcripts of unknown function have recently been identified. In this study, we report a novel mechanism that relies on transcription of noncoding RNA prt (pho1-repressing transcript) regulating expression of the pho1 gene. A product of this gene, Pho1, is a major secreted phosphatase needed for uptake of extracellular phosphate in fission yeast. prt is produced from the promoter located upstream of the pho1 gene in response to phosphate, and its transcription leads to deposition of RNAi-dependent H3K9me2 across the pho1 locus. In contrast, phosphate starvation leads to loss of H3K9me2 and pho1 induction. Strikingly, deletion of Clr4, a H3K9 methyltransferase, results in faster pho1 induction in response to phosphate starvation. We propose a new role for noncoding transcription in establishing transient heterochromatin to mediate an effective transcriptional response to environmental stimuli. RNAi recruitment to prt depends on the RNA-binding protein Mmi1. Importantly, we found that the exosome complex and Mmi1 are required for transcription termination and the subsequent degradation of prt but not pho1 mRNA. Moreover, in mitotic cells, transcription termination of meiotic RNAs also relies on this mechanism. We propose that exosome-dependent termination constitutes a specialized system that primes transcripts for degradation to ensure their efficient elimination. PMID:24493644

  17. The fission yeast DASH complex is essential for satisfying the spindle assembly checkpoint induced by defects in the inner-kinetochore proteins.

    PubMed

    Kobayashi, Yasuyo; Saitoh, Shigeaki; Ogiyama, Yuki; Soejima, Saeko; Takahashi, Kohta

    2007-03-01

    Spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance system for chromosome missegregation. We isolated fission yeast Hos2, a component of the Dam1/DASH complex, as a multicopy suppressor of temperature-sensitive (ts) growth of nnf1-495 mutant that exhibits the minichromosome instability (mis) phenotype, producing lethal aneuploids without prominent mitotic delay. It remains elusive why SAC is satisfied in mis mutants despite the occurrence of missegregation. We found that Hos2 binds to the inner-kinetochore regions in both prometaphase and metaphase. Hos2 is essential for kinetochore localization of Dis1, a microtubule (MT) associated Dis1/XMAP215/TOG family protein that is required for proper MT dynamics. Cells lacking DASH exhibit cold-sensitive (cs) growth with the defective in sister-chromatid disjoining (dis) phenotype, which is characterized by hyper-condensed sister-chromatid pairs and elongated spindle MTs. Although DASH-deficient cells are viable at high temperatures, DASH-deletion transforms all the inner-kinetochore mis mutants so far tested into a constitutively active state of SAC, leading to the dis phenotype. We also discovered that Hos2 over-expression commonly suppresses growth retardation in a variety of inner-kinetochore mutants. These genetic interactions highlight the DASH-action(s) in satisfying SAC when aneuploids are formed during mitosis in the inner-kinetochore-defective mis mutants. PMID:17352737

  18. Citrinin-induced fluidization of the plasma membrane of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Blaskó, Ágnes; Mike, Nóra; Gróf, Pál; Gazdag, Zoltán; Czibulya, Zsuzsanna; Nagy, Lívia; Kunsági-Máté, Sándor; Pesti, Miklós

    2013-09-01

    Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. In the present study, its acute toxicity and effects on the plasma membrane of Schizosaccharomyces pombe were investigated. The minimum inhibitory concentration of CTN against the yeast cells proved to be 500 μM. Treatment with 0, 250, 500 or 1000 μM CTN for 60 min resulted in a 0%, 2%, 21% or 100% decrease, respectively, in the survival rate of the cell population. Treatment of cells with 0, 100, 500 or 1000 μM CTN for 20 min induced decrease in the phase-transition temperature of the 5-doxylstearic acid-labeled plasma membrane to 16.51, 16.04, 14.18 or 13.98°C, respectively as measured by electron paramagnetic resonance spectroscopy. This perturbation was accompanied by the efflux of essential K⁺ from the cells. The existence of an interaction between CTN and glutathione was detected for the first time by spectrofluorometry. Our observations may suggest a direct interaction of CTN with the free sulfhydryl groups of the integral proteins of the plasma membrane, leading to dose-dependent membrane fluidization. The change in fluidity disturbed the ionic homeostasis, contributing to the death of the cells, which is a novel aspect of CTN cytotoxicity. PMID:23851147

  19. Characterization of the ptr5{sup +} gene involved in nuclear mRNA export in fission yeast

    SciTech Connect

    Watanabe, Nobuyoshi; Ikeda, Terumasa; Mizuki, Fumitaka; Tani, Tokio

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We cloned the ptr5{sup +} gene involved in nuclear mRNA export in fission yeast. Black-Right-Pointing-Pointer The ptr5{sup +} gene was found to encode nucleoporin 85 (Nup85). Black-Right-Pointing-Pointer Seh1p and Mlo3p are multi-copy suppressors for the ptr5 mutation. Black-Right-Pointing-Pointer Ptr5p/Nup85p functions in nuclear mRNA export through the mRNA export factor Rae1p. Black-Right-Pointing-Pointer Ptr5p/Nup85p interacts genetically with pre-mRNA splicing factors. -- Abstract: To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A){sup +} RNA transport] 1 to 11, which accumulate poly(A){sup +} RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5-1 mutant shows dots- or a ring-like accumulation of poly(A){sup +} RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5{sup +} gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5-1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5-1 mutation. In addition, we found that the ptr5-1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5-1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

  20. The hairpin region of Ndc80 is important for the kinetochore recruitment of Mph1/MPS1 in fission yeast.

    PubMed

    Chmielewska, Aldona Ewa; Tang, Ngang Heok; Toda, Takashi

    2016-03-01

    The establishment of proper kinetochore-microtubule attachments facilitates faithful chromosome segregation. Incorrect attachments activate the spindle assembly checkpoint (SAC), which blocks anaphase onset via recruitment of a cohort of SAC components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to kinetochores. KNL1, a component of the outer kinetochore KMN network (KNL1/Mis12 complex/Ndc80 complex), acts as a platform for Bub1 and Bub3 localization upon its phosphorylation by Mph1/MPS1. The Ndc80 protein, a major microtubule-binding site, is critical for MPS1 localization to the kinetochores in mammalian cells. Here we characterized the newly isolated mutant ndc80-AK01 in fission yeast, which contains a single point mutation within the hairpin region. This hairpin connects the preceding calponin-homology domain with the coiled-coil region. ndc80-AK01 was hypersensitive to microtubule depolymerizing reagents with no apparent growth defects without drugs. Subsequent analyses indicated that ndc80-AK01 is defective in SAC signaling, as mutant cells proceeded into lethal cell division in the absence of microtubules. Under mitotic arrest conditions, all SAC components (Ark1/Aurora B, Mph1, Bub1, Bub3, Mad3, Mad2 and Mad1) did not localize to the kinetochore. Further genetic analyses indicated that the Ndc80 hairpin region might act as a platform for the kinetochore recruitment of Mph1, which is one of the most upstream SAC components in the hierarchy. Intriguingly, artificial tethering of Mph1 to the kinetochore fully restored checkpoint signaling in ndc80-AK01 cells, further substantiating the notion that Ndc80 is a kinetochore platform for Mph1. The hairpin region of Ndc80, therefore, plays a critical role in kinetochore recruitment of Mph1. PMID:26900649

  1. Effects of the tumor inhibitory triterpenoid avicin G on cell integrity, cytokinesis, and protein ubiquitination in fission yeast

    PubMed Central

    Gutterman, Jordan U.; Lai, Hong T.; Yang, Peirong; Haridas, Valsala; Gaikwad, Amos; Marcus, Stevan

    2005-01-01

    Avicins comprise a class of triterpenoid compounds that exhibit tumor inhibitory activity. Here we show that avicin G is inhibitory to growth of the fission yeast Schizosaccharomyces pombe. S. pombe cells treated with a lethal concentration of avicin G (20 μM) exhibited a shrunken morphology, indicating that avicin G adversely affects cell integrity. Cells treated with a sublethal concentration of avicin G (6.5 μM) exhibited a strong cytokinesis-defective phenotype (multiseptated cells), as well as cell morphology defects. These phenotypes bear resemblance to those resulting from loss of Rho1 GTPase function in S. pombe. Indeed, Rho1-deficient S. pombe cells were strongly hypersensitive to avicin G, suggesting that the compound may perturb Rho1-dependent processes. Consistent with previously observed effects in human Jurkat T cells, avicin G treatment resulted in hyperaccumulation of ubiquitinated proteins in S. pombe cells. Interestingly, proteasome-defective S. pombe mutants were not markedly hypersensitive to avicin G, whereas an anaphase-promoting complex (mitotic ubiquitin ligase) mutant exhibited avicin G resistance, suggesting that the increase in levels of ubiquitinated proteins resulting from avicin G treatment may be due to increased protein ubiquitination, rather than inhibition of 26S proteasome activity. Mutants defective in the cAMP/PKA pathway also exhibited resistance to avicin G. Our results suggest that S. pombe will be a useful model organism for elucidating molecular targets of avicin G and serve as a guide to clinical application where dysfunctional aspects of Rho and/or ubiquitination function have been demonstrated as in cancer, fibrosis, and inflammation. PMID:16118282

  2. Rho1 GTPase and PKC Ortholog Pck1 Are Upstream Activators of the Cell Integrity MAPK Pathway in Fission Yeast

    PubMed Central

    Sánchez-Mir, Laura; Soto, Teresa; Franco, Alejandro; Madrid, Marisa; Viana, Raúl A.; Vicente, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2014-01-01

    In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms. PMID:24498240

  3. The hairpin region of Ndc80 is important for the kinetochore recruitment of Mph1/MPS1 in fission yeast

    PubMed Central

    Chmielewska, Aldona Ewa; Tang, Ngang Heok; Toda, Takashi

    2016-01-01

    ABSTRACT The establishment of proper kinetochore-microtubule attachments facilitates faithful chromosome segregation. Incorrect attachments activate the spindle assembly checkpoint (SAC), which blocks anaphase onset via recruitment of a cohort of SAC components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to kinetochores. KNL1, a component of the outer kinetochore KMN network (KNL1/Mis12 complex/Ndc80 complex), acts as a platform for Bub1 and Bub3 localization upon its phosphorylation by Mph1/MPS1. The Ndc80 protein, a major microtubule-binding site, is critical for MPS1 localization to the kinetochores in mammalian cells. Here we characterized the newly isolated mutant ndc80-AK01 in fission yeast, which contains a single point mutation within the hairpin region. This hairpin connects the preceding calponin-homology domain with the coiled-coil region. ndc80-AK01 was hypersensitive to microtubule depolymerizing reagents with no apparent growth defects without drugs. Subsequent analyses indicated that ndc80-AK01 is defective in SAC signaling, as mutant cells proceeded into lethal cell division in the absence of microtubules. Under mitotic arrest conditions, all SAC components (Ark1/Aurora B, Mph1, Bub1, Bub3, Mad3, Mad2 and Mad1) did not localize to the kinetochore. Further genetic analyses indicated that the Ndc80 hairpin region might act as a platform for the kinetochore recruitment of Mph1, which is one of the most upstream SAC components in the hierarchy. Intriguingly, artificial tethering of Mph1 to the kinetochore fully restored checkpoint signaling in ndc80-AK01 cells, further substantiating the notion that Ndc80 is a kinetochore platform for Mph1. The hairpin region of Ndc80, therefore, plays a critical role in kinetochore recruitment of Mph1. PMID:26900649

  4. Specific biomarkers for stochastic division patterns and starvation-induced quiescence under limited glucose levels in fission yeast

    PubMed Central

    Pluskal, Tomáš; Hayashi, Takeshi; Saitoh, Shigeaki; Fujisawa, Asuka; Yanagida, Mitsuhiro

    2011-01-01

    Glucose as a source of energy is centrally important to our understanding of life. We investigated the cell division–quiescence behavior of the fission yeast Schizosaccharomyces pombe under a wide range of glucose concentrations (0–111 mm). The mode of S. pombe cell division under a microfluidic perfusion system was surprisingly normal under highly diluted glucose concentrations (5.6 mm, 1/20 of the standard medium, within human blood sugar levels). Division became stochastic, accompanied by a curious division-timing inheritance, in 2.2–4.4 mm glucose. A critical transition from division to quiescence occurred within a narrow range of concentrations (2.2–1.7 mm). Under starvation (1.1 mm) conditions, cells were mostly quiescent and only a small population of cells divided. Under fasting (0 mm) conditions, division was immediately arrested with a short chronological lifespan (16 h). When cells were first glucose starved prior to fasting, they possessed a substantially extended lifespan (∼14 days). We employed a quantitative metabolomic approach for S. pombe cell extracts, and identified specific metabolites (e.g. biotin, trehalose, ergothioneine, S-adenosyl methionine and CDP-choline), which increased or decreased at different glucose concentrations, whereas nucleotide triphosphates, such as ATP, maintained high concentrations even under starvation. Under starvation, the level of S-adenosyl methionine increased sharply, accompanied by an increase in methylated amino acids and nucleotides. Under fasting, cells rapidly lost antioxidant and energy compounds, such as glutathione and ATP, but, in fasting cells after starvation, these and other metabolites ensuring longevity remained abundant. Glucose-starved cells became resistant to 40 mm H2O2 as a result of the accumulation of antioxidant compounds. PMID:21306563

  5. Genetic Evidence for Phospholipid-Mediated Regulation of the Rab GDP-Dissociation Inhibitor in Fission Yeast

    PubMed Central

    Ma, Yan; Kuno, Takayoshi; Kita, Ayako; Nabata, Toshiya; Uno, Satoshi; Sugiura, Reiko

    2006-01-01

    We have previously identified mutant alleles of genes encoding two Rab proteins, Ypt3 and Ryh1, through a genetic screen using the immunosuppressant drug FK506 in fission yeast. In the same screen, we isolated gdi1-i11, a mutant allele of the essential gdi1+ gene encoding Rab GDP-dissociation inhibitor. In gdi1-i11, a conserved Gly267 was substituted by Asp. The Gdi1G267D protein failed to extract Rabs from membrane and Rabs were depleted from the cytosolic fraction in the gdi1-i11 mutant cells. Consistently, the Gdi1G267D protein was found mostly in the membrane fraction, whereas wild-type Gdi1 was found in both the cytosolic and the membrane fraction. Notably, overexpression of spo20+, encoding a phosphatidylcholine/phosphatidylinositol transfer protein, rescued gdi1-i11 mutation, but not ypt3-i5 or ryh1-i6. The gdi1-i11 and spo20-KC104 mutations are synthetically lethal, and the wild-type Gdi1 failed to extract Rabs from the membrane in the spo20-KC104 mutant. The phosphatidylinositol-transfer activity of Spo20 is dispensable for the suppression of the gdi1-i11 mutation, suggesting that the phosphatidylcholine-transfer activity is important for the suppression. Furthermore, knockout of the pct1+ gene encoding a choline phosphate cytidyltransferase rescued the gdi1-i11 mutation. Together, our findings suggest that Spo20 modulates Gdi1 function via regulation of phospholipid metabolism of the membranes. PMID:16980382

  6. Tor Signaling Regulates Transcription of Amino Acid Permeases through a GATA Transcription Factor Gaf1 in Fission Yeast

    PubMed Central

    Liu, Qingbin; Qi, Yao; Manabe, Ri-ichiroh; Furuyashiki, Tomoyuki

    2015-01-01

    In the fission yeast, two Tor isoforms, Tor1 and Tor2, oppositely regulate gene expression of amino acid permeases. To elucidate the transcriptional machinery for these regulations, here we have employed the cap analysis of gene expression (CAGE), a method of analyzing expression profiles and identifying transcriptional start sites (TSSs). The loss of Tor1 decreased, and Tor2 inhibition by its temperature sensitive mutation increased, mRNA expression of isp5+, per1+, put4+ and SPBPB2B2.01. In contrast, the loss of Tor1 increased, and Tor2 inhibition decreased, the expression of cat1+. These changes were confirmed by semi-quantitative RT-PCR. These opposite effects by the loss of Tor1 and Tor2 inhibition appeared to occur evenly across multiple TSSs for the respective genes. The motif discovery analysis based on the CAGE results identified the GATA motifs as a potential cis-regulatory element for Tor-mediated regulation. In the luciferase reporter assay, the loss of Tor1 reduced, and Tor2 inhibition and nitrogen depletion increased, the activity of isp5+ promoter as well as that of a GATAAG reporter. One of the GATAAG motifs in isp5+ promoter was critical for its transcriptional activity, and a GATA transcription factor Gaf1 was critical for the activities of isp5+ promoter and the GATAAG reporter. Furthermore, Tor2 inhibition and nitrogen depletion induced nuclear localization of Gaf1 from the cytosol and its dephosphorylation. These results suggest that Tor2 inhibition, which is known to be induced by nitrogen depletion, promotes nuclear localization of Gaf1, thereby inducing isp5+ transcription through Gaf1 binding to the GATAAG motif in its promoter. Since Gaf1 was also critical for transcription of per1+ and put4+, Tor-Gaf1 signaling may coordinate transcription of multiple amino acid permeases according to nutrient availability. PMID:26689777

  7. Fission yeast LAMMER kinase Lkh1 regulates the cell cycle by phosphorylating the CDK-inhibitor Rum1

    SciTech Connect

    Yu, Eun-Young; Lee, Ju-Hee; Kang, Won-Hwa; Park, Yun-Hee; Kim, Lila; Park, Hee-Moon

    2013-03-01

    Highlights: ► Deletion of lkh1{sup +} made cells pass the G1/S phase faster than the wild type. ► Lkh1 can interact with a cyclin-dependent kinase inhibitor (CKI) Rum1. ► Lkh1 can phosphorylate Rum1 to activate its CKI activity. ► Thr110 was confirmed as the Lkh1-dependent phosphorylation site of Rum1. ► Positive acting mechanism for the Rum1 activation is reported for the first time. - Abstract: In eukaryotes, LAMMER kinases are involved in various cellular events, including the cell cycle. However, no attempt has been made to investigate the mechanisms that underlie the involvement of LAMMER kinase. In this study, we performed a functional analysis of LAMMER kinase using the fission yeast, Schizosaccharomyces pombe. FACS analyses revealed that deletion of the gene that encodes the LAMMER kinase Lkh1 made mutant cells pass through the G1/S phase faster than their wild-type counterparts. Co-immunoprecipitation and an in vitro kinase assay also revealed that Lkh1 can interact with and phosphorylate Rum1 to activate this molecule as a cyclin-dependent kinase inhibitor, which blocks cell cycle progression from the G1 phase to the S phase. Peptide mass fingerprinting and kinase assay with Rum1{sup T110A} confirmed T110 as the Lkh1-dependent phosphorylation residue. In this report we present for the first time a positive acting mechanism that is responsible for the CKI activity of Rum1, in which the LAMMER kinase-mediated phosphorylation of Rum1 is involved.

  8. Biochemical and Genetic Conservation of Fission Yeast Dsk1 and Human SR Protein-Specific Kinase 1

    PubMed Central

    Tang, Zhaohua; Kuo, Tiffany; Shen, Jenny; Lin, Ren-Jang

    2000-01-01

    Arginine/serine-rich (RS) domain-containing proteins and their phosphorylation by specific protein kinases constitute control circuits to regulate pre-mRNA splicing and coordinate splicing with transcription in mammalian cells. We present here the finding that similar SR networks exist in Schizosaccharomyces pombe. We previously showed that Dsk1 protein, originally described as a mitotic regulator, displays high activity in phosphorylating S. pombe Prp2 protein (spU2AF59), a homologue of human U2AF65. We now demonstrate that Dsk1 also phosphorylates two recently identified fission yeast proteins with RS repeats, Srp1 and Srp2, in vitro. The phosphorylated proteins bear the same phosphoepitope found in mammalian SR proteins. Consistent with its substrate specificity, Dsk1 forms kinase-competent complexes with those proteins. Furthermore, dsk1+ gene determines the phenotype of prp2+ overexpression, providing in vivo evidence that Prp2 is a target for Dsk1. The dsk1-null mutant strain became severely sick with the additional deletion of a related kinase gene. Significantly, human SR protein-specific kinase 1 (SRPK1) complements the growth defect of the double-deletion mutant. In conjunction with the resemblance of dsk1+ and SRPK1 in sequence homology, biochemical properties, and overexpression phenotypes, the complementation result indicates that SRPK1 is a functional homologue of Dsk1. Collectively, our studies illustrate the conserved SR networks in S. pombe consisting of RS domain-containing proteins and SR protein-specific kinases and thus establish the importance of the networks in eucaryotic organisms. PMID:10629038

  9. Genome-wide screen of fission yeast mutants for sensitivity to 6-azauracil, an inhibitor of transcriptional elongation.

    PubMed

    Zhou, Huan; Liu, Qi; Shi, Tianfang; Yu, Yao; Lu, Hong

    2015-10-01

    6-Azauracil (6 AU) inhibits enzymes in nucleoside synthesis and depletes the intracellular GTP/UTP pool. Mutations in transcriptional elongation machinery, as well as mutations in a variety of other pathways, exaggerate the growth defect of cells in the presence of 6 AU. Thus, identification of mutations that render cells sensitive to 6 AU will benefit study on the basis of 6 AU-sensitive phenotype. Here we performed a genome-wide screen of a fission yeast deletion library. Of 3235 single-gene deletions, 66 mutants displayed at least 50% drop of fitness in the presence of 6 AU and 60 mutants were reported for the first time; five deletions showed synthetic decrease of fitness when combined with deletion of set3(+) , which encodes a transcriptional regulator. Genes conferring tolerance to 6 AU were enriched in various processes, especially in chromosome segregation. Accordingly, genes encoding subunits of CLRC complex and spindle pole body were over-represented. Mutants were subjected to an in vivo transcript length-dependent reporter assay to assess the potential roles of deleted genes in transcriptional elongation. As with the deletions known to affect elongation, nab2Δ, nxt1Δ, rhp18Δ, SPAC24C9.08Δ, clr3Δ and ncs1Δset3Δ mutants exhibited defects in expressing long transcripts. New 6 AU-sensitive mutants identified here will help to elucidate the mechanism of action of 6 AU in the cells. Meanwhile, our study revealed novel genes potentially involved in transcriptional elongation and provided valuable targets for transcription study. PMID:26173815

  10. Glucose monitoring in fission yeast via the Gpa2 galpha, the git5 Gbeta and the git3 putative glucose receptor.

    PubMed Central

    Welton, R M; Hoffman, C S

    2000-01-01

    The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase. The resulting cAMP signal activates protein kinase A (PKA). PKA inhibits glucose starvation-induced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase. We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six "upstream" genes required for adenylate cyclase activation. The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guanine-nucleotide binding protein (Galpha) while git5 encodes a Gbeta subunit. Multicopy suppression studies with gpa2(+) previously indicated that S. pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Galpha followed by Gbetagamma. We show here that an activated allele of gpa2 (gpa2(R176H), carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model. We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor. A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation. Since the git3 deletion is fully suppressed by the gpa2(R176H) allele with respect to fbp1 transcription, git3 appears to encode a G protein-coupled glucose receptor responsible for adenylate cyclase activation in S. pombe. PMID:11014802

  11. Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly.

    PubMed

    Masuda, Hirohisa; Toda, Takashi

    2016-06-01

    In fission yeast, γ-tubulin ring complex (γTuRC)-specific components Gfh1(GCP4), Mod21(GCP5), and Alp16(GCP6) are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1(MOZART1), a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16(GCP6) promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1(GCP4) and Mod21(GCP5) are not required for Alp16(GCP6)-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16(GCP6) and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation. PMID:27053664

  12. Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly

    PubMed Central

    Masuda, Hirohisa; Toda, Takashi

    2016-01-01

    In fission yeast, γ-tubulin ring complex (γTuRC)–specific components Gfh1GCP4, Mod21GCP5, and Alp16GCP6 are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1MOZART1, a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16GCP6 promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1GCP4 and Mod21GCP5 are not required for Alp16GCP6-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16GCP6 and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation. PMID:27053664

  13. Fission yeast Scm3 mediates stable assembly of Cnp1/CENP-A into centromeric chromatin.

    PubMed

    Williams, Jessica S; Hayashi, Takeshi; Yanagida, Mitsuhiro; Russell, Paul

    2009-02-13

    Mis16 and Mis18 are subunits of a protein complex required for incorporation of the histone H3 variant CenH3 (Cnp1/CENP-A) into centromeric chromatin in Schizosaccharomyces pombe and mammals. How the Mis16-Mis18 complex performs this function is unknown. Here, we report that the Mis16-Mis18 complex is required for centromere localization of Scm3(Sp), a Cnp1-binding protein related to Saccharomyces cerevisiae Scm3. Scm3(Sp) is required for centromeric localization of Cnp1, while Scm3(Sp) localizes at centromeres independently of Cnp1. Like the Mis16-Mis18 complex but unlike Cnp1, Scm3(Sp) dissociates from centromeres during mitosis. Inactivation of Scm3(Sp) or Mis18 increases centromere localization of histones H3 and H2A/H2B, which are largely absent from centromeres in wild-type cells. Whereas S. cerevisiae Scm3 is proposed to replace histone H2A/H2B in centromeric nucleosomes, the dynamic behavior of S. pombe Scm3 suggests that it acts as a Cnp1 assembly/maintenance factor that directly mediates the stable deposition of Cnp1 into centromeric chromatin. PMID:19217403

  14. Fission Yeast Scm3 Mediates Stable Assembly of Cnp1 (CENP-A) into Centromeric Chromatin

    PubMed Central

    Williams, Jessica S.; Hayashi, Takeshi; Yanagida, Mitsuhiro; Russell, Paul

    2009-01-01

    Summary Mis16 and Mis18 are subunits of a protein complex required for incorporation of the histone H3 variant CenH3 (Cnp1/CENP-A) into centromeric chromatin in Schizosaccharomyces pombe and mammals. How the Mis16-Mis18 complex performs this function is unknown. Here we report that the Mis16-Mis18 complex is required for centromere localization of Scm3Sp, a Cnp1-binding protein related to Saccharomyces cerevisiae Scm3. Scm3Sp is required for centromeric localization of Cnp1, whilst Scm3Sp localizes at centromeres independently of Cnp1. Like the Mis16-Mis18 complex but unlike Cnp1, Scm3Sp dissociates from centromeres during mitosis. Inactivation of Scm3Sp or Mis18 increases centromere localization of histones H3 and H2A/H2B, which are largely absent from centromeres in wild type cells. Whereas S. cerevisiae Scm3 is proposed to replace histone H2A/H2B in centromeric nucleosomes, the dynamic behavior of S. pombe Scm3 suggests that it acts as a Cnp1 assembly/maintenance factor that directly mediates the stable deposition of Cnp1 into centromeric chromatin. PMID:19217403

  15. RNA degradation in fission yeast mitochondria is stimulated by a member of a new family of proteins that are conserved in lower eukaryotes.

    PubMed

    Wiesenberger, Gerlinde; Speer, Falk; Haller, Günter; Bonnefoy, Nathalie; Schleiffer, Alexander; Schafer, Bernd

    2007-03-30

    We report here on the role of open reading frame (ORF) SPCC1183.04c of Schizosaccharomyces pombe in mitochondrial RNA metabolism. A mutant deleted for this ORF on chromosome III accumulates mitochondrial transcripts with the exception of the cob mRNA. A detailed Northern blot analysis showed that the effect results from a decrease in RNA degradation but not from RNA processing deficiencies. Overexpression of the SPCC1183.04c gene in a S. pombe wild-type strain is characterized by slow growth at 37 degrees C on non-fermentable carbon sources and a significant reduction of steady-state levels of mitochondrial transcripts. A NCBI BLASTP search with the amino acid sequence deduced from the S. pombe gene identified significant similarity to a number of proteins in fungi (e.g. Ascomycota, Basidiomycota) and in some non-fungal eukaryotes (e.g. ciliate, slime mold, red algae). By heterologous expression of SPCC1183.04c in a Saccharomyces cerevisiae pet127Delta strain, we demonstrate that the fission yeast protein and Pet127p from S. cerevisiae function similarly: The fission yeast gene complemented the respiratory defect associated with the pet127Delta allele and partially restored the RNA processing phenotype. Although it lacks any recognizable targeting signal, the S. pombe protein is imported into S. cerevisiae mitochondria in vivo. We conclude from our results that the fission yeast SPCC1183.04c gene is a member of a new protein family that functions to stimulate mitochondrial RNA degradation, a function that is conserved within the mitochondria of lower eukaryotes but seems to have been replaced by alternative pathways in metazoans and higher plants. PMID:17292401

  16. Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

    2007-11-30

    Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe.

  17. Multi-domain CGFS-type glutaredoxin Grx4 regulates iron homeostasis via direct interaction with a repressor Fep1 in fission yeast

    SciTech Connect

    Kim, Kyoung-Dong; Kim, Hyo-Jin; Lee, Kyung-Chang; Roe, Jung-Hye

    2011-05-20

    Research highlights: {yields} Monothiol glutaredoxin Grx4 allows Fep1-mediated de-repression of iron uptake genes at low iron. {yields} Grx4 directly interacts with Fep1 in vivo and in vitro. {yields} The Cys172 in the CGFS motif of Grx4 is necessary for cell proliferation and iron regulation. {yields} The Cys172 of Grx4 is required for normal interaction with Fep1. -- Abstract: The fission yeast Schizosaccharomyces pombe contains two CGFS-type monothiol glutaredoxins, Grx4 and Grx5, which are localized primarily in the nucleus and mitochondria, respectively. We observed involvement of Grx4 in regulating iron-responsive gene expression, which is modulated by a repressor Fep1. Lack of Grx4 caused defects not only in growth but also in the expression of both iron-uptake and iron-utilizing genes regardless of iron availability. In order to unravel how Grx4 is involved in Fep1-mediated regulation, interaction between them was investigated. Co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) revealed that Grx4 physically interacts with Fep1 in vivo. BiFC revealed localized nuclear dots produced by interaction of Grx4 with Fep1. Mutation of cysteine-172 in the CGFS motif to serine (C172S) produced effects similarly observed under Grx4 depletion, such as the loss of iron-dependent gene regulation and the absence of nuclear dots in BiFC analysis. These results suggest that the ability of Grx4 to bind iron, most likely Fe-S cofactor, could be critical in interacting with and modulating the activity of Fep1.

  18. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Yeast Extract Hydrolysate from... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1246 Yeast Extract Hydrolysate from... exemption from the requirement of a tolerance for residues of the biochemical pesticide Yeast...

  19. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Yeast Extract Hydrolysate from... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1246 Yeast Extract Hydrolysate from... exemption from the requirement of a tolerance for residues of the biochemical pesticide Yeast...

  20. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Yeast Extract Hydrolysate from... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1246 Yeast Extract Hydrolysate from... exemption from the requirement of a tolerance for residues of the biochemical pesticide Yeast...

  1. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Yeast Extract Hydrolysate from... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1246 Yeast Extract Hydrolysate from... exemption from the requirement of a tolerance for residues of the biochemical pesticide Yeast...

  2. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Yeast Extract Hydrolysate from... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1246 Yeast Extract Hydrolysate from... exemption from the requirement of a tolerance for residues of the biochemical pesticide Yeast...

  3. The Splicing Factor U2AF Small Subunit Is Functionally Conserved between Fission Yeast and Humans

    PubMed Central

    Webb, Christopher J.; Wise, Jo Ann

    2004-01-01

    The small subunit of U2AF, which functions in 3′ splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AFSM) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AFSM depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues. PMID:15121844

  4. Chromosome conformation maps in fission yeast reveal cell cycle dependent sub nuclear structure

    PubMed Central

    Grand, Ralph S.; Pichugina, Tatyana; Gehlen, Lutz R.; Jones, M. Beatrix; Tsai, Peter; Allison, Jane R.; Martienssen, Robert; O'Sullivan, Justin M.

    2014-01-01

    Successful progression through the cell cycle requires spatial and temporal regulation of gene transcript levels and the number, positions and condensation levels of chromosomes. Here we present a high resolution survey of genome interactions in Schizosaccharomyces pombe using synchronized cells to investigate cell cycle dependent changes in genome organization and transcription. Cell cycle dependent interactions were captured between and within S. pombe chromosomes. Known features of genome organization (e.g. the clustering of telomeres and retrotransposon long terminal repeats (LTRs)) were observed throughout the cell cycle. There were clear correlations between transcript levels and chromosomal interactions between genes, consistent with a role for interactions in transcriptional regulation at specific stages of the cell cycle. In silico reconstructions of the chromosome organization within the S. pombe nuclei were made by polymer modeling. These models suggest that groups of genes with high and low, or differentially regulated transcript levels have preferred positions within the S. pombe nucleus. We conclude that the S. pombe nucleus is spatially divided into functional sub-nuclear domains that correlate with gene activity. The observation that chromosomal interactions are maintained even when chromosomes are fully condensed in M phase implicates genome organization in epigenetic inheritance and bookmarking. PMID:25342201

  5. Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7.

    PubMed

    Edamatsu, M

    2016-01-01

    Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubule-binding properties of this protein in vitro. PMID:26909973

  6. Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

    PubMed Central

    Huang, Junqi; Huang, Yinyi; Yu, Haochen; Subramanian, Dhivya; Padmanabhan, Anup; Thadani, Rahul; Tao, Yaqiong; Tang, Xie; Wedlich-Soldner, Roland

    2012-01-01

    In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly. PMID:23185032

  7. Characterization of nuclear pore complex components in fission yeast Schizosaccharomyces pombe

    PubMed Central

    Asakawa, Haruhiko; Yang, Hui-Ju; Yamamoto, Takaharu G; Ohtsuki, Chizuru; Chikashige, Yuji; Sakata-Sogawa, Kumiko; Tokunaga, Makio; Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2014-01-01

    The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8–47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC. PMID:24637836

  8. Histone Chaperone Asf1 Plays an Essential Role in Maintaining Genomic Stability in Fission Yeast

    PubMed Central

    Tanae, Katsuhiro; Horiuchi, Tomitaka; Matsuo, Yuzy; Katayama, Satoshi; Kawamukai, Makoto

    2012-01-01

    The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or disassembly), histone exchange, regulation of transcription, and chromatin silencing in several organisms. To investigate the essential functions of Asf1 in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 different kinase genes to identify synthetic lethal combinations. The asf1-33 mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the asf1-33 mutant at the restrictive temperature, indicating that the DNA damage checkpoint was activated in the asf1-33 mutant. DNA damage occured in the asf1-33 mutant, with degradation of the chromosomal DNA observed through pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1+ strain at the restrictive temperature, suggesting that asf1 mutations also caused a defect in overall chromatin structure. The Asf1-33 mutant protein was mislocalized and incapable of binding histones. Furthermore, histone H3 levels at the centromeric outer repeat region were decreased in the asf1-33 mutant and heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. Taken together, these results clearly indicate that Asf1 plays an essential role in maintaining genomic stability in S. pombe. PMID:22291963

  9. Polypeptone induces dramatic cell lysis in ura4 deletion mutants of fission yeast.

    PubMed

    Matsuo, Yuzy; Nishino, Kouhei; Mizuno, Kouhei; Akihiro, Takashi; Toda, Takashi; Matsuo, Yasuhiro; Kaino, Tomohiro; Kawamukai, Makoto

    2013-01-01

    Polypeptone is widely excluded from Schizosaccharomyces pombe growth medium. However, the reasons why polypeptone should be avoided have not been documented. Polypeptone dramatically induced cell lysis in the ura4 deletion mutant when cells approached the stationary growth phase, and this phenotype was suppressed by supplementation of uracil. To determine the specificity of this cell lysis phenotype, we created deletion mutants of other genes involved in de novo biosynthesis of uridine monophosphate (ura1, ura2, ura3, and ura5). Cell lysis was not observed in these gene deletion mutants. In addition, concomitant disruption of ura1, ura2, ura3, or ura5 in the ura4 deletion mutant suppressed cell lysis, indicating that cell lysis induced by polypeptone is specific to the ura4 deletion mutant. Furthermore, cell lysis was also suppressed when the gene involved in coenzyme Q biosynthesis was deleted. This is likely because Ura3 requires coenzyme Q for its activity. The ura4 deletion mutant was sensitive to zymolyase, which mainly degrades (1,3)-beta-D glucan, when grown in the presence of polypeptone, and cell lysis was suppressed by the osmotic stabiliser, sorbitol. Finally, the induction of cell lysis in the ura4 deletion mutant was due to the accumulation of orotidine-5-monophosphate. Cell wall integrity was dramatically impaired in the ura4 deletion mutant when grown in the presence of polypeptone. Because ura4 is widely used as a selection marker in S. pombe, caution needs to be taken when evaluating phenotypes of ura4 mutants. PMID:23555823

  10. Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance.

    PubMed

    Hamasaki-Katagiri, Nobuko; Molchanova, Tatiana; Takeda, Kazuyo; Ames, James B

    2004-03-26

    The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization

  11. A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase.

    PubMed Central

    Lundgren, K; Allan, S; Urushiyama, S; Tani, T; Ohshima, Y; Frendewey, D; Beach, D

    1996-01-01

    The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis. We undertook a study of this gene to understand more fully the general requirements for entry into mitosis. Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature. We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8. DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases. The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors. Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature. Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8. We have shown that cdc28 and prp8 are allelic. These results suggest a connection between pre-mRNA splicing and progression through the cell cycle. Images PMID:8862522

  12. SUMOylation regulates telomere length by targeting the shelterin subunit Tpz1(Tpp1) to modulate shelterin-Stn1 interaction in fission yeast.

    PubMed

    Miyagawa, Keisuke; Low, Ross S; Santosa, Venny; Tsuji, Hiroki; Moser, Bettina A; Fujisawa, Shiho; Harland, Jennifer L; Raguimova, Olga N; Go, Andrew; Ueno, Masaru; Matsuyama, Akihisa; Yoshida, Minoru; Nakamura, Toru M; Tanaka, Katsunori

    2014-04-22

    Telomeres protect DNA ends of linear eukaryotic chromosomes from degradation and fusion, and ensure complete replication of the terminal DNA through recruitment of telomerase. The regulation of telomerase is a critical area of telomere research and includes cis regulation by the shelterin complex in mammals and fission yeast. We have identified a key component of this regulatory pathway as the SUMOylation [the covalent attachment of a small ubiquitin-like modifier (SUMO) to target proteins] of a shelterin subunit in fission yeast. SUMOylation is known to be involved in the negative regulation of telomere extension by telomerase; however, how SUMOylation limits the action of telomerase was unknown until now. We show that SUMOylation of the shelterin subunit TPP1 homolog in Schizosaccharomyces pombe (Tpz1) on lysine 242 is important for telomere length homeostasis. Furthermore, we establish that Tpz1 SUMOylation prevents telomerase accumulation at telomeres by promoting recruitment of Stn1-Ten1 to telomeres. Our findings provide major mechanistic insights into how the SUMOylation pathway collaborates with shelterin and Stn1-Ten1 complexes to regulate telomere length. PMID:24711392

  13. ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

    SciTech Connect

    Fujita, Atsushi

    2008-02-01

    Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6{sup +}, encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6{delta} cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p, for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast.

  14. Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

    PubMed Central

    Zhao, Y; Cao, J; O'Gorman, M R; Yu, M; Yogev, R

    1996-01-01

    The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions. PMID:8709199

  15. Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).

    PubMed Central

    Haas, A; Wickner, W

    1996-01-01

    In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles. We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p. Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro. Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion. We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion. Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p. Images PMID:8670830

  16. Identification and characterization of srp1, a gene of fission yeast encoding a RNA binding domain and a RS domain typical of SR splicing factors.

    PubMed Central

    Gross, T; Richert, K; Mierke, C; Lützelberger, M; Käufer, N F

    1998-01-01

    The SR protein family is involved in constitutive and regulated pre-mRNA splicing and has been found to be evolutionarily conserved in metazoan organisms. In contrast, the genome of the unicellular yeast Saccharomyces cerevisiae does not contain genes encoding typical SR proteins. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD respectively, and a RS (arginine/serine-rich) domain which gave the family its name. We have now cloned from the fission yeast Schizosaccharomyces pombe the gene srp1. This gene is the first yeast gene encoding a protein with typical features of mammalian SR protein family members. The gene is not essential for growth. We show that overexpression of the RNA binding domain inhibits pre-mRNA splicing and that the highly conserved sequence RDAEDA in the RBD is involved. Overexpression of Srp1 containing mutations in the RS domain also inhibits pre-mRNA splicing activity. Furthermore, we show that overexpression of Srp1 and overexpression of the mammalian SR splicing factor ASF/SF2 suppress the pre-mRNA splicing defect of the temperature-sensitive prp4-73 allele. prp4 encodes a protein kinase involved in pre-mRNA splicing. These findings are consistent with the notion that Srp1 plays a role in the splicing process. PMID:9421507

  17. Mes1 controls the meiosis I to meiosis II transition by distinctly regulating the anaphase-promoting complex/cyclosome coactivators Fzr1/Mfr1 and Slp1 in fission yeast

    PubMed Central

    Kimata, Yuu; Kitamura, Kenji; Fenner, Nicola; Yamano, Hiroyuki

    2011-01-01

    Meiosis is a specialized form of cell division generating haploid gametes and is dependent upon protein ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C). Accurate control of the APC/C during meiosis is important in all eukaryotic cells and is in part regulated by the association of coactivators and inhibitors. We previously showed that the fission yeast meiosis-specific protein Mes1 binds to a coactivator and inhibits APC/C; however, regulation of the Mes1-mediated APC/C inhibition remains elusive. Here we show how Mes1 distinctively regulates different forms of the APC/C. We study all the coactivators present in the yeast genome and find that only Slp1/Cdc20 is essential for meiosis I progression. However, Fzr1/Mfr1 is a critical target for Mes1 inhibition because fzr1Δ completely rescues the defect on the meiosis II entry in mes1Δ cells. Furthermore, cell-free studies suggest that Mes1 behaves as a pseudosubstrate for Fzr1/Mfr1 but works as a competitive substrate for Slp1. Intriguingly, mutations in the D-box or KEN-box of Mes1 increase its recognition as a substrate by Fzr1, but not by Slp1. Thus Mes1 interacts with two coactivators in a different way to control the activity of the APC/C required for the meiosis I/meiosis II transition. PMID:21389117

  18. Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion

    PubMed Central

    Osman, Christof; Noriega, Thomas R.; Okreglak, Voytek; Fung, Jennifer C.; Walter, Peter

    2015-01-01

    Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin–dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome. PMID:25730886

  19. A Dynamin-Actin Interaction Is Required for Vesicle Scission during Endocytosis in Yeast

    PubMed Central

    Palmer, Sarah E.; Smaczynska-de Rooij, Iwona I.; Marklew, Christopher J.; Allwood, Ellen G.; Mishra, Ritu; Johnson, Simeon; Goldberg, Martin W.; Ayscough, Kathryn R.

    2015-01-01

    Summary Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that—like wild-type Vps1—it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission. PMID:25772449

  20. The CENP-A N-Tail Confers Epigenetic Stability to Centromeres via the CENP-T Branch of the CCAN in Fission Yeast

    PubMed Central

    Folco, H. Diego; Campbell, Christopher S.; May, Karen M.; Espinoza, Celso A.; Oegema, Karen; Hardwick, Kevin G.; Grewal, Shiv I. S.; Desai, Arshad

    2014-01-01

    Summary In most eukaryotes, centromeres are defined epigenetically by presence of the histone H3 variant CENP-A [1-3]. CENP-A containing chromatin recruits the constitutive centromere-associated network (CCAN) of proteins, which in turn directs assembly of the outer kinetochore to form microtubule attachments and ensure chromosome segregation fidelity [4-6]. While the mechanisms that load CENP-A at centromeres are being elucidated, the functions of its divergent N-terminal tail remain enigmatic [7-12]. Here, we employ the well-studied fission yeast centromere [13-16] to investigate the function of the CENP-A (Cnp1) N-tail. We show that alteration of the N-tail did not affect Cnp1 loading at centromeres, outer kinetochore formation, or spindle checkpoint signaling, but nevertheless elevated chromosome loss. N-Tail mutants exhibited synthetic lethality with an altered centromeric DNA sequence, with rare survivors harboring chromosomal fusions in which the altered centromere was epigenetically inactivated. Elevated centromere inactivation was also observed for N-tail mutants with unaltered centromeric DNA sequences. N-tail mutants specifically reduced localization of the CCAN proteins Cnp20/CENP-T and Mis6/CENP-I, but not Cnp3/CENP-C. Overexpression of Cnp20/CENP-T suppressed defects in an N-tail mutant, suggesting a link between reduced CENP-T recruitment and the observed centromere inactivation phenotype. Thus, the Cnp1 N-tail promotes epigenetic stability of centromeres in fission yeast, at least in part via recruitment of the CENP-T branch of the CCAN. PMID:25619765

  1. The CENP-A N-tail confers epigenetic stability to centromeres via the CENP-T branch of the CCAN in fission yeast.

    PubMed

    Folco, H Diego; Campbell, Christopher S; May, Karen M; Espinoza, Celso A; Oegema, Karen; Hardwick, Kevin G; Grewal, Shiv I S; Desai, Arshad

    2015-02-01

    In most eukaryotes, centromeres are defined epigenetically by presence of the histone H3 variant CENP-A [1-3]. CENP-A-containing chromatin recruits the constitutive centromere-associated network (CCAN) of proteins, which in turn directs assembly of the outer kinetochore to form microtubule attachments and ensure chromosome segregation fidelity [4-6]. Whereas the mechanisms that load CENP-A at centromeres are being elucidated, the functions of its divergent N-terminal tail remain enigmatic [7-12]. Here, we employ the well-studied fission yeast centromere [13-16] to investigate the function of the CENP-A (Cnp1) N-tail. We show that alteration of the N-tail does not affect Cnp1 loading at centromeres, outer kinetochore formation, or spindle checkpoint signaling but nevertheless elevates chromosome loss. N-tail mutants exhibited synthetic lethality with an altered centromeric DNA sequence, with rare survivors harboring chromosomal fusions in which the altered centromere was epigenetically inactivated. Elevated centromere inactivation was also observed for N-tail mutants with unaltered centromeric DNA sequences. N-tail mutants specifically reduced localization of the CCAN proteins Cnp20/CENP-T and Mis6/CENP-I, but not Cnp3/CENP-C. Overexpression of Cnp20/CENP-T suppressed defects in an N-tail mutant, suggesting a link between reduced CENP-T recruitment and the observed centromere inactivation phenotype. Thus, the Cnp1 N-tail promotes epigenetic stability of centromeres in fission yeast, at least in part via recruitment of the CENP-T branch of the CCAN. PMID:25619765

  2. The effect of coenzyme Q10 included by γ-cyclodextrin on the growth of fission yeast studied by microscope Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Nishida, Tatsuro; Kaino, Tomohiro; Ikarashi, Ryo; Nakata, Daisuke; Terao, Keiji; Ando, Masahiro; Hamaguchi, Hiro-o.; Kawamukai, Makoto; Yamamoto, Tatsuyuki

    2013-09-01

    The inclusion complex of coenzyme Q10 (CoQ10) by γ-cyclodextrin (γ-CD), CoQ10-CD complex, was recently developed. The addition of the CoQ10-CD complex recovered the growth of a fission yeast mutant strain, Δdps1, which otherwise cannot grow well due to the lack of coenzyme Q producing ability. However, the oxygen consumption rate of this strain was not restored by the addition of the CoQ10-CD complex. The addition of two other anti-oxidative reagents, glutathione and ascorbic acid, also recovered the growth of the Δdps1 strain as well. These results indicated that the recovery of the growth of Δdps1 was brought about by the anti-oxidative property of CoQ10. The intensity of Raman spectra of Δdps1 at 1602 cm-1, which is prominently observed for the wild type of the fission yeast, was compared between before and after addition of the CoQ10-CD complex. The signal was very weakly observed for Δdps1 and did not increase in intensity by the addition of the CoQ10-CD complex. These results suggested the recovery of the growth of Δdps1 was brought about not by the restoration of respiration function of Δdps1 but by the anti-oxidative property of CoQ10 to result in the decrease in the oxidative stress.

  3. The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

    PubMed Central

    Wang, Ning; Wang, Mo; Zhu, Yi-Hua; Grosel, Timothy W.; Sun, Daokun; Kudryashov, Dmitri S.; Wu, Jian-Qiu

    2015-01-01

    Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast. PMID:25411334

  4. Secretion is required for late events in the cell-fusion pathway of mating yeast.

    PubMed

    Grote, Eric

    2010-06-01

    Secretory vesicles accumulate adjacent to the contact site between the two cells of a yeast mating pair before they fuse, but there is no direct evidence that secretion is required to complete fusion. In this study, temperature-sensitive secretion (sec(ts)) mutants were used to investigate the role of secretion in yeast cell fusion. Cell fusion arrested less than 5 minutes after inhibiting secretion. This rapid fusion arrest was not an indirect consequence of reduced mating pheromone signaling, mating-pair assembly or actin polarity. Furthermore, secretion was required to complete cell fusion when it was transiently inhibited by addition and removal of the lipophilic styryl dye, FM4-64. These results indicate that ongoing secretion is required for late events in the cell-fusion pathway, which include plasma-membrane fusion and the completion of cell-wall remodeling, and they demonstrate a just-in-time delivery mechanism for the cell-fusion machinery. PMID:20460435

  5. PRIMED: PRIMEr database for deleting and tagging all fission and budding yeast genes developed using the open-source genome retrieval script (GRS).

    PubMed

    Cummings, Michael T; Joh, Richard I; Motamedi, Mo

    2015-01-01

    The fission (Schizosaccharomyces pombe) and budding (Saccharomyces cerevisiae) yeasts have served as excellent models for many seminal discoveries in eukaryotic biology. In these organisms, genes are deleted or tagged easily by transforming cells with PCR-generated DNA inserts, flanked by short (50-100 bp) regions of gene homology. These PCR reactions use especially designed long primers, which, in addition to the priming sites, carry homology for gene targeting. Primer design follows a fixed method but is tedious and time-consuming especially when done for a large number of genes. To automate this process, we developed the Python-based Genome Retrieval Script (GRS), an easily customizable open-source script for genome analysis. Using GRS, we created PRIMED, the complete PRIMEr D atabase for deleting and C-terminal tagging genes in the main S. pombe and five of the most commonly used S. cerevisiae strains. Because of the importance of noncoding RNAs (ncRNAs) in many biological processes, we also included the deletion primer set for these features in each genome. PRIMED are accurate and comprehensive and are provided as downloadable Excel files, removing the need for future primer design, especially for large-scale functional analyses. Furthermore, the open-source GRS can be used broadly to retrieve genome information from custom or other annotated genomes, thus providing a suitable platform for building other genomic tools by the yeast or other research communities. PMID:25643023

  6. Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

    PubMed Central

    Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A.

    2016-01-01

    Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3ʹ end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. PMID:27172183

  7. Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast.

    PubMed

    Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A

    2016-01-01

    Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3' end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. PMID:27172183

  8. Constitutive Tor2 Activity Promotes Retention of the Amino Acid Transporter Agp3 at Trans-Golgi/Endosomes in Fission Yeast

    PubMed Central

    Liu, Qingbin; Ma, Yan; Zhou, Xin; Furuyashiki, Tomoyuki

    2015-01-01

    Amino acid transporters are located at specific subcellular compartments, and their localizations are regulated by the extracellular availability of amino acids. In yeast, target of rapamycin (TOR) activation induces the internalization of amino acid transporters located at the plasma membrane. However, whether and how TOR signaling regulates other amino acid transporters located at intracellular compartments remains unknown. Here, we demonstrate that in the fission yeast, the TOR inhibitor Torin–1 induces the transfer of several yellow fluorescent protein (YFP)-fused intracellular amino acid transporters, including Agp3, Isp5, Aat1, and Put4, from trans-Golgi/endosomes into the vacuoles. By contrast, the localizations of YFP-fused Can1, Fnx1, and Fnx2 transporter proteins were unaffected upon Torin–1 treatment. There are two TOR isoforms in fission yeast, Tor1 and Tor2. Whereas tor1 deletion did not affect the Torin-1-induced transfer of Agp3-YFP, Tor2 inhibition using a temperature-sensitive mutant induced the transfer of Agp3-YFP to the vacuolar lumen, similar to the effects of Torin–1 treatment. Tor2 inhibition also induced the transfer of the YFP-fused Isp5, Aat1, and Put4 transporter proteins to the vacuoles, although only partial transfer of the latter two transporters was observed. Under nitrogen depletion accompanied by reduced Tor2 activity, Agp3-YFP was transferred from the trans-Golgi/endosomes to the plasma membrane and then to the vacuoles, where it was degraded by the vacuolar proteases Isp6 and Psp3. Mutants with constitutively active Tor2 showed delayed transfer of Agp3-YFP to the plasma membrane upon nitrogen depletion. Cells lacking Tsc2, a negative regulator of Tor2, also showed a delay in this process in a Tor2-dependent manner. Taken together, these findings suggest that constitutive Tor2 activity is critical for the retention of amino acid transporters at trans-Golgi/endosomes. Moreover, nitrogen depletion suppresses Tor2 activity

  9. Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria.

    PubMed Central

    Esteves, Telma C; Echtay, Karim S; Jonassen, Tanya; Clarke, Catherine F; Brand, Martin D

    2004-01-01

    Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes [Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609-613] and for increasing the binding of the activator retinoic acid to UCP1 [Tomás, Ledesma and Rial (2002) FEBS Lett. 526, 63-65]. In the present study, yeast ( Saccharomyces cerevisiae ) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria. Wild-type yeast strain and two mutant strains (CENDeltaCOQ3 and CENDeltaCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene. UCP1 activity was measured as fatty acid-dependent, GDP-sensitive proton conductance in mitochondria isolated from the cells. The activity of UCP1 was similar in both Q-containing and -deficient yeast mitochondria. We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria. PMID:14680474

  10. Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase δ.

    PubMed

    Pagliuso, Alessandro; Valente, Carmen; Giordano, Lucia Laura; Filograna, Angela; Li, Guiling; Circolo, Diego; Turacchio, Gabriele; Marzullo, Vincenzo Manuel; Mandrich, Luigi; Zhukovsky, Mikhail A; Formiggini, Fabio; Polishchuk, Roman S; Corda, Daniela; Luini, Alberto

    2016-01-01

    Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself). PMID:27401954

  11. Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase δ

    PubMed Central

    Pagliuso, Alessandro; Valente, Carmen; Giordano, Lucia Laura; Filograna, Angela; Li, Guiling; Circolo, Diego; Turacchio, Gabriele; Marzullo, Vincenzo Manuel; Mandrich, Luigi; Zhukovsky, Mikhail A.; Formiggini, Fabio; Polishchuk, Roman S.; Corda, Daniela; Luini, Alberto

    2016-01-01

    Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself). PMID:27401954

  12. Expression and secretion of a CB4-1 scFv-GFP fusion protein by fission yeast.

    PubMed

    Naumann, Julia Maria; Küttner, Gabriele; Bureik, Matthias

    2011-01-01

    There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv-GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv-GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv-GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion. PMID:20617397

  13. Distinct Amino Acid Compositional Requirements for Formation and Maintenance of the [PSI+] Prion in Yeast

    PubMed Central

    MacLea, Kyle S.; Paul, Kacy R.; Ben-Musa, Zobaida; Waechter, Aubrey; Shattuck, Jenifer E.; Gruca, Margaret

    2014-01-01

    Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions. PMID:25547291

  14. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    SciTech Connect

    Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  15. The Loss of Lam2 and Npr2-Npr3 Diminishes the Vacuolar Localization of Gtr1-Gtr2 and Disinhibits TORC1 Activity in Fission Yeast

    PubMed Central

    Ma, Ning; Ma, Yan; Nakashima, Akio; Kikkawa, Ushio; Furuyashiki, Tomoyuki

    2016-01-01

    In mammalian cells, mTORC1 activity is regulated by Rag GTPases. It is thought that the Ragulator complex and the GATOR (GAP activity towards Rags) complex regulate RagA/B as its GDP/GTP exchange factor (GEF) and GTPase-activating protein (GAP), respectively. However, the functions of components in these complexes remain elusive. Using fission yeast as a model organism, here we found that the loss of Lam2 (SPBC1778.05c), a homolog of a Ragulator component LAMTOR2, as well as the loss of Gtr1 or Gtr2 phenocopies the loss of Npr2 or Npr3, homologs of GATOR components Nprl2 or Nprl3, respectively. These phenotypes were rescued by TORC1 inhibition using pharmacological or genetic means, and the loss of Lam2, Gtr1, Gtr2, Npr2 or Npr3 disinhibited TORC1 activity under nitrogen depletion, as measured by Rps6 phosphorylation. Consistently, overexpression of GDP-locked Gtr1S20L or GTP-locked Gtr2Q60L, which suppress TORC1 activity in budding yeast, rescued the growth defect of Δgtr1 cells or Δgtr2 cells, respectively, and the loss of Lam2, Npr2 or Npr3 similarly diminished the vacuolar localization and the protein levels of Gtr1 and Gtr2. Furthermore, Lam2 physically interacted with Npr2 and Gtr1. These findings suggest that Lam2 and Npr2-Npr3 function together as a tether for GDP-bound Gtr1 to the vacuolar membrane, thereby suppressing TORC1 activity for multiple cellular functions. PMID:27227887

  16. Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division.

    PubMed

    Wu, Yue-Xuan; Greene, Lois E; Masison, Daniel C; Eisenberg, Evan

    2005-09-01

    Propagation of the yeast prion [PSI+], a self-replicating aggregated form of Sup35p, requires Hsp104. One model to explain this phenomenon proposes that, in the absence of Hsp104, Sup35p aggregates enlarge but fail to replicate thus becoming diluted out as the yeast divide. To test this model, we used live imaging of Sup35p-GFP to follow the changes that occur in [PSI+] cells after the addition of guanidine to inactivate Hsp104. After guanidine addition there was initially an increase in aggregation of Sup35p-GFP; but then, before the yeast divided, the aggregates began to dissolve, and after approximately 6 h the Sup35-GFP looked identical to the Sup35-GFP in [psi+] cells. Although plating studies showed that the yeast were still [PSI+], this reduction in aggregation suggested that curing of [PSI+] by inactivation of Hsp104 might be independent of cell division. This was tested by measuring the rate of curing of [PSI+] cells in both dividing and nondividing cells. Cell division was inhibited by adding either alpha factor or farnesol. Remarkably, with both of these methods, we found that the rate of curing was not significantly affected by cell division. Thus, cell division is not a determining factor for curing [PSI+] by inactivating Hsp104 with guanidine. Rather, curing apparently occurs because Sup35-GFP polymers slowly depolymerize in the absence of Hsp104 activity. Hsp104 then counteracts this curing possibly by catalyzing formation of new polymers. PMID:16123122

  17. Monitoring of spectroscopic changes of a single trapped fission yeast cell by using a Raman tweezers set-up

    NASA Astrophysics Data System (ADS)

    Başar, G.; Kın, S.

    2008-10-01

    We demonstrate an improvement of the sensitivity of a Raman tweezers set-up, which combines optical tweezers with Raman spectroscopy. The system was tested by taking the Raman spectrum of a 4.6 μm diameter polystyrene sphere trapped in an aqueous solution. The improvement of sensitivity of the set-up was achieved by adjusting the trap depth for maximum signal to noise ratio (SNR). The maximum SNR was obtained by investigating the Raman peak of a trapped polystyrene sphere at 1001 cm -1 according to trap depth. With this system, a single trapped living Schizosaccharomyces Pombe yeast cell was sensitively monitored by taking the kinetic Raman spectra for more than 2 h. The relative intensity decrease in amide I and amide III bands, frequency increase in amide I band together with alterations in tyrosine marker band around 850 cm -1 was observed, which indicates alterations in the hydration state of protein by time progressing.

  18. ICRF-193, an anticancer topoisomerase II inhibitor, induces arched telophase spindles that snap, leading to a ploidy increase in fission yeast.

    PubMed

    Nakazawa, Norihiko; Mehrotra, Rajesh; Arakawa, Orie; Yanagida, Mitsuhiro

    2016-09-01

    ICRF-193 [meso-4,4-(2,3-butanediyl)-bis(2,6-piperazinedione)] is a complex-stabilizing inhibitor of DNA topoisomerase II (topo II) that is used as an effective anticancer drug. ICRF-193 inhibits topo II catalytic activity in vitro and blocks nuclear division in vivo. Here, we examined the effects of ICRF-193 treatment on chromatin behavior and spindle dynamics using detailed live mitotic cell analysis in the fission yeast, Schizosaccharomyces pombe. Time-lapse movie analysis showed that ICRF-193 treatment leads to an elongation of presumed chromatin fibers connected to kinetochores during mid-mitosis. Anaphase spindles begin to arch, and eventually spindle poles come together abruptly, as if the spindle snapped at the point of spindle microtubule overlap in telophase. Segregating chromosomes appeared as elastic clumps and subsequently pulled back and merged. The snapped spindle phenotype was abolished by microtubule destabilization after thiabendazole treatment, accompanied by unequal chromosome segregation or severe defects in spindle extension. Thus, we conclude that ICRF-193-treated, unseparated sister chromatids pulling toward opposite spindle poles produce the arched and snapped telophase spindle. ICRF-193 treatment increased DNA content, suggesting that the failure of sister chromatids to separate properly in anaphase, causes the spindle to break in telophase, resulting in polyploidization. PMID:27458047

  19. Targeting of SUMO substrates to a Cdc48–Ufd1–Npl4 segregase and STUbL pathway in fission yeast

    PubMed Central

    Køhler, Julie Bonne; Tammsalu, Triin; Jørgensen, Maria Mønster; Steen, Nana; Hay, Ronald Thomas; Thon, Geneviève

    2015-01-01

    In eukaryotes, the conjugation of proteins to the small ubiquitin-like modifier (SUMO) regulates numerous cellular functions. A proportion of SUMO conjugates are targeted for degradation by SUMO-targeted ubiquitin ligases (STUbLs) and it has been proposed that the ubiquitin-selective chaperone Cdc48/p97-Ufd1-Npl4 facilitates this process. However, the extent to which the two pathways overlap, and how substrates are selected, remains unknown. Here we address these questions in fission yeast through proteome-wide analyses of SUMO modification sites. We identify over a thousand sumoylated lysines in a total of 468 proteins and quantify changes occurring in the SUMO modification status when the STUbL or Ufd1 pathways are compromised by mutations. The data suggest the coordinated processing of several classes of SUMO conjugates, many dynamically associated with centromeres or telomeres. They provide new insights into subnuclear organization and chromosome biology, and, altogether, constitute an extensive resource for the molecular characterization of SUMO function and dynamics. PMID:26537787

  20. RNA Cytidine Acetyltransferase of Small-Subunit Ribosomal RNA: Identification of Acetylation Sites and the Responsible Acetyltransferase in Fission Yeast, Schizosaccharomyces pombe

    PubMed Central

    Taoka, Masato; Ishikawa, Daisuke; Nobe, Yuko; Ishikawa, Hideaki; Yamauchi, Yoshio; Terukina, Goro; Nakayama, Hiroshi; Hirota, Kouji; Takahashi, Nobuhiro; Isobe, Toshiaki

    2014-01-01

    The eukaryotic small-subunit (SSU) ribosomal RNA (rRNA) has two evolutionarily conserved acetylcytidines. However, the acetylation sites and the acetyltransferase responsible for the acetylation have not been identified. We performed a comprehensive MS-based analysis covering the entire sequence of the fission yeast, Schizosaccharomyces pombe, SSU rRNA and identified two acetylcytidines at positions 1297 and 1815 in the 3′ half of the rRNA. To identify the enzyme responsible for the cytidine acetylation, we searched for an S. pombe gene homologous to TmcA, a bacterial tRNA N-acetyltransferase, and found one potential candidate, Nat10. A temperature-sensitive strain of Nat10 with a mutation in the Walker A type ATP-binding motif abolished the cytidine acetylation in SSU rRNA, and the wild-type Nat10 supplemented to this strain recovered the acetylation, providing evidence that Nat10 is necessary for acetylation of SSU rRNA. The Nat10 mutant strain showed a slow-growth phenotype and was defective in forming the SSU rRNA from the precursor RNA, suggesting that cytidine acetylation is necessary for ribosome assembly. PMID:25402480

  1. Plasticity and epigenetic inheritance of centromere-specific histone H3 (CENP-A)-containing nucleosome positioning in the fission yeast.

    PubMed

    Yao, Jianhui; Liu, Xingkun; Sakuno, Takeshi; Li, Wenzhu; Xi, Yuanxin; Aravamudhan, Pavithra; Joglekar, Ajit; Li, Wei; Watanabe, Yoshinori; He, Xiangwei

    2013-06-28

    Nucleosomes containing the specific histone H3 variant CENP-A mark the centromere locus on each chromatin and initiate kinetochore assembly. For the common type of regional centromeres, little is known in molecular detail of centromeric chromatin organization, its propagation through cell division, and how distinct organization patterns may facilitate kinetochore assembly. Here, we show that in the fission yeast S. pombe, a relatively small number of CENP-A/Cnp1 nucleosomes are found within the centromeric core and that their positioning relative to underlying DNA varies among genetically homogenous cells. Consistent with the flexible positioning of Cnp1 nucleosomes, a large portion of the endogenous centromere is dispensable for its essential activity in mediating chromosome segregation. We present biochemical evidence that Cnp1 occupancy directly correlates with silencing of the underlying reporter genes. Furthermore, using a newly developed pedigree analysis assay, we demonstrated the epigenetic inheritance of Cnp1 positioning and quantified the rate of occasional repositioning of Cnp1 nucleosomes throughout cell generations. Together, our results reveal the plasticity and the epigenetically inheritable nature of centromeric chromatin organization. PMID:23661703

  2. Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast*

    PubMed Central

    Yao, Jianhui; Liu, Xingkun; Sakuno, Takeshi; Li, Wenzhu; Xi, Yuanxin; Aravamudhan, Pavithra; Joglekar, Ajit; Li, Wei; Watanabe, Yoshinori; He, Xiangwei

    2013-01-01

    Nucleosomes containing the specific histone H3 variant CENP-A mark the centromere locus on each chromatin and initiate kinetochore assembly. For the common type of regional centromeres, little is known in molecular detail of centromeric chromatin organization, its propagation through cell division, and how distinct organization patterns may facilitate kinetochore assembly. Here, we show that in the fission yeast S. pombe, a relatively small number of CENP-A/Cnp1 nucleosomes are found within the centromeric core and that their positioning relative to underlying DNA varies among genetically homogenous cells. Consistent with the flexible positioning of Cnp1 nucleosomes, a large portion of the endogenous centromere is dispensable for its essential activity in mediating chromosome segregation. We present biochemical evidence that Cnp1 occupancy directly correlates with silencing of the underlying reporter genes. Furthermore, using a newly developed pedigree analysis assay, we demonstrated the epigenetic inheritance of Cnp1 positioning and quantified the rate of occasional repositioning of Cnp1 nucleosomes throughout cell generations. Together, our results reveal the plasticity and the epigenetically inheritable nature of centromeric chromatin organization. PMID:23661703

  3. A Recombinationally Repressed Region between Mat2 and Mat3 Loci Shares Homology to Centromeric Repeats and Regulates Directionality of Mating-Type Switching in Fission Yeast

    PubMed Central

    Grewal, SIS.; Klar, AJS.

    1997-01-01

    Cells of the fission yeast Schizosaccharomyces pombe switch mating type by replacing genetic information at the transcriptionally active mat1 locus with sequences copied from one of two closely linked silent loci, mat2-P or mat3-M. By a process referred to as directionality of switching, cells predominantly switch to the opposite mat1 allele; the mat1-P allele preferentially recombines with mat3, while mat1-M selects the mat2. In contrast to efficient recombination at mat1, recombination within the adjoining mat2-mat3 interval is undetectable. We defined the role of sequences between mat2 and mat3, designated the K-region, in directionality as well as recombinational suppression. Cloning and sequencing analysis revealed that a part of the K-region is homologous to repeat sequences present at centromeres, which also display transcriptional and recombinational suppression. Replacement of 7.5 kb of the K-region with the ura4(+) gene affected directionality in a variegated manner. Analysis of the swi6-mod locus, which was previously shown to affect directionality, in KΔ::ura4(+) strains suggested the existence of at least two overlapping directionality mechanisms. Our work furthers the model that directionality is regulated by cell-type-specific organization of the heterochromatin-like structure in the mating-type region and provides evidence that the K-region contributes to silencing of the mat2-mat3 interval. PMID:9258669

  4. F-BAR domain protein Rga7 collaborates with Cdc15 and Imp2 to ensure proper cytokinesis in fission yeast.

    PubMed

    Martín-García, Rebeca; Coll, Pedro M; Pérez, Pilar

    2014-10-01

    F-BAR domain proteins act as linkers between the cell cortex and cytoskeleton, and are involved in membrane binding and bending. Rga7 is one of the seven F-BAR proteins present in the fission yeast Schizosaccharomyces pombe. In addition to the F-BAR domain in the N-terminal region, Rga7 possesses a Rho GTPase-activating protein (GAP) domain at its C-terminus. We show here that Rga7 is necessary to prevent fragmentation of the contracting ring and incorrect septum synthesis. Accordingly, cultures of cells lacking Rga7 contain a higher percentage of dividing cells and more frequent asymmetric or aberrant septa, which ultimately might cause cell death. The Rga7 F-BAR domain is necessary for the protein localization to the division site and to the cell tips, and also for the Rga7 roles in cytokinesis. In contrast, Rga7 GAP catalytic activity seems to be dispensable. Moreover, we demonstrate that Rga7 cooperates with the two F-BAR proteins Cdc15 and Imp2 to ensure proper cytokinesis. We have also detected association of Rga7 with Imp2, and its binding partners Fic1 and Pxl1. Taken together, our findings suggest that Rga7 forms part of a protein complex that coordinates the late stages of cytokinesis. PMID:25052092

  5. The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.

    PubMed

    Li, Yujie; Christensen, Jenna R; Homa, Kaitlin E; Hocky, Glen M; Fok, Alice; Sees, Jennifer A; Voth, Gregory A; Kovar, David R

    2016-06-01

    The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction. PMID:27075176

  6. The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

    PubMed Central

    Li, Yujie; Christensen, Jenna R.; Homa, Kaitlin E.; Hocky, Glen M.; Fok, Alice; Sees, Jennifer A.; Voth, Gregory A.; Kovar, David R.

    2016-01-01

    The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction. PMID:27075176

  7. Set3 contributes to heterochromatin integrity by promoting transcription of subunits of Clr4-Rik1-Cul4 histone methyltransferase complex in fission yeast

    PubMed Central

    Yu, Yao; Zhou, Huan; Deng, Xiaolong; Wang, Wenchao; Lu, Hong

    2016-01-01

    Heterochromatin formation in fission yeast depends on RNAi machinery and histone-modifying enzymes. One of the key histone-modifying complexes is Clr4-Rik1-Cul4 methyltransferase complex (CLRC), which mediates histone H3K9 methylation, a hallmark for heterochromatin. CLRC is composed of the Clr4 histone methyltransferase, Rik1, Raf1, Raf2 and Pcu4. However, transcriptional regulation of the CLRC subunits is not well understood. In this study, we identified Set3, a core subunit of the Set3/Hos2 histone deacetylase complex (Set3C), as a contributor to the integrity and silencing of heterochromatin at centromeres, telomeres and silent mating-type locus. This novel role of Set3 relies on its PHD finger, but is independent of deacetylase activity or structural integrity of Set3C. Set3 is not located to the centromeric region. Instead, Set3 is targeted to the promoters of clr4+ and rik1+, probably through its PHD finger. Set3 promotes transcription of clr4+ and rik1+. Consistently, the protein levels of Clr4 and Rik1 were reduced in the set3Δ mutant. The heterochromatin silencing defect in the set3Δ mutant could be rescued by overexpressing of clr4+ or rik1+. Our study suggests transcriptional activation of essential heterochromatin factors underlies the tight regulation of heterochromatin integrity. PMID:27538348

  8. Centromere mapping functions for aneuploid meiotic products: Analysis of rec8, rec10 and rec11 mutants of the fission yeast Schizosaccharomyces pombe.

    PubMed Central

    Krawchuk, M D; Wahls, W P

    1999-01-01

    Recent evidence suggests that the position of reciprocal recombination events (crossovers) is important for the segregation of homologous chromosomes during meiosis I and sister chromatids during meiosis II. We developed genetic mapping functions that permit the simultaneous analysis of centromere-proximal crossover recombination and the type of segregation error leading to aneuploidy. The mapping functions were tested in a study of the rec8, rec10, and rec11 mutants of fission yeast. In each mutant we monitored each of the three chromosome pairs. Between 38 and 100% of the chromosome segregation errors in the rec8 mutants were due to meiosis I nondisjunction of homologous chromosomes. The remaining segregation errors were likely the result of precocious separation of sister chromatids, a previously described defect in the rec8 mutants. Between 47 and 100% of segregation errors in the rec10 and rec11 mutants were due to nondisjunction of sister chromatids during meiosis II. In addition, centromere-proximal recombination was reduced as much as 14-fold or more on chromosomes that had experienced nondisjunction. These results demonstrate the utility of the new mapping functions and support models in which sister chromatid cohesion and crossover position are important determinants for proper chromosome segregation in each meiotic division. PMID:10471699

  9. A metabolic strategy to enhance long-term survival by Phx1 through stationary phase-specific pyruvate decarboxylases in fission yeast

    PubMed Central

    Kim, Ji-Yoon; Kim, Eun-Jung; Lopez-Maury, Luis; Bähler, Jürg; Roe, Jung-Hye

    2014-01-01

    In the fission yeast Schizosaccharomyces pombe, the stationary phase-specific transcription factor Phx1 contributes to long-term survival, stress tolerance, and meiosis. We identified Phx1-dependent genes through transcriptome analysis, and further analyzed those related with carbohydrate and thiamine metabolism, whose expression decreased in Δphx1. Consistent with mRNA changes, the level of thiamine pyrophosphate (TPP) and TPP-utilizing pyruvate decarboxylase activity that converts pyruvate to acetaldehyde were also reduced in the mutant. Therefore, Phx1 appears to shift metabolic flux by diverting pyruvate from the TCA cycle and respiration to ethanol fermentation. Among the four predicted genes for pyruvate decarboxylase, only the Phx1-dependent genes (pdc201+ and pdc202+) contributed to long-term survival as judged by mutation and overexpression studies. These findings indicate that the Phx1-mediated long-term survival is achieved primarily through increasing the synthesis and activity of pyruvate decarboxylase. Consistent with this hypothesis, we observed that Phx1 curtailed respiration when cells entered stationary phase. Introduction of Δphx1 mutation compromised the long-lived phenotypes of Δpka1 and Δsck2 mutants that are devoid of pro-aging kinases of nutrient-signalling pathways, and of the Δpyp1 mutant with constitutively activated stress-responsive kinase Sty1. Therefore, achievement of long-term viability through both nutrient limitation and anti-stress response appears to be dependent on Phx1. PMID:25102102

  10. A metabolic strategy to enhance long-term survival by Phx1 through stationary phase-specific pyruvate decarboxylases in fission yeast.

    PubMed

    Kim, Ji-Yoon; Kim, Eun-Jung; Lopez-Maury, Luis; Bähler, Jürg; Roe, Jung-Hye

    2014-07-01

    In the fission yeast Schizosaccharomyces pombe, the stationary phase-specific transcription factor Phx1 contributes to long-term survival, stress tolerance, and meiosis. We identified Phx1-dependent genes through transcriptome analysis, and further analyzed those related with carbohydrate and thiamine metabolism, whose expression decreased in ∆phx1. Consistent with mRNA changes, the level of thiamine pyrophosphate (TPP) and TPP-utilizing pyruvate decarboxylase activity that converts pyruvate to acetaldehyde were also reduced in the mutant. Therefore, Phx1 appears to shift metabolic flux by diverting pyruvate from the TCA cycle and respiration to ethanol fermentation. Among the four predicted genes for pyruvate decarboxylase, only the Phx1-dependent genes (pdc201+ and pdc202+) contributed to long-term survival as judged by mutation and overexpression studies. These findings indicate that the Phx1-mediated long-term survival is achieved primarily through increasing the synthesis and activity of pyruvate decarboxylase. Consistent with this hypothesis, we observed that Phx1 curtailed respiration when cells entered stationary phase. Introduction of Δphx1 mutation compromised the long-lived phenotypes of Δpka1 and Δsck2 mutants that are devoid of pro-aging kinases of nutrient-signalling pathways, and of the Δpyp1 mutant with constitutively activated stress-responsive kinase Sty1. Therefore, achievement of long-term viability through both nutrient limitation and anti-stress response appears to be dependent on Phx1. PMID:25102102

  11. The Protein Level of Rev1, a TLS Polymerase in Fission Yeast, Is Strictly Regulated during the Cell Cycle and after DNA Damage

    PubMed Central

    Uchiyama, Masashi; Terunuma, Junko; Hanaoka, Fumio

    2015-01-01

    Translesion DNA synthesis provides an alternative DNA replication mechanism when template DNA is damaged. In fission yeast, Eso1 (polη), Kpa1/DinB (polκ), Rev1, and Polζ (a complex of Rev3 and Rev7) have been identified as translesion synthesis polymerases. The enzymatic characteristics and protein-protein interactions of these polymerases have been intensively characterized; however, how these proteins are regulated during the cell cycle remains unclear. Therefore, we examined the cell cycle oscillation of translesion polymerases. Interestingly, the protein levels of Rev1 peaked during G1 phase and then decreased dramatically at the entry of S phase; this regulation was dependent on the proteasome. Temperature-sensitive proteasome mutants, such as mts2-U31 and mts3-U32, stabilized Rev1 protein when the temperature was shifted to the restrictive condition. In addition, deletion of pop1 or pop2, subunits of SCF ubiquitin ligase complexes, upregulated Rev1 protein levels. Besides these effects during the cell cycle, we also observed upregulation of Rev1 protein upon DNA damage. This upregulation was abolished when rad3, a checkpoint protein, was deleted or when the Rev1 promoter was replaced with a constitutive promoter. From these results, we hypothesize that translesion DNA synthesis is strictly controlled through Rev1 protein levels in order to avoid unwanted mutagenesis. PMID:26147350

  12. Regulation of unbalanced redox homeostasis induced by the expression of wild-type HIV-1 viral protein R (NL4-3Vpr) in fission yeast.

    PubMed

    Gazdag, Zoltán; Stromájer-Rácz, Timea; Belagyi, Joseph; Zhao, Richard Y; Elder, Robert T; Virág, Eszter; Pesti, Miklós

    2015-09-01

    The wild-type viral protein R (Vpr) of human immunodeficiency virus type 1 exerts multiple effects on cellular activities during infection, including the induction of cell cycle G2 arrest and the death of human cells and cells of the fission yeast Schizosaccharomyces pombe. In this study, wild-type Vpr (NL4-3Vpr) integrated as a single copy gene in S. pombe chromosome was used to investigate the molecular impact of Vpr on cellular oxidative stress. NL4-3Vpr triggered an atypical response in early (14-h), and a wellregulated oxidative stress response in late (35-h) log-phase cultures. Specifically, NL4-3Vpr expression induced oxidative stress in the 14-h cultures leading, to decreased levels of superoxide anion (O(2)(·-)), hydroxyl radical (·OH) and glutathione (GSH), and significantly decreased activities of catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase. In the 35-h cultures, elevated levels of O(2)(·-) and peroxides were accompanied by increased activities of most antioxidant enzymes, suggesting that the Vpr-induced unbalanced redox state of the cells might contribute to the adverse effects in HIV-infected patients. PMID:26344028

  13. Set3 contributes to heterochromatin integrity by promoting transcription of subunits of Clr4-Rik1-Cul4 histone methyltransferase complex in fission yeast.

    PubMed

    Yu, Yao; Zhou, Huan; Deng, Xiaolong; Wang, Wenchao; Lu, Hong

    2016-01-01

    Heterochromatin formation in fission yeast depends on RNAi machinery and histone-modifying enzymes. One of the key histone-modifying complexes is Clr4-Rik1-Cul4 methyltransferase complex (CLRC), which mediates histone H3K9 methylation, a hallmark for heterochromatin. CLRC is composed of the Clr4 histone methyltransferase, Rik1, Raf1, Raf2 and Pcu4. However, transcriptional regulation of the CLRC subunits is not well understood. In this study, we identified Set3, a core subunit of the Set3/Hos2 histone deacetylase complex (Set3C), as a contributor to the integrity and silencing of heterochromatin at centromeres, telomeres and silent mating-type locus. This novel role of Set3 relies on its PHD finger, but is independent of deacetylase activity or structural integrity of Set3C. Set3 is not located to the centromeric region. Instead, Set3 is targeted to the promoters of clr4(+) and rik1(+), probably through its PHD finger. Set3 promotes transcription of clr4(+) and rik1(+). Consistently, the protein levels of Clr4 and Rik1 were reduced in the set3Δ mutant. The heterochromatin silencing defect in the set3Δ mutant could be rescued by overexpressing of clr4(+) or rik1(+). Our study suggests transcriptional activation of essential heterochromatin factors underlies the tight regulation of heterochromatin integrity. PMID:27538348

  14. Loss of Apm1, the μ1 Subunit of the Clathrin-Associated Adaptor-Protein-1 Complex, Causes Distinct Phenotypes and Synthetic Lethality with Calcineurin Deletion in Fission Yeast

    PubMed Central

    Kita, Ayako; Sugiura, Reiko; Shoji, Hiromi; He, Yi; Deng, Lu; Lu, Yabin; Sio, Susie O.; Takegawa, Kaoru; Sakaue, Motoyoshi; Shuntoh, Hisato; Kuno, Takayoshi

    2004-01-01

    Calcineurin is a highly conserved regulator of Ca2+ signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl- homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1+ gene that encodes a homolog of the mammalian μ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Δapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Δapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Δapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events. PMID:15047861

  15. Yeast genes required for conversion of grape precursors to varietal thiols in wine.

    PubMed

    Santiago, Margarita; Gardner, Richard C

    2015-08-01

    Three varietal thiols are important for the tropical fruit aromas of Sauvignon blanc: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexanol (3MH) and its acetylated derivative 3-mercaptohexyl acetate (3MHA). These thiols are produced by yeast during fermentation from precursors in grape juice. Here we identify genes in Saccharomyces cerevisiae that are required for the transport and cleavage of two thiol precursors: cysteine-4MMP and glutathione-3MH. A full-length copy of IRC7 is absolutely required for the cleavage of both precursors in the tested strains; the deleted form of the enzyme found in most yeast strains is incapable of converting these compounds into detectable thiols. By using strains that overexpress full-length IRC7, we further show that the glutathione transporter OPT1 and the transpeptidase CIS2 are also required for conversion of glut-3MH to its varietal thiol. No transporter for cys-4MMP was identified: a strain deleted for all nine known cysteine transport genes was still capable of converting cys-4MMP to its varietal thiol, and was also able to take up cysteine at high concentrations. Based on these results, we conclude that cysteine and glutathione precursors make a relatively minor contribution to 3MH production from most grape juices. PMID:26038341

  16. Kip3, the yeast kinesin-8, is required for clustering of kinetochores at metaphase

    PubMed Central

    Wargacki, Megan M; Tay, Jessica C; Muller, Eric G; Asbury, Charles L

    2010-01-01

    In Saccharomyces cerevisiae, chromosome congression clusters kinetochores on either side of the spindle equator at metaphase. Many organisms require one or more kinesin-8 molecular motors to achieve chromosome alignment. the yeast kinesin-8, Kip3, has been well studied in vitro but a role in chromosome congression has not been reported. We investigated Kip3's role in this process using semi-automated, quantitative fluorescence microscopy and time-lapse imaging and found that Kip3 is required for congression. Deletion of KIP3 increases inter-kinetochore distances and increases the variability in the position of sister kinetochores along the spindle axis during metaphase. Kip3 does not regulate spindle length and is not required for kinetochore-microtubule attachment. Instead, Kip3 clusters kinetochores on the metaphase spindle by tightly regulating kinetochore microtubule lengths. PMID:20603597

  17. Region of Yeast TAF 130 Required for TFIID To Associate with Promoters

    PubMed Central

    Mencía, Mario; Struhl, Kevin

    2001-01-01

    TFIID, a multiprotein complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs), associates specifically with core promoters and nucleates the assembly the RNA polymerase II transcription machinery. In yeast cells, TFIID is not generally required for transcription, although it plays an important role at many promoters. Understanding of the specific functions and physiological roles of individual TAFs within TFIID has been hampered by the fact that depletion or thermal inactivation of individual TAFs generally results in dissociation of the TFIID complex. We describe here C-terminally deleted derivatives of yeast TAF130 that assemble into normal TFIID complexes but are transcriptionally inactive in vivo. In vivo, these mutant TFIID complexes are dramatically reduced in their ability to associate with all promoters tested. In vitro, a TFIID complex containing a deleted form of TAF130 associates poorly with DNA, but it is unaffected for interacting with transcriptional activation domains. These results suggest that the C-terminal region of TAF130 is required for TFIID to associate with promoters. PMID:11158301

  18. Mitochondrial Fusion in Yeast Requires the Transmembrane GTPase Fzo1p

    PubMed Central

    Hermann, Greg J.; Thatcher, John W.; Mills, John P.; Hales, Karen G.; Fuller, Margaret T.; Nunnari, Jodi; Shaw, Janet M.

    1998-01-01

    Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion. PMID:9786948

  19. Structural variants of yeast prions show conformer-specific requirements for chaperone activity

    PubMed Central

    Stein, Kevin C.; True, Heather L.

    2016-01-01

    Summary Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1, and its human ortholog Hdj1, had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation. PMID:25060529

  20. Interaction of APC/C-E3 Ligase with Swi6/HP1 and Clr4/Suv39 in Heterochromatin Assembly in Fission Yeast*S⃞♦

    PubMed Central

    Dubey, Rudra Narayan; Nakwal, Nandni; Bisht, Kamlesh Kumar; Saini, Ashok; Haldar, Swati; Singh, Jagmohan

    2009-01-01

    Heterochromatin assembly in fission yeast is initiated by binding of Swi6/HP1 to the Lys-9-dimethylated H3 followed by spreading via cooperative recruitment of Swi6/HP1. Recruitment of Cohesin by Swi6/HP1 further stabilizes the heterochromatin structure and integrity. Subsequently, polyubiquitylation of Cut2 by anaphase-promoting complex-cyclosome (APC/C)-ubiquitin-protein isopeptide ligase (E3 ligase) followed by degradation of Cut2 releases Cut1, which cleaves the Rad21 subunit of Cohesin, facilitating sister chromatid separation during mitosis. Here, we demonstrate a surprising role of APC/C in assembly of heterochromatin and silencing at mating type, centromere, and ribosomal DNA loci. Coincidentally with the loss of silencing, recruitment of Swi6, H3-Lys-9-Me2, and Clr4 at dg-dh repeats at cen1 and the K region of mat locus is abrogated in mutants cut4, cut9, and nuc2. Surprisingly, both Cut4 and Cut9 are also highly enriched at these regions in wild type and depleted in swi6Δ mutant. Cut4 and Cut9 interact directly with Swi6/HP1 and Clr4, whereas the mutant Cut4 does not, suggesting that a direct physical interaction of APC subunits Cut4 and Cut9 with Swi6 and Clr4 is instrumental in heterochromatin assembly. The silencing defect in APC mutants is causally related to ubiquitylation activity of APC-E3 ligase. Like swi6 mutant, APC mutants are also defective in Cohesin recruitment and exhibit defects like lagging chromosomes, chromosome loss, and aberrant recombination in the mat region. In addition, APC mutants exhibit a bidirectional expression of dh repeats, suggesting a role in the RNA interference pathway. Thus, APC and heterochromatin proteins Swi6 and Clr4 play a mutually cooperative role in heterochromatin assembly, thereby ensuring chromosomal integrity, inheritance, and segregation during mitosis and meiosis. PMID:19117951

  1. Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast.

    PubMed

    Xu, Yong-Jie

    2016-04-01

    DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2(+) sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling are seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside the nucleus, a previously unknown mechanism. PMID:26746798

  2. Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan

    2010-09-03

    Research highlights: {yields} cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. {yields} Pka1 phosphorylation is further induced by physiological stresses. {yields} Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. {yields} Results suggest that cAMP-independent Pka1 phosphorylation is stimulatory in nature. -- Abstract: In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1{Delta} cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1{Delta} cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1{sup +} or cyr1{Delta} S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.

  3. Mitochondrial fission is required for cardiomyocyte hypertrophy mediated by a Ca2+-calcineurin signaling pathway

    PubMed Central

    Pennanen, Christian; Parra, Valentina; López-Crisosto, Camila; Morales, Pablo E.; del Campo, Andrea; Gutierrez, Tomás; Rivera-Mejías, Pablo; Kuzmicic, Jovan; Chiong, Mario; Zorzano, Antonio; Rothermel, Beverly A.; Lavandero, Sergio

    2014-01-01

    ABSTRACT Cardiomyocyte hypertrophy has been associated with diminished mitochondrial metabolism. Mitochondria are crucial organelles for the production of ATP, and their morphology and function are regulated by the dynamic processes of fusion and fission. The relationship between mitochondrial dynamics and cardiomyocyte hypertrophy is still poorly understood. Here, we show that treatment of cultured neonatal rat cardiomyocytes with the hypertrophic agonist norepinephrine promotes mitochondrial fission (characterized by a decrease in mitochondrial mean volume and an increase in the relative number of mitochondria per cell) and a decrease in mitochondrial function. We demonstrate that norepinephrine acts through α1-adrenergic receptors to increase cytoplasmic Ca2+, activating calcineurin and promoting migration of the fission protein Drp1 (encoded by Dnml1) to mitochondria. Dominant-negative Drp1 (K38A) not only prevented mitochondrial fission, it also blocked hypertrophic growth of cardiomyocytes in response to norepinephrine. Remarkably, an antisense adenovirus against the fusion protein Mfn2 (AsMfn2) was sufficient to increase mitochondrial fission and stimulate a hypertrophic response without agonist treatment. Collectively, these results demonstrate the importance of mitochondrial dynamics in the development of cardiomyocyte hypertrophy and metabolic remodeling. PMID:24777478

  4. Mitochondrial fission is required for cardiomyocyte hypertrophy mediated by a Ca2+-calcineurin signaling pathway.

    PubMed

    Pennanen, Christian; Parra, Valentina; López-Crisosto, Camila; Morales, Pablo E; Del Campo, Andrea; Gutierrez, Tomás; Rivera-Mejías, Pablo; Kuzmicic, Jovan; Chiong, Mario; Zorzano, Antonio; Rothermel, Beverly A; Lavandero, Sergio

    2014-06-15

    Cardiomyocyte hypertrophy has been associated with diminished mitochondrial metabolism. Mitochondria are crucial organelles for the production of ATP, and their morphology and function are regulated by the dynamic processes of fusion and fission. The relationship between mitochondrial dynamics and cardiomyocyte hypertrophy is still poorly understood. Here, we show that treatment of cultured neonatal rat cardiomyocytes with the hypertrophic agonist norepinephrine promotes mitochondrial fission (characterized by a decrease in mitochondrial mean volume and an increase in the relative number of mitochondria per cell) and a decrease in mitochondrial function. We demonstrate that norepinephrine acts through α1-adrenergic receptors to increase cytoplasmic Ca(2+), activating calcineurin and promoting migration of the fission protein Drp1 (encoded by Dnml1) to mitochondria. Dominant-negative Drp1 (K38A) not only prevented mitochondrial fission, it also blocked hypertrophic growth of cardiomyocytes in response to norepinephrine. Remarkably, an antisense adenovirus against the fusion protein Mfn2 (AsMfn2) was sufficient to increase mitochondrial fission and stimulate a hypertrophic response without agonist treatment. Collectively, these results demonstrate the importance of mitochondrial dynamics in the development of cardiomyocyte hypertrophy and metabolic remodeling. PMID:24777478

  5. Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission

    PubMed Central

    Richter, Viviane; Palmer, Catherine S.; Osellame, Laura D.; Singh, Abeer P.; Elgass, Kirstin; Stroud, David A.; Sesaki, Hiromi

    2014-01-01

    Mitochondrial fission is important for organelle transport, inheritance, and turnover, and alterations in fission are seen in neurological disease. In mammals, mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a cytosolic guanosine triphosphatase that polymerizes and constricts the organelle. Recruitment of Drp1 to mitochondria involves receptors including Mff, MiD49, and MiD51. MiD49/51 form foci at mitochondrial constriction sites and coassemble with Drp1 to drive fission. Here, we solved the crystal structure of the cytosolic domain of human MiD51, which adopts a nucleotidyltransferase fold. Although MiD51 lacks catalytic residues for transferase activity, it specifically binds guanosine diphosphate and adenosine diphosphate. MiD51 mutants unable to bind nucleotides were still able to recruit Drp1. Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci. MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus. PMID:24515348

  6. Dissection of the essential steps for condensin accumulation at kinetochores and rDNAs during fission yeast mitosis.

    PubMed

    Nakazawa, Norihiko; Nakamura, Takahiro; Kokubu, Aya; Ebe, Masahiro; Nagao, Koji; Yanagida, Mitsuhiro

    2008-03-24

    The condensin complex has a fundamental role in chromosome dynamics. In this study, we report that accumulation of Schizosaccharomyces pombe condensin at mitotic kinetochores and ribosomal DNAs (rDNAs) occurs in multiple steps and is necessary for normal segregation of the sister kinetochores and rDNAs. Nuclear entry of condensin at the onset of mitosis requires Cut15/importin alpha and Cdc2 phosphorylation. Ark1/aurora and Cut17/Bir1/survivin are needed to dock the condensin at both the kinetochores and rDNAs. Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites. Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs. The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells. PMID:18362178

  7. Microtubule capture by the cleavage apparatus is required for proper spindle positioning in yeast.

    PubMed

    Kusch, Justine; Meyer, Anne; Snyder, Michael P; Barral, Yves

    2002-07-01

    Cell division is the result of two major cytoskeletal events: partition of the chromatids by the mitotic spindle and cleavage of the cell by the cytokinetic apparatus. Spatial coordination of these events ensures that each daughter cell inherits a nucleus. Here we show that, in budding yeast, capture and shrinkage of astral microtubules at the bud neck is required to position the spindle relative to the cleavage apparatus. Capture required the septins and the microtubule-associated protein Kar9. Like Kar9-defective cells, cells lacking the septin ring failed to position their spindle correctly and showed an increased frequency of nuclear missegregation. Microtubule attachment at the bud neck was followed by shrinkage and a pulling action on the spindle. Enhancement of microtubule shrinkage at the bud neck required the Par-1-related, septin-dependent kinases (SDK) Hsl1 and Gin4. Neither the formin Bnr1 nor the actomyosin contractile ring was required for either microtubule capture or microtubule shrinkage. Together, our results indicate that septins and septin-dependent kinases may coordinate microtubule and actin functions in cell division. PMID:12101122

  8. A cell cycle-regulated GATA factor promotes centromeric localization of CENP-A in fission yeast.

    PubMed

    Chen, Ee Sin; Saitoh, Shigeaki; Yanagida, Mitsuhiro; Takahashi, Kohta

    2003-01-01

    CENP-A, the centromere-specific histone H3 variant, plays a crucial role in organizing kinetochore chromatin for precise chromosome segregation. We have isolated Ams2, a Daxx-like motif-containing GATA factor, and histone H4, as multicopy suppressors of cnp1-1, an S. pombe CENP-A mutant. While depletion of Ams2 results in the reduction of CENP-A binding to the centromere and chromosome missegregation, increasing its dosage restores association of a CENP-A mutant protein with centromeres. Conversely, overexpression of CENP-A or histone H4 suppresses an ams2 disruptant. The intracellular amount of Ams2 thus affects centromeric nucleosomal constituents. Ams2 is abundant in S phase and associates with chromatin, including the central centromeres through binding to GATA-core sequences. Ams2 is thus a cell cycle-regulated GATA factor that is required for centromere function. PMID:12535531

  9. MEI4, a meiosis-specific yeast gene required for chromosome synapsis.

    PubMed Central

    Menees, T M; Ross-MacDonald, P B; Roeder, G S

    1992-01-01

    The MEI4 gene product is required for meiotic induction of recombination and viable spore production in the yeast Saccharomyces cerevisiae. DNA sequence analysis shows that the MEI4 gene encodes a 450-amino-acid protein bearing no homology to any previously identified protein. The MEI4 coding region is interrupted by a small intron located near the 5' end of the gene. Efficient splicing of the MEI4 transcript is not dependent on the MER1 protein, which is required for splicing the transcript of another meiotic gene, MER2. Expression of a mei4::lacZ fusion gene is meiosis-specific and depends on both heterozygosity at the mating-type locus and nutrient limitation. Northern (RNA) blot hybridization analysis suggests that MEI4 gene expression is regulated at the level of transcription. A functional MEI4 gene is not required for meiotic induction of transcription of the MER1, MER2, MEK1, RED1, SPO11, or RAD50 gene. Cytological analysis of mei4 mutant strains during meiotic prophase demonstrates that the chromosomes form long axial elements that fail to undergo synapsis. The meiosis II division is delayed in mei4 strains. Images PMID:1545815

  10. A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast

    PubMed Central

    Hirashima, Kyotaro; Iwaki, Tomoko; Takegawa, Kaoru; Giga-Hama, Yuko; Tohda, Hideki

    2006-01-01

    The technologies for chromosome modification developed to date are not satisfactorily universal, owing to the typical requirements for special enzymes and sequences. In the present report, we propose a new approach for chromosome modification in Schizosaccharomyces pombe that does not involve any special enzymes or sequences. This method, designated the ‘Latour system’, has wide applicability with extremely high efficiency, although both the basic principle and the operation are very simple. We demonstrate the ability of the Latour system to discriminate essential genes, with a long chromosomal area of 100 kb containing 33 genes deleted simultaneously and efficiently. Since no foreign sequences are retained after deletion using the Latour system, this system can be repeatedly applied at other sites. Provided that a negative selectable marker is available, the Latour system relies solely upon homologous recombination, which is highly conserved in living organisms. For this reason, it is expected that the system will be applicable to various yeasts. PMID:16434698

  11. Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF

    PubMed Central

    Tafforeau, Lionel; Le Blastier, Sophie; Bamps, Sophie; Dewez, Monique; Vandenhaute, Jean; Hermand, Damien

    2006-01-01

    We describe a new member of the F-box family, Pof14, which forms a canonical, F-box dependent SCF (Skp1, Cullin, F-box protein) ubiquitin ligase complex. The Pof14 protein has intrinsic instability that is abolished by inactivation of its Skp1 interaction motif (the F-box), Skp1 or the proteasome, indicating that Pof14 stability is controlled by an autocatalytic mechanism. Pof14 interacts with the squalene synthase Erg9, a key enzyme in ergosterol metabolism, in a membrane-bound complex that does not contain the core SCF components. pof14 transcription is induced by hydrogen peroxide and requires the Pap1 transcription factor and the Sty1 MAP kinase. Pof14 binds to and decreases Erg9 activity in vitro and a pof14 deletion strain quickly loses viability in the presence of hydrogen peroxide due to its inability to repress ergosterol synthesis. A pof14 mutant lacking the F-box and an skp1-3 ts mutant behave as wild type in the presence of oxidant showing that Pof14 function is independent of SCF. This indicates that modulation of ergosterol level plays a key role in adaptation to oxidative stress. PMID:17016471

  12. C-terminal anchoring of mid1p to membranes stabilizes cytokinetic ring position in early mitosis in fission yeast.

    PubMed

    Celton-Morizur, Séverine; Bordes, Nicole; Fraisier, Vincent; Tran, Phong T; Paoletti, Anne

    2004-12-01

    mid1p is a key factor for the central positioning of the cytokinetic ring in Schizosaccharomyces pombe. In interphase and early mitosis, mid1p forms a medial cortical band overlying the nucleus, which may represent a landmark for cytokinetic ring assembly. It compacts before anaphase into a tight ring with other cytokinetic ring components. We show here that mid1p binds to the medial cortex by at least two independent means. First, mid1p C-terminus association with the cortex requires a putative amphipathic helix adjacent to mid1p nuclear localization sequence (NLS), which is predicted to insert directly into the lipid bilayer. This association is stabilized by the polybasic NLS. mid1p mutated within the helix and the NLS forms abnormal filaments in early mitosis that are not properly anchored to the medial cortex. Misplaced rings assemble in late mitosis, indicating that mid1p C-terminus binding to membranes stabilizes cytokinetic ring position. Second, the N terminus of mid1p has the ability to associate faintly with the medial cortex and is sufficient to form tight rings. In addition, we show that mid1p oligomerizes. We propose that membrane-bound oligomers of mid1p assemble recruitment "platforms" for cytokinetic ring components at the medial cortex and stabilize the ring position during its compaction. PMID:15572668

  13. Transient Receptor Potential (TRP) and Cch1-Yam8 Channels Play Key Roles in the Regulation of Cytoplasmic Ca2+ in Fission Yeast

    PubMed Central

    Ma, Yan; Sugiura, Reiko; Koike, Atsushi; Ebina, Hidemine; Sio, Susie O.; Kuno, Takayoshi

    2011-01-01

    The regulation of cytoplasmic Ca2+ is crucial for various cellular processes. Here, we examined the cytoplasmic Ca2+ levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca2+ level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca2+ caused a dose-dependent increase in the cytoplasmic Ca2+ level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca2+ peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca2+ increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca2+ increase is abolished by the addition of the Ca2+ chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca2+ channel complex, indicating that the synergistic increase is caused by the Ca2+ influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca2+ influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the

  14. Genetic and Metabolomic Dissection of the Ergothioneine and Selenoneine Biosynthetic Pathway in the Fission Yeast, S. pombe, and Construction of an Overproduction System

    PubMed Central

    Pluskal, Tomáš; Ueno, Masaru; Yanagida, Mitsuhiro

    2014-01-01

    Ergothioneine is a small, sulfur-containing metabolite (229 Da) synthesized by various species of bacteria and fungi, which can accumulate to millimolar levels in tissues or cells (e.g. erythrocytes) of higher eukaryotes. It is commonly marketed as a dietary supplement due to its proposed protective and antioxidative functions. In this study we report the genes forming the two-step ergothioneine biosynthetic pathway in the fission yeast, Schizosaccharomyces pombe. We identified the first gene, egt1+ (SPBC1604.01), by sequence homology to previously published genes from Neurospora crassa and Mycobacterium smegmatis. We showed, using metabolomic analysis, that the Δegt1 deletion mutant completely lacked ergothioneine and its precursors (trimethyl histidine/hercynine and hercynylcysteine sulfoxide). Since the second step of ergothioneine biosynthesis has not been characterized in eukaryotes, we examined four putative homologs (Nfs1/SPBC21D10.11c, SPAC11D3.10, SPCC777.03c, and SPBC660.12c) of the corresponding mycobacterial enzyme EgtE. Among deletion mutants of these genes, only one (ΔSPBC660.12c, designated Δegt2) showed a substantial decrease in ergothioneine, accompanied by accumulation of its immediate precursor, hercynylcysteine sulfoxide. Ergothioneine-deficient strains exhibited no phenotypic defects during vegetative growth or quiescence. To effectively study the role of ergothioneine, we constructed an egt1+ overexpression system by replacing its native promoter with the nmt1+ promoter, which is inducible in the absence of thiamine. We employed three versions of the nmt1 promoter with increasing strength of expression and confirmed corresponding accumulations of ergothioneine. We quantified the intracellular concentration of ergothioneine in S. pombe (0.3, 157.4, 41.6, and up to 1606.3 µM in vegetative, nitrogen-starved, glucose-starved, and egt1+-overexpressing cells, respectively) and described its gradual accumulation under long-term quiescence

  15. Fatty Acid Desaturase Mutants of Yeast: Growth Requirements and Electron Spin Resonance Spin-Label Distribution

    PubMed Central

    Wisnieski, Bernadine J.; Kiyomoto, Richard K.

    1972-01-01

    Two respiratory-sufficient and one respiratory-deficient (nuclear petite) strains of yeast Δ9-desaturase mutants were analyzed to determine which fatty acids would serve as replacements for the naturally occurring fatty acids, 16:1 Δ9cis and 18:1 Δ9cis. The requirement can be satisfied by several fatty acids differing in double-bond position, steric configuration, chain length, and degree of unsaturation. The features common to growth-supporting fatty acids are presented and the effects of varying the carbon source and temperature are considered. In addition, we illustrate several pitfalls encountered in membrane studies which exploit lipid-requiring organisms. Since the membrane fatty acid composition of these mutants can be modified readily, electron spin resonance spectroscopy is used to compare membranes of mutant strains enriched for different fatty acids. The lipid distribution pattern of the most commonly employed electron spin resonance spin-label, 12-nitroxide stearate, was ascertained and compared to that of 18:1 Δ9cis. PMID:4333377

  16. A yeast TCP-1-like protein is required for actin function in vivo.

    PubMed Central

    Vinh, D B; Drubin, D G

    1994-01-01

    We previously identified the ANC2 gene in a screen for mutations that enhance the defects caused by yeast actin mutations. Here we report that ANC2 is an essential gene that encodes a member of the TCP-1 family. TCP-1-related proteins are subunits of cytosolic heteromeric protein complexes referred to as chaperonins. These complexes can bind to newly synthesized actin and tubulin in vitro and can convert these proteins into an assembly-competent state. We show that anc2-1 mutants contain abnormal and disorganized actin structures, are defective in cellular morphogenesis, and are hypersensitive to the microtubule inhibitor benomyl. Furthermore, overexpression of wild-type Anc2p ameliorates defects in actin organization and cell growth caused by actin overproduction. Mutations in BIN2 and BIN3, two other genes that encode TCP-1-like proteins, also enhance the phenotypes of actin mutants. Taken together, these findings demonstrate that TCP-1-like proteins are required for actin and tubulin function in vivo. Images PMID:7916461

  17. Genome-Wide Functional Profiling Reveals Genes Required for Tolerance to Benzene Metabolites in Yeast

    PubMed Central

    North, Matthew; Tandon, Vickram J.; Thomas, Reuben; Loguinov, Alex; Gerlovina, Inna; Hubbard, Alan E.; Zhang, Luoping; Smith, Martyn T.; Vulpe, Chris D.

    2011-01-01

    Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease. PMID:21912624

  18. Constitutive turnover of histone H2A.Z at yeast promoters requires the preinitiation complex

    PubMed Central

    Tramantano, Michael; Sun, Lu; Au, Christy; Labuz, Daniel; Liu, Zhimin; Chou, Mindy; Shen, Chen; Luk, Ed

    2016-01-01

    The assembly of the preinitiation complex (PIC) occurs upstream of the +1 nucleosome which, in yeast, obstructs the transcription start site and is frequently assembled with the histone variant H2A.Z. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, conditional mutants were used to block PIC assembly. A quantitative ChIP-seq approach, which allows detection of global occupancy change, was employed to measure H2A.Z occupancy. Blocking PIC assembly resulted in promoter-specific H2A.Z accumulation, indicating that the PIC is required to evict H2A.Z. By contrast, H2A.Z eviction was unaffected upon depletion of INO80, a remodeler previously reported to displace nucleosomal H2A.Z. Robust PIC-dependent H2A.Z eviction was observed at active and infrequently transcribed genes, indicating that constitutive H2A.Z turnover is a general phenomenon. Finally, sites with strong H2A.Z turnover precisely mark transcript starts, providing a new metric for identifying cryptic and alternative sites of initiation. DOI: http://dx.doi.org/10.7554/eLife.14243.001 PMID:27438412

  19. Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes

    PubMed Central

    1996-01-01

    In order for secretion to progress, ER-derived transport vesicles must target to, and fuse with the cis-Golgi compartment. These processes have been reconstituted using highly enriched membrane fractions and partially purified soluble components. The functionally active yeast Golgi membranes that have been purified are highly enriched in the cis- Golgi marker enzymes alpha 1,6 mannosyltransferase and GDPase. Fusion of transport vesicles with these membranes requires both GTP and ATP hydrolysis, and depends on cytosolic and peripheral membrane proteins. At least two protein fractions from yeast cytosol are required for the reconstitution of ER-derived vesicle fusion. Soluble fractions prepared from temperature-sensitive mutants revealed requirements for the Ypt1p, Sec19p, Sly1p, Sec7p, and Uso1 proteins. A model for the sequential involvement of these components in the targeting and fusion reaction is proposed. PMID:8636207

  20. Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p.

    PubMed Central

    Babst, M; Sato, T K; Banta, L M; Emr, S D

    1997-01-01

    In a late-Golgi compartment of the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y (CPY) are actively sorted away from the secretory pathway and transported to the vacuole via a pre-vacuolar, endosome-like intermediate. The vacuolar protein sorting (vps) mutant vps4 accumulates vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar pre-vacuolar compartment. The VPS4 gene has been cloned and found to encode a 48 kDa protein which belongs to the protein family of AAA-type ATPases. The Vps4 protein was purified and shown to exhibit an N-ethylmaleimide-sensitive ATPase activity. A single amino acid change within the AAA motif of Vps4p yielded a protein that lacked ATPase activity and did not complement the protein sorting or morphological defects of the vps4 delta1 mutant. Indeed, when expressed at normal levels in wild-type cells, the mutant vps4 gene acted as a dominant-negative allele. The phenotypic characterization of a temperature-sensitive vps4 allele showed that the immediate consequence of loss of Vps4p function is a defect in vacuolar protein delivery. In this mutant, precursor CPY was not secreted but instead accumulated in an intracellular compartment, presumably the pre-vacuolar endosome. Electron microscopy revealed that upon temperature shift, exaggerated stacks of curved cisternal membranes (aberrant endosome) also accumulated in the vps4ts mutant. Based on these and other observations, we propose that Vps4p function is required for efficient transport out of the pre-vacuolar endosome. PMID:9155008

  1. Assessing the Mechanisms Responsible for Differences between Nitrogen Requirements of Saccharomyces cerevisiae Wine Yeasts in Alcoholic Fermentation

    PubMed Central

    Brice, Claire; Sanchez, Isabelle; Tesnière, Catherine

    2014-01-01

    Nitrogen is an essential nutrient for Saccharomyces cerevisiae wine yeasts during alcoholic fermentation, and its abundance determines the fermentation rate and duration. The capacity to ferment under conditions of nitrogen deficiency differs between yeasts. A characterization of the nitrogen requirements of a set of 23 strains revealed large differences in their fermentative performances under nitrogen deficiency, and these differences reflect the nitrogen requirements of the strains. We selected and compared two groups of strains, one with low nitrogen requirements (LNRs) and the other with high nitrogen requirements (HNRs). A comparison of various physiological traits indicated that the differences are not related to the ability to store nitrogen or the protein content. No differences in protein synthesis activity were detected between strains with different nitrogen requirements. Transcriptomic analysis revealed expression patterns specific to each of the two groups of strains, with an overexpression of stress genes in HNR strains and a stronger expression of biosynthetic genes in LNR strains. Our data suggest that differences in glycolytic flux may originate from variations in nitrogen sensing and signaling under conditions of starvation. PMID:24334661

  2. Rap1p telomere association is not required for mitotic stability of a C3TA2 telomere in yeast

    PubMed Central

    Alexander, Mary Kate; Zakian, Virginia A.

    2003-01-01

    Telomeric DNA usually consists of a repetitive sequence: C1–3A/TG1–3 in yeast, and C3TA2/T2AG3 in vertebrates. In yeast, the sequence-specific DNA- binding protein Rap1p is thought to be essential for telomere function. In a tlc1h mutant, the templating region of the telomerase RNA gene is altered so that telomerase adds the vertebrate telomere sequence instead of the yeast sequence to the chromosome end. A tlc1h strain has short but stable telomeres and no growth defect. We show here that Rap1p and the Rap1p-associated Rif2p did not bind to a telomere that contains purely vertebrate repeats, while the TG1–3 single-stranded DNA binding protein Cdc13p and the normally non-telomeric protein Tbf1p did bind this telomere. A chromosome with one entirely vertebrate-sequence telomere had a wild-type loss rate, and the telomere was maintained at a short but stable length. However, this telomere was unable to silence a telomere-adjacent URA3 gene, and the strain carrying this telomere had a severe defect in meiosis. We conclude that Rap1p localization to a C3TA2 telomere is not required for its essential mitotic functions. PMID:12660174

  3. The C-Terminal Domain of Yeast PCNA Is Required for Physical And Functional Interactions With Cdc9 DNA Ligase

    SciTech Connect

    Vijayakumar, S.; Chapados, B.R.; Schmidt, K.H.; Kolodner, R.D.; Tainer, J.A.; Tomkinson, A.E.

    2007-07-13

    There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the {beta}-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the coordinated, sequential action of these enzymes.

  4. Recognition of yeast by murine macrophages requires mannan but not glucan.

    PubMed

    Keppler-Ross, Sabine; Douglas, Lois; Konopka, James B; Dean, Neta

    2010-11-01

    The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake. PMID:20833894

  5. The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast.

    PubMed Central

    Peculis, B A; Greer, C L

    1998-01-01

    Accurate and efficient processing of pre-rRNA is critical to the accumulation of mature functional ribosomal subunits for maintenance of cell growth. Processing requires numerous factors which act in trans as well as RNA sequence/ structural elements which function in cis. To examine the latter, we have used directed mutagenesis and expression of mutated pre-rRNAs in yeast. Specifically, we tested requirements for formation of an ITS2-proximal stem on processing, a structure formed by an interaction between sequences corresponding to the 3' end of 5.8S rRNA and the 5' end of 25S. Pre-rRNA processing is inhibited in templates encoding mutations that prevent the formation of the ITS2-proximal stem. Compensatory, double mutations, which alter the sequence of this region but restore the structure of the stem, also restore processing, although at lower efficiency. This reduction in efficiency is reflected in decreased levels of mature 5.8S and 25S rRNA and increased levels of 35S pre-rRNA and certain processing intermediates. This phenotype is reminiscent of the biochemical depletion of U8 snoRNA in vertebrates for which the ITS2-proximal stem has been proposed as a potential site for interaction with U8 RNP. Thus, formation of the ITS2-proximal stem may be a requirement common to yeast and vertebrate pre-rRNA processing. PMID:9848657

  6. The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins.

    PubMed Central

    Nothwehr, S F; Bryant, N J; Stevens, T H

    1996-01-01

    Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its PMID:8649377

  7. Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo

    PubMed Central

    Kuo, Min-Hao; Zhou, Jianxin; Jambeck, Per; Churchill, Mair E.A.; Allis, C. David

    1998-01-01

    Gcn5p is a transcriptional coactivator required for correct expression of various genes in yeast. Several transcriptional regulators, including Gcn5p, possess intrinsic histone acetyltransferase (HAT) activity in vitro. However, whether the HAT activity of any of these proteins is required for gene activation remains unclear. Here, we demonstrate that the HAT activity of Gcn5p is critical for transcriptional activation of target genes in vivo. Core histones are hyperacetylated in cells overproducing functional Gcn5p, and promoters of Gcn5p-regulated genes are associated with hyperacetylated histones upon activation by low-copy Gcn5p. Point mutations within the Gcn5p catalytic domain abolish both promoter-directed histone acetylation and Gcn5p-mediated transcriptional activation. These data provide the first in vivo evidence that promoter-specific histone acetylation, catalyzed by functional Gcn5p, plays a critical role in gene activation. PMID:9499399

  8. Papulacandin B resistance in budding and fission yeasts: isolation and characterization of a gene involved in (1,3)beta-D-glucan synthesis in Saccharomyces cerevisiae.

    PubMed Central

    Castro, C; Ribas, J C; Valdivieso, M H; Varona, R; del Rey, F; Duran, A

    1995-01-01

    Papulacandin B, an antifungal agent that interferes with the synthesis of yeast cell wall (1,3)beta-D-glucan, was used to isolate resistant mutants in Schizosaccharomyces pombe and Saccharomyces cerevisiae. The resistance to papulacandin B always segregated as a recessive character that defines a single complementation group in both yeasts (pbr1+ and PBR1, respectively). Determination of several kinetic parameters of (1,3)beta-D-glucan synthase activity revealed no differences between S. pombe wild-type and pbr1 mutant strains except in the 50% inhibitory concentration for papulacandin B of the synthases (about a 50-fold increase in mutant activity). Inactivation of the synthase activity of both yeasts after in vivo treatment with the antifungal agent showed that mutant synthases were more resistant than the corresponding wild-type ones. Detergent dissociation of the S. pombe synthase into soluble and particulate fractions and subsequent reconstitution indicated that the resistance character of pbr1 mutants resides in the particulate fraction of the enzyme. Cloning and sequencing of PBR1 from S. cerevisiae revealed a gene identical to others recently reported (FKS1, ETG1, CWH53, and CND1). Its disruption leads to reduced levels of both (1,3)beta-D-glucan synthase activity and the alkali-insoluble cell wall fraction. Transformants containing the PBR1 gene reverse the defect in (1,3)beta-D-glucan synthase. It is concluded that Pbr1p is probably part of the (1,3)beta-D-glucan synthase complex. PMID:7592316

  9. A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth.

    PubMed Central

    Muller, E G

    1996-01-01

    A glutathione reductase null mutant of Saccharomyces cerevisiae was isolated in a synthetic lethal genetic screen for mutations which confer a requirement for thioredoxin. Yeast mutants that lack glutathione reductase (glr1 delta) accumulate high levels of oxidized glutathione and have a twofold increase in total glutathione. The disulfide form of glutathione increases 200-fold and represents 63% of the total glutathione in a glr1 delta mutant compared with only 6% in wild type. High levels of oxidized glutathione are also observed in a trx1 delta, trx2 delta double mutant (22% of total), in a glr1 delta, trx1 delta double mutant (71% of total), and in a glr1 delta, trx2 delta double mutant (69% of total). Despite the exceptionally high ratio of oxidized/reduced glutathione, the glr1 delta mutant grows with a normal cell cycle. However, either one of the two thioredoxins is essential for growth. Cells lacking both thioredoxins and glutathione reductase are not viable under aerobic conditions and grow poorly anaerobically. In addition, the glr1 delta mutant shows increased sensitivity to the thiol oxidant diamide. The sensitivity to diamide was suppressed by deletion of the TRX2 gene. The genetic analysis of thioredoxin and glutathione reductase in yeast runs counter to previous studies in Escherichia coli and for the first time links thioredoxin with the redox state of glutathione in vivo. Images PMID:8930901

  10. Phosphorylation of Elp1 by Hrr25 Is Required for Elongator-Dependent tRNA Modification in Yeast

    PubMed Central

    Abdel-Fattah, Wael; Jablonowski, Daniel; Di Santo, Rachael; Thüring, Kathrin L.; Scheidt, Viktor; Hammermeister, Alexander; ten Have, Sara; Helm, Mark; Schaffrath, Raffael; Stark, Michael J. R.

    2015-01-01

    Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase. PMID:25569479

  11. Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast.

    PubMed

    Gurunathan, Sangiliyandi; David, Doris; Gerst, Jeffrey E

    2002-02-15

    Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis. PMID:11847108

  12. Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation

    PubMed Central

    Colinet, Anne-Sophie; Sengottaiyan, Palanivelu; Deschamps, Antoine; Colsoul, Marie-Lise; Thines, Louise; Demaegd, Didier; Duchêne, Marie-Clémence; Foulquier, François; Hols, Pascal; Morsomme, Pierre

    2016-01-01

    Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca2+ exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca2+ uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn2+ to the cells. PMID:27075443

  13. Exonucleolysis is required for nuclear mRNA quality control in yeast THO mutants.

    PubMed

    Assenholt, Jannie; Mouaikel, John; Andersen, Kasper R; Brodersen, Ditlev E; Libri, Domenico; Jensen, Torben Heick

    2008-11-01

    Production of aberrant messenger ribonucleoprotein particles (mRNPs) is subject to quality control (QC). In yeast strains carrying mutations of the THO complex, transcription induction triggers a number of interconnected QC phenotypes: (1) rapid degradation of several mRNAs; (2) retention of a fraction of THO-dependent mRNAs in transcription site-associated foci; and (3) formation of a high molecular weight DNA/protein complex in the 3'-ends of THO target genes. Here, we demonstrate that the 3'-5' exonucleolytic domain of the nuclear exosome factor Rrp6p is necessary for establishing all QC phenotypes associated with THO mutations. The N terminus of Rrp6p is also important presumably through its binding to the Rrp6p co-factor Rrp47p. Interestingly, the 3'-5' exonucleolytic activity of Dis3p, the only other active exonuclease of the nuclear exosome, can also contribute to RNA QC in THO mutants, while other nuclear 3'-5' exonucleases cannot. Our data show that exonucleolytic attack by the nuclear exosome is needed both for provoking mRNP QC and for its ensuing elimination of faulty RNA. PMID:18824516

  14. Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol

    PubMed Central

    van Rossum, Harmen M.; Kozak, Barbara U.; Niemeijer, Matthijs S.; Dykstra, James C.; Luttik, Marijke A. H.; van Maris, Antonius J. A.

    2016-01-01

    ABSTRACT In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle. PMID:27143389

  15. Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression.

    PubMed Central

    Kraakman, L S; Winderickx, J; Thevelein, J M; De Winde, J H

    1999-01-01

    In baker's yeast (Saccharomyces cerevisiae) the hexokinases PI (Hxk1) and PII (Hxk2) are required for triggering of the activation of the Ras-cAMP pathway and catabolite repression. Specifically, Hxk2 is essential for the establishment of glucose repression, whereas either Hxk1 or Hxk2 can sustain fructose repression. Previous studies have suggested that the extent of glucose repression is inversely correlated with hexokinase catalytic activity and hence with an adequate elevation of intracellular sugar phosphate levels. However, several lines of evidence indicate that glucose 6-phosphate is not the trigger of catabolite repression in yeast. In the present study we employed site-directed mutagenesis of amino acids important for the binding of sugar and ATP, for efficient phosphoryl transfer and for the closure of the substrate-binding cleft, to obtain an insight into the structural requirements of Hxk2 for sugar-induced signalling. We show that the ATP-binding Lys-111 is not essential for catalysis in vivo or for signal triggering. Substitution of the catalytic-centre Asp-211 caused loss of catalytic activity, but high-affinity sugar binding was retained. However, this was not sufficient to cause cAMP activation nor catabolite repression. Mutation of Ser-158 abrogated glucose-induced, but not fructose-induced, repression. Moreover, 2-deoxyglucose sustained repression despite an extremely low catalytic activity. We conclude that the establishment of catabolite repression is dependent on the onset of the phosphoryl transfer reaction on hexokinase and is probably related to the stable formation of a transition intermediate and concomitant conformational changes within the enzyme. In contrast, the role of Hxk2 in Ras-cAMP activation seems to be directly connected to its catalytic function. The implications of this model are discussed. PMID:10493925

  16. Targeting of Chitin Synthase 3 to Polarized Growth Sites in Yeast Requires Chs5p and Myo2p

    PubMed Central

    Santos, Beatriz; Snyder, Michael

    1997-01-01

    Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycledependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p. PMID:9008706

  17. Regulation of lipid metabolism: a tale of two yeasts

    PubMed Central

    Raychaudhuri, Sumana; Young, Barry P.; Espenshade, Peter J.; Loewen, Christopher J.R.

    2015-01-01

    Eukaryotic cells synthesize multiple classes of lipids by distinct metabolic pathways in order to generate membranes with optimal physical and chemical properties. As a result, complex regulatory networks are required in all organisms to maintain lipid and membrane homeostasis as well as to rapidly and efficiently respond to cellular stress. The unicellular nature of yeast makes it particularly vulnerable to environmental stress and yeast has evolved elaborate signaling pathways to maintain lipid homeostasis. In this article we highlight the recent advances that have been made using the budding and fission yeasts and we discuss potential roles for the unfolded protein response (UPR) and the SREBP-Scap pathways in coordinate regulation of multiple lipid classes. PMID:22694927

  18. Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast

    PubMed Central

    Cisneros-Barroso, Eugenia; Yance-Chávez, Tula; Kito, Ayako; Sugiura, Reiko; Gómez-Hierro, Alba; Giménez-Zaragoza, David; Aligue, Rosa

    2014-01-01

    Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calcium-activated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca2+/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca2+. Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca2+. Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca2+. Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca2+, due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe. PMID:25081204

  19. Functional analysis of the fission yeast Prp4 protein kinase involved in pre-mRNA splicing and isolation of a putative mammalian homologue.

    PubMed Central

    Gross, T; Lützelberger, M; Weigmann, H; Klingenhoff, A; Shenoy, S; Käufer, N F

    1997-01-01

    The prp4 gene of Schizosaccharomyces pombe encodes a protein kinase. A physiological substrate is not yet known. A mutational analysis of prp4 revealed that the protein consists of a short N-terminal domain, containing several essential motifs, which is followed by the kinase catalytic domain comprising the C-terminus of the protein. Overexpression of N-terminal mutations disturbs mitosis and produces elongated cells, Using a PCR approach, we isolated a putative homologue of Prp4 from human and mouse cells. The mammalian kinase domain is 53% identical to the kinase domain of Prp4. The short N-terminal domains share <20% identical amino acids, but contain conserved motifs. A fusion protein consisting of the N-terminal region from S. pombe followed by the mammalian kinase domain complements a temperature-sensitive prp4 mutation of S. pombe. Prp4 and the recombinant yeast/mouse protein kinase phosphorylate the human SR splicing factor ASF/SF2 in vitro in its RS domain. PMID:9102632

  20. Requirements and potential development pathways for fission energy supply infrastructures of the 21st century - a systems viewpoint.

    SciTech Connect

    Wade, D. C.

    1999-06-14

    Using an energy supply systems approach, we envision attributes and characteristic needs of a future global fission-based energy supply infrastructure, enumerate potential pathways for meeting those needs, and identify the underlying enabling science and technology developments for R and D efforts to meet the needs.

  1. Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast.

    PubMed

    Chatterjee, Debashree; Sanchez, Ana M; Goldgur, Yehuda; Shuman, Stewart; Schwer, Beate

    2016-07-01

    Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt-pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m(6)A-modified RNA. PMID:27165520

  2. Swa2, the yeast homolog of mammalian auxilin, is specifically required for the propagation of the prion variant [URE 3‐1

    PubMed Central

    Troisi, Elizabeth M.; Rockman, Michael E.; Nguyen, Phil P.; Oliver, Emily E.

    2015-01-01

    Summary Yeast prions require a core set of chaperone proteins including Sis1, Hsp70 and Hsp104 to generate new amyloid templates for stable propagation, yet emerging studies indicate that propagation of some prions requires additional chaperone activities, demonstrating chaperone specificity beyond the common amyloid requirements. To comprehensively assess such prion‐specific requirements for the propagation of the [URE 3] prion variant [URE 3‐1], we screened 12 yeast cytosolic J‐proteins, and here we report a novel role for the J‐protein Swa2/Aux1. Swa2 is the sole yeast homolog of the mammalian protein auxilin, which, like Swa2, functions in vesicle‐mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. We found that, in addition to Sis1, [URE 3‐1] is specifically dependent upon Swa2, but not on any of the 11 other J‐proteins. Further, we show that [URE 3‐1] propagation requires both a functional J‐domain and the tetratricopeptide repeat (TPR) domain, but surprisingly does not require Swa2‐clathrin binding. Because the J‐domain of Swa2 can be replaced with the J‐domains of other proteins, our data strongly suggest that prion‐chaperone specificity arises from the Swa2 TPR domain and supports a model where Swa2 acts through Hsp70, most likely to provide additional access points for Hsp104 to promote prion template generation. PMID:26031938

  3. Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA.

    PubMed Central

    Alexandrov, Andrei; Martzen, Mark R; Phizicky, Eric M

    2002-01-01

    7-methylguanosine (m7G) modification of tRNA occurs widely in eukaryotes and bacteria, is nearly always found at position 46, and is one of the few modifications that confers a positive charge to the base. Screening of a Saccharomyces cerevisiae genomic library of purified GST-ORF fusion proteins reveals two previously uncharacterized proteins that copurify with m7G methyltransferase activity on pre-tRNA(Phe). ORF YDL201w encodes Trm8, a protein that is highly conserved in prokaryotes and eukaryotes and that contains an S-adenosylmethionine binding domain. ORF YDR165w encodes Trm82, a less highly conserved protein containing putative WD40 repeats, which are often implicated in macromolecular interactions. Neither protein has significant sequence similarity to yeast Abd1, which catalyzes m7G modification of the 5' cap of mRNA, other than the methyltransferase motif shared by Trm8 and Abd1. Several lines of evidence indicate that both Trm8 and Trm82 proteins are required for tRNA m7G-methyltransferase activity: Extracts derived from strains lacking either gene have undetectable m7G methyltransferase activity, RNA from strains lacking either gene have much reduced m7G, and coexpression of both proteins is required to overproduce activity. Aniline cleavage mapping shows that Trm8/Trm82 proteins modify pre-tRNAPhe at G46, the site that is modified in vivo. Trm8 and Trm82 proteins form a complex, as affinity purification of Trm8 protein causes copurification of Trm82 protein in approximate equimolar yield. This functional two-protein family appears to be retained in eukaryotes, as expression of both corresponding human proteins, METTL1 and WDR4, is required for m7G-methyltransferase activity. PMID:12403464

  4. Spontaneous Fission

    DOE R&D Accomplishments Database

    Segre, Emilio

    1950-11-22

    The first attempt to discover spontaneous fission in uranium was made by [Willard] Libby, who, however, failed to detect it on account of the smallness of effect. In 1940, [K. A.] Petrzhak and [G. N.] Flerov, using more sensitive methods, discovered spontaneous fission in uranium and gave some rough estimates of the spontaneous fission decay constant of this substance. Subsequently, extensive experimental work on the subject has been performed by several investigators and will be quoted in the various sections. [N.] Bohr and [A.] Wheeler have given a theory of the effect based on the usual ideas of penetration of potential barriers. On this project spontaneous fission has been studied for the past several years in an effort to obtain a complete picture of the phenomenon. For this purpose the spontaneous fission decay constants {lambda} have been measured for separated isotopes of the heavy elements wherever possible. Moreover, the number {nu} of neutrons emitted per fission has been measured wherever feasible, and other characteristics of the spontaneous fission process have been studied. This report summarizes the spontaneous fission work done at Los Alamos up to January 1, 1945. A chronological record of the work is contained in the Los Alamos monthly reports.

  5. Pexophagy in yeasts.

    PubMed

    Oku, Masahide; Sakai, Yasuyoshi

    2016-05-01

    Pexophagy, selective degradation of peroxisomes via autophagy, is the main system for reducing organelle abundance. Elucidation of the molecular machinery of pexophagy has been pioneered in studies of the budding yeast Saccharomyces cerevisiae and the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha. Recent analyses using these yeasts have elucidated the molecular machineries of pexophagy, especially in terms of the interactions and modifications of the so-called adaptor proteins required for guiding autophagic membrane biogenesis on the organelle surface. Based on the recent findings, functional relevance of pexophagy and another autophagic pathway, mitophagy (selective autophagy of mitochondria), is discussed. We also discuss the physiological importance of pexophagy in these yeast systems. PMID:26409485

  6. Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

    PubMed Central

    Lauder, S; Bankmann, M; Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1996-01-01

    Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected. PMID:8943333

  7. STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo.

    PubMed Central

    Zufferey, R; Knauer, R; Burda, P; Stagljar, I; te Heesen, S; Lehle, L; Aebi, M

    1995-01-01

    N-linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid-linked core-oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid-linked core-oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N-linked glycosylation. Besides novel mutants deficient in the assembly of the lipid-linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3-3 cells very little glycosyl transfer occurs from incomplete lipid-linked oligosaccharide, whereas the transfer of full-length Glc3Man9GlcNAc2 is hardly affected as compared with wild-type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex. Images PMID:7588624

  8. Membrane Fission Reactions of the Mammalian ESCRT Pathway

    PubMed Central

    McCullough, John; Colf, Leremy A.; Sundquist, Wesley I.

    2014-01-01

    The endosomal sorting complexes required for transport (ESCRT) pathway was initially defined in yeast genetic screens that identified the factors necessary to sort membrane proteins into intraluminal endosomal vesicles. Subsequent studies have revealed that the mammalian ESCRT pathway also functions in a series of other key cellular processes, including formation of extracellular microvesicles, enveloped virus budding, and the abscission stage of cytokinesis. The core ESCRT machinery comprises Bro1 family proteins and ESCRT-I, ESCRT-II, ESCRT-III, and VPS4. Site-specific adaptors recruit these soluble factors to assemble on different cellular membranes, where they carry out membrane fission reactions. ESCRT-III proteins form filaments that draw membranes together from the cytoplasmic face, and mechanistic models have been advanced to explain how ESCRT-III filaments and the VPS4 ATPase can work together to catalyze membrane fission. PMID:23527693

  9. Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone.

    PubMed Central

    Cyert, M S; Thorner, J

    1992-01-01

    By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo. Images PMID:1321337

  10. Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore.

    PubMed

    Measday, Vivien; Hailey, Dale W; Pot, Isabelle; Givan, Scott A; Hyland, Katherine M; Cagney, Gerard; Fields, Stan; Davis, Trisha N; Hieter, Philip

    2002-01-01

    The budding yeast kinetochore is composed of an inner and outer protein complex, which binds to centromere (CEN) DNA and attaches to microtubules. We performed a genetic synthetic dosage lethality screen to identify novel kinetochore proteins in a collection of chromosome transmission fidelity mutants. Our screen identified several new kinetochore-related proteins including YLR381Wp/Ctf3p, which is a member of a conserved family of centromere-binding proteins. Ctf3p interacts with Mcm22p, Mcm16p, and the outer kinetochore protein Ctf19p. We used chromatin immunoprecipitation to demonstrate that Ctf3p, Mcm22p, and Mcm16p bind to CEN DNA in a Ctf19p-dependent manner. In addition, Ctf3p, Mcm22p, and Mcm16p have a localization pattern similar to other kinetochore proteins. The fission yeast Ctf3p homolog, Mis6, is required for loading of a CENP-A centromere specific histone, Cnp1, onto centromere DNA. We find however that Ctf3p is not required for loading of the budding yeast CENP-A homolog, Cse4p, onto CEN DNA. In contrast, Ctf3p and Ctf19p fail to bind properly to the centromere in a cse4-1 mutant strain. We conclude that the requirements for CENP-A loading onto centromere DNA differ in fission versus budding yeast. PMID:11782448

  11. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  12. Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis

    PubMed Central

    Urbanek, Agnieszka N.; Allwood, Ellen G.; Smith, Adam P.; Booth, Wesley I.; Ayscough, Kathryn R.

    2015-01-01

    During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis. PMID:26312755

  13. Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation*

    PubMed Central

    Fernández-Pevida, Antonio; Rodríguez-Galán, Olga; Díaz-Quintana, Antonio; Kressler, Dieter; de la Cruz, Jesús

    2012-01-01

    Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process. PMID:22995916

  14. Fission Yeast SCYL1/2 Homologue Ppk32: A Novel Regulator of TOR Signalling That Governs Survival during Brefeldin A Induced Stress to Protein Trafficking.

    PubMed

    Kowalczyk, Katarzyna M; Petersen, Janni

    2016-05-01

    Target of Rapamycin (TOR) signalling allows eukaryotic cells to adjust cell growth in response to changes in their nutritional and environmental context. The two distinct TOR complexes (TORC1/2) localise to the cell's internal membrane compartments; the endoplasmic reticulum (ER), Golgi apparatus and lysosomes/vacuoles. Here, we show that Ppk32, a SCYL family pseudo-kinase, is a novel regulator of TOR signalling. The absence of ppk32 expression confers resistance to TOR inhibition. Ppk32 inhibition of TORC1 is critical for cell survival following Brefeldin A (BFA) induced stress. Treatment of wild type cells with either the TORC1 specific inhibitor rapamycin or the general TOR inhibitor Torin1 confirmed that a reduction in TORC1 activity promoted recovery from BFA induced stress. Phosphorylation of Ppk32 on two residues that are conserved within the SCYL pseudo-kinase family are required for this TOR inhibition. Phosphorylation on these sites controls Ppk32 protein levels and sensitivity to BFA. BFA induced ER stress does not account for the response to BFA that we report here, however BFA is also known to induce Golgi stress and impair traffic to lysosomes. In summary, Ppk32 reduce TOR signalling in response to BFA induced stress to support cell survival. PMID:27191590

  15. Fission Yeast SCYL1/2 Homologue Ppk32: A Novel Regulator of TOR Signalling That Governs Survival during Brefeldin A Induced Stress to Protein Trafficking

    PubMed Central

    Petersen, Janni

    2016-01-01

    Target of Rapamycin (TOR) signalling allows eukaryotic cells to adjust cell growth in response to changes in their nutritional and environmental context. The two distinct TOR complexes (TORC1/2) localise to the cell’s internal membrane compartments; the endoplasmic reticulum (ER), Golgi apparatus and lysosomes/vacuoles. Here, we show that Ppk32, a SCYL family pseudo-kinase, is a novel regulator of TOR signalling. The absence of ppk32 expression confers resistance to TOR inhibition. Ppk32 inhibition of TORC1 is critical for cell survival following Brefeldin A (BFA) induced stress. Treatment of wild type cells with either the TORC1 specific inhibitor rapamycin or the general TOR inhibitor Torin1 confirmed that a reduction in TORC1 activity promoted recovery from BFA induced stress. Phosphorylation of Ppk32 on two residues that are conserved within the SCYL pseudo-kinase family are required for this TOR inhibition. Phosphorylation on these sites controls Ppk32 protein levels and sensitivity to BFA. BFA induced ER stress does not account for the response to BFA that we report here, however BFA is also known to induce Golgi stress and impair traffic to lysosomes. In summary, Ppk32 reduce TOR signalling in response to BFA induced stress to support cell survival. PMID:27191590

  16. Xlf1 is required for DNA repair by nonhomologous end joining in Schizosaccharomyces pombe.

    PubMed

    Cavero, Santiago; Chahwan, Charly; Russell, Paul

    2007-02-01

    The accurate repair of DNA double-strand breaks is essential for cell survival and maintenance of genome integrity. Here we describe xlf1+, a gene in the fission yeast Schizosaccharomyces pombe that is required for repair of double-strand breaks by nonhomologous end joining during G1 phase of the cell cycle. Xlf1 is the ortholog of budding yeast Nej1 and human XLF/Cernunnos proteins. PMID:17151234

  17. Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1 Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

    PubMed Central

    Neely, Lori A.; Hoffman, Charles S.

    2000-01-01

    A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site. PMID:10938120

  18. Rif1: A Conserved Regulator of DNA Replication and Repair Hijacked by Telomeres in Yeasts

    PubMed Central

    Mattarocci, Stefano; Hafner, Lukas; Lezaja, Aleksandra; Shyian, Maksym; Shore, David

    2016-01-01

    Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoans, reveal that Rif1 also functions more globally, both in the temporal control of DNA replication and in DNA repair. Rif1 proteins are large and characterized by N-terminal HEAT repeats, predicted to form an elongated alpha-helical structure. In addition, all Rif1 homologs contain two short motifs, abbreviated RVxF/SILK, that are implicated in recruitment of the PP1 (yeast Glc7) phosphatase. In yeasts the RVxF/SILK domains have been shown to play a role in control of DNA replication initiation, at least in part through targeted de-phosphorylation of proteins in the pre-Replication Complex. In human cells Rif1 is recruited to DNA double-strand breaks through an interaction with 53BP1 where it counteracts DNA resection, thus promoting repair by non-homologous end-joining. This function requires the N-terminal HEAT repeat-containing domain. Interestingly, this domain is also implicated in DNA end protection at un-capped telomeres in yeast. We conclude by discussing the deployment of Rif1 at telomeres in yeasts from both an evolutionary perspective and in light of its recently discovered global functions. PMID:27066066

  19. Yeast Infections

    MedlinePlus

    ... antibiotics, it can multiply and cause an infection. Yeast infections affect different parts of the body in different ways: Thrush is a yeast infection that causes white patches in your mouth Candida ...

  20. Comparative Functional Genomic Analysis Identifies Distinct and Overlapping Sets of Genes Required for Resistance to Monomethylarsonous Acid (MMAIII) and Arsenite (AsIII) in Yeast

    PubMed Central

    Jo, William J.; Loguinov, Alex; Wintz, Henri; Chang, Michelle; Smith, Allan H.; Kalman, Dave; Zhang, Luoping; Smith, Martyn T.; Vulpe, Chris D.

    2009-01-01

    Arsenic is a human toxin and carcinogen commonly found as a contaminant in drinking water. Arsenite (AsIII) is the most toxic inorganic form, but recent evidence indicates that the metabolite monomethylarsonous acid (MMAIII) is even more toxic. We have used a chemical genomics approach to identify the genes that modulate the cellular toxicity of MMAIII and AsIII in the yeast Saccharomyces cerevisiae. Functional profiling using homozygous deletion mutants provided evidence of the requirement of highly conserved biological processes in the response against both arsenicals including tubulin folding, DNA double-strand break repair, and chromatin modification. At the equitoxic doses of 150μM MMAIII and 300μM AsIII, genes related to glutathione metabolism were essential only for resistance to the former, suggesting a higher potency of MMAIII to disrupt glutathione metabolism than AsIII. Treatments with MMAIII induced a significant increase in glutathione levels in the wild-type strain, which correlated to the requirement of genes from the sulfur and methionine metabolic pathways and was consistent with the induction of oxidative stress. Based on the relative sensitivity of deletion strains deficient in GSH metabolism and tubulin folding processes, oxidative stress appeared to be the primary mechanism of MMAIII toxicity whereas secondary to tubulin disruption in the case of AsIII. Many of the identified yeast genes have orthologs in humans that could potentially modulate arsenic toxicity in a similar manner as their yeast counterparts. PMID:19635755

  1. Singlet exciton fission photovoltaics.

    PubMed

    Lee, Jiye; Jadhav, Priya; Reusswig, Philip D; Yost, Shane R; Thompson, Nicholas J; Congreve, Daniel N; Hontz, Eric; Van Voorhis, Troy; Baldo, Marc A

    2013-06-18

    Singlet exciton fission, a process that generates two excitons from a single photon, is perhaps the most efficient of the various multiexciton-generation processes studied to date, offering the potential to increase the efficiency of solar devices. But its unique characteristic, splitting a photogenerated singlet exciton into two dark triplet states, means that the empty absorption region between the singlet and triplet excitons must be filled by adding another material that captures low-energy photons. This has required the development of specialized device architectures. In this Account, we review work to develop devices that harness the theoretical benefits of singlet exciton fission. First, we discuss singlet fission in the archetypal material, pentacene. Pentacene-based photovoltaic devices typically show high external and internal quantum efficiencies. They have enabled researchers to characterize fission, including yield and the impact of competing loss processes, within functional devices. We review in situ probes of singlet fission that modulate the photocurrent using a magnetic field. We also summarize studies of the dissociation of triplet excitons into charge at the pentacene-buckyball (C60) donor-acceptor interface. Multiple independent measurements confirm that pentacene triplet excitons can dissociate at the C60 interface despite their relatively low energy. Because triplet excitons produced by singlet fission each have no more than half the energy of the original photoexcitation, they limit the potential open circuit voltage within a solar cell. Thus, if singlet fission is to increase the overall efficiency of a solar cell and not just double the photocurrent at the cost of halving the voltage, it is necessary to also harvest photons in the absorption gap between the singlet and triplet energies of the singlet fission material. We review two device architectures that attempt this using long-wavelength materials: a three-layer structure that uses

  2. Genes Required for Survival in Microgravity Revealed by Genome-Wide Yeast Deletion Collections Cultured during Spaceflight

    PubMed Central

    Nislow, Corey; Lee, Anna Y.; Allen, Patricia L.; Giaever, Guri; Smith, Andrew; Gebbia, Marinella; Stodieck, Louis S.; Hammond, Jeffrey S.; Birdsall, Holly H.; Hammond, Timothy G.

    2015-01-01

    Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness. PMID:25667933

  3. Genes required for survival in microgravity revealed by genome-wide yeast deletion collections cultured during spaceflight.

    PubMed

    Nislow, Corey; Lee, Anna Y; Allen, Patricia L; Giaever, Guri; Smith, Andrew; Gebbia, Marinella; Stodieck, Louis S; Hammond, Jeffrey S; Birdsall, Holly H; Hammond, Timothy G

    2015-01-01

    Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness. PMID:25667933

  4. Zebrafish ("Danio rerio") endomembrane antiporter similar to a yeast cation/H(+) transporter is required for neural crest development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CAtion/H (+) eXchangers (CAXs) are integral membrane proteins that transport Ca (2+) or other cations by exchange with protons. While several yeast and plant CAX proteins have been characterized, no functional analysis of a vertebrate CAX homologue has yet been reported. In this study, we further ch...

  5. A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast.

    PubMed Central

    Burd, C G; Peterson, M; Cowles, C R; Emr, S D

    1997-01-01

    The vacuolar protein-sorting (VPS) pathway of Saccharomyces cerevisiae mediates localization of proteins from the trans-Golgi to the vacuole via a prevacuolar endosome compartment. Mutations in class D vacuolar protein-sorting (vps) genes affect vesicle-mediated Golgi-to-endosome transport and result in secretion of vacuolar proteins. Temperature-sensitive-for-function (tsf) and dominant negative mutations in PEP12, encoding a putative SNARE vesicle receptor on the endosome, and tsf mutations in VAC1, a gene implicated in vacuole inheritance and vacuolar protein sorting, were constructed and used to demonstrate that Pep12p and Vac1p are components of the VPS pathway. The sequence of Vac1p contains two putative zinc-binding RING motifs, a zinc finger motif, and a coiled-coil motif. Site-directed mutations in the carboxyl-terminal RING motif strongly affected vacuolar protein sorting. Vac1p was found to be tightly associated with membranes as a monomer and in a large SDS-resistant complex. By using Pep12p affinity chromatography, we found that Vac1p, Vps45p (SEC1 family member), and Sec18p (yeast N-ethyl maleimide-sensitive factor, NSF) bind Pep12p. Consistent with a functional role for this complex in vacuolar protein sorting, double pep12tsfvac1tsf and pep12tsf vps45tsf mutants exhibited synthetic Vps- phenotypes, the tsf phenotype of the vac1tsf mutant was rescued by overexpression of VPS45 or PEP12, overexpression of a dominant pep12 allele in a sec18-1 strain resulted in a severe synthetic growth defect that was rescued by deletion of PEP12 or VAC1, and subcellular fractionation of vac1 delta cells revealed a striking change in the fractionation of Pep12p and Vps21p, a rab family GTPase required for vacuolar protein sorting. The functions of Pep12p, Vps45p, and Vps21p indicate that key aspects of Golgi-to-endosome trafficking are similar to other vesicle-mediated transport steps, although the role of Vac1p suggests that there are also novel components of the VPS

  6. The budding yeast amphiphysin complex is required for contractile actin ring (CAR) assembly and post-contraction GEF-independent accumulation of Rho1-GTP.

    PubMed

    Cundell, Michael John; Price, Clive

    2014-01-01

    The late events of the budding yeast cell division cycle, cytokinesis and cell separation, require the assembly of a contractile actomyosin ring (CAR), primary and secondary septum formation followed by enzymatic degradation of the primary septum. Here we present evidence that demonstrates a role for the budding yeast amphiphysin complex, a heterodimer comprising Rvs167 and Rvs161, in CAR assembly and cell separation. The iqg1-1 allele is synthetically lethal with both rvs167 and rvs161 null mutations. We show that both Iqg1 and the amphiphysin complex are required for CAR assembly in early anaphase but cells are able to complete assembly in late anaphase when these activities are, respectively, either compromised or absent. Amphiphysin dependent CAR assembly is dependent upon the Rvs167 SH3 domain, but this function is insufficient to explain the observed synthetic lethality. Dosage suppression of the iqg1-1 allele demonstrates that endocytosis is required for the default cell separation pathway in the absence of CAR contraction but is unlikely to be required to maintain viability. The amphiphysin complex is required for normal, post-mitotic, localization of Chs3 and the Rho1 GEF, Rom2, which are responsible for secondary septum deposition and the accumulation of GTP bound Rho1 at the bud neck. It is concluded that a failure of polarity establishment in the absence of CAR contraction and amphiphysin function leads to loss of viability as a result of the consequent cell separation defect. PMID:24874185

  7. Resveratrol Modulates Mitochondria Dynamics in Replicative Senescent Yeast Cells

    PubMed Central

    Wang, Yu-Han; Chang, Ko-Wei; Chen, Ying-Chieh; Chang, Chuang-Rung

    2014-01-01

    Mitochondria form a reticulum network dynamically fuse and divide in the cell. The balance between mitochondria fusion and fission is correlated to the shape, activity and integrity of these pivotal organelles. Resveratrol is a polyphenol antioxidant that can extend life span in yeast and worm. This study examined mitochondria dynamics in replicative senescent yeast cells as well as the effects of resveratrol on mitochondria fusion and fission. Collecting cells by biotin-streptavidin sorting method revealed that majority of the replicative senescent cells bear fragmented mitochondrial network, indicating mitochondria dynamics favors fission. Resveratrol treatment resulted in a reduction in the ratio of senescent yeast cells with fragmented mitochondria. The readjustment of mitochondria dynamics induced by resveratrol likely derives from altered expression profiles of fusion and fission genes. Our results demonstrate that resveratrol serves not only as an antioxidant, but also a compound that can mitigate mitochondria fragmentation in replicative senescent yeast cells. PMID:25098588

  8. Crossing over during Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in budding yeast.

    PubMed Central

    Zalevsky, J; MacQueen, A J; Duffy, J B; Kemphues, K J; Villeneuve, A M

    1999-01-01

    Formation of crossovers between homologous chromosomes during Caenorhabditis elegans meiosis requires the him-14 gene. Loss of him-14 function severely reduces crossing over, resulting in lack of chiasmata between homologs and consequent missegregation. Cytological analysis showing that homologs are paired and aligned in him-14 pachytene nuclei, together with temperature-shift experiments showing that him-14 functions during the pachytene stage, indicate that him-14 is not needed to establish pairing or synapsis and likely has a more direct role in crossover formation. him-14 encodes a germline-specific member of the MutS family of DNA mismatch repair (MMR) proteins. him-14 has no apparent role in MMR, but like its Saccharomyces cerevisiae ortholog MSH4, has a specialized role in promoting crossing over during meiosis. Despite this conservation, worms and yeast differ significantly in their reliance on this pathway: whereas worms use this pathway to generate most, if not all, crossovers, yeast still form 30-50% of their normal number of crossovers when this pathway is absent. This differential reliance may reflect differential stability of crossover-competent recombination intermediates, or alternatively, the presence of two different pathways for crossover formation in yeast, only one of which predominates during nematode meiosis. We discuss a model in which HIM-14 promotes crossing over by interfering with Holliday junction branch migration. PMID:10545458

  9. Subunit interactions in yeast transcription/repair factor TFIIH. Requirement for Tfb3 subunit in nucleotide excision repair.

    PubMed

    Feaver, W J; Huang, W; Gileadi, O; Myers, L; Gustafsson, C M; Kornberg, R D; Friedberg, E C

    2000-02-25

    A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2. These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome. PMID:10681587

  10. A Genome-Wide Screen Identifies Yeast Genes Required for Tolerance to Technical Toxaphene, an Organochlorinated Pesticide Mixture

    PubMed Central

    Gaytán, Brandon D.; Loguinov, Alex V.; Peñate, Xenia; Lerot, Jan-Michael; Chávez, Sebastián; Denslow, Nancy D.; Vulpe, Chris D.

    2013-01-01

    Exposure to toxaphene, an environmentally persistent mixture of chlorinated terpenes previously utilized as an insecticide, has been associated with various cancers and diseases such as amyotrophic lateral sclerosis. Nevertheless, the cellular and molecular mechanisms responsible for these toxic effects have not been established. In this study, we used a functional approach in the model eukaryote Saccharomyces cerevisiae to demonstrate that toxaphene affects yeast mutants defective in (1) processes associated with transcription elongation and (2) nutrient utilization. Synergistic growth defects are observed upon exposure to both toxaphene and the known transcription elongation inhibitor mycophenolic acid (MPA). However, unlike MPA, toxaphene does not deplete nucleotides and additionally has no detectable effect on transcription elongation. Many of the yeast genes identified in this study have human homologs, warranting further investigations into the potentially conserved mechanisms of toxaphene toxicity. PMID:24260565

  11. Benchmarking nuclear fission theory

    SciTech Connect

    Bertsch, G. F.; Loveland, W.; Nazarewicz, W.; Talou, P.

    2015-05-14

    We suggest a small set of fission observables to be used as test cases for validation of theoretical calculations. Thus, the purpose is to provide common data to facilitate the comparison of different fission theories and models. The proposed observables are chosen from fission barriers, spontaneous fission lifetimes, fission yield characteristics, and fission isomer excitation energies.

  12. Fission Spectrum

    DOE R&D Accomplishments Database

    Bloch, F.; Staub, H.

    1943-08-18

    Measurements of the spectrum of the fission neutrons of 25 are described, in which the energy of the neutrons is determined from the ionization produced by individual hydrogen recoils. The slow neutrons producing fission are obtained by slowing down the fast neutrons from the Be-D reaction of the Stanford cyclotron. In order to distinguish between fission neutrons and the remaining fast cyclotron neutrons both the cyclotron current and the pusle amplifier are modulated. A hollow neutron container, in which slow neutrons have a lifetime of about 2 milliseconds, avoids the use of large distances. This method results in much higher intensities than the usual modulation arrangement. The results show a continuous distribution of neutrons with a rather wide maximum at about 0.8 MV falling off to half of its maximum value at 2.0 MV. The total number of netrons is determined by comparison with the number of fission fragments. The result seems to indicate that only about 30% of the neutrons have energies below .8 MV. Various tests are described which were performed in order to rule out modification of the spectrum by inelastic scattering. Decl. May 4, 1951

  13. Yeast linker histone Hho1p is required for efficient RNA polymerase I processivity and transcriptional silencing at the ribosomal DNA

    PubMed Central

    Levy, Anat; Eyal, Miri; Hershkovits, Gitit; Salmon-Divon, Mali; Klutstein, Michael; Katcoff, Don Jay

    2008-01-01

    Nucleosome core particles in eukaryotes are linked by a stretch of DNA that is usually associated with a linker histone. Here, we show in yeast, that the presence of yeast linker histone Hho1p represses expression of a pol II transcribed gene (MET15) embedded in the rDNA. In vivo deletions of Hho1p sequences showed that the second globular domain is sufficient for that repression, whereas the presence of the N terminus is required for its derepression. In contrast, a run-on assay confirmed by a ChIP experiment showed that Hho1p is required for maximal pol I processivity during rDNA transcription. Psoralen accessibility experiments indicated that Hho1p is necessary for normal rDNA compaction. DNA array expression analysis comparing RNA transcripts in wild-type and hho1 strains before and after a heat-shock showed that Hho1p is necessary to achieve wild-type mRNA levels of transcripts that encode ribosomal components. Taken together, our results suggest that Hho1p is involved in rDNA compaction, and like core histones, is required for efficient rDNA transcription by pol I. PMID:18687885

  14. Mitochondrial-morphology-targeted breeding of industrial yeast strains for alcohol fermentation.

    PubMed

    Kitagaki, Hiroshi

    2009-07-01

    Since mitochondrial genes are repressed under high glucose and low O2, and these conditions correspond to the conditions in which yeast cells are exposed during alcohol fermentation, the existence and structure of yeast mitochondria during alcohol fermentation have not been elucidated. Yeast mitochondria can be observed throughout brewing of sake (Japanese rice wine) and fragment during brewing. Furthermore, it has been revealed that Fis1 [fission 1 (mitochondrial outer membrane) homologue (Saccharomyces cerevisiae)], which is a transmembrane protein with its C-terminal anchor embedded in the outer membrane of mitochondria, is required for fragmentation of yeast mitochondria during sake brewing. By utilizing this knowledge, a fis1 disruptant of a sake yeast strain has been generated that has a networked mitochondrial structure throughout sake brewing. It transpired that this strain produces a high content of malate, which imparts a crisp acidic taste, during sake brewing. This strategy is a useful and a completely novel strategy towards developing a new yeast strain which produces a high content of malate in sake, and mitochondrial morphology has now emerged as a promising target for the breeding of practical industrial strains. PMID:19476438

  15. Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance

    PubMed Central

    Malo, Mackenzie E.; Postnikoff, Spike D.L.; Arnason, Terra G.; Harkness, Troy A.A.

    2016-01-01

    The Saccharomyces cerevisiae Forkhead Box (Fox) orthologs, Forkheads (Fkh) 1 and 2, are conserved transcription factors required for stress response, cell cycle progression and longevity. These yeast proteins play a key role in mitotic progression through activation of the ubiquitin E3 ligase Anaphase Promoting Complex (APC) via transcriptional control. Here, we used genetic and molecular analyses to demonstrate that the APC E3 activity is necessary for mitotic Fkh1 protein degradation and subsequent cell cycle progression. We report that Fkh1 protein degradation occurs specifically during mitosis, requires APCCdc20 and proteasome activity, and that a stable Fkh1 mutant reduces normal chronological lifespan, increases genomic instability, and increases sensitivity to stress. Our data supports a model whereby cell cycle progression through mitosis and G1 requires the targeted degradation of Fkh1 by the APC. This is significant to many fields as these results impact our understanding of the mechanisms underpinning the control of aging and cancer. PMID:27099939

  16. Fission modelling with FIFRELIN

    NASA Astrophysics Data System (ADS)

    Litaize, Olivier; Serot, Olivier; Berge, Léonie

    2015-12-01

    The nuclear fission process gives rise to the formation of fission fragments and emission of particles (n,γ , e-) . The particle emission from fragments can be prompt and delayed. We present here the methods used in the FIFRELIN code, which simulates the prompt component of the de-excitation process. The methods are based on phenomenological models associated with macroscopic and/or microscopic ingredients. Input data can be provided by experiment as well as by theory. The fission fragment de-excitation can be performed within Weisskopf (uncoupled neutron and gamma emission) or a Hauser-Feshbach (coupled neutron/gamma emission) statistical theory. We usually consider five free parameters that cannot be provided by theory or experiments in order to describe the initial distributions required by the code. In a first step this set of parameters is chosen to reproduce a very limited set of target observables. In a second step we can increase the statistics to predict all other fission observables such as prompt neutron, gamma and conversion electron spectra but also their distributions as a function of any kind of parameters such as, for instance, the neutron, gamma and electron number distributions, the average prompt neutron multiplicity as a function of fission fragment mass, charge or kinetic energy, and so on. Several results related to different fissioning systems are presented in this work. The goal in the next decade will be i) to replace some macroscopic ingredients or phenomenological models by microscopic calculations when available and reliable, ii) to be a support for experimentalists in the design of detection systems or in the prediction of necessary beam time or count rates with associated statistics when measuring fragments and emitted particle in coincidence iii) extend the model to be able to run a calculation when no experimental input data are available, iv) account for multiple chance fission and gamma emission before fission, v) account for the

  17. Histidine Methylation of Yeast Ribosomal Protein Rpl3p Is Required for Proper 60S Subunit Assembly

    PubMed Central

    Al-Hadid, Qais; Roy, Kevin; Munroe, William; Dzialo, Maria C.; Chanfreau, Guillaume F.

    2014-01-01

    Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine 243, a residue that contacts the 25S rRNA near the P site. Rpl3p methylation is dependent upon the presence of Hpm1p, a candidate seven-beta-strand methyltransferase. In this study, we elucidated the biological activities of Hpm1p in vitro and in vivo. Amino acid analyses reveal that Hpm1p is responsible for all of the detectable protein histidine methylation in yeast. The modification is found on a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nucleus-containing organelle fraction but was not detected in proteins of the ribosome-free cytosol fraction. In vitro assays demonstrate that Hpm1p has methyltransferase activity on ribosome-associated but not free Rpl3p, suggesting that its activity depends on interactions with ribosomal components. hpm1 null cells are defective in early rRNA processing, resulting in a deficiency of 60S subunits and translation initiation defects that are exacerbated in minimal medium. Cells lacking Hpm1p are resistant to cycloheximide and verrucarin A and have decreased translational fidelity. We propose that Hpm1p plays a role in the orchestration of the early assembly of the large ribosomal subunit and in faithful protein producti