Mkh1, a MEK kinase required for cell wall integrity and proper response to osmotic and temperature stress in Schizosaccharomyces pombe.

We have identified a Schizosaccharomyces pombe gene, mkh1, that encodes a MEK kinase (MEKK) homolog. The coding region of mkh1 is contained within a single exon encoding a 1,116-amino-acid protein. The putative catalytic domain of Mkh1 is 54% identical to the catalytic domain of S. cerevisiae Bck1, the most closely related protein. Deletion of mkh1 did not significantly affect cell growth or division under standard conditions. However, mkh1delta cell growth was inhibited by high KCl or NaCl concentrations. mkh1delta cells required a longer time to reenter the cell cycle after prolonged stationary-phase arrest. Also, mkh1delta cells exhibited a round cell shape, while overexpression of Mkh1 resulted in an elongated cell shape. mkh1delta cells exhibited a more dramatic phenotype when grown in nutrient-limiting conditions at high temperature or in hyperosmotic medium. In such conditions, completion of cytokinesis was inhibited, resulting in the growth of pseudohyphal filaments with multiple septa and nuclei. Also, mkh1delta cells were hypersensitive to beta-glucanase treatment. Together these results suggest that Mkh1 regulates cell morphology, cell wall integrity, salt resistance, cell cycle reentry from stationary-phase arrest, and filamentous growth in response to stress. These phenotypes are essentially identical to those exhibited by cells lacking Pmk1/Spm1, a recently identified mitogen-activated protein kinase. Our evidence suggests that Pmk1/Spm1 acts downstream from Mkh1 in a common pathway. Our results also suggest that Mkh1 and Pck2 act independently to maintain cell wall integrity, cell morphology, and salt resistance but act in opposition to regulate filamentous growth.     

Schizosaccharomyces pombe ehs1p is involved in maintaining cell wall integrity and in calcium uptake

The Schizosaccharomyces pombe mutant ehs1-1 mutant was isolated on the basis of its hypersensitivity to Echinocandin and Calcofluor White, which inhibit cell wall synthesis. The mutant shows a thermosensitive growth phenotype that is suppressed in the presence of an osmotic stabiliser. The mutant also showed other cell wall-associated phenotypes, such as enhanced sensitivity to enzymatic cell wall degradation and an imbalance in polysaccharide synthesis. The ehs1 + gene encodes a predicted integral membrane protein that is 30% identical to Saccharomyces cerevisiae Mid1p, a protein that has been proposed to form part of a calcium channel. As expected for such a function, we found that ehs1+ is involved in intracellular Ca2+ accumulation. High external Ca2+ concentrations suppressed all phenotypes associated with the ehs1 null mutation, suggesting that the cell integrity defects of ehs1 mutants result from inadequate levels of calcium in the cell. We observed a genetic relationship between ehs1+ and the protein kinase C homologue pck2+. pck2+ suppressed all phenotypes of ehs1-1 mutant cells. Overproduction of pck2p is deleterious to wild-type cells, increasing 1,3-beta-D-glucan synthase activity and promoting accumulation of extremely high levels of Ca2+. The lethality associated with pck2p, the increase in 1,3-beta-D-glucan synthase production and the strong Ca2+ accumulation are all dependent on the presence of ehs1p. Our results suggest that in fission yeast ehs1p forms part of a calcium channel that is involved in the cell wall integrity pathway that includes the kinase pck2p.     

Cell cycle regulation in Schizosaccharomyces pombe

Cdc2, a cyclin-dependent kinase, controls cell cycle progression in fission yeast. New details of Cdc2 regulation and function have been uncovered in recent studies. These studies involve cyclins that associate with Cdc2 in G1-phase and the proteins that regulate inhibitory phosphorylation of Cdc2 during S-phase and G2-phase. Recent investigations have also provided a better understanding of proteins that regulate DNA replication and that are directly or indirectly controlled by Cdc2.     

Interaction of Epe1 with the heterochromatin assembly pathway in Schizosaccharomyces pombe

Epe1 is a JmjC domain protein that antagonizes heterochromatization in Schizosaccharomyces pombe. Related JmjC domain proteins catalyze a histone demethylation reaction that depends on Fe(II) and alpha-ketoglutarate. However, no detectable demethylase activity is associated with Epe1, and its JmjC domain lacks conservation of Fe(II)-binding residues. We report that Swi6 recruits Epe1 to heterochromatin and that overexpression of epe1+, like mutations in silencing genes or overexpression of swi6+, upregulates expression of certain genes. A significant overlap was observed between the lists of genes that are upregulated by overexpression of epe1+ and those that are upregulated by mutations in histone deacetylase genes. However, most of the common genes are not regulated by Clr4 histone methyltransferase. This suggests that Epe1 interacts with the heterochromatin assembly pathway at the stage of histone deacetylation. Mutational inactivation of Epe1 downregulates approximately 12% of S. pombe genes, and the list of these genes overlaps significantly with the lists of genes that are upregulated by mutations in silencing genes and genes that are hyperacetylated at their promoter regions in clr6-1 mutants. We propose that an interplay between the repressive HDACs activity and Epe1 helps to regulate gene expression in S. pombe.     

Novel Rho GTPase involved in cytokinesis and cell wall integrity in the fission yeast Schizosaccharomyces pombe

The Rho family of GTPases is present in all eukaryotic cells from yeast to mammals; they are regulators in signaling pathways that control actin organization and morphogenetic processes. In yeast, Rho GTPases are implicated in cell polarity processes and cell wall biosynthesis. It is known that Rho1 and Rho2 are key proteins in the construction of the cell wall, an essential structure that in Schizosaccharomyces pombe is composed of beta-glucan, alpha-glucan, and mannoproteins. Rho1 regulates the synthesis of 1,3-beta-D-glucan by activation of the 1,3-beta-D-glucan synthase, and Rho2 regulates the synthesis of alpha-glucan by the 1,3-alpha-D-glucan synthase Mok1. Here we describe the characterization of another Rho GTPase in fission yeast, Rho4. rho4Delta cells are viable but display cell separation defects at high temperature. In agreement with this observation, Rho4 localizes to the septum. Overexpression of rho4(+) causes lysis and morphological defects. Several lines of evidence indicate that both rho4(+) deletion or rho4(+) overexpression result in a defective cell wall, suggesting an additional role for Rho4 in cell wall integrity. Rho4Delta cells also accumulate secretory vesicles around the septum and are defective in actin polarization. We propose that Rho4 could be involved in the regulation of the septum degradation during cytokinesis.     

Pap1-mediated regulation of thioredoxin gene from Schizosaccharomyces pombe

The genomic DNA encoding thioredoxin (TRX) was previously isolated from the fission yeast Schizosaccharomyces pombe. In this investigation, regulation of the S. pombe TRX gene was studied in lacZ translational fusions. The synthesis of beta-galactosidase from the fusion plasmid pYKT24 was significantly enhanced by treatments with cadmium chloride, zinc chloride, and high temperatures. Synthesis of beta-galactosidase from the fusion plasmid was significantly decreased by higher concentrations (5 microM, 10 microM) of mercuric chloride, whereas it was enhanced by its lower concentration (1 microM). Diamide affected the synthesis of beta-galactosidase in the same manner with mercuric chloride. However, high osmolarity had no effect on the beta-galactosidase synthesis from the fusion plasmid pYKT24. Various fusion plasmids were constructed to carry serially deleted upstream regions of the TRX gene. Pap1 mediates the regulation of the S. pombe TRX gene. The upstream region, between 987 and 1,270 bp from the translational initiation point, is responsible for the regulation.     

Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe

The nuclear envelope is essential for compartmentalizing the nucleus from the cytoplasm in all eukaryotic cells. There is a tremendous flux of both RNA and proteins across the nuclear envelope, which is intact throughout the entire cell cycle of yeasts but breaks down during mitosis of animal cells. Transport across the nuclear envelope requires the recognition of cargo molecules by receptors, docking at the nuclear pore, transit through the nuclear pore, and then dissociation of the cargo from the receptor. This process depends on the RanGTPase system, transport receptors, and the nuclear pore complex. We provide an overview of the nuclear transport process, with particular emphasis on the fission yeast Schizosaccharomyces pombe, including strategies for predicting and experimentally verifying the signals that determine the sub-cellular localization of a protein of interest. We also describe a variety of reagents and experimental strategies, including the use of mutants and chemical inhibitors, to study nuclear protein import, nuclear protein export, nucleocytoplasmic protein shuttling, and mRNA export in fission yeast. The RanGTPase and its regulators also play an essential transport independent role in nuclear envelope re-assembly after mitosis in animal cells and in the maintenance of nuclear envelope integrity at mitosis in S. pombe. Several experimental strategies and reagents for studying nuclear size, nuclear shape, the localization of nuclear pores, and the integrity of the nuclear envelope in living fission yeast cells are described.     

Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage

The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

Schizosaccharomyces pombe Ddb1 is functionally linked to the replication checkpoint pathway

Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the Deltaddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Deltaddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in Deltaddb1 strain. The results suggest that many of the defects observed in Deltaddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1.     

Activation and regulation of the Spc1 stress-activated protein kinase in Schizosaccharomyces pombe.

Spc1, an osmotic-stress-stimulated mitogen-activated protein kinase (MAPK) homolog in the fission yeast Schizosaccharomyces pombe, is required for the induction of mitosis and survival in high-osmolarity conditions. Spc1, also known as Sty1, is activated by Wis1 MAPK kinase and inhibited by Pyp1 tyrosine phosphatase. Spc1 is most closely related to Saccharomyces cerevisiae Hog1 and mammalian p38 kinases. Whereas Hog1 is specifically responsive to osmotic stress, we report here that Spc1 is activated by multiple forms of stress, including high temperature and oxidative stress. In this regard Spc1 is more similar to mammalian p38. Activation of Spc1 is crucial for survival of various forms of stress. Spc1 regulates expression of genes encoding stress-related proteins such as glycerol-3-phosphate dehydrogenase (gpd1+) and trehalose-6-phosphate synthase (tps1+). Spc1 also promotes expression of pyp2+, which encodes a tyrosine phosphatase postulated as a negative regulator of Spc1. This proposal is supported by the finding that Spc1 associates with Pyp2 in vivo and that the amount of Spc1 tyrosine phosphorylation is lower in a Pyp2-overproducing strain than in the wild type. Moreover, the level of stress-stimulated gpd1+ expression is higher in delta pyp2 mutants than in the wild type. These findings demonstrate that Spc1 promotes expression of genes involved in stress survival and that of regulation may be commonly employed to modulate MAPK signal transduction pathways in eukaryotic species.     

Rga5p is a specific Rho1p GTPase-activating protein that regulates cell integrity in Schizosaccharomyces pombe

Schizosaccharomyces pombe Rho1p regulates (1,3)beta-d-glucan synthesis and is required for cell integrity maintenance and actin cytoskeleton organization, but nothing is known about the regulation of this protein. At least nine different S. pombe genes code for proteins predicted to act as Rho GTPase-activating proteins (GAPs). The results shown in this paper demonstrate that the protein encoded by the gene named rga5+ is a GAP specific for Rho1p. rga5+ overexpression is lethal and causes morphological alterations similar to those reported for Rho1p inactivation. rga5+ deletion is not lethal and causes a mild general increase in cell wall biosynthesis and morphological alterations when cells are grown at 37 degrees C. Upon mild overexpression, Rga5p localizes to growth areas and possesses both in vivo and in vitro GAP activity specific for Rho1p. Overexpression of rho1+ in rga5Delta cells is lethal, with a morphological phenotype resembling that of the overexpression of the constitutively active allele rho1G15V. In addition (1,3)beta-d-glucan synthase activity, regulated by Rho1p, is increased in rga5Delta cells and decreased in rga5-overexpressing cells. Moreover, the increase in (1,3)beta-d-glucan synthase activity caused by rho1+ overexpression is considerably higher in rga5Delta than in wild-type cells. Genetic interactions suggest that Rga5p is also important for the regulation of the other known Rho1p effectors, Pck1p and Pck2p.     

Cloning, expression and regulation of Schizosaccharomyces pombe gene encoding thioltransferase

The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction. The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast-Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1. The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1. The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11261 Da. Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S. pombe containing plasmid pEH1 and pYEH1, respectively. The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless beta-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso-N-acetylpenicillamine.     

Recombinational repair in Schizosaccharomyces pombe: role in maintaining genomic integrity

Recombinational repair was first detected in budding yeast Saccharomyces cerevisiae and was also studied in fission yeast Schizosaccharomyces pombe over the recent decade. The discovery of Sch. pombe homologs of the S. cerevisiae RAD52 genes made it possible not only to identify and to clone their vertebrate counterparts, but also to study in detail the role of DNA recombination in certain cell processes. For instance, recombinational repair was shown to play a greater role in maintaining genome integrity in fission yeast and in vertebrates compared with S. cerevisiae. The present state of the problem of recombinational double-strand break repair in fission yeast is considered with a focus on comparisons between Sch. pombe and higher eukaryotes. The role of double-strand break repair in maintaining genome stability is discussed.