Opposite effects of tor1 and tor2 on nitrogen starvation responses in fission yeast

The TOR protein kinases exhibit a conserved role in regulating cellular growth and proliferation. In the fission yeast two TOR homologs are present. tor1(+) is required for starvation and stress responses, while tor2(+) is essential. We report here that Tor2 depleted cells show a phenotype very similar to that of wild-type cells starved for nitrogen, including arrest at the G(1) phase of the cell cycle, induction of nitrogen-starvation-specific genes, and entrance into the sexual development pathway. The phenotype of tor2 mutants is in a striking contrast to the failure of tor1 mutants to initiate sexual development or arrest in G(1) under nitrogen starvation conditions. Tsc1 and Tsc2, the genes mutated in the human tuberous sclerosis complex syndrome, negatively regulate the mammalian TOR via inactivation of the GTPase Rheb. We analyzed the genetic relationship between the two TOR genes and the Schizosaccharomyces pombe orthologs of TSC1, TSC2, and Rheb. Our data suggest that like in higher eukaryotes, the Tsc1-2 complex negatively regulates Tor2. In contrast, the Tsc1-2 complex and Tor1 appear to work in parallel, both positively regulating amino acid uptake through the control of expression of amino acid permeases. Additionally, either Tsc1/2 or Tor1 are required for growth on a poor nitrogen source such as proline. Mutants lacking Tsc1 or Tsc2 are highly sensitive to rapamycin under poor nitrogen conditions, suggesting that the function of Tor1 under such conditions is sensitive to rapamycin. We discuss the complex genetic interactions between tor1(+), tor2(+), and tsc1/2(+) and the implications for rapamycin sensitivity in tsc1 or tsc2 mutants.     

The serine/threonine kinase Cmk2 is required for oxidative stress response in fission yeast

Cmk2, a fission yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, is essential for oxidative stress response. Cells lacking cmk2 gene were specifically sensitive to oxidative stress conditions. Upon stress, Cmk2 was phosphorylated in vivo, and this phosphorylation was dependent on the stress-activated MAPK Sty1/Spc1. Co-precipitation assays demonstrated that Cmk2 binds Sty1. Furthermore, in vivo or in vitro activated Sty1 was able to phosphorylate Cmk2, and the phosphorylation occurred at the C-terminal regulatory domain at Thr-411. Cell lethality caused by overexpression of Wis1 MAPK kinase was abolished by deletion of cmk2 or by mutation of Thr-411 of Cmk2. Taken together, our data suggest that Cmk2 acts downstream of Sty1 and is an essential kinase for oxidative stress responses.     

The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses

Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in eukaryotes. In the yeast Saccharomyces cerevisiae, four distinct ubiquitin-coding loci have been described. UBI1, UBI2, and UBI3 each encode hybrid proteins in which ubiquitin is fused to unrelated sequences. The fourth gene, UBI4, contains five ubiquitin-coding elements in a head-to-tail arrangement, and thus encodes a polyubiquitin precursor protein. A precise, oligonucleotide-directed deletion of UBI4 was constructed in vitro and substituted in the yeast genome in place of the wild-type allele. ubi4 deletion mutants are viable as vegetative cells, grow at wild-type rates, and contain wild-type levels of free ubiquitin under exponential growth conditions. However, although ubi4/UBI4 diploids can form four initially viable spores, the two ubi4 spores within the ascus lose viability extremely rapidly, apparently a novel phenotype in yeast. Furthermore, ubi4/ubi4 diploids are sporulation-defective. ubi4 mutants are also hypersensitive to high temperatures, starvation, and amino acid analogs. These three conditions, while diverse in nature, are all known to induce stress proteins. Expression of the UBI4 gene is similarly induced by either heat stress or starvation. These results indicate that UBI4 is specifically required for the resistance of cells to stress, and that ubiquitin is an essential component of the stress response system.     

rqh1+, a fission yeast gene related to the Bloom''s and Werner''s syndrome genes, is required for reversible S phase arrest.

In eukaryotic cells, S phase can be reversibly arrested by drugs that inhibit DNA synthesis or DNA damage. Here we show that recovery from such treatments is under genetic control and is defective in fission yeast rqh1 mutants. rqh1+, previously known as hus2+, encodes a putative DNA helicase related to the Escherichia coli RecQ helicase, with particular homology to the gene products of the human BLM and WRN genes and the Saccharomyces cerevisiae SGS1 gene. BLM and WRN are mutated in patients with Bloom's syndrome and Werner's syndrome respectively. Both syndromes are associated with genomic instability and cancer susceptibility. We show that, like BLM and SGS1, rqh1+ is required to prevent recombination and that in fission yeast suppression of inappropriate recombination is essential for reversible S phase arrest.     

The highly conserved protein methyltransferase, Skb1, is a mediator of hyperosmotic stress response in the fission yeast Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is required for cell viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe. Previous genetic studies suggested that a presumptive protein methyltransferase, Skb1, functions as a positive modulator of Shk1. However, unlike Shk1, Skb1 is not required for viability or mating of S. pombe cells and contributes only modestly to the regulation of cell morphology under normal growth conditions. Here we demonstrate that Skb1 plays a more significant role in regulating cell growth and polarity under conditions of hyperosmotic stress. We provide evidence that the inability of skb1Delta cells to properly maintain cell polarity in hyperosmotic conditions results from inefficient subcellular targeting of F-actin. We show that Skb1 localizes to cell ends, sites of septation, and nuclei of S. pombe cells. Hyperosmotic shock results in substantial delocalization of Skb1 from cell ends and nuclei, as well as stimulation of Skb1 protein methyltransferase activity. Taken together, our results demonstrate a new role for Skb1 as a mediator of hyperosmotic stress response in fission yeast. We show that the protein methyltransferase activity of the human Skb1 homolog, Skb1Hs, is also stimulated by hyperosmotic stress in fission yeast, providing evidence for evolutionary conservation of a role for Skb1-related proteins as mediators of hyperosmotic stress response, as well as mechanisms involved in regulating this novel class of protein methyltransferases.     

The protein kinase kin1 is required for cellular symmetry in fission yeast

The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused. The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation. Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p. Using a deletion allele (kin1Delta), we show that kin1 mutation promotes a delay in septation. Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling. Abnormal shaped interphase kin1Delta cells misplace F-actin patches and the premitotic nucleus. Thus, mitotic kin1Delta cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus. The resulting asymmetric cell division produces daughter cells with distinct shapes. Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules. Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell.     

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage.

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.     

The fission yeast ran GTPase is required for microtubule integrity

The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.     

bgs2+, a sporulation-specific glucan synthase homologue is required for proper ascospore wall maturation in fission yeast

The formation of the ascospore cell wall of Schizosaccharomyces pombe requires the co-ordinated activity of enzymes involved in the biosynthesis of its components, such as glucans. We have cloned the bgs2+ gene. bgs2+ belongs to the glucan synthase family of S. pombe and is homologous to the Saccharomyces cerevisiae FKS1 and FKS2 genes. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth, but homozygous bgs2Delta diploids do show a sporulation defect. Although meiosis takes place normally, ascospores are unable to mature, and their wall differs from that of wild-type ascospores. Moreover, bgs2Delta zygotes were not able to release ascospores spontaneously, and the ascospores were unable to germinate. We show that expression of bgs2+ is restricted to sporulation and that a bgs2-green fluorescent protein (GFP) fusion protein localizes to the ascospore envelope. The glucan synthase activity in sporulating diploids bearing a bgs2 deletion was diminished in comparison with that of the wild-type diploids, a fact that underscores the importance of the bgs2+ gene and glucan synthesis for the proper formation and maturation of the ascospore wall.     

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

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.     

The fission yeast Map4 protein is a novel adhesin required for mating

Cell adhesion is required for many cellular processes. In fungi, cell-cell contact during mating, flocculation or virulence is mediated by adhesins, which typically are glycosyl phosphatidyl inositol (GPI)-modified cell wall glycoproteins. Proteins with internal repeats (PIR) are surface proteins involved in the response to stress. In Schizosaccharomyces pombe no adhesins or PIR proteins have been described. Here we study the S. pombe Map4p, which defines a new class of surface protein that is not GPI-modified and has a serine/threonine rich domain and internal repeats that differ from those present in PIR proteins. Map4p is a mating type-specific adhesin required for mating in h(+) cells and enhances cell adhesion when overexpressed.     

A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast

The chromodomain is a conserved motif that functions in the epigenetic control of gene expression. Here, we report the functional characterization of a chromodomain protein, Chp1, in the heterochromatin assembly in fission yeast. We show that Chp1 is a structural component of three heterochromatic regions-centromeres, the mating-type region, and telomeres-and that its localization in these regions is dependent on the histone methyltransferase Clr4. Although deletion of the chp1(+) gene causes centromere-specific decreases in Swi6 localization and histone H3-K9 methylation, we show that the role of Chp1 is not exclusive to the centromeres. We found that some methylation persists in native centromeric regions in the absence of Chp1, which is also true for the mating-type region and telomeres, and determined that Swi6 and Chp2 are critical to maintaining this residual methylation. We also show that Chp1 participates in the establishment of repressive chromatin in all three chromosomal regions. These results suggest that different heterochromatic regions share common structural properties, and that centromeric heterochromatin requires Chp1-mediated establishment steps differently than do other heterochromatic regions.     

A fission yeast RCC1-related protein is required for the mitosis to interphase transition.

The isolation and characterization of the mutant dcdts (defect in chromatin decondensation) has led to the identification of two conserved proteins required for the re-establishment of the interphase state following the completion of mitosis. The gene that rescues the dcdts mutant encodes a protein similar to the human chromatin binding protein, RCC1. A suppressor of dcdts encodes a protein nearly identical to the human GTP-binding protein, RAN, encoded by the TC4 gene. These results indicate that completion of mitosis is regulated at least in part by a GTPase molecular switch. The gene and suppressor of dcdts are identical to the previously described Schizosaccharomyces pombe genes pim1 (premature initiation of mitosis) and spi1 (suppressor of pim), but the dcdts mutant does not enter mitosis prematurely, a phenotype that has been reported for the pim1-46ts mutant. Based on our studies we propose that the pim1 gene product is required for regulating chromatin condensation with a primary role at the end of mitosis and pleiotropic effects on other aspects of cell behavior.     

Rapamycin specifically interferes with the developmental response of fission yeast to starvation.

Rapamycin is a microbial macrolide which belongs to a family of immunosuppressive drugs that suppress the immune system by blocking stages of signal transduction in T lymphocytes. In Saccharomyces cerevisiae cells, as in T lymphocytes, rapamycin inhibits growth and cells become arrested at the G1 stage of the cell cycle. Rapamycin is also an effective antifungal agent, affecting the growth of yeast and filamentous fungi. Unexpectedly, we observed that rapamycin has no apparent effect on the vegetative growth of Schizosaccharomyces pombe. Instead, the drug becomes effective only when cells experience starvation. Under such conditions, homothallic wild-type cells will normally mate and undergo sporulation. In the presence of rapamycin, this sexual development process is strongly inhibited and cells adopt an alternative physiological option and enter stationary phase. Rapamycin strongly inhibits sexual development of haploid cells prior to the stage of sexual conjugation. In contrast, the drug has only a slight inhibitory effect on the sporulation of diploid cells. A genetic approach was applied to identify the signal transduction pathway that is inhibited by rapamycin. The results indicate that either rapamycin did not suppress the derepression of sexual development of strains in which adenylate cyclase was deleted or the cyclic AMP-dependent protein kinase encoded by pka1 was mutated. Nor did rapamycin inhibit the unscheduled meiosis observed in pat1-114 mutants. Overexpression of ras1+, an essential gene for sexual development, did not rescue the sterility of rapamycin-treated cells. However, expression of the activated allele, ras1Val17, antagonized the effect of rapamycin and restored the ability of the cells to respond to mating signals in the presence of the drug. We discuss possible mechanisms for the inhibitory effect of rapamycin on sexual development in S. pombe.