Functional complementation of the Schizosaccharomyces pombe wis1 mutant by Arabidopsis MEK1 and non-catalytic enhancement by CTR1.

Arabidopsis thaliana MEK1 encodes a MAPKK homolog whose role in plants is currently unknown. High (but not low) expression of MEK1 rescued the Deltawis1 (MAPKK) mutant of the Schizosaccharomyces pombe Win1/Wis4-Wis1-Sty1 stress-activated MAPK pathway. Rescue was dependent upon upstream and downstream components of the pathway, suggesting that MEK1 might function in a homologous MAPK pathway in plants. When MEK1 was expressed at a low level, rescue of Deltawis1 was achieved by co-expressing Arabidopsis CTR1 (a putative MAPKK kinase (MAPKKK)). CTR1 constructs alone did not rescue the pathway, indicating that CTR1 augmented MEK1 function. Further data indicated that this enhancement was not due to CTR1 kinase activity.     

The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases

S. pombe dis mutants block mitotic chromosome disjunction in a manner reminiscent of aneuploidy formation, and belong to three distinct genes, dis1-dis3. We cloned two independent genomic DNAs that complemented both the cold-sensitive and caffeine-hypersensitive phenotype of dis2-11. These genes, dis2+ and a suppressor sds21+, encode proteins (calculated MW 37,000) with similar predicted amino acid sequences. dis2+ and sds21+ have overlapping functions, and disruptants are lethal only when both genes are disrupted. The gene products identified by anti-dis2 serum are enriched in nuclei. By hybridization, we obtained two cDNA clones from mouse and one genomic clone from S. cerevisiae; the latter complements S. pombe dis2-11. These dis2+ and similar polypeptides of yeasts and mouse are found to be highly homologous (75%-90% identical) to rabbit protein phosphatase 1. The implications of these findings are discussed with regard to mitotic control.     

Mcs4 mitotic catastrophe suppressor regulates the fission yeast cell cycle through the Wik1-Wis1-Spc1 kinase cascade.

Spc1 in Schizosaccharomyces pombe is a member of the stress-activated protein kinase family, an evolutionary conserved subfamily of mitogen-activated protein kinases (MAPKs). Spc1 is activated by a MAPK kinase homologue, Wis1, and negatively regulated by Pyp1 and Pyp2 tyrosine phosphatases. Mutations in the spc1+ and wis1+ genes cause a G2 cell cycle delay that is exacerbated during stress. Herein, we describe two upstream regulators of the Wis1-Spc1 cascade. wik1+ (Wis1 kinase) was identified from its homology to budding yeast SSK2, which encodes a MAPKK kinase that regulates the HOG1 osmosensing pathway. Delta wik1 cells are impaired in stress-induced activation of Spc1 and show a G2 cell cycle delay and osmosensitive growth. Moreover, overproduction of a constitutively active form of Wik1 induces hyperactivation of Spc1 in wis1(+)-dependent manner, suggesting that Wik1 regulates Spc1 through activation of Wis1. A mutation of mcs4+ (mitotic catastrophe suppressor) was originally isolated as a suppressor of the mitotic catastrophe phenotype of a cdc2-3w wee1-50 double mutant. We have found that mcs4- cells are defective at activation of Spc1 in response to various forms of stress. Epistasis analysis has placed Mcs4-upstream of Wik1 in the Spc1 activation cascade. These results indicate that Mcs4 is part of a sensor system for multiple environmental signals that modulates the timing of entry into mitosis by regulating the Wik1-Wis1-Spc1 kinase cascade. Inactivation of the sensor system delays the onset of mitosis and rescues lethal premature mitosis in cdc2-3w wee1-50 cells.     

The Fission Yeast Mitotic Regulator win1+ Encodes an MAP Kinase Kinase Kinase That Phosphorylates and Activates Wis1 MAP Kinase Kinase in Response to High Osmolarity

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1⁺ and wis4⁺, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1⁺ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.     

Identification of sds21 in fission yeast in an inhibitor-resistant high molecular mass protein phosphatase-1 complex.

While characterizing the type-1 protein phosphatases sds21 and dis2 in fission yeast (Schizosaccharomyces pombe) a novel high molecular mass protein was identified with serine/threonine phosphatase activity (referred to as PP-R) that was resistant to a panel of characteristic inhibitors of protein phosphatases. Purification of the native sds21 catalytic isoform of protein phosphatase-1 (PP-1) from an S. pombe knockout strain lacking dis2 (deltadis2) resulted predominantly in identification of PP-R. To test the hypothesis that the catalytic activity of PP-R comprised sds21, a parallel purification was performed of PP-1 activity from an S. pombe knockout strain lacking sds21 (deltasds21). Both deltasds21 and deltadis2 strains exhibited similar protein phosphatase activity profiles as determined by DEAE-sepharose, Mono-Q and Superdex gel filtration chromatography. However, the peak of protein phosphatase activity from deltasds21 S. pombe that co-migrated with PP-R from deltadis2 S. pombe exhibited the sensitivity to a panel of inhibitors that was characteristic of a type-1 protein phosphatase. These data suggest that the catalytic subunit of PP-R comprises sds21 and that the resistance to inhibitors may originate from structural differences between dis2 and sds21 isoforms. A key structural feature present in sds21, but lacking in dis2, is a classical phosphorylation consensus sequence surrounding serine-145 of sds21. The previous hypothesis was that PP-1 activity among several lower eukaryotes may be regulated directly by cAMP-dependent protein kinase (PKA) phosphorylation. However, this study demonstrated that recombinant sds21 is not a target for PKA in vitro. The constrained configuration of the putative PKA site on the PP-1 holoenzyme may restrict its ability to be targeted by PKA.     

Identification and characterization of a novel gene, hos3+, the function of which is necessary for growth under high osmotic stress in fission yeast

hos3 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos3-M26 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. The hos3+ gene was cloned and identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. A hos3delta strain, which we then constructed, had the phenotype of high osmolarity sensitivity, as in the case of the original hos3-M26 mutant. More interestingly, when these hos- cells were grown in the non-permissive growth condition in the presence of 2 M glucose, we found that unusually many septated cells were accumulated after a prolonged incubation. A multicopy suppressor gene for hos- mutations was also isolated and identified as the dsk1+ gene encoding a protein kinase, which was previously suggested to be implicated in a process of the mitotic regulation of S. pombe. The function of the hos3+ gene is discussed from these results.     

Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast

hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.     

Role of fission yeast primase catalytic subunit in the replication checkpoint

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.     

Selective inhibition of MAPKK Wis1 in the stress-activated MAPK cascade of Schizosaccharomyces pombe by novel berberine derivatives.

Intracellular molecular targets of novel berberine derivatives, HWY 289 and HWY 336, were identified by a screen of a variety of mutants in fission yeast Schizosaccharomyces pombe. HWY 289 and HWY 336 completely inhibited the proliferation of wild type as well as various mutant fission yeast cells (minimal inhibitory concentrations were 29.52 microm for HWY 289 and 11.83 microm for HWY 336), but did not affect the proliferation of Wis1 mitogen-activated protein kinase kinase (MAPKK) deletion mutants. In addition, HWY 289 with an IC(50) value of 7.3 microm or HWY 336 with IC(50) of 5.7 microm specifically inhibited in vitro kinase activities of purified Wis1, whereas either compound did not affect the activities of other kinases in the mitogen-activated protein kinase (MAPK) cascades of fission yeast. These genetic and biochemical results demonstrate the high degree of specificity of HWY 289 and HWY 336 to MAPKK Wis1 and suggest that the cytotoxicity of these compounds is not simply due to the inhibition of Wis1 kinase activity. High salt wash experiments have shown that strong noncovalent binding occurs between Wis1 and either HWY 289 or HWY 336. The preincubation of Wis1 kinase with ATP did not affect the inhibition of Wis1 by HWY 289 and HWY 336, but when Wis1 was preincubated with MBP, a protein substrate, Wis1 kinase activity was no longer inhibited. These observations demonstrate that HWY 289/HWY 336 do inhibit Wis1 kinase, not by binding to the ATP-binding site but by disturbing the binding of substrate to the kinase. Target validation of the complex of HWY 289/HWY 336 and Wis1 kinase will provide important clues for the mechanism of specific cytotoxicity of these compounds in S. pombe. On a broader aspect, it would create an initiative to further modify and develop compounds that selectively inhibit kinases and cause cytotoxicity in various MAPK cascades including those of mammals.     

Mcs4, a Two-Component System Response Regulator Homologue, Regulates the Schizosaccharomyces Pombe Cell Cycle Control

The Schizosaccharomyces pombe cdc2-3w wee1-50 double mutant displays a temperature-sensitive lethal phenotype termed mitotic catastrophe. Six mitotic catastrophe suppressor (mcs1-6) genes were identified in a genetic screen designed to identify regulators of cdc2. Mutations in mcs1-6 suppress the cdc2-3w wee1-50 temperature-sensitive growth defect. Here, the cloning of mcs4 is described. The mcs4 gene product displays significant sequence homology to members of the two-component system response regulator protein family. Strains carrying the mcs4 and cdc25 mutations display a synthetic osmotic lethal phenotype along with an inability to grow on minimal synthetic medium. These phenotypes are suppressed by a mutation in wee1. In addition, the wis1 gene, encoding a stress-activated mitogen-activated protein kinase kinase, was identified as a dosage suppressor in this screen. These findings link the two-component signal transduction system to stress response and cell cycle control in S. pombe.     

Mitotic regulation of protein phosphatases by the fission yeast sds22 protein

BACKGROUND: Cell cycle progression requires the activity of protein kinases and phosphatases at critical points in the cell cycle in all eukaryotes. We have previously reported that the dis2(+) and sds2(+) genes of fission yeast encode redundant catalytic subunits of a type 1-like protein phosphatase. The sds22(+) gene was shown to be essential for cell viability and to interact genetically with dis2(+) and sds21(+). RESULTS: Here we show by immunoprecipitation that the sds22 protein physically interacts with the dis2 and sds21 proteins, and that sds22-associated phosphatase activity has altered substrate specificity, The loss of sds22 function by a temperature sensitive mutation leads to cell cycle arrest at mid-mitosis, at which point cdc2-dependent histone Hl kinase activity is high while sds22-dependent H1 phosphatase activity is low. To examine the unusual properties of sds22 protein structure, we analyzed a collection of sds22 deletion and point mutants by a variety of functional criteria. CONCLUSION: We propose that sds22 is a regulatory subunit of the dis2/sds21 phosphatase catalytic subunits and that sds22-bound phosphatase carries a key phosphatase activity essential for the progression from metaphase to anaphase. Mutational analysis indicates that dis2/sds21 interacts with the central repetitive domain of sds22, while the C-terminal and central regions of sds22 may be involved in subcellular targeting and the N-terminus is important for stability.     

Role of exonucleolytic degradation in group I intron homing in phage T4

The Schizosaccharomyces pombe checkpoint gene named rad3(+) encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. Here we describe a novel role for Rad3 in the control of telomere integrity. Mutations in the rad3(+) gene alleviated telomeric silencing and produced shortened lengths in the telomere repeat tracts. Genetic analysis revealed that the other checkpoint rad mutations rad1, rad17, and rad26 belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening. tel1(+), another ATM homologue in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1(+) caused no obvious changes in the telomeric DNA structure. Our results demonstrate a novel role of the S. pombe ATM homologues that is independent of chk1(+) and cds1(+).     

A novel gene, ecl1(+), extends the chronological lifespan in fission yeast.

We have identified a novel gene from Schizosaccharomyces pombe that we have named ecl1(+) (extender of the chronological lifespan). When ecl1(+) is provided on a high-copy number plasmid, it extends the viability of both the Deltasty1 MAP kinase mutant and the wild-type cells after entry into the stationary phase. ecl1(+) encodes an 80-amino acid polypeptide that had not been annotated in the current database. The ecl1(+)-mRNA increases transiently when the growth phase is changed from the log phase to the stationary phase. The Ecl1 protein is localized in the nucleus. Calorie restriction extends the chronological lifespan of wild-type and Deltaecl1 cells but not ecl1(+)-overproducing cells. The Deltapka1 mutant shows little, if any, additional extension of viability when Ecl1 is overproduced. The ste11(+) gene that is negatively controlled by Pka1 is up regulated when Ecl1 is overproduced. From these results we propose that the effect of Ecl1 overproduction may be mainly linked to and negatively affects the Pka1-dependent pathway.     

The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex.

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation. In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated. One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity). Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants. The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum. Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase. These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.     

Counteractive roles of protein phosphatase 2C (PP2C) and a MAP kinase kinase homolog in the osmoregulation of fission yeast.

With the goal of discovering the cellular functions of type 2C protein phosphatases, we have cloned and analyzed two ptc (phosphatase two C) genes, ptc2+ and ptc3+, from the fission yeast Schizosaccharomyces pombe. Together with the previously identified ptc1+ gene, the enzymes encoded by these genes account for approximately 90% of the measurable PP2C activity in fission yeast cells. No obvious growth defects result from individual disruptions of ptc genes, but a delta ptc1 delta ptc3 double mutant displays aberrant cell morphology and temperature-sensitive cell lysis that is further accentuated in a delta ptc1 delta ptc2 delta ptc3 triple mutant. These phenotypes are almost completely suppressed by the presence of osmotic stabilizers, strongly indicating that PP2C has an important role in osmoregulation. Genetic suppression of delta ptc1 delta ptc3 lethality identified two loci, mutations of which render cells hypersensitive to high-osmolarity media. One locus is identical to wis1+, encoding a MAP kinase kinase (MEK) homolog. The Wis1 sequence is most closely related to the Saccharomyces cerevisiae MEK encoded by PBS2, which is required for osmoregulation. These data indicate that divergent yeasts have functionally conserved MAP kinase pathways, which are required to increase intracellular osmotic concentrations in response to osmotic stress. Moreover, our observations implicate PP2C enzymes as also having an important role in signal transduction processes involved in osmoregulation, probably acting to negatively regulate the osmosensing signal that is transmitted through Wis1 MAP kinase kinase.     

Requirement for PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase is lessened by the dosage increase of a novel gene sds23+.

Ubiquitin-dependent proteolysis is required for the onset of anaphase. We show that protein dephosphorylation by protein phosphatase 1 (PP1) is also essential for initiating anaphase in fission yeast. PP1 may directly or indirectly regulate the 20S cyclosome/APC (anaphase-promoting complex) required for anaphase-promoting proteolysis. Using anti-phosphopeptide antibodies, PP1 is shown to be dephosphorylated at the C-terminus, upon the onset of anaphase, for reactivation. sds23+, a novel gene, is a multicopy suppressor for mutations in PP1 and the 20S cyclosome/APC, implying that the gene dosage increase can relieve the requirement for PP1 and the cyclosome/APC for the onset of anaphase. The sds23+ gene is not essential for cell viability, but a mutant with the gene deleted cannot form colonies at 22 and 36 degrees C. In the sds23 deletion mutant, the progression of anaphase and cytokinesis is retarded and cell shape is aberrant. These defects are overcome by plasmids carrying the genes encoding subunits of the 20S cyclosome/APC or PP1. These results demonstrate functions other than promoting anaphase for the components of the 20S cyclosome/APC and also a close functional relationship of Sds23 with PP1 and 20S cyclosome/APC.     

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.