Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

Modeling the control of DNA replication in fission yeast

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.

Negative regulation of mitosis in fission yeast by the Shk1interacting protein Skb1 and its human homolog, Skb1Hs

We previously provided evidence that the protein encoded by the highly conserved skb1 gene is a putative regulator of Shk1, a p21Cdc42/Rac-activated kinase (PAK) homolog in the fission yeast Schizosaccharomyces pombe. skb1 null mutants are viable and competent for mating but less elongate than wild-type S. pombe cells, whereas cells that overexpress skb1 are hyperelongated. These phenotypes suggest a possible role for Skb1 as a mitotic inhibitor. Here we show genetic interactions of both skb1 and shk1 with genes encoding key mitotic regulators in S. pombe. Our results indicate that Skb1 negatively regulates mitosis by a mechanism that is independent of the Cdc2-activating phosphatase Cdc25 but that is at least partially dependent on Shk1 and the Cdc2 inhibitory kinase Wee1. We provide biochemical evidence for association of Skb1 and Shk1 with Cdc2 in S. pombe, suggesting that Skb1 and Shk1 inhibit mitosis through interaction with the Cdc2 complex, rather than by an indirect mechanism. These results provide evidence of a previously undescribed role for PAK-related protein kinases as mitotic inhibitors. We also describe the cloning of a human homolog of skb1, SKB1Hs, and show that it can functionally replace skb1 in S. pombe. Thus, the molecular functions of Skb1-related proteins have likely been substantially conserved through evolution.

Liz1p, a Novel Fission Yeast Membrane Protein, Is Required for Normal Cell Division When Ribonucleotide Reductase Is Inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1+, which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1− cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1+ encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

The Win1 Mitotic Regulator Is a Component of the Fission Yeast Stress-activated Sty1 MAPK Pathway

The fission yeast Sty1 mitogen-activated protein (MAP) kinase (MAPK) and its activator the Wis1 MAP kinase kinase (MAPKK) are required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAPKs, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, UV light, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have recently identified two upstream regulators of the Wis1 MAPKK, namely the Wak1 MAPKKK and the Mcs4 response regulator. Cells lacking Mcs4 or Wak1, however, are able to proliferate under stressful conditions and undergo sexual differentiation, suggesting that additional pathway(s) control the Wis1 MAPKK. We now show that this additional signal information is provided, at least in part, by the Win1 mitotic regulator. We show that Wak1 and Win1 coordinately control activation of Sty1 in response to multiple environmental stresses, but that Wak1 and Win1 perform distinct roles in the control of Sty1 under poor nutritional conditions. Our results suggest that the stress-activated Sty1 MAPK integrates information from multiple signaling pathways.

Active Nucleocytoplasmic Shuttling Required for Function and Regulation of Stress-Activated Kinase Spc1/StyI in Fission Yeast

Transcriptional induction of many stress-response genes is dependent on stress-induced nuclear accumulation of stress-activated protein kinases (SAPKs). In the fission yeast Schizosaccharomyces pombe, nuclear accumulation of the SAPK Spc1 (also known as StyI) requires activating phosphorylation catalyzed by the SAPK kinase Wis1; however, it is unknown whether the localization of Spc1 is regulated by nuclear transport factors. Herein are reported studies that show that Spc1 localization is regulated by active transport mechanisms during osmotic stress. Nuclear import of Spc1 requires Pim1, a homologue of the guanine nucleotide exchange factor RCC1 that is essential for nucleocytoplasmic shuttling of proteins. Nuclear export of Spc1 is regulated by the export factor Crm1. An Spc1–Crm1 complex forms as Spc1 is exported from the nucleus. Wis1 and the tyrosine phosphatases Pyp1 and Pyp2 that inactivate Spc1 are excluded from the nucleus by a Crm1-independent mechanism; hence the nuclear import of Spc1 leads to transient isolation from its regulatory proteins. Thus, active nucleocytoplasmic shuttling is required for both the function and regulation of Spc1 during the osmotic shock response.

Ssp1 Promotes Actin Depolymerization and Is Involved in Stress Response and New End Take-Off Control in Fission Yeast

The ssp1 gene encodes a protein kinase involved in alteration of cell polarity in Schizosaccharomyces pombe. ssp1 deletion causes stress sensitivity, reminiscent of defects in the stress-activated MAP kinase, Spc1; however, the two protein kinases do not act through the same pathway. Ssp1 is localized mainly in the cytoplasm, but after a rise in external osmolarity it is rapidly recruited to the plasma membrane, preferentially to active growth zones and septa. Loss of Ssp1 function inhibits actin relocalization during osmotic stress, in cdc3 and cdc8 mutant backgrounds, and in the presence of latrunculin A, implicating Ssp1 in promotion of actin depolymerization. We propose a model in which Ssp1 can be activated independently of Spc1 and can partially compensate for its loss. The ssp1 deletion mutant exhibited monopolar actin distribution, but new end take-off (NETO) could be induced in these cells by exposure to KCl or to latrunculin A pulse treatment. This treatment induced NETO in cdc10 cells arrested in G1 but not in tea1 cells. This suggests that cells that contain intact cell end markers are competent to undergo NETO throughout interphase, and Ssp1 is involved in generating the NETO stimulus by enlarging the actin monomer pool.

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role of ste9+ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10ts, cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase.

Cyclin B Proteolysis and the Cyclin-dependent Kinase Inhibitor rum1p Are Required for Pheromone-induced G1 Arrest in Fission Yeast

The blocking of G1 progression by fission yeast pheromones requires inhibition of the cyclin-dependent kinase cdc2p associated with the B-cyclins cdc13p and cig2p. We show that cyclosome-mediated degradation of cdc13p and cig2p is necessary for down-regulation of B-cyclin–associated cdc2p kinase activity and for phermone-induced G1 arrest. The cyclin-dependent kinase inhibitor rum1p is also required to maintain this G1 arrest; it binds both cdc13p and cig2p and is specifically required for cdc13p proteolysis. We propose that rum1p acts as an adaptor targeting cdc13p for degradation by the cyclosome. In contrast, the cig2p–cdc2p kinase can be down-regulated, and the cyclin cig2p can be proteolyzed independently of rum1p. We suggest that pheromone signaling inhibits the cig2p–cdc2p kinase, bringing about a transient G1 arrest. As a consequence, rum1p levels increase, thus inhibiting and inducing proteolysis of the cdc13p–cdc2p kinase; this is necessary to maintain G1 arrest. We have also shown that pheromone-induced transcription occurs only in G1 and is independent of rum1p.

Heat Stress Activates Fission Yeast Spc1/StyI MAPK by a MEKK-Independent Mechanism

Fission yeast Spc1/StyI MAPK is activated by many environmental insults including high osmolarity, oxidative stress, and heat shock. Spc1/StyI is activated by Wis1, a MAPK kinase (MEK), which is itself activated by Wik1/Wak1/Wis4, a MEK kinase (MEKK). Spc1/StyI is inactivated by the tyrosine phosphatases Pyp1 and Pyp2. Inhibition of Pyp1 was recently reported to play a crucial role in the oxidative stress and heat shock responses. These conclusions were based on three findings: 1) osmotic, oxidative, and heat stresses activate Spc1/StyI in wis4 cells; 2) oxidative stress and heat shock activate Spc1/StyI in cells that express Wis1AA, in which MEKK consensus phosphorylation sites were replaced with alanine; and 3) Spc1/StyI is maximally activated in Δpyp1 cells. Contrary to these findings, we report: 1) Spc1/StyI activation by osmotic stress is greatly reduced in wis4 cells; 2) wis1-AA and Δwis1 cells have identical phenotypes; and 3) all forms of stress activate Spc1/StyI in Δpyp1 cells. We also report that heat shock, but not osmotic or oxidative stress, activate Spc1 in wis1-DD cells, which express Wis1 protein that has the MEKK consensus phosphorylation sites replaced with aspartic acid. Thus osmotic and oxidative stress activate Spc1/StyI by a MEKK-dependent process, whereas heat shock activates Spc1/StyI by a novel mechanism that does not require MEKK activation or Pyp1 inhibition.

Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2+ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2+; overexpression of atb2+ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

Multiple Domains of Fission Yeast Cdc19p (MCM2) Are Required for Its Association with the Core MCM Complex

The members of the MCM protein family are essential eukaryotic DNA replication factors that form a six-member protein complex. In this study, we use antibodies to four MCM proteins to investigate the structure of and requirements for the formation of fission yeast MCM complexes in vivo, with particular regard to Cdc19p (MCM2). Gel filtration analysis shows that the MCM protein complexes are unstable and can be broken down to subcomplexes. Using coimmunoprecipitation, we find that Mis5p (MCM6) and Cdc21p (MCM4) are tightly associated with one another in a core complex with which Cdc19p loosely associates. Assembly of Cdc19p with the core depends upon Cdc21p. Interestingly, there is no obvious change in Cdc19p-containing MCM complexes through the cell cycle. Using a panel of Cdc19p mutants, we find that multiple domains of Cdc19p are required for MCM binding. These studies indicate that MCM complexes in fission yeast have distinct substructures, which may be relevant for function.

Effects of Genome Position and the DNA Damage Checkpoint on the Structure and Frequency of sod2 Gene Amplification in Fission Yeast

The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing both sod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2 amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications of sod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.

Mutational Effect of Fission Yeast Polα on Cell Cycle Events

Polα is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations in polα+. Germinating spores carrying either a deletion of polα+ (polαΔ) or a structurally intact but catalytically dead polα mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polα, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with a polαts allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polαts in cdc20 (polε mutant) background arrested with a cdc phenoytpe, not a polαts-like phenotype. At 25°C, replication perturbation caused by polαts alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36°C, replication disruption caused by polαts alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polα is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polαts mutations induce differential downstream kinase responses.

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.