Npp106p, a Schizosaccharomyces pombe nucleoporin similar to Saccharomyces cerevisiae Nic96p, functionally interacts with Rae1p in mRNA export.

To identify components of the mRNA export machinery in Schizosaccharomyces pombe, a screen was developed to identify mutations that were synthetically lethal with the conditional mRNA export allele rae1-167. Mutations defining three complementation groups were isolated, and here we report the characterization of npp106 (for nuclear pore protein of 106 kDa). This gene encodes a predicted protein that has significant similarity to the Nic96p nucleoporin of Saccharomyces cerevisiae. Consistent with Npp106p being a nucleoporin, a functional green fluorescent protein (GFP)-tagged Npp106p localized to the nuclear periphery. In contrast to NIC96, the npp106 gene is not essential. Moreover, a delta npp106 mutant did not show cytoplasmic mislocalization of a simian virus 40 nuclear localization signal-GFP-LacZ reporter protein, and a fraction of cells had accumulation of poly(A)+ RNA in the nucleus. A consequence of the synthetic lethality between rae1-167 and npp106-1 was the accumulation of poly(A)+ RNA in the nucleus when cells were grown under synthetic lethal conditions. In addition to npp106-1, which is a nonsense mutation that truncates the protein at amino acid 292, the delta npp106 mutation was synthetically lethal with rae1-167, suggesting that the synthetic lethality is a consequence of the loss of a function of npp106. We further demonstrate that a region between amino acids 74 and 348 of Npp106p is required for complementation of the synthetic lethality. These results uncover a potential direct or indirect involvement of Npp106p in mRNA export.     

Analysis of the cps1 gene provides evidence for a septation checkpoint in Schizosaccharomyces pombe

The fission yeast gene cps1, which encodes the catalytic subunit of β-glucan synthase, was isolated in a screen for mutants that show an increase in ploidy at the restrictive temperature. cps1 mutants display defects in both polarity and septation at the permissive temperature, and become swollen and multinucleate at the restrictive temperature. Analysis of the interaction of cps1 with other mutations suggests the existence of a septation checkpoint, which requires the activity of the protein kinase wee1 for function.     

Schizosaccharomyces pombe Hsk1p is a potential cds1p target required for genome integrity

The fission yeast Hsk1p kinase is an essential activator of DNA replication. Here we report the isolation and characterization of a novel mutant allele of the gene. Consistent with its role in the initiation of DNA synthesis, hsk1(ts) genetically interacts with several S-phase mutants. At the restrictive temperature, hsk1(ts) cells suffer abnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replication arrest. Interestingly, hsk1(ts) mutants released to the restrictive temperature after early S-phase arrest in hydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormal mitosis. These findings indicate a second role for hsk1 subsequent to HU arrest. Consistent with a later S-phase role, hsk1(ts) is synthetically lethal with Deltarqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed by Deltacds1 (RAD53 kinase) mutants. We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro. These results indicate that the Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replication initiation for normal mitosis.     

Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.     

The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe

Abstract: Plants and the fission yeast Schizosaccharomyces pombe synthesize small cadmium-binding peptides, called phytochelatins, in response to cadmium. Derived from glutathione (GSH: λ-Glu-Cys-Gly), they have the general structure (λ-Glu-Cys) n Gly, where n is 2–11. In order to study the biosynthesis of phytochelatins, we used the mutagen N -methyl- N '-nitro- N nitrosoguanidine (MNNG) to select mutants with a lowered GSH content. GSH-deficient mutants show a Cd-sensitive phenotype, whereas resistance to Cu is only slightly influenced. These Cd-sensitive mutants contain 2–15% of the wild-type GSH level. For three mutants a lowered activity of λ-glutamylcysteine synthetase was measured. One of the mutants was transformed to Cd-resistance and the complementing fragment was analyzed further. The complementing fragment hybridized with chromosome III. In the transformants, GSH content was restored up to wild-type levels, whereas the activity of λ-glutamylcysteine synthetase was significantly increased compared with the wild-type. Possible mechanisms for Cd-resistance in the transformants are discussed.     

The alpha-glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe

BACKGROUND INFORMATION: In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall. RESULTS: We identified a novel gene in Schizosaccharomyces pombe, namely the agn1(+) gene that has homology to fungal 1,3-alpha-glucanases (mutanases). Disruption of the agn1(+) gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1(+) is responsible for the 1,3-alpha-glucanase activity, which shows a maximum at the end of septation. CONCLUSIONS: Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.     

Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe.

Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin-based contractile ring. A division septum is formed centripetally, concomitant with ring constriction. Although several genes essential for cytokinesis have been described previously, enzymes that participate in the assembly of the division septum have not been identified. Here we describe a temperature-sensitive mutation, drc1-191, that prevents division septum assembly and causes mutant cells to arrest with a stable actomyosin ring. Unlike the previously characterized cytokinesis mutants, which undergo multiple mitotic cycles, drc1-191 is the first cytokinesis mutant that arrests with two interphase nuclei. Interestingly, unlike drc1-191, drc1-null mutants proceed through multiple mitotic cycles, leading to the formation of large cells with many nuclei. drc1 is allelic to cps1, which encodes a 1,3-beta-glucan synthase subunit. We conclude that Drc1p/Cps1p is not required for cell elongation and cell growth, but plays an essential role in assembly of the division septum. Furthermore, it appears that constriction of the actomyosin ring might depend on assembly of the division septum. We discuss possible mechanisms that account for the differences in the phenotypes of the drc1-191 and the drc1-null mutants and also reflect the potential links between Drc1p and other cytokinesis regulators.     

Cloning and characterization of the kinesin-related protein,Krp1p,in Schizosaccharomyces pombe

Kinesin have been cloned in many organisms. They played important roles in the transport of cell organelles, polarized growth, and secretion. We report here the identification of a kinesin-related protein in Schizosaccharomyces pombe, which was named kinesin-related protein (Krplp). The primer sequences were driven from the highly conserved area of the kinesin genes in other organisms. We cloned kinesin genes from S. pombe using the PCR technique. Sequence analysis revealed that krp1+ has a 1,665 bp open-reading frame (ORF) that encoded a protein that consisted of 554 amino acids with a molecular weight of 61,900. It is homologous to the proteins that belong to the kinesin heavy chain (KHC) superfamily [GenBank accession No. AF156966 (genomic DNA) and AF247188 (mRNA)]. To characterize Krplp, the gene was disrupted and overexpressed in S. pombe. Cells that contained a krp1+ null allele were viable. Overexpression of Krp1p resulted in the inhibition of mitotic growth; cells became elongated, branched, and formed aberrant septa. To identify proteins that interact with Krplp, the yeast two-hybrid system was used. As a result, the novel protein, designated kinesin associated protein (Kap1p), was identified and showed structural homology to the proteins of the myosin family (GenBank accession No. AF351206). The data from the overexpression and two-hybrid study of Krplp may provide information that Krplp can have roles in cytokinesis with myosin.     

Mex67p of Schizosaccharomyces pombe interacts with Rae1p in mediating mRNA export

We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Deltamex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A)(+) RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 Deltamex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149-505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149-505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149-505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.     

Nif1, a novel mitotic inhibitor in Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, the activity of the M-phase-inducing Cdc2/Cdc13 cyclin-dependent kinase is inhibited by Wee1 and Mik1 tyrosine kinases, and activated by Cdc25 and Pyp3 tyrosine phosphatases. Cdc2/Cdc13 activity is also indirectly regulated by the approximately 70 kDa Nim1 (Cdrl) serine/threonine kinase, which promotes mitosis by inhibiting Wee1 via direct phosphorylation. To understand better the function and regulation of Nim1, the yeast two-hybrid system was used to isolate S.pombe cDNA clones encoding proteins that interact with Nim1. Sixteen of the 17 cDNA clones were derived from the same gene, named nif1 + (nim1 interacting factor-1). Nif1 is a novel approximately 75 kDa protein containing a leucine zipper motif. The Nif1-Nim1 interaction requires a small region of Nim1 that immediately follows the N-terminal catalytic domain. This region is required for Nim1 activity both in vivo and in vitro. delta nif1 mutants are approximately 10% smaller than wild type, indicating that Nif1 is involved in inhibiting the onset of mitosis. Consistent with this proposal, overproduction of Nif1 was found to cause a cell elongation phenotype that is very similar to delta nim1 mutants. Nif1 overproduction causes cell cycle arrest in cells that are partly defective for Cdc25 activity, but has no effect in delta nim1 or delta wee1 mutants. Nif1 also inhibits Nim1-mediated phosphorylation of Wee1 in an insect cell expression system. These observations strongly suggest that Nif1 negatively regulates the onset of mitosis by a novel mechanism, namely inhibiting Nim1 kinase.     

Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe

The Schizosaccharomyces pombe Cdc4 protein is required for the formation and function of the contractile ring, presumably acting as a myosin light chain. By using NMR spectroscopy, we demonstrate that purified Cdc4p is a monomeric protein with two structurally independent domains, each exhibiting a fold reminiscent of the EF-hand class of calcium-binding proteins. Although Cdc4p has one potentially functional calcium-binding site, it does not bind calcium in vitro. Three variants of Cdc4p containing single point mutations responsible for temperature-sensitive arrest of the cell cycle at cytokinesis (Gly-19 to Glu, Gly-82 to Asp, and Gly-107 to Ser) were also characterized by NMR and circular dichroism spectroscopy. In each case, the amino acid substitution only leads to small perturbations in the conformation of the protein. Furthermore, thermal unfolding studies indicate that, like wild-type Cdc4p, the three mutant forms are all extremely stable, remaining completely folded at temperatures significantly above those causing failure of cytokinesis in intact cells. Therefore, the altered phenotype must arise directly from a disruption of the function of Cdc4p rather than indirectly through a disruption of its overall structure. Several mutant alleles of Cdc4p also show interallelic complementation in diploid cells. This phenomenon can be explained if Cdcp4 has more than one essential function or, alternatively, if two mutant proteins assemble to form a functional complex. Based on the structure of Cdc4p, possible models for interallelic complementation including interactions with partner proteins and the formation of a myosin complex with Cdc4p fulfilling the role of both an essential and regulatory light chain are proposed.     

Isolation and characterization of Nrf1p, a novel negative regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ts) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1(+), encoded an approximately 15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Deltanrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1(+) also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.     

Cloning and regulation of Schizosaccharomyces pombe thi2, a gene involved in thiamine biosynthesis.

Biosyntheses of the pyrimidine and thiazole moieties of the thiamine molecule occur by separate pathways. In Schizosaccharomyces pombe, a gene, thi2, is responsible for thiazole synthesis [Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We have cloned a 3.1-kb genomic S. pombe fragment which can functionally complement a thi2 mutant. The fragment maps genetically at the thi2 site, indicating that it carries thi2. As shown by Northern hybridization analysis, the appearance of thi2 mRNA levels is repressed when cells are grown in the presence of thiamine and 5-(2-hydroxyethyl)-4-methylthiazole. The thi3 gene involved in the biosynthesis of the pyrimidine moiety, is also regulated by thiamine [Maundrell, J. Biol. Chem. 265 (1990) 10857-10864; Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We previously identified and analyzed four regulatory genes (tnr1, tnr2, tnr3, and thi1) that are responsible for the regulation of thi3 [Schweingruber et al., Genetics (1992) in press]. Mutants defective in these regulatory genes affect expression of thi2 in a similar way to thi3. This indicates that biosynthesis of the pyrimidine and thiazole moieties are under common genetic control in S. pombe.     

std1, a Gene Involved in Glucose Transport in Schizosaccharomyces pombe

A wild-type strain, Sp972 h⁻, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [¹⁴C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S. pombe, unlike in Saccharomyces cerevisiae. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617–2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.     

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.     

Gef1p and Scd1p,the Two GDP-GTP exchange factors for Cdc42p,form a ring structure that shrinks during cytokinesis in Schizosaccharomyces pombe

Fission yeast Cdc42p, a small GTPase of the Rho family, is essential for cell proliferation and maintenance of the rod-like cell morphology. Scd1/Ral1p is a GDP-GTP exchange factor (GEF) for Cdc42p. This study and a parallel study by others establish that Gef1p is another GEF for Cdc42p. Deletions of gef1 and scd1 are synthetically lethal, generating round dead cells, and hence mimic the phenotype of cdc42 deletion. Gef1p is localized mainly to the cell division site. Scd1p is also there, but it is also detectable in other parts of the cell, including the nucleus, growing ends, and the tips of conjugation tubes. Gef1p and Scd1p form a ring structure at the cell division site, which shrinks during cytokinesis following the contraction of the actomyosin ring. Formation of the Gef1p/Scd1p ring apparently depends on the integrity of the actomyosin ring. In turn, recruitment of Cdc42p to the cell division site follows the shrinking Gef1p/Scd1p ring; the Cdc42p accumulates like a closing iris. These observations suggest that Gef1p and Scd1p may have a role in mediating between contraction of the actomyosin ring and formation of the septum, by recruiting active Cdc42p to the septation site.     

Tpr1, a Schizosaccharomyces pombe protein involved in potassium transport.

The Schizosaccharomyces pombe Tpr1 was isolated as suppressor of the Saccharomyces cerevisiae Delta trk1,2 potassium uptake deficient phenotype. Tpr1, for tetratrico peptide repeat, encodes a 1039 amino acid residues protein with several reiterated TPR units displaying significant homology to p150(TSP), a recently identified phosphoprotein of mouse, to S. cerevisiae CTR9 and to related sequences of human, Caenorhabditis elegans, Methanoccocus jannaschii and Arabidopsis thaliana. Expression of Tpr1 restored growth on 0.2 mM K(+) media, induced K(+) transport with a K(T) of 4.6 mM and resumed inward currents of -90 pA at -250 mV (pH 7.2) conducting K(+) and other alkali-metal ions. The tetratrico peptide repeat is a degenerate motif of 34 amino acids that is repeated several times within TPR-containing proteins and has been suggested to mediate protein-protein interactions. The sequence and putative binding properties of Tpr1 suggest the protein unlikely as transporter but involved in the enhancement of K(+) uptake via conventional carriers.