The kelch repeat protein,Tea1, is a potential substrate target of the p21-activated kinase,Shk1, in the fission yeast,Schizosaccharomyces pombe

The p21-activated kinase (PAK) homolog, Shk1, is a critical component of a multifunctional Ras/Cdc42/PAK complex required for viability, polarized growth and cell shape, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. Substrate targets of the Shk1 kinase have not previously been described. Here we show that the S. pombe cell polarity factor, Tea1, is directly phosphorylated by Shk1 in vitro. We demonstrate further that Tea1 is phosphorylated in S. pombe cells and that its level of phosphorylation is significantly reduced in cells defective in Shk1 function. Consistent with a role for Tea1 as a potential downstream effector of Shk1, we show that a tea1 null mutation rescues the Shk1 hyperactivity-induced lethal phenotype caused by loss of function of the essential Shk1 inhibitor, Skb15. All phenotypes associated with Skb15 loss, including defects in actin cytoskeletal organization, chromosome segregation, and cytokinesis, are suppressed by tea1 Delta, suggesting that Tea1 is a potential mediator of multiple Shk1 functions. S. pombe cells carrying a weak hypomorphic allele of shk1 together with a tea1 Delta mutation exhibit a cytokinesis defective phenotype that is significantly more severe than that observed in the respective single mutants, providing evidence that Shk1 and Tea1 cooperate to regulate cytokinesis. In addition, we show that S. pombe cells carrying the orb2-34 allele of shk1 exhibit a pattern of monopolar growth similar to that observed in tea1 Delta cells, suggesting that Shk1 and Tea1 may regulate one or more common processes involved in the regulation of polarized cell growth. Taken together, our results strongly implicate Tea1 as a potential substrate-effector of the Shk1 kinase.     

HIV-1 Vpr induces defects in mitosis, cytokinesis, nuclear structure, and centrosomes

Human immunodeficiency virus type 1 (HIV-1) Vpr is a 15-kDa accessory protein that contributes to several steps in the viral replication cycle and promotes virus-associated pathology. Previous studies demonstrated that Vpr inhibits G2/M cell cycle progression in both human cells and in the fission yeast Schizosaccharomyces pombe. Here, we report that, upon induction of vpr expression, fission yeast exhibited numerous defects in the assembly and function of the mitotic spindle. In particular, two spindle pole body proteins, sad1p and the polo kinase plo1p, were delocalized in vpr-expressing yeast cells, suggesting that spindle pole body integrity was perturbed. In addition, nuclear envelope structure, contractile actin ring formation, and cytokinesis were also disrupted. Similar Vpr-induced defects in mitosis and cytokinesis were observed in human cells, including aberrant mitotic spindles, multiple centrosomes, and multinucleate cells. These defects in cell division and centrosomes might account for some of the pathological effects associated with HIV-1 infection.     

The Schizosaccharomyces pombe aurora-related kinase Ark1 interacts with the inner centromere protein Pic1 and mediates chromosome segregation and cytokinesis

The chromosomal passenger proteins aurora-B, survivin, and inner centromere protein (INCENP) have been implicated in coordinating chromosome segregation with cell division. This work describes the interplay between aurora, survivin, and INCENP orthologs in the fission yeast Schizosaccharomyces pombe and defines their roles in regulating chromosome segregation and cytokinesis. We describe the cloning and characterization of the aurora-related kinase gene ark1(+), demonstrating that it is an essential gene required for sister chromatid segregation. Cells lacking Ark1p exhibit the cut phenotype, DNA fragmentation, and other defects in chromosome segregation. Overexpression of a kinase-defective version of Ark1, Ark1-K147R, inhibits cytokinesis, with cells exhibiting an elongated, multiseptate phenotype. Ark1p interacts physically and/or genetically with the survivin and INCENP orthologs Bir1p and Pic1p. We identified Pic1p in a two-hybrid screen for Ark1-K147R interacting partners and went on to map domains in both proteins that mediate their binding. Pic1p residues 925-972 are necessary and sufficient for Ark1p binding, which occurs through the kinase domain. As with Ark1-K147R, overexpression of Ark1p-binding fragments of Pic1p leads to multiseptate phenotypes. We also provide evidence that the dominant-negative effect of Ark1-K147R requires Pic1p binding, indicating that the formation of Ark1p-Pic1p complexes is required for the execution of cytokinesis.     

Schizosaccharomyces pombe RanGAP homolog, SpRna1, is required for centromeric silencing and chromosome segregation

We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP. Resulting Sprna1ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation. In addition to Sprna1+ and Spksp1+, the clr4+ (histone H3-K9 methyltransferase), the S. pombe gene, SPAC25A8.01c, designated snf2SR+ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1+ (S. pombe Ran homolog), rescued a lethality of Sprna1ts. Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres. Consistently, Sprna1ts was defective in gene-silencing at the centromeres. But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1ts strains, indicating SpRna1 in general did not function for a heterochromatin formation. snf2SR+ rescued a centromeric silencing defect and Deltaclr4+ was synthetic lethal with Sprna1ts. Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR. Loss of SpRna1 activity, therefore, caused chromosome missegregation.     

Identification and characterization of two novel proteins affecting fission yeast gamma-tubulin complex function

The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe gamma-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S. pombe.     

Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation

Cdc14-like phosphatases regulate a variety of cell cycle events by dephosphorylating CDK sites. Their cell cycle-dependent changes in localization may be important to carry out distinct functions. Work in budding and fission yeast suggested that Cdc14-like phosphatases are inhibited by nucleolar sequestration. In S. cerevisiae, Cdc14p is released from the nucleolus by the FEAR network and Cdk1, whereas the S. pombe CDC14-like phosphatase Clp1p (also known as Flp1p) is released at mitotic entry by an unknown mechanism. The mitotic exit network (MEN) in S. cerevisiae and its homologous network, the septation initiation network (SIN), in S. pombe act through an unknown mechanism to keep the phosphatase out of the nucleolus in late mitosis. SIN-dependent cytoplasmic maintenance of Clp1p is thought to be essential for the cytokinesis checkpoint, which blocks further rounds of nuclear division until cytokinesis is completed. By targeting Clp1p to the nucleus or the cytoplasm, we demonstrate distinct functions for these pools of Clp1p in chromosome segregation and cytokinesis, respectively. Our results further suggest that the SIN does not keep Clp1p out of the nucleolus by regulating nucleolar affinity, as proposed for S. cerevisiae Cdc14p, but instead, Clp1p may be regulated by nuclear import/export.     

S. pombe Pbh1p: an inhibitor of apoptosis domain containing protein is essential for chromosome segregation

Proteins containing the baculovirus inhibitor of apoptosis repeats (BIR domains) have been identified in a wide range of species. BIR domain containing proteins are thought to inhibit caspases and thereby cause inhibition of apoptosis. A BIR domain containing protein has been recently identified by the Schizosaccharomyces pombe genome sequencing project. However, caspase-like proteins have not been found in yeasts, suggesting that the BIR domain containing proteins might play a fundamental role in cell regulation, in addition to their well-characterized role in inhibition of apoptosis. In this study, we have characterized Pbh1p, an S. pombe BIR domain containing protein. Construction and analysis of a null mutant in pbh1+ revealed that pbh1+ is essential for cell viability. Moreover, cells devoid of Pbh1p are defective in chromosome condensation and chromosome segregation. Thus, proper chromosome segregation requires the function of Pbh1p. Over-production of Pbh1p led to abnormalities in mitosis and cytokinesis, suggesting that the levels of Pbh1p are important for regulation of mitosis and cytokinesis.     

Roles of the DYRK kinase Pom2 in cytokinesis, mitochondrial morphology, and sporulation in fission yeast

Pom2 is predicted to be a dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) related to Pom1 in Schizosaccharomyces pombe. DYRKs share a kinase domain capable of catalyzing autophosphorylation on tyrosine and exogenous phosphorylation on serine/threonine residues. Here we show that Pom2 is functionally different from the well-characterized Pom1, although they share 55% identity in the kinase domain and the Pom2 kinase domain functionally complements that of Pom1. Pom2 localizes to mitochondria throughout the cell cycle and to the contractile ring during late stages of cytokinesis. Overexpression but not deletion of pom2 results in severe defects in cytokinesis, indicating that Pom2 might share an overlapping function with other proteins in regulating cytokinesis. Gain and loss of function analyses reveal that Pom2 is required for maintaining mitochondrial morphology independently of microtubules. Intriguingly, most meiotic pom2Δ cells form aberrant asci with meiotic and/or forespore membrane formation defects. Taken together, Pom2 is a novel DYRK kinase involved in regulating cytokinesis, mitochondrial morphology, meiosis, and sporulation in fission yeast.     

Inactivation of the pre-mRNA cleavage and polyadenylation factor Pfs2 in fission yeast causes lethal cell cycle defects

Faithful chromosome segregation is fundamentally important for the maintenance of genome integrity and ploidy. By isolating conditional mutants defective in chromosome segregation in the fission yeast Schizosaccharomyces pombe, we identified a role for the essential gene pfs2 in chromosome dynamics. In the absence of functional Pfs2, chromosomal attachment to the mitotic spindle was defective, with consequent chromosome missegregation. Under these circumstances, multiple intracellular foci of spindle checkpoint proteins Bub1 and Mad2 were seen, and deletion of bub1 exacerbated the mitotic defects and the loss of cell viability that resulted from the loss of pfs2 function. Progression from G1 into S phase following release from nitrogen starvation also required pfs2+ function. The product of the orthologous Saccharomyces cerevisiae gene PFS2 is a component of a multiprotein complex required for 3'-end cleavage and polyadenylation of pre-mRNAs and, in keeping with the conservation of this essential function, an S. pombe pfs2 mutant was defective in mRNA 3'-end processing. Mutations in pfs2 were suppressed by overexpression of the putative mRNA 3'-end cleavage factor Cft1. These data suggest unexpected links between mRNA 3'-end processing and chromosome replication and segregation.     

Functional characterization of the fission yeast phosphatidylserine synthase gene, pps1, reveals novel cellular functions for phosphatidylserine

To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1Delta mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1Delta cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1Delta mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1Delta cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.     

RNAi-directed assembly of heterochromatin in fission yeast

Heterochromatin is an epigenetically heritable and conserved feature of eukaryotic chromosomes with important roles in chromosome segregation, genome stability, and gene regulation. The formation of heterochromatin involves an ordered array of chromatin changes, including histone deacetylation, histone H3-lysine 9 methylation, and recruitment of histone binding proteins such as Swi6/HP1. Recent discoveries have uncovered a role for the RNA interference (RNAi) pathway in heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and other eukaryotes. Purification of two RNAi complexes, RITS and RDRC, from fission yeast has provided further insight into the mechanism of RNAi-mediated heterochromatin assembly. These discoveries have given rise to a model in which small interfering RNA molecules act as specificity factors that initiate epigenetic chromatin modifications and double strand RNA synthesis at specific chromosome regions.     

Ibd1p, a possible spindle pole body associated protein, regulates nuclear division and bud separation in Saccharomyces cerevisiae

The proper spatial and temporal coordination of mitosis and cytokinesis is essential for maintaining genomic integrity. We describe the identification and characterization of the Saccharomyces cerevisiae IBD1 gene, which encodes a novel protein that regulates the proper nuclear division and bud separation. IBD1 was identified by the limited homology to byr4, a dosage-dependent regulator of cytokinesis in Schizosaccharomyces pombe. IBD1 is not an essential gene, and the knock-out cells show no growth defects except for the reduced mating efficiency [1]. However, upon ectopic expression from an inducible promoter, IBD1 is lethal to the cell and leads to abnormal nuclear division and bud separation. In detail, approximately 90% of the IBD1 overexpressing cells arrest at large bud stages with dividing or divided nuclei. In some IBD1 overexpressing cells, spindle elongation and chromosome separation occur within the mother cell, leading to anucleated and binucleate daughter cells. The anucleated cell can not bud, but the binucleate cell proceeds through another cell cycle(s) to produce a cell with multiple nuclei and multiple buds. Observations of the F-actin and chitin rings in the IBD1 overexpressing cells reveal that these cells lose the polarity for bud site selection and growth or attain the hyper-polarity for growth. Consistent with the phenotypes, the IBD1 overexpressing cells contain a broad range of DNA content, from 2 to 4 N or more. A functional Ibd1p-GFP fusion protein localizes to a single dot at the nuclear DNA boundary in the divided nuclei or to double dots in dividing nuclei, suggesting its localization on the spindle pole body (SPB). The cross-species expressions of IBD1 in S. pombe and byr4 in S. cerevisiae cause defects in shape, implicating the presence of a conserved mechanism for the control of cytokinesis in eukaryotes. We propose that Ibd1p is an SPB associated protein that links proper nuclear division to cytokinesis and bud separation.