Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function.

Eukaryotic cell cycle progression requires the periodic activation and inactivation of a protein-serine/threonine kinase which in fission yeast is encoded by the cdc2+ gene. The activity of this gene product, p34cdc2, is controlled by numerous interactions with other proteins and by its phosphorylation state. In fission yeast, p34cdc2 is phosphorylated on two sites, one of which has been identified as Tyr15. Dephosphorylation of Tyr15 regulates the initiation of mitosis. To understand more completely the regulation of p34cdc2 kinase activity, we have identified the second site of phosphorylation as Thr167, a residue conserved amongst all p34cdc2 homologues. By analysing the phenotypes of cells expressing various position 167 mutations and performing in vitro experiments, we establish that Thr167 phosphorylation is required for p34cdc2 kinase activity at mitosis and is involved in the association of p34cdc2 with cyclin B. Dephosphorylation of Thr167 might also play a role in the exit from mitosis.     

Sce3, a suppressor of the Schizosaccharomyces pombe septation mutant cdc11, encodes a putative RNA-binding protein.

In the fission yeast Schizosaccharomyces pombe, the cdc11 gene is required for the initiation of septum formation at the end of mitosis. The sce3 gene was cloned as a multi-copy suppressor of the heat-sensitive mutant cdc11-136. When over-expressed, it rescues all mutants of cdc11 and also a heat-sensitive allele of cdc14, but not the cdc14 null mutant. Deletion shows that sce3 is not essential for cell proliferation. It encodes a putative RNA-binding protein which shows homology to human eIF4B. Immunolocalisation indicates that Sce3p is located predominantly in the cytoplasm. Elevated expression of sce3 increases the steady-state level of cdc14 mRNA. Possible mechanisms of its action are discussed.     

The dihydroceramide desaturase is not essential for cell viability in Schizosaccharomyces pombe

Recent studies have identified a new family of desaturase-like polypeptide sequences in many higher eukaryotes. Functional characterisation of one member of this family, from Schizosaccharomyces pombe, revealed the enzyme to be a sphingolipid desaturase. This S. pombe gene designated SDCB3b8.07c was identified as the dihydroceramide Delta(4)-desaturase, responsible for the synthesis of sphingosine. Homologous recombination was used to disrupt the endogenous S. pombe dihydroceramide Delta(4)-desaturase. Surprisingly, this had no effect on cell viability, indicating that sphingosine may not be crucial for normal S. pombe functions. This observation has implications for our understanding of the role of sphingosine and its phosphorylated metabolite sphingosine-1-phosphate in lower eukaryotes.     

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.     

Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.     

The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe

Plo1-associated casein kinase activity peaked during mitosis before septation. Phosphatase treatment abolished this activity. Mitotic Plo1 activation had a requirement for prior activation of M-phase promoting factor (MPF), suggesting that Plo1 does not act as a mitotic trigger kinase to initiate MPF activation during mitotic commitment. A link between Plo1 and the septum initiating network (SIN) has been suggested by the inability of plo1 Delta cells to septate and the prolific septation following plo1(+) overexpression. Interphase activation of Spg1, the G protein that modulates SIN activity, induced septation but did not stimulate Plo1-associated kinase activity. Conversely, SIN inactivation did not affect the mitotic stimulation of Plo1-associated kinase activity. plo1.ts4 cells formed a misshapen actin ring, but rarely septated at 36 degrees C. Forced activation of Spg1 enabled plo1.ts4 mutant cells, but not cells with defects in the SIN component Sid2, to convert the actin ring to a septum. The ability of plo1(+) overexpression to induce septation was severely compromised by SIN inactivation. We propose that Plo1 acts before the SIN to control septation.     

The Schizosaccharomyces pombe cdc14 gene is required for septum formation and can also inhibit nuclear division.

A conditional heat-sensitive mutation in the cdc14 gene of the fission yeast Schizosaccharomyces pombe results in failure to form a septum. Cells become highly elongated and multinucleate as growth and nuclear division continue in the absence of cell division. This article describes the cloning of the cdc14 gene and the identification of its product, a protein of 240 amino acids, p28cdc14. A null allele of the cdc14 gene shows that the gene is essential for septum formation and completion of the cell-division cycle. Overexpression of the gene product, p28cdc14, causes cell-cycle arrest in late G2 before mitosis. Cells leaking past the block activate p34cdc2 kinase and show condensed chromosomes, but the normal rearrangements of the microtubules and microfilaments that are associated with the transition from interphase to mitosis do not occur. Overexpression of p28cdc14 in mutants, in which the timing of mitosis is altered, suggests that these effects may be mediated upstream of the mitotic inhibitor wee1. These data are consistent with the idea that p28cdc14 may play a role in both the initiation of mitosis and septum formation and, by doing so, be part of the mechanism that coordinates these two cell-cycle events.     

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.     

Regulation of cdc2 activity in Schizosaccharomyces pombe: the role of phosphorylation.

The cdc2 protein kinase, first identified as a cell cycle gene required for transition into the S- and M-phases of budding and fission yeast, has been shown to act as a key component in the regulation of the eukaryotic cell cycle. The periodic activation of cdc2 kinase, which is required for entry into M-phase, is regulated by subunit association with cyclin B, the cdc25, wee1, mik1 gene products and differential phosphorylation of the cdc2 protein. Phosphorylation at Tyr 15 inhibits activation of the cdc2/cdc13 complex whereas phosphorylation of Thr 167 is required for kinase activity.     

Schizosaccharomyces pombe Noc3 is essential for ribosome biogenesis and cell division but not DNA replication

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.     

Schizosaccharomyces pombe rsm1 genetically interacts with spmex67, which is involved in mRNA export

We have previously isolated three synthetic lethal mutants from Schizosaccharomyces pombe in order to identify mutations in the genes that are functionally linked to spmex67 with respect to mRNA export. A novel rsm1 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMexl. The rsm1 gene contains no introns and encodes a 296 amino-acid-long protein with the RING finger domain, a C3HC4 in the N-terminal half. The deltarsm1 null mutant is viable, but it showed a slight poly(A)+ RNA accumulation in the nucleus. Also, the combination of deltarsm1 and deltaspmex67 mutations confers synthetic lethality that is accompanied by the severe poly(A)+ RNA export defect. These results suggest that rsm1 is involved in mRNA export from the nucleus.     

Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides

The adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is localized to the plasma membrane of the cell. The enzyme utilizes Mn2+/ATP as substrate and free Mn2+ ions as an effector. Unlike the baker yeast Saccharomyces cerevisiae, S. pombe adenylyl cyclase does not utilize Mg2+/ATP as substrate and the activity is not stimulated by guanyl nucleotides. The optimal pH for the S. pombe adenylyl cyclase activity is 6.0. The activity dependence on ATP is cooperative with a Hill coefficient of 1.68 +/- 0.14.