PXA domain-containing protein Pxa1 is required for normal vacuole function and morphology in Schizosaccharomyces pombe

PhoX homology (PX) domain-containing proteins play critical roles in vesicular trafficking, protein sorting, and lipid modification in eukaryotic cells. Several proteins with PX domains contain an associated domain termed PXA (PX-associated). Although PXA domain-containing proteins are required for some important cellular processes, the function of the PXA domain is unknown. We identified three PXA domain-containing proteins in Schizosaccharomyces pombe. S. pombe Pxa1p (SPAC5D6.07c) contained only the PXA domain, not the PX domain. To elucidate the role of the PXA domain in eukaryotic cells, we constructed and characterized a disruption mutant, pxa1. The pxa1 disruptant contained enlarged vacuoles and exhibited mislocalization of vacuolar carboxypeptidase Y (CPY). The conversion rate from pro- to mature-CPY was greatly impaired in pxa1 cells, and fluorescence microscopy indicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. The mutants were also deficient in vacuolar sorting of a multivesicular body (MVB) marker, a ubiquitin-GFP-carboxypeptidase S (Ub-GFP-CPS) fusion protein. Taken together, these results indicate that Pxa1 protein is required for normal vacuole function and morphology in S. pombe.     

Schizosaccharomyces pombe histone acetyltransferase Mst1 (KAT5) is an essential protein required for damage response and chromosome segregation

Schizosaccharomyces pombe Mst1 is a member of the MYST family of histone acetyltransferases and is the likely ortholog of Saccharomyces cerevisiae Esa1 and human Tip60 (KAT5). We have isolated a temperature-sensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA-damaging agents and to the spindle poison thiabendazole. mst1 has an increased frequency of Rad22 repair foci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function, including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hip1 (Sc Hir1), and the Msc1 protein related to a family of histone demethylases. mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.     

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.     

CUE domain-containing protein Vps901 is required for vacuolar protein transport in Schizosaccharomyces pombe

The functions of two Schizosaccharomyces pombe Vps9-like genes, SPBC4F6.10/vps901(+) and SPBC29A10.11c/vps902(+), were characterized. Genomic sequence analysis predicted that Vps901p contains a VPS9 domain, whereas cDNA analyses revealed that Vps901p contains a CUE domain (coupling of ubiquitin to ER degradation) in its C-terminal region. Deletion of vps901(+) resulted in mis-sorting and secretion of S. pombe vacuolar carboxypeptidase Cpy1p, whereas deletion of vps902(+) had no effect, suggesting that only Vps901p functions in vacuolar protein transport in S. pombe. Deletion of vps901(+) further produced pleiotropic phenotypes, including vacuolar homotypic fusion and endocytosis defects. Heterologous expression of the budding yeast VPS9 gene corrected the CPY mis-sorting defect in vps901Δ cells. These findings suggest that the VPS9 domain of Vps901p is required for vacuolar protein trafficking in S. pombe.     

Nuclear RanGAP is required for the heterochromatin assembly and is reciprocally regulated by histone H3 and Clr4 histone methyltransferase in Schizosaccharomyces pombe

Although the Ran GTPase-activating protein RanGAP mainly functions in the cytoplasm, several lines of evidence indicate a nuclear function of RanGAP. We found that Schizosaccharomyces pombe RanGAP, SpRna1, bound the core of histone H3 (H3) and enhanced Clr4-mediated H3-lysine 9 (K9) methylation. This enhancement was not observed for methylation of the H3-tail containing K9 and was independent of SpRna1-RanGAP activity, suggesting that SpRna1 itself enhances Clr4-mediated H3-K9 methylation via H3. Although most SpRna1 is in the cytoplasm, some cofractionated with H3. Sprna1(ts) mutations caused decreases in Swi6 localization and H3-K9 methylation at all three heterochromatic regions of S. pombe. Thus, nuclear SpRna1 seems to be involved in heterochromatin assembly. All core histones bound SpRna1 and inhibited SpRna1-RanGAP activity. In contrast, Clr4 abolished the inhibitory effect of H3 on the RanGAP activity of SpRna1 but partially affected the other histones. SpRna1 formed a trimeric complex with H3 and Clr4, suggesting that nuclear SpRna1 is reciprocally regulated by histones, especially H3, and Clr4 on the chromatin to function for higher order chromatin assembly. We also found that SpRna1 formed a stable complex with Xpo1/Crm1 plus Ran-GTP, in the presence of H3.     

The Schizosaccharomyces pombe Cdc7 protein kinase required for septum formation is a client protein of Cdc37

Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7. Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.     

SGT1-HSP90 complex is required for CENP-A deposition at centromeres

The centromere plays an essential role in accurate chromosome segregation, and defects in its function lead to aneuploidy and thus cancer. The centromere-specific histone H3 variant CENP-A is proposed to be the epigenetic mark of the centromere, as active centromeres require CENP-A–containing nucleosomes to direct the recruitment of multiple kinetochore proteins. CENP-A K124 ubiquitylation, mediated by CUL4A-RBX1-COPS8 E3 ligase activity, is required for CENP-A deposition at the centromere. However, the mechanism that controls the E3 ligase activity of the CUL4A-RBX1-COPS8 complex remains obscure. We have discovered that the SGT1-HSP90 complex is required for recognition of CENP-A by COPS8. Thus, the SGT1-HSP90 complex contributes to the E3 ligase activity of the CUL4A complex that is necessary for CENP-A ubiquitylation and CENP-A deposition at the centromere.     

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.     

The gld1 + gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 ⁺), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1Δ cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 ⁺ is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 ⁺ and dak2 ⁺ encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1Δ strain showed a more severe reduction of growth on glycerol and DHA than the dak2Δ strain because the expression of dak1 ⁺ mRNA was higher than that of dak2 ⁺. In wild-type S. pombe, expression of the gld1 ⁺, dak1 ⁺, and dak2 ⁺ genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 ⁺ was regulated by glucose repression and that it was derepressed in scr1Δ and tup12Δ strains.     

Xlf1 is required for DNA repair by nonhomologous end joining in Schizosaccharomyces pombe

The accurate repair of DNA double-strand breaks is essential for cell survival and maintenance of genome integrity. Here we describe xlf1+, a gene in the fission yeast Schizosaccharomyces pombe that is required for repair of double-strand breaks by nonhomologous end joining during G1 phase of the cell cycle. Xlf1 is the ortholog of budding yeast Nej1 and human XLF/Cernunnos proteins.     

Identification of a SNARE protein required for vacuolar protein transport in Schizosaccharomyces pombe

Intracellular vesicle trafficking is mediated by a set of SNARE proteins in eukaryotic cells. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. A search of the Schizosaccharomyces pombe genome database revealed a total of 17 SNARE-related genes. Although no homologs of Vam3p, Nyv1p, and Vam7p have been found in S. pombe, we identified one SNARE-like protein that is homologous to S. cerevisiae Pep12p. However, the disruptants transport vacuolar hydrolase CPY (SpCPY) to the vacuole normally, suggesting that the Pep12 homolog is not required for vacuolar protein transport in S. pombe cells. To identify the SNARE protein(s) involved in Golgi-to-vacuole protein transport, we have deleted four SNARE homolog genes in S. pombe. SpCPY was significantly missorted to the cell surface on deletion of one of the SNARE proteins, Fsv1p (SPAC6F12.03c), with no apparent S. cerevisiae ortholog. In addition, sporulation, endocytosis, and in vivo vacuolar fusion appear to be normal in fsv1Delta cells. These results showed that Fsv1p is mainly involved in vesicle-mediated protein transport between the Golgi and vacuole in S. pombe cells.     

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.     

The tetraspan protein Dni1p is required for correct membrane organization and cell wall remodelling during mating in Schizosaccharomyces pombe

In fungi, success of mating requires that both cells agglutinate, modify their extracellular envelopes, and fuse their plasma membranes and nuclei to produce a zygote. Here we studied the role of the Schizosaccharomyces pombe Dni1 protein in the cell fusion step of mating. Dni1p is a tetraspan protein bearing a conserved cystein motif similar to that present in fungal claudin-related proteins. Dni1p expression is induced during mating and Dni1p concentrates as discrete patches at the cell–cell contact area and along the mating bridge. Proper Dni1p localization depends on Fus1p, actin and integrity of lipid rafts. In dni1 Δ mutants, cell differentiation and agglutination are as efficient as in the wild-type strain, but cell fusion is significantly reduced at temperatures above 25°C. We found that the defect in cell fusion was not associated with an altered cytoskeleton, with an abnormal distribution of Fus1p, or with a defect in calcium accumulation, but with a severe disorganization of the plasma membrane and cell wall at the area of cell–cell contact. These results show that Dni1p plays a relevant role in co-ordinating membrane organization and cell wall remodelling during mating, a function that has not been described for other proteins in the fission yeast.