Project description:We have identified a family of six hexose transporter genes (Ght1 to Ght6) in the fission yeast Schizosaccharomyces pombe. Sequence homology to Saccharomyces cerevisiae and mammalian hexose transporters (Hxtp and GLUTp, respectively) and secondary-structure predictions of 12 transmembrane domains for each of the Ght proteins place them into the sugar porter subfamily within the major facilitator superfamily. Interestingly, among this sugar porter family, the emerging S. pombe hexose transporter family clusters are separate from monosaccharide transporters of other yeasts (S. cerevisiae, Kluyveromyces lactis, and Candida albicans) and of humans, suggesting that these proteins form a distinct structural family of hexose transporters. Expression of the Ght1, Ght2, Ght5, and Ght6 genes in the S. cerevisiae mutant RE700A may functionally complement its D-glucose uptake-deficient phenotype. Northern blot analysis and reverse transcription-PCR showed that among all Ght's of S. pombe, Ght5 is the most prominently expressed hexose transporter. Ght1p, Ght2p, and Ght5p displayed significantly higher specificities for D-glucose than for D-fructose. Analysis of the previously described S. pombe D-glucose transport-deficient mutant YGS-5 revealed that this strain is defective in the Ght1, Ght5, and Ght6 genes. Based on an analysis of three S. pombe strains bearing single or double mutations in Ght3 and Ght4, we conclude that the Ght3p function is required for D-gluconate transport in S. pombe. The function of Ght4p remains to be clarified. Ght6p exhibited a slightly higher affinity to D-fructose than to D-glucose, and among the Ght's it is the transporter with the highest specificity for D-fructose.
Project description:The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.
Project description:We describe a new chronological lifespan (CLS) assay for the yeast Schizosaccharomyces pombe. Yeast CLS assays monitor the loss of cell viability in a culture over time, and this new assay shows a continuous decline in viability without detectable regrowth until all cells in the culture are dead. Thus, the survival curve is not altered by the generation of mutants that can grow during the experiments, and one can monitor the entire lifespan of a strain until the number of viable cells has decreased over 10(6)-fold. This CLS assay recapitulates the evolutionarily conserved features of lifespan shortening by over nutrition, lifespan extension by caloric restriction, increased stress resistance of calorically restricted cells and lifespan control by the AKT kinases. Both S. pombe AKT kinase orthologs regulate CLS: loss of sck1(+) extended lifespan in over nutrition conditions, loss of sck2(+) extended lifespan under both normal and over nutrition conditions, and loss of both genes showed that sck1(+) and sck2(+) control different longevity pathways. The longest-lived S. pombe cells showed the most efficient cell cycle exit, demonstrating that caloric restriction links these two processes. This new S. pombe CLS assay will provide a valuable tool for aging research.
Project description:The Spt5-Spt4 complex regulates early transcription elongation by RNA polymerase II and has an imputed role in pre-mRNA processing via its physical association with mRNA capping enzymes. Here we characterize the Schizosaccharomyces pombe core Spt5-Spt4 complex as a heterodimer and map a trypsin-resistant Spt4-binding domain within the Spt5 subunit. A genetic analysis of Spt4 in S. pombe revealed it to be inessential for growth at 25 degrees C-30 degrees C but critical at 37 degrees C. These results echo the conditional spt4Delta growth phenotype in budding yeast, where we find that Saccharomyces cerevisiae and S. pombe Spt4 are functionally interchangeable. Complementation of S. cerevisiae spt4Delta and a two-hybrid assay for Spt4-Spt5 interaction provided a readout of the effects of 33 missense and truncation mutations on S. pombe Spt4 function in vivo, which were interpreted in light of the recent crystal structure of S. cerevisiae Spt4 fused to a fragment of Spt5. Our results highlight the importance of the Spt4 Zn2+-binding residues--Cys12, Cys15, Cys29, and Asp32--and of Ser57, a conserved constituent of the Spt4-Spt5 interface. The 990-amino acid S. pombe Spt5 protein has an exceptionally regular carboxyl-terminal domain (CTD) composed of 18 nonapeptide repeats. We find that as few as three nonamer repeats sufficed for S. pombe growth, but only when Spt4 was present. Synthetic lethality of the spt5(1-835) spt4Delta double mutant at 34 degrees C suggests that interaction of Spt4 with the central domain of Spt5 overlaps functionally with the Spt5 CTD.
Project description:<h4>Background</h4>Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date.<h4>Methodology/principal findings</h4>We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe.<h4>Conclusions/significance</h4>We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/).
Project description:Glutathione synthetase predicted from the reported gene sequence from Schizosaccharomyces pombe is substantially smaller than the equivalent protein predicted from the cDNAs sequenced from Arabidopsis thaliana, Saccharomyces cerevisiae and other eukaryotes. Sequence alignments of the proteins encoded by the cDNA clones for glutathione synthetase from Arabidopsis and S. pombe show that the Arabidopsis protein contains 200 extra amino acids at the N-terminus. In order to test if this sequence is essential in the function of the protein, the full-length Arabidopsis protein and as two N-terminal deletions (Delta67-71 and Delta67-200) were expressed in S. pombe mutant MN101, which lacks endogenous glutathione synthetase activity. Although the wild-type plant cDNA could complement the yeast mutation, neither deletion mutant was able to restore glutathione-dependent cadmium resistance. When the three proteins were expressed as fusion proteins in Escherichia coli, they accumulated to the same level, but only the plasmid containing the full-length cDNA, pFLAG222, produced detectable enzyme activity in vitro. These results suggested that the N-terminus of the Arabidopsis glutathione synthetase is essential for its function and opened up the possibility that there was a sequencing error in the reported S. pombe sequence. Therefore the gsh2 sequence from wild-type S. pombe and the mutant strain MN101 were determined. The wild-type S. pombe gsh2 encodes a protein that is about the same length as that found in Arabidopsis, and the MN101 mutation involves a frameshift mutation early in the glutathione synthetase reading frame.
Project description:The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.
Project description:The fission yeast Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway. Elevated cAMP levels activate protein kinase A (PKA) to inhibit transcription of genes involved in sexual development and gluconeogenesis, including the fbp1(+) gene, which encodes fructose-1,6-bisphosphatase. Glucose-mediated activation of PKA requires the function of nine glucose-insensitive transcription (git) genes, encoding adenylate cyclase, the PKA catalytic subunit, and seven "upstream" proteins required for glucose-triggered adenylate cyclase activation. We describe the cloning and characterization of the git10(+) gene, which is identical to swo1(+) and encodes the S. pombe Hsp90 chaperone protein. Glucose repression of fbp1(+) transcription is impaired by both git10(-) and swo1(-) mutant alleles of the hsp90(+) gene, as well as by chemical inhibition of Hsp90 activity and temperature stress to wild-type cells. Unlike the swo1(-) mutant alleles, the git10-201 allele supports cell growth at 37 degrees , while severely reducing glucose repression of an fbp1-lacZ reporter, suggesting a separation-of-function defect. Sequence analyses of three swo1(-) alleles and the one git10(-) allele indicate that swo1(-) mutations alter core functional domains of Hsp90, while the git10(-) mutation affects the Hsp90 central domain involved in client protein binding. These results suggest that Hsp90 plays a specific role in the S. pombe glucose/cAMP pathway.
Project description:Mediator is an evolutionary conserved coregulator complex required for transcription of almost all RNA polymerase II-dependent genes. The Schizosaccharomyces pombe Mediator consists of two dissociable components-a core complex organized into a head and middle domain as well as the Cdk8 regulatory subcomplex. In this work we describe a functional characterization of the S. pombe Mediator. We report the identification of the S. pombe Med20 head subunit and the isolation of ts alleles of the core head subunit encoding med17+. Biochemical analysis of med8(ts), med17(ts), Deltamed18, Deltamed20 and Deltamed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell-surface proteins. Head domain-associated mutants display a hyphal growth phenotype due to defective expression of factors required for cell separation regulated by transcription factor Ace2. Comparison with Saccharomyces cerevisiae Mediator expression data reveals that these functionally distinct modules are conserved between S. pombe and S. cerevisiae.
Project description:Essential nucleoporins Nup98 and Nup96 are coded by a single open reading frame, and produced by autopeptidase cleavage. The autocleavage site of Nup98-Nup96 is highly conserved in a wide range of organisms. To understand the importance of autocleavage, we examined a mutant that produces the Nup98-Nup96 joint molecule as a sole protein product of the nup189 (+) gene in the fission yeast Schizosaccharomyces pombe. Cells expressing only the joint molecule were found to be viable. This result indicates that autocleavage of Nup98-Nup96 is dispensable for cell growth, at least under normal culture conditions in S. pombe.