K+ fluxes in Schizosaccharomyces pombe

All living cells accumulate high concentrations of K+ in order to keep themselves alive. To this end they have developed a great diversity of transporters. The internal level of K+ is the result of the net balance between the activities of the K+ influx and the K+ efflux transporters. Potassium fluxes have been extensively studied and characterized in Saccharomyces cerevisiae. However, this is not the case in the fission yeast and, in addition, the information available indicates that both yeasts present substantial and interesting differences. In this paper we have reviewed and summarized the information on K+ fluxes in Schizosaccharomyces pombe. We have included some unpublished results recently obtained in our laboratory and, in particular, we have highlighted the significant differences found between the well-known yeast S. cerevisiae and the fission yeast Sch. pombe.     

Identification of 24-methylene-24,25-dihydrolanosterol as a precursor of ergosterol in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus

Abstract Study of the plasma membrane sterol composition in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus revealed the presence of ergosterol, lanosterol, dehydroergosterol, fecosterol, episterol and 24-methylene-24,25-dihydrolanosterol (eburicol), a C-31 derivative. The growth of both yeasts in the presence of ketoconazole led to a decrease by 85% of the ergosterol content while the levels of lanosterol and eburicol increased. This suggests that in the biosynthetic pathway of ergosterol in Schizosaccharomyces species, the transmethylation process on the C-24 may occur directly on lanosterol and not only on zymosterol. On the other hand, it cannot be excluded that in the genus Schizosaccharomyces two routes exist from lanosterol to ergosterol: the classical one via a direct C-14, C-4 demethylation of lanosterol and the second one via the formation of a C-31 derivative followed by demethylations.     

Carbon and energy balances in cell-recycle cultures of Schizosaccharomyces pombe

A strain of the fission yeast Schizosaccharomyces pombe was aerobically grown in a cell-recycle fermentor under various operating conditions, i.e., different bleeding rates and various separate feed rates of glucose and basal medium. Carbon and energy balances were analyzed during steady-state culture regimes, allowing growth yields and maintenance coefficients to be determined under glucose-limited and glucose-excess environments. Special attention was given to the metabolic shift from purely oxidative to respirofermentative glucose catabolism resulting from a change in the growth-limiting factor. No maintenance requirements for the carbon source and for energy were observed during glucose-limited culture regimes and oxidative catabolism. Under glucose excess and respirofermentative metabolism, the m(G) coefficient was shown to be growth-linked, whereas the enhancement of the apparent m(e) coefficient observed for increased residual glucose concentrations could be assigned to a decline in the ATP yield. (c) 1993 John Wiley & Sons, Inc.     

Identification of Open Reading Frames in Schizosaccharomyces pombe cDNAs

A total of 214 non-overlapping cDNA clones from Schizosaccharomycespombe were selected and completely sequenced. The clones notpreviously reported were divided into the following three groups:1) homologous to Saccharomyces cerevisiae genes (139 clones);2) homologous to genes from other organisms but not to thosefrom Sac. cerevisiae (4 clones); and 3) no similar sequences(40 clones). Among the 31 sequences identical to those in thepublic databases, 4 genes have regions corresponding to introns.Protein sequences which had homologs both in budding yeast andmammals were compared with those from Sac. cerevisiae and mammals.The search revealed that the evolutionary distances among thesespecies are similar at least with genes of this category.     

Pgt1, a glutathione transporter from the fission yeast Schizosaccharomyces pombe

The Schizosaccharomyces pombe ORF, SPAC29B12.10c, a predicted member of the oligopeptide transporter (OPT) family, was identified as a gene encoding the S. pombe glutathione transporter ( Pgt1 ) by a genetic strategy that exploited the requirement of the cys1a Δ strain of S. pombe (which is defective in cysteine biosynthesis) for either cysteine or glutathione, for growth. Disruption of the ORF in the cys1a Δ strain led to an inability to grow on glutathione as a source of cysteine. Cloning and subsequent biochemical characterization of the ORF revealed that a high-affinity transporter for glutathione ( K _m=63 μM) that was found to be localized to the plasma membrane. The transporter was specific for glutathione, as significant inhibition in glutathione uptake could be observed only by either reduced or oxidized glutathione, or glutathione conjugates, but not by dipeptides or tripeptides. Furthermore, although glu–cys–gly, an analogue of glutathione (γ-glu–cys–gly), could be utilized as a sulphur source, the growth was not Pgt1 dependent. This further underlined the specificity of this transporter for glutathione. The strong repression of pgt1⁺ expression by cysteine suggested a role in scavenging glutathione from the extracellular environment for the maintenance of sulphur homeostasis in this yeast.     

Apoptosis and lipoapoptosis in the fission yeast Schizosaccharomyces pombe

Yeasts being simple eukaryotes are established genetic systems that are often employed to solve important biological questions. Recently, it has become evident that certain cell death programs exist in these unicellular organisms. For example, it has been shown recently that strains of the fission yeast Schizosaccharomyces pombe deficient in triacylglycerol synthesis undergo cell death with prominent apoptotic markers. This minireview is intended to discuss key developments that have rendered fission yeast useful both as a tool and as a model for apoptosis and lipoapoptosis research. It is attempted to delineate a putative signaling pathway leading to the execution of lipoapoptosis in the fission yeast. Although in its infancy, apoptosis research in the fission yeast promises exciting breakthroughs in the near future.     

Synthesis and degradation of polyphosphate in the fission yeast Schizosaccharomyces pombe: mutations in phosphatase genes do not affect polyphosphate metabolism

The fission yeast Schizosaccharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on arginine as the nitrogen source. Upon transfer to a medium without phosphate, polyphosphate was degraded and served as an endogenous phosphate reserve. When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphosphate than they had contained before starvation, a phenomenon known as over-compensation. Strains carrying mutated structural genes for three different phosphatases, pho1, pho2 or pho3, degraded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again.     

The POL1 gene from the fission yeast, Schizosaccharomyces pombe, shows conserved amino acid blocks specific for eukaryotic DNA polymerases alpha

Summary The POL1 gene of the fission yeast, Schizosaccharomyces pombe, was isolated using a POL1 gene probe from the budding yeast Saccharomyces cerevisiae, cloned and sequenced. This gene is unique and located on chromosome II. It includes a single 91 by intron and is transcribed into a mRNA of about 4500 nucleotides. The predicted protein coded for by the S. pombe POL1 gene is 1405 amino acid long and its calculated molecular weight is about 160000 daltons. This peptide contains seven amino acid blocks conserved among several DNA polymerases from different organisms and shares overall 37% and 34% identity with DNA polymerases alpha from S. cerevisiae and human cells, respectively. These results indicate that this gene codes for the S. pombe catalytic subunit of DNA polymerase alpha. The comparisons with human DNA polymerase alpha and with the budding yeast DNA polymerases alpha, delta and epsilon reveal conserved blocks of amino acids which are structurally and/or functionally specific only for eukaryotic alpha-type DNA polymerases.     

Characterization of a Schizosaccharomyces pombe morphological mutant altered in the galactomannan content

In a search for Schizosaccharomyces pombe mutants resistant to the antifungal agent papulacandin B, a morphological mutant was isolated. The mutant is round shaped in contrast to the rod shaped parental strain. This morphological defect segregated as a recessive Mendelian character and was not observed in other papulacandin B resistant mutants belonging to the same complementation group. The mutation mapped in the right arm of S. pombe chromosome III very close to pap1 marker. Mutant cell walls were more susceptible to alkali extraction and Novozyme degradation than those from the wild-type. A specific reduction in the cell wall galactomannan fraction was the only significant difference detected as compared to the wild-type strain. Levels of beta (1,3)-glucan and mannan synthases as well as other enzymic periplasmic mannoproteins were very similar in wild type and mutant strains.     

Involvement of Moc1 in Sexual Development and Survival of Schizosaccharomyces pombe

The moc1 gene in Schizosaccharomyces pombe was found as to overcome sterility caused by high expression of adenylyl cyclase. The moc1 gene was found to be identical with sds23 and psp1. Although psp1 has been reported to be essential for growth, sds23 has not been. To clarify this apparent discrepancy, we first assessed independently the phenotypes of the moc1 disruptant. We confirmed that the deletion mutant of moc1 is sterile, sensitive to high salt, and grows slowly at higher and lower temperatures, and that mutant cells are elongated. Besides these phenotypes, we found that viability of the moc1 disruptant was rapidly lost at the stationary phase. We confirmed that the Moc1 protein is phosphorylated in the stationary phase and also under nitrogen-starved conditions. We examined the significance of this phosphorylation of Moc1 by creating the S333A or S333D mutant Moc1. Interestingly, while S333D mutant Moc1 is lower in inducing sexual development, S333A mutant Moc1 is higher in this than the wild type, suggesting that phosphorylation of Moc1 affects sexual development. The other phenotypes, such as sensitivity to high salt and higher temperature and elongation of cells, were not affected by mutation of S333A nor S333D. We found that Moc1-GFP localized to both the cytosol and the nucleus during mitotic growth, but accumulated in the nucleus in mating cells and then enriched in spores, and that this localization shift was negatively regulated by the cAMP pathway. This and the observations above suggest that nuclear localized Moc1 is an inducer of sexual development. Thus, in addition to the roles of moc1/sds23/psp1 in mitosis and stress response, it is also important for the survival and sexual development of fission yeast, but phosphorylation of Moc1 only affects the sexual development.     

Identification and characterization of a gene required for α1,2-mannose extension in the O-linked glycan synthesis pathway in Schizosaccharomyces pombe

The KTR α1,2-mannosyltransferase gene family of Saccharomyces cerevisiae is responsible not only for outer-chain modifications of N -linked oligosaccharides but also for elongation of O -linked mannose residues. To identify genes involved in the elongation step of O -linked oligosaccharide chains in Schizosaccharomyces pombe , we characterized six genes, omh1 ⁺ –omh6 ⁺, that share significant sequence similarity to the S. cerevisiae KTR family. Six deletion strains were constructed, each carrying a single disrupted omh allele. All strains were viable, indicating that none of the omh genes was essential. Heterologous expression of a chitinase from S. cerevisiae in the omh mutants revealed that O -glycosylation of chitinase had decreased in omh1 Δ cells, but not in the other mutants, indicating that the other omh genes do not appear to be required for O -glycan synthesis. Addition of the second α1,2-linked mannose residue was blocked in omh1 Δ cells. An Omh1–GFP fusion protein was found to be localized in the Golgi apparatus. These results indicate that Omh1p plays a major role in extending α1,2-linked mannose in the O -glycan pathway in S. pombe .     

粟酒裂殖酵母全基因组中含信号肽蛋白质的研究

对粟酒裂殖酵母全基因组3条染色体上的4,997个蛋白序列进行了全局性的分析,利用signalP3.0软件分析这些蛋白的N-末端信号肽序列, 预测有N-末端分泌信号肽序列的蛋白196个;利用TMpred 软件分析跨膜结构, 预测跨膜蛋白117个; 使用PrositeScan程序分析膜脂蛋白的脂结合位点, 预测有膜脂结合蛋白13个, 进而预测分泌性蛋白序列66个。使用Target P分析66个分泌蛋白的蛋白序列, 研究这些蛋白在细胞中的定位。这些分泌蛋白的功能涉及粟酒裂殖酵母的营养、生殖、细胞间以及细胞与环境间的交流等许多方面, 对细胞的生存和繁殖有重要意义, 在系统生物学的研究中有重要参考价值。粟酒裂殖酵母分泌组的研究也将为粟酒裂殖酵母作为药物筛选模型以及开发为外源蛋白表达的宿主提供基础。     

The proteolytic system of the fission yeast Schizosaccharomyces pombe

Proteinase and peptidase activities of the fission yeast Schizosaccharomyces pombe were investigated. Several intracellular proteolytic enzymes were found: two endoproteinases, one carboxypeptidase, one aminopeptidase and one dipeptidyl-aminopeptidase. In addition, proteinase inhibitors were detected. In fresh crude extracts an activation procedure is needed to measure maximal activities of endoproteinases and carboxypeptidase, whose level is markedly dependent on growth medium composition and on growth phase, while aminopeptidase and dipeptidyl-aminopeptidase activities are very little, if at all, regulated by the carbon source.     

裂殖酵母Cnb1参与胞质分裂过程

丝/苏氨酸特异性钙调磷酸酶(Calcineurin, CN)是一种在真核生物中广泛存在的蛋白, 是参与转录调控的重要分子。裂殖酵母中的CN是由催化亚基Ppb1和调节亚基Cnb1组成的异源二聚体。文章报道了裂殖酵母中cnb1+的缺失引起细胞生长速度缓慢, 产生多隔膜现象, 胞质分裂受阻滞。胞质分裂过程中, Cnb1与Ppb1组成CN复合物, 与收缩环在分裂平面上共定位, 并与收缩环一起收缩。cnb1Δ菌株的隔膜成熟过程存在缺陷, 微管出现纵穿隔膜的现象。上述结果说明Cnb1可能参与隔膜的成熟过程。此外, 还检测了cnb1D菌株中胞裂蛋白的信号。胞裂蛋白包括Spn1、Spn2、Spn3和Spn4, 它们是引导隔膜降解的重要分子。结果显示, 在cnb1D菌株中, 80%左右的细胞在隔膜处缺失Spn2和Spn3的信号, 20%左右的细胞缺失Spn1和Spn4的信号。由于胞裂蛋白的蛋白表达量在cnb1D中没有降低, 因此胞裂蛋白信号的消失不是转录缺陷引起的, 这暗示Cnb1可能采用了不依赖转录的方式来调控胞裂蛋白环的稳定性。以上结果提示, Cnb1可能通过影响隔膜的成熟及胞裂蛋白环的稳定性参与调节裂殖酵母的胞质分裂过程。     

Fission stories: using PomBase to understand Schizosaccharomyces pombe biology

PomBase (www.pombase.org), the model organism database (MOD) for the fission yeast Schizosaccharomyces pombe, supports research within and beyond the S. pombe community by integrating and presenting genetic, molecular, and cell biological knowledge into intuitive displays and comprehensive data collections. With new content, novel query capabilities, and biologist-friendly data summaries and visualization, PomBase also drives innovation in the MOD community.     

Adaptive response of Schizosaccharomyces pombe to hydrogen peroxide

Abstract Schizosaccharomyces pombe becomes resistant to killing by high concentration of hydrogen peroxide and other severe stresses including oxidants, high temperature and high concentration of ethanol when pretreated with nonlethal levels of hydrogen peroxide. In the presence of the protein synthesis inhibitor, cycloheximide, during hydrogen peroxide pretreatment, the cell obtained partial resistance to a higher level of hydrogen peroxide. The partial resistance to hydrogen peroxide in the presence of cycloheximide was acquired within 30 min of pretreatment but complete resistance obtained with de novo protein synthesis was not attained before 45 min of pretreatment. During adaptation to hydrogen peroxide, at least 15 polypeptides are induced, as analyzed by two-dimensional gel electrophoresis. Catalase activity is induced eight-fold by treatment with a nonlethal level of hydrogen peroxide.     

粟酒裂殖酵母中ppr基因缺失引起细胞絮凝的机制研究

粟酒裂殖酵母中PPR蛋白在线粒体和叶绿体的RNA代谢中起重要作用,ppr基因的缺失会引起线粒体功能异常(ppr5除外)。旨在揭示粟酒裂殖酵母中ppr基因缺失引起细胞絮凝的机制。利用含有半乳糖的培养基分别培养WT、Δppr3、Δppr4、Δppr6和Δppr10,观察野生型和突变体的生长表型;通过添加不同的金属离子及改变细胞表面pH来探究诱导这类突变体出现絮凝的因素;采用RT-PCR技术检测缺陷菌中编码细胞表面絮凝因子及细胞壁重构酶基因的表达情况。研究结果表明:ppr3、ppr4、ppr6和ppr10的敲除会引起由Ca²⁺依赖型半乳糖结合蛋白参与的细胞絮凝;不同的pH和不同金属离子对这些缺陷菌的细胞絮凝有影响;ppr3、ppr4、ppr6和ppr10的缺失会使细胞内编码絮凝因子和细胞壁重构酶基因表达上调。线粒体功能异常会诱导絮凝基因表达上调和细胞壁属性发生改变,从而产生絮凝现象。     

Candida albicans exoglucanase as a reporter gene in Schizosaccharomyces pombe

The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37 degrees C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.     

Activation of neutral trehalase by fermentable sugars and cAMP in the fission yeast Schizosaccharomyces pombe

Abstract Derepressed cells of Schizosaccharomyces pombe 972 h⁻ suspended in the presence of glucose or other fermentable sugars displayed a transient activation of trehalase which was not blocked by cycloheximide. Repressed cells were unable to show glucose-induced trehalase stimulation. Nitrogen sources, protonophores or uncouplers failed to produce direct trehalase activation but increased the activity of the enzyme in the presence of glucose. Exogenous cAMP induced a rapid and pronounced stimulation of trehalase in both repressed and derepressed cells suggesting that the response to glucose includes activation of adenylate cyclase as part of a cAMP signalling pathway that increases the catalytic activity of trehalase by enzyme modification.