Fission yeast cytoskeletons and cell polarity factors: connecting at the cortex

Cell polarity is a fundamental property of cells from unicellular to multicellular organisms. Most of the time, it is essential so that the cells can achieve their function. The fission yeast Schizosaccharomyces pombe is a powerful genetic model organism for studying the molecular mechanisms of the cell polarity process. Indeed, S. pombe cells are rod-shaped and cell growth is restricted at the poles. The accurate localization of the cell growth machinery at the cell cortex, which involves the actin cytoskeleton, depends on cell polarity pathways that are temporally and spatially regulated. The importance of interphase microtubules and cell polarity factors acting at the cortex of cell ends in this process has been shown. Here, we review recent advances in knowledge of molecular pathways leading to the establishment of a cellular axis in fission yeast. We also describe the role of cortical proteins and mitotic cytoskeletal rearrangements that control the symmetry of cell division.     

Physical and Chemical Properties of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Some physical and chemical properties of the extracellular lipase from the thermophilic fungus, Humico la lanuginosa S–38, were investigated. The results were as follows: Sedimentation coefficient was 2.4 × 10^?13 (cm-g/sec-dyne); diffusion coefficient was 8.8 × 10^?7 (cm²/sec); and frictional coefficient was 1.22. Molecular weight was 27,500±500 and α-helix content was 18.9%. The number of amino acid residues contained in 1 mole of protein of Humicola lipase was 224. Sugar and lipid were not detected. The effect of calcium ion and denaturing reagents, such as urea, sodium dodecyl sulfate and dithiothreitol, on the thermostability of Humicola lipase was examined. It was concluded that the thermostability of Humicola lipase was not influenced by protective cofactors but was attributable to the enzyme itself. Some properties of enzyme structure which were concerned with the thermostability of Humicola lipase are also discussed.     

Sulfur amino acid metabolism in Schizosaccharomyces pombe: occurrence of two O-acetylhomoserine sulfhydrylases and the lack of the reverse transfulfuration pathway

Abstract The fission yeast Schizosaccharomyces pombe has a unique organization of sulfur amino acid metabolism: it has two distinct O -acetylhomoserine sulfhydrylases (homocysteine synthases). Similar to Enterobacteriaceae, S. pombe lacks cystathionine β-synthase and cystathionine γ-lyase - the enzymes of the reverse transsulfuration pathway, by which methionine is readily metabolized to cysteine - a likely effector in the sulfur metabolite repression system. Consequently no repression of sulfate assimilation is observed when methionine is added to the growth medium.     

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.     

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.     

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.     

Ca~(2+)在粟酒裂殖酵母细胞周期时相中的作用

以粟酒裂殖酵母(Schizosaccharomyces pombe)为研究材料,研究了Ca~(2+)在细胞周期时相中的作用。当外源Ca~(2+)浓度在0.5-20 mmol/L范围内,随Ca~(2+)浓度增加,细胞增殖速度加快,延滞期逐渐缩短。但SD-Ca（CaCl2省略）并不能终止Sch. pombe的细胞周期。采用缺氮对群体细胞进行同步化,并以EGTA 螯合培养介质中低浓度的Ca~(2+),Sch. pombe 细胞增殖被完全抑制,细胞流式法测定结果表明：细胞周期被终止在G1期。分析认为Ca~(2+) 对Sch. pombe 细胞增殖是必不可少的,外源Ca~(2+)在G1期向S期转化过程中起着关键性的作用。     

外源钙调素对粟酒裂殖酵母细胞增殖的影响

外源钙调素（CaM）对粟酒裂殖酵母（Schizosaccharomycespombe）细胞增殖的影响。实验结果表明外源CaM能明显抑制粟酒裂殖酵母细胞的增殖,其作用方式是延长了粟酒裂殖酵母细胞生长的延滞期。抗粟酒裂殖酵母CaM抗体、TFP及Phenyl-SepharoseCL－4B能降低CaM对细胞生长的抑制作用,而Ca2+及Ca2+螫合剂EGTA对CaM的抑制作用均无影响。以上结果提示,外源CaM对粟酒裂殖酵母细胞增殖的抑制作用可能是由于胞外CaM激活了细胞膜上的Ca2+泵,使胞内Ca2+浓度降低所致。     

TFP刺激裂殖酵母细胞钙离子内流的质膜效应

ＴＦＰ（１０－１００μｍｏｌ／Ｌ）可引起裂殖酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ）胞外Ｃａ２＋内流,ＴＦＰ浓度不同,促进Ｃａ２＋内流程度也不一样,５０μｍｏｌ／ＬＴＦＰ的促进作用最大。并且ＴＦＰ浓度越大,Ｃａ２＋内流出现峰值也越早,１０、２０、５０、１００μｍｏｌ／ＬＴＦＰ处理后,胞内总钙出现峰值时间分别为４５、４５、３０、１５分钟。胞外Ｈ＋浓度也会对ＴＦＰ引起的Ｃａ２＋内流产生不同影响,缓冲液的ｐＨ值为６．０时最有利于ＴＦＰ引起胞内Ｃａ２＋含量增加,碱性条件下ＴＦＰ的效果最不明显。由ＴＦＰ引起的Ｃａ２＋内流增加要比单一地增加外钙浓度效果好得多,ＴＦＰ在１０μｍｏｌ／Ｌ浓度的外钙条件下引起的胞内钙含量数值比１０００μｍｏｌ／Ｌ的外钙条件而无ＴＦＰＴ所引起的胞内钙含量还要高５３．９％。缓冲液中加入０．８％的钙离子通道阻断剂ＬａＣ１３或溶液中无葡萄糖的存在,ＴＦＰ的促进作用消失,说明ＴＦＰ促进Ｃａ２＋内流是通过钙离子通道来完成的并需要能量参与。     

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.     

Purification and Some Properties of Cyclomaltodextrin Glucanotransferase from Bacillus circulans

Cyclomaltodextrin glucanotransferase was purified from B. circulans C31 through two successive steps of starch and Biogel column chromatography. The enzyme was purified up to 90-fold with a 30% yield. Its molecular weight was around 103,000. The purified enzyme converted 28% of the soluble starch to β-cyclodextrin at pH 7.0 and a substrate concentration of 5%. The optimum pH for the enzyme was found to be 5.5. The optimum temperature was 60°C. The enzyme optimum was stable from pH 5.5~9.0 and up to 50°C.     

TFP刺激裂殖酵母细胞钙离子内流的质膜效应*

ＴＦＰ（１０－１００μｍｏｌ／Ｌ）可引起裂殖酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ）胞外Ｃａ２＋内流,ＴＦＰ浓度不同,促进Ｃａ２＋内流程度也不一样,５０μｍｏｌ／ＬＴＦＰ的促进作用最大。并且ＴＦＰ浓度越大,Ｃａ２＋内流出现峰值也越早,１０、２０、５０、１００μｍｏｌ／ＬＴＦＰ处理后,胞内总钙出现峰值时间分别为４５、４５、３０、１５分钟。胞外Ｈ＋浓度也会对ＴＦＰ引起的Ｃａ２＋内流产生不同影响,缓冲液的ｐＨ值为６．０时最有利于ＴＦＰ引起胞内Ｃａ２＋含量增加,碱性条件下ＴＦＰ的效果最不明显。由ＴＦＰ引起的Ｃａ２＋内流增加要比单一地增加外钙浓度效果好得多,ＴＦＰ在１０μｍｏｌ／Ｌ浓度的外钙条件下引起的胞内钙含量数值比１０００μｍｏｌ／Ｌ的外钙条件而无ＴＦＰＴ所引起的胞内钙含量还要高５３．９％。缓冲液中加入０．８％的钙离子通道阻断剂ＬａＣ１３或溶液中无葡萄糖的存在,ＴＦＰ的促进作用消失,说明ＴＦＰ促进Ｃａ２＋内流是通过钙离子通道来完成的并需要能量参与。     

Possible Anti-tumor Promoting Properties of Marine Algae and In Vivo Activity of Wakame Seaweed Extract

The cAMP pathway and the Ras pathway are the two major pathways to sexual development in the fission yeast Schizosaccharomyces pombe. To understand the с AMP pathway or the related pathway, we analyzed mutants that display a phenotype similar to cyrl-, that is, hyper-sporulation. Nine mutants termed sam (sporulation abnormal mutant), which are highly inclined to sexual development despite the presence of nitrogen sources, were partially characterized. Cyclic AMP was detected in all nine sam mutant cells, and over-expression of the adenylyl cyclase gene (cyrl) failed to suppress the hyper-sporulation phenotype of these sam mutants, suggesting that none of the sam mutants were likely to be allelic to cyrl. Epistatic tests of sam mutants showed that they were divided into two dominant and seven recessive mutants. Dominants were able to make spores in sam/sam⁺ heterodiploid cells upon abundant nutrients. Both two dominant mutants bypassed the inability to make spores in rasl deficient diploid cells, suppressed the deficiency to execute sporulation in byr2 deficient diploid cells, but failed to suppress the byr1 deficiency. Two dominant mutations seem not to occur within the byr2 gene.     

Purification of a Lipolytic Acyl-hydrolase from Phaseolus vulgaris Leaves by Affinity Chromatography on Palmitoylated Gauze and Its Properties

A lipolytic acyl-hydrolase was purified about 220-fold from the homogenate of the leaves of Phaseolus vulgaris L. cv. Kurodane-kinugasa by acetone precipitation, affinity chromatography on a palmitoylated gauze column and isoelectric focusing. The purified enzyme showed a single protein band by polyacrylamide gel disc electrophoresis. The enzyme had an isoelectric point of 4.4 and a molecular weight of about 90,000. It had pH optima of 5.5 and 6.5, and Km values of 0.24 and 0.53 mm for monogalactosyldiacylglycerol and phosphatidylcholine, respectively. The pH dependences were changed by Triton X–100. No separation of these two hydrolyzing activities were achieved, and the ratio of the specific activity of galactolipase to that of phospholipase (about 3/1) remained constant throughout the purification procedures. Both the activities changed in parallel with each other by the addition of reagents and by heat treatment. The enzyme clearly catalyzed the deacylation of the several classes of glyco- and phospholipids. These results suggest that a single enzyme is responsible for both the activities.     

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.     

Cloning and sequencing of a gene encoding pyruvate kinase from Schizosaccharomyces pombe; implications for quaternary structure and regulation of the enzyme

Abstract A cDNA encoding pyruvate kinase from Schizosaccharomyces pombe has been isolated from a lambda ZAPII library. This cDNA was sequenced and found to contain an open reading frame of 1524 nucleotides, giving a predicted protein subunit M _r of 55 470. The sequence shows a high degree of identity with other pyruvate kinase sequences, with residues implicated in the binding of substrate and metal ion co-factors conserved. However, there are significant differences in the putative subunit interface and effector binding regions which may account for the unusual quaternary structure and regulatory properties of the S. pombe enzyme.