Sexual co-flocculation by heterothallic cells of the fission yeast Schizosaccharomyces pombe modulated by medium constituents

Novel simple synthetic media for inducing sexual co-flocculation in a short time after mixing heterothallic fission-yeast (Schizosaccharomyces pombe) cells of h^- and h⁺ were devised; The most effective of these, mannose synthetic medium (MSM), contains 0.4% mannose as a carbon source in addition to galactose, KH₂PO₄ (pH4.0) and 4 vitamins. The addition of galactose to the medium suppressed the asexual self-flocculation but rather promoted the sexual co-flocculation. By transferring and mixing h^- and h⁺ cells grown in malt-extract broth plus galactose into MSM, these heterothallic strains were revealed to be sexually ready through a long period of the log to stationary phases. Furthermore, a variety of C sources and NH₄Cl at various concentrations in various media were examined for their effects upon sexual co-flocculation, conjugation and sporulation; it was found that the sugar concentration strictly affected the progress of the sequence of sexual reproduction at 26°C but not 30°C and that sexual co-flocculation of the heterothallic strains was induced only under lower concentrations of C and N source than that for the homothallic one.     

Complex Formation,Phosphorylation, and Localization of Protein Kinase A of Schizosaccharomyces pombe upon Glucose Starvation

Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.     

HIV-1 Vpr-induced cell death in Schizosaccharomyces pombe is reminiscent of apoptosis

Human immunodeficiency virus type 1 （HIV-1） Vpr induces cell death in mammalian and fssion yeast cells, suggesting that Vpr may affect a conserved cellular process. It is unclear, however, whether Vpr-induced yeast cell death mimics Vpr-mediated apoptosis in mammalian cells. We have recently identified a number of Vpr suppressors that not only suppress Vpr-induced cell death in fission yeast, but also block Vpr-induced apoptosis in mammalian cells. These findings suggest that Vpr-induced cell death in yeast may resemble some of the apoptotic processes of mammalian cells. The goal of this study was to develop and validate a fission yeast model system for future studies of apoptosis. Similar to Vpr-induced apoptosis in mammalian cells, we show here that Vpr in fission yeast promotes phosphatidylserine externalization and induces hyperpolarization of mitochondria, leading to changes of mitochondrial membrane potential. Moreover, Vpr triggers production of reactive oxygen species （ROS）, indicating that the apoptotic-like cell death might be mediated by ROS. Interestingly, Vpr induces unique morphologic changes in mitochondria that may provide a simple marker for measuring the apoptotic-like process in fission yeast. To verify this possibility, we tested two Vpr suppressors （EF2 and Hspl6） that suppress Vpr-induced apoptosis in mammalian cells in addition to a newly identified Vpr suppressor （Skpl）. All three proteins abolished cell death mediated by Vpr and restored normal mitochondrial morphology in the yeast cells. In conclusion, Vpr-induced cell death in fission yeast resembles the mammalian apoptotic process. Fission yeast may thus potentially be used as a simple model organism for the future study of the apoptotic-like process induced by Vpr and other proapoptotic agents.     

Hydrocellulase of Aspergillus aculeatus

Conditions for extracellular production of vitamin B₆ compounds (B₆), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leu1 strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B₆ and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B₆ and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H₂O₂ increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and in contrast, increased pyridoxine.     

tRNA production links nutrient conditions to the onset of sexual differentiation through the TORC1 pathway

Target of rapamycin (TOR) kinase controls cell growth and metabolism in response to nutrient availability. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1) promotes vegetative growth and inhibits sexual differentiation in the presence of ample nutrients. Here, we report the isolation and characterization of mutants with similar phenotypes as TORC1 mutants, in that they initiate sexual differentiation even in nutrient‐rich conditions. In most mutants identified, TORC1 activity is downregulated and the mutated genes are involved in tRNA expression or modification. Expression of tRNA precursors decreases when cells undergo sexual differentiation. Furthermore, overexpression of tRNA precursors prevents TORC1 downregulation upon nitrogen starvation and represses the initiation of sexual differentiation. Based on these observations, we propose that tRNA precursors operate in the S. pombe TORC1 pathway to switch growth mode from vegetative to reproductive.     

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.     

Pom1 gradient buffering through intermolecular auto‐phosphorylation

Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod‐shaped Schizosaccharomyces pombe cells, the DYRK‐family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4‐phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto‐phosphorylation. Here, we demonstrate that Pom1 auto‐phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system's properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto‐phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super‐linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration‐dependent autocatalysis may be a widely used simple feedback to buffer biological activities.     

Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose

The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.     

Distinct levels in Pom1 gradients limit Cdr2 activity and localization to time and position division

Where and when cells divide are fundamental questions. In rod-shaped fission yeast cells, the DYRK-family kinase Pom1 is organized in concentration gradients from cell poles and controls cell division timing and positioning. Pom1 gradients restrict to mid-cell the SAD-like kinase Cdr2, which recruits Mid1/Anillin for medial division. Pom1 also delays mitotic commitment through Cdr2, which inhibits Wee1. Here, we describe quantitatively the distributions of cortical Pom1 and Cdr2. These reveal low profile overlap contrasting with previous whole-cell measurements and Cdr2 levels increase with cell elongation, raising the possibility that Pom1 regulates mitotic commitment by controlling Cdr2 medial levels. However, we show that distinct thresholds of Pom1 activity define the timing and positioning of division. Three conditions—a separation-of-function Pom1 allele, partial downregulation of Pom1 activity, and haploinsufficiency in diploid cells—yield cells that divide early, similar to pom1 deletion, but medially, like wild-type cells. In these cells, Cdr2 is localized correctly at mid-cell. Further, Cdr2 overexpression promotes precocious mitosis only in absence of Pom1. Thus, Pom1 inhibits Cdr2 for mitotic commitment independently of regulating its localization or cortical levels. Indeed, we show Pom1 restricts Cdr2 activity through phosphorylation of a C-terminal self-inhibitory tail. In summary, our results demonstrate that distinct levels in Pom1 gradients delineate a medial Cdr2 domain, for cell division placement, and control its activity, for mitotic commitment.     

Dipeptidyl aminopeptidase yspI mutants of Schizosaccharomyces pombe: Genetic mapping of dpa1+ on chromosome III

Abstract A mutant strain of Schizosaccharomyces pombe lacking dipeptidyl aminopeptidase yspI was isolated from a strain already defective in aminopeptidase activity by means of a staining technique with the chromogenic substrate ala-pro-4-methoxy-β-naphthylamide to screen colonies for the absence of the enzyme. The defect segregated 2⁺ :2⁻ in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Gene dosage experiments indicated that the mutation resides in the structural gene of dipeptidyl aminopeptidase yspI, dpa 1⁺. The dpa 1⁺ gene was located on chromosome III by using l m- fluorophen-ylalanine-induced haploidization and mitotic analysis. dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.     

A Rapidly Labeled RNA Component Isolated from Posterior Silkgland of Silkworm

The effects of NaCl and heating temperature on the gel-forming ability of legumin were studied. The addition of NaCl progressively increased the denaturation temperature of legumin. Heating to around the denaturation point, i.e., below the onset temperature (zone 1), between the onset and maximal temperatures (zone 2), between the maximal and final temperatures (zone 3), and above the final temperature (zone 4), affected both the gel-forming ability and gel properties. No gel was formed in zone 1, while the gel was harder in zone 3 than in zones 2 and 4. The gel hardness gradually decreased with increasing temperature in zone 4. Differences in the viscoelastic and microstructural properties between gels heated at various temperatures around the denaturation point were observed.     

Eisosomes Regulate Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Cortical Clusters and Mitogen-activated Protein (MAP) Kinase Signaling upon Osmotic Stress

Eisosomes are multiprotein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate (PI(4,5)P₂) by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P₂ regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast Schizosaccharomyces pombe. Activation of the MAPK Pmk1 was impaired by mutations in the phosphatidylinositol (PI) 5-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P₂ clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI 5-kinase Its3 and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P₂ clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.     

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。     

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。     

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.