Normal Yeast Cells Research Articles

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells cultivated aerobically or anaerobically

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells were examined on the basis of the cell growth rate, NAD(P)H concentration, reactive oxygen production, plasma membrane H+-ATPase activity, and ethanol production. In aerobically or anaerobically cultured yeast cells, NAD(P)H concentration decreased with increasing concentration of menadione, and the recovery of NAD(P)H concentration was proportional to the cell growth rate. However, there was no relationship among the inhibition of the cell growth and reactive oxygen production, plasma membrane H+-ATPase activity, and ethanol production. Among them, ethanol production showed resistance to the cytotoxicity of menadione, suggesting the resistance of glycolysis to menadione. The growth inhibitory effect of menadione depended on the rapid decrease and the recovery of NAD(P)H rather than production of reactive oxygen species regardless of aerobic culture or anaerobic culture and presence or absence of mitochondrial function. The recovery of NAD(P)H concentration after the addition of menadione might depend on menadione-resistant glycolytic enzymes.

Learning from the Uncontrollable

Research papers are usually revisionist histories, imagined accounts driven by impeccable logic and unbroken experimental success, which in no way reflect the slowness, messiness, and serendipity of real life in the lab. To give a different hopefully more balanced perspective than what is often told, I want to tell you the story behind some of the discoveries that shaped my early career.“In reality, of course, I had stumbled on the control by complete accident.”

Regulating Global Sumoylation by a MAP Kinase Hog1 and Its Potential Role in Osmo-Tolerance in Yeast

Sumoylation, a post-translational protein modification by small ubiquitin-like modifier (SUMO), has been implicated in many stress responses. Here we analyzed the potential role of sumoylation in osmo-response in yeast. We find that osmotic stress induces rapid accumulation of sumoylated species in normal yeast cells. Interestingly, disruption of MAP kinase Hog1 leads to a much higher level of accumulation of sumoylated conjugates that are independent of new protein synthesis. We also find that the accumulation of sumoylated species is dependent on a SUMO ligase Siz1. Notably, overexpression of SIZ1 in HOG1-disruption mutants (hog1Δ) but not in wild type cells leads to a markedly increased and prolonged accumulation of sumoylated species. Examination of osmo-tolerance of yeast mutants that display either an increase or a decrease in the global sumoylation level revealed an inverse relationship between accumulation of sumoylated conjugates and osmo-tolerance. Further investigation has shown that many of the sumoylated species induced by hyperosmotic stress are actually poly-sumoylated. Together, these findings indicate that abnormal accumulation of poly-sumoylated conjugates is harmful for osmo-tolerance in yeast, and suggest that Hog1 promotes adaptation to hyperosmotic stress partially via regulation of global sumoylation level.

Preparation of Organic Selenium Yeast by Fed-Batch Fermentation

Selenium is highly proper prepare essential element for the development of mammals and its incorporation in yeast could serve to Selenium enriched yeast. Production of Selenium (Se) enriched yeast was studied by fed-batch fermentation technology using synthetic nutrients to minimize the influence of sulfur and glucose used as the carbon sources. The effect of Selenium (Se) on yeast growth was also evaluated by employing. Se-adapted yeast cultures in continuous fermentation. Fedbatch fermentation with a total of 100 ppm of Sodium selenite in the medium (Na2SeO3), yielded 32.09 g L−1 of dry yeast cell biomass containing 3758 ± 6.5 ppm of organic Se. The resultant product had the Methylene Blue Reduction Test (MBRT) timing of > 25 min, indicating the highest accumulation of organic Se. The elemental analysis showed the sulfur content was reduced by 37% in selenium yeast (Se-yeast) than the normal yeast produced without Se supplementation. There was a reduction in the experimental biomass yield of Se-yeast by 18 g L−1 than the theoretical biomass yield in response to the Se addition. A morphological analysis of the normal and Se enriched yeast cells using scanning electron microscopy indicated a reduction in cell size by 0.88 μm in response to Se supplementation.

Making Magnetic Yeast

This study demonstrates that normal yeast cells can be magnetized, and identifies local redox control via carbon metabolism and iron supply as key factors involved in magnetization.

The Role of the Contractile Ring during Cytokinesis

Cytokinesis in animals and fungi involves constriction of an actomyosin contractile ring, but the constriction mechanisms and the role of the ring are not established. The constriction rate could be determined only by the properties and internal dynamics of the ring itself (a “dynamically autonomous” mechanism) as suggested by recent experiments on C. elegans embryos. Alternatively, the ring constriction rate could be set by coupled processes that occur simultaneously with constriction (a “dynamically coupled” mechanism). In fission yeast constriction occurs simultaneously with septation, the poorly understood process of cell wall growth in the wake of the constricting ring. To isolate the ring constriction mechanism, we combined mathematical modeling with experiments on fission yeast protoplasts which lack cell wall and adopt a rounded shape. Protoplasts assembled functional contractile rings that constricted without septation by sliding along the plasma membrane without dividing the cell. Because we could manipulate the shapes of protoplast cells, we could test the influence of cell shape on ring constriction dynamics and distinguish between dynamically autonomous and dynamically coupled constriction dynamics. In compressed protoplasts that had a partially flattened shape, contractile rings adopted characteristic bent shapes during constriction that were in remarkably close quantitative and parameter-free agreement with a mathematical model that assumed the ring produces tension but its constriction rate is set by the sliding of ring anchors in the membrane. Thus, ring constriction in fission yeast protoplasts is dynamically coupled: the ring does not set its own constriction rate. Dynamically autonomous models could not reproduce our experimental observations. Our results suggest that in normal yeast cells the constriction rate is determined by the septum growth rate or other coupled process and the role of the ring could be to exert tension on the septum to regulate its growth.

Molecular Display Technology Using Yeast—Arming Technology—

A technology to display proteins or peptides on living organisms has been developed over the last two decades. So-called "Molecular display (Arming) technology" or "Cell surface engineering" has been one of the important tools for analyzing and understanding protein function, or for screening of novel clones from libraries. In addition, it endows cells with novel abilities that cannot be added by conventional genetic recombination. In particular, the yeast Saccharomyces cerevisiae has a lot of advantages in molecular display technology. Normal yeast cells can be transformed into a wide variety of "arming yeasts" that have catalytic functions, affinity binding to valuable ligands, bioremediation properties, bio-monitoring properties, etc. This review describes the background, applications, and representative achievements of molecular display technology of yeast.

Depletion of the Yeast Nuclear Exosome Subunit Rrp6 Results in Accumulation of Polyadenylated RNAs in a Discrete Domain within the Nucleolus

Recent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)(+) RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)(+) RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome.

What a Cell Should Know (But May Not)

Normal yeast cells may not know whether DNA replication is complete before starting mitosis. How does the cell cycle progress in an orderly manner?

Can eukaryotic cells monitor the presence of unreplicated DNA?

Completion of DNA replication before mitosis is essential for genome stability and cell viability. Cellular controls called checkpoints act as surveillance mechanisms capable of detecting errors and blocking cell cycle progression to allow time for those errors to be corrected. An important question in the cell cycle field is whether eukaryotic cells possess mechanisms that monitor ongoing DNA replication and make sure that all chromosomes are fully replicated before entering mitosis, that is whether a replication-completion checkpoint exists. From recent studies with smc5–smc6 mutants it appears that yeast cells can enter anaphase without noticing that replication in the ribosomal DNA array was unfinished. smc5–smc6 mutants are proficient in all known cellular checkpoints, namely the S phase checkpoint, DNA-damage checkpoint, and spindle checkpoint, thus suggesting that none of these checkpoints can monitor the presence of unreplicated segments or the unhindered progression of forks in rDNA. Therefore, these results strongly suggest that normal yeast cells do not contain a DNA replication-completion checkpoint.

Role of Malassezia yeast (Pityrosporum) in seborrhoeic dermatitis (SD).

The objective of this study was to find out the prevalence of Pityrosporum species in the patients with seborrheic dermatitis (SD) and compare the colonization rate with normal healthy individuals. Comparative study. Dermatology Department, Military Hospital and Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi from 01 March 1996 to 28 February 1997. Fifty patients with clinically diagnosed seborrheic dermatitis were included in this study. Fifty normal healthy individual with matched age and gender were included as control subjects. Three samples from each effected sites were taken from 2 cm2 area. Identical sites in control subjects were also sampled. These samples were examined under the microscope after treating with 10% potassium hydroxide and staining with parker blue black ink for the presence of Pityrosporum yeast cells. The specimen were also inoculated in special lipid enriched media, incubated at 37 degrees C for 4-6 days. Out of 50 patients, Pityrosporum yeast cells were seen microscopically in 37 (74%), and the cultures were positive in 43 (86%). Among the normal individuals yeast cells were seen microscopically in 23 (46%) and the cultures were positive in 22 (44%). The colonization rate of Pityrosporum species was higher in the seborrheic dermatitis patients. It might be playing a causative role in the aetiology of this disease.

Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap

Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MS(n) spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the "lock mass" by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.

The Glutamine Residue of the Conserved GGQ Motif in Saccharomyces cerevisiae Release Factor eRF1 Is Methylated by the Product of the YDR140w Gene

Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to release factor (RF) activity in both kingdoms. In bacteria, the Gln residue of the motif in RF1 and RF2 is modified to N(5)-methyl-Gln by the S-adenosyl l-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae release factor eRF1, the first time that N(5)-methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial release factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl l-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1.eRF3.GTP.

Evaluation of Radioprotective Action of Compounds Using Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae serves as a model eukaryotic system to screen radioprotectors that can be used primarily in radiotherapy as well as in occupational workers in nuclear and allied industries. A number of antioxidants are suggested to be radioprotectors by virtue of their ability to quench reactive oxygen species, but their radioprotective action has not been investigated so far. In this study, a number of antioxidants were tested for their efficacy in radioprotection using yeast cells. Sulfhydryl compounds (disulfiram at 100 and 200 microM) and reduced glutathione (10 and 100 mM), purified compounds of plant origin, such as curcumin (1 mM, 10 mM, and 100 mM), quercetin (100 and 500 microM), rutin (100 and 200 microM), ellagic acid (100,200, and 500 microM) and gallic acid (100 and 500 microM)-were studied. The results revealed that all compounds, except gallic acid, specifically protected normal yeast cells from gamma-radiation damage. Using rad 52 mutants, which lack recombinational DNA repair pathway, it has been found that protection was solely brought about by reducing DNA damage rather than by interfering with DNA repair. Results with DNA repair polymerase further substantiated this contention. We conclude that simple eukaryotic yeast cells can serve as a test system not only for rapid screening of radiomodifiers but also to study their mode of action.

The effects of lipid extraction on the immunomodulatory activity of Malassezia species in vitro.

Malassezia spp. are believed to be the causative agents of pityriasis versicolor and are strongly implicated in seborrhoeic dermatitis. The yeast also forms part of the normal human cutaneous microflora. We have previously shown that when Malassezia yeast cells are incubated with human peripheral blood mononuclear cells (PBMCs), they are capable of reducing the levels of pro-inflammatory cytokines produced. In order to test the hypothesis that this immunoevasive phenomenon may be related to the unusually high level of lipid in the Malassezia yeast cell wall, we have compared the immunomodulatory capacity of normal and lipid-depleted yeast cells. Stationary phase yeast cells of Malassezia sympodialis, M. globosa and M. restricta were treated with chloroform/methanol to extract the surface lipids. The lipid-depleted and non-depleted yeast cells were then co-cultured with human PBMCs from three different human donors at a ratio of 20 yeasts per leukocyte for 24 h. The levels of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha were then determined by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that extraction of lipid reversed the yeast cell capacity to reduce the levels of pro-inflammatory cytokines. The levels of IL-1beta, IL-6 and TNF-alpha produced in response to lipid-extracted Malassezia of all three species were either no different from or significantly greater (P < 0.05; ANOVA) than the constitutive control levels. These results suggest that the lipid microfibrillar layer of Malassezia may prevent the yeast cells from inducing inflammation and provide an explanation for the normal commensal status of the organism on human skin. The hypothesis that the lipid layer is absent or altered in seborrhoeic dermatitis may provide an explanation for the inflammatory nature of this dermatosis.

Changes in membrane electrical parameters of yeast following chemical treatment for protoplast isolation

The yeast strains Saccharomyces species and Candida tropicalis were used. In each case, the electrorotation spectra were recorded for normal untreated yeast cells, β-mercaptoethanol-treated cells and isolated protoplasts. In order to obtain realistic information on the passive electrical membrane parameters associated with each step of protoplast isolation, a theoretical analysis of the simulated electrorotation spectra for shelled ellipsoids was performed. As a consequence, the parameters chosen for fitting of the experimental spectra were the electrical conductivities of the cell compartments, the membrane permittivity and the scaling factor. β-Mercaptoethanol was found to have significant action at the membrane level: (1) at high concentration (2%), σ m was increased dramatically; (2) in mild incubation conditions (0.1%), σ m was decreased slightly. Treatment with the enzyme complex led to a decrease in internal conductivity, probably due to a loss of the ionic equilibrium balance.

Changes of the compositional asymmetry of phospholipids associated to the increment in the membrane surface potential

The contribution of phosphatidylinositol (PI) and phosphatidylserine (PS) to the outer negative membrane surface potential was studied in normal, PS-rich and PI-rich yeast cells. Under carefully defined conditions; PS and PE were quantified by using the non-penetrating chemical probe trinitrobenzenesulfonic acid (TNBS) and the PI by degradation with a specific phospholipase C. An asymmetric distribution of phospholipids in the plasma membrane with more PS (80–90%), PI (70–85%) in the inner leaflet was found. When compared to normal cells there were 3-times more PI and 2-times more PS in the outer leaflet of the PI-rich and PS-rich cells. These values are consistent with the two-times increased surface potential in these cells. Interestingly, the contribution of PI was around twice the contribution of PS to the surface potential in the cells studied. When compared to normal cells there was a two-times increased accessibility of PS to TNBS in the PI-rich cells and the accessibility of PI to phospholipase C was also increased two-times in the PS-rich cells, while the proportion of derivatized PE was similar in all cells. Taking into account that the amount of PI is similar in normal cells and PS-rich cells and the amount of PS is similar in PI-rich cells and normal cells, a charge driven transbilayer transport of acidic phospholipids can be proposed.

PIF1: a DNA helicase in yeast mitochondria.

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix-localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single-stranded (ss) DNA-dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5' to 3' direction with respect to the ss DNA on which it binds first.

37] Sugar transport in normal and mutant yeast cells

This chapter focuses on the sugar transport in normal and mutant yeast cell. Most studies of sugar transport in yeasts have been carried out with baker's yeast, Saccharomyces cerevisiae , which exhibits a variety of sugar transport processes––namely, facilitated diffusion, active transport, and, possibly, group translocation by transport-associated phosphorylation. The high- and low-affinity processes for monosaccharide uptake in S. cerevisiae may differ with respect to mechanism as well as kinetics. The low-affinity process is dearly due to a constitutive, facilitated diffusion mechanism; however, the high-affinity process may be mediated by a group translocation mechanism––namely, a transport-associated phosphorylation. Transport is customarily studied in washed cells suspended in distilled water or buffered salt solutions. Radiolabeled sugars are added to the cell suspensions, and samples are filtered and washed on glass microfiber or membrane filters. Glass microfiber filters are less expensive and allow much higher filtration rates than membrane filters.

Mutations in the pho80 gene confer permeability to 5'-mononucleotides in Saccharomyces cerevisiae.

Yeast mutants permeable to dTMP (tup) were selected and two new complementation groups (tup5 and tup7) were identified. Assay of the levels of both acid and alkaline phosphatase in cells grown under either repressing (5 mM PO4(-3) or derepressing (0.03 mM PO4(-3) conditions indicated that, in general, tup mutations cause cells to be defective in their regulation of phosphatase synthesis. In addition, three of the tup mutations (tup1, tup4 and tup7) displayed markedly elevated rates of inorganic phosphate transport. The tup7 locus was found to be tightly centromere-linked on the right arm of chromosome XV, and was shown to be allelic with the pho80 regulatory locus on the basis of both genetic and biochemical criteria. Analysis of other mutations known to affect phosphatase levels (pho) indicated that some also conferred permeability to dTMP. Possible allelic relationships between tup genes and certain of these pho mutations are discussed. Regardless of the culture conditions, wild-type strains were not permeable to dTMP; in contrast, it was found in the course of this work that normal yeast cells were permeable to dUMP and that dUMP permeability was regulated by the concentration of inorganic phosphate present in the medium used to grow the cells. Thus, permeability to 5'-mononucleotides appears to be under coordinate control with phosphatase synthesis.