The Sir4 C-terminal coiled coil is required for telomeric and mating type silencing in Saccharomyces cerevisiae

Saccharomyces cerevisiae Sir4p plays important roles in silent chromatin at telomeric and silent mating type loci. The C terminus of Sir4p (Sir4CT) is critical for its functions in vivo because over-expression or deletion of Sir4CT fragments disrupts normal telomeric structure and abolishes the telomere position effect. The 2.5A resolution X-ray crystal structure of an Sir4CT fragment (Sir4p 1217-1358) reveals a 72 residue homodimeric, parallel coiled coil, burying an extensive 3600A(2) of surface area. The crystal structure is consistent with results of protein cross-linking and analytical ultracentrifugation results demonstrating that Sir4CT exists as a dimer in solution. Disruption of the coiled coil in vivo by point mutagenesis results in total derepression of telomeric and HML silent mating marker genes, suggesting that coiled coil dimerization is essential for Sir4p-mediated silencing. In addition to the coiled coil dimerization interface (Sir4CC interface), a crystallographic interface between pairs of coiled coils is significantly hydrophobic and buries 1228A(2) of surface area (interface II). Remarkably, interface II mutants are deficient in telomeric silencing but not in mating type silencing in vivo. However, point mutants of interface II do not affect the oligomerization state of Sir4CT in solution. These results are consistent with the hypothesis that interface II mimics a protein interface between Sir4p and one of its protein partners that is essential for telomeric silencing but not mating type silencing.     

Metabolism of lysine-chromium complex in Saccharomyces cerevisiae

Saccharomyces cerevisiae which cannot utilize lysine as a sole nitrogen source is shown to metabolize a Lysine 3 Cr³⁺ (1:1) complex synthesized, as a combined nitrogen and carbon source. It induces rapid uptake of lysine and prevents loss of viability, in contrast with free lysine. That complexation with trivalent chromium has the effect of profoundly influencing intracellular distribution and metabolism of the liganded amino acid is demonstrated.     

Genetic analysis of inducible sexual agglutination ability in the yeast Saccharomyces cerevisiae

Genetic regulation of the inducibility of sexual agglutination ability in the yeast Saccharomyces cerevisiae was studied. Detailed analysis of the degree of sexual agglutination was carried out; it showed that a greater number of genes are involved in the regulation of inducible sexual agglutination in strain H1-0 than previously assumed. Although dominancy of inducible phenotype over constitutive was confirmed, the effectiveness of one gene changing the constitutive phenotype to the inducible seemed to be somewhat low. Quantity per cell of agglutination substances responsible for sexual agglutination increased as the agglutination ability became greater.     

Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women

Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare.The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus.

Subjects and methods

Vaginal samples were collected from a total of262 (asymptomaticandsymptomatic) women with vaginitis attending the centre of family planning of General hospital ofPiraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae.

Results

A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker’s yeast.

Conclusions

Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy.     

Functional evaluation of serine 252 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase mutant Ser252Ala, affecting the conserved Walker A serine residue, was characterized to elucidate the role of this serine residue. The substitution did not result in changes in the protein structure, as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analysis of the mutated enzyme in both directions of the main reaction and in the two secondary reactions showed an approximately 50-fold increase in apparent K(m) for oxaloacetate with minor alterations in the other kinetic parameters. These results show that the hydroxyl group of serine 252 is required for proper oxaloacetate interaction.     

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae

Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.     

Immunologically related proteins in cytoplasmic and mitochondrial ribosomes of yeast Saccharomyces cerevisiae

Summary Ribosomal proteins from the cytoplasm and mitochondria of the yeast Saccharomyces cerevisiae were compared by immunoblotting techniques. Antibodies raised against cytoplasmic ribosomal proteins cross-react with five mitochondrial ribosomal proteins, four of which are located in the large and one in the small mitochondrial subunits. The possible existence of common ribosomal proteins for cytoplasmic and mitochondrial ribosomes is discussed.Abbreviations cyto cytoplasmic - mito mitochondrial     

Degradation of RNA during the autolysis of Saccharomyces cerevisiae produces predominantly ribonucleotides

Autolytic degradation of yeast RNA occurs in many foods and beverages and can impact on the sensory quality of the product, but the resulting complex mixture of nucleotides, nucleosides and nucleobases has not been properly characterised. In this study, yeast autolysis was induced by incubating cell suspensions of Saccharomyces cerevisiae at 30–60 °C (pH 7.0), and at pH 4.0–7.0 (40 °C) for 10–14 days, and the RNA degradation products formed during the process were determined by reversed-phase HPLC. Up to 95% of cell RNA was degraded, with consequent leakage into the extracellular environment of mainly 3′-, 5′- and 2′-ribonucleotides, and lesser amounts of polynucleotides, ribonucleosides and nucleobases. The rate of RNA degradation and the composition of the breakdown products varied with temperature and pH. RNA degradation was fastest at 50 °C (pH 7.0). Autolysis at lower temperatures (30 °C and 40 °C) and at pH 5.0 and 6.0 favoured the formation of 3′-nucleotides, whereas autolysis at 40 °C and 50 °C (pH 7.0) favoured 5′- and 2′-nucleotides. The best conditions for the formation of the two flavour-enhancing nucleotides, 5′-AMP and 5′-GMP, were 50 °C (pH 7.0) and pH 4.0 (40 °C), respectively.     

Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells

Sex-specific agglutinins from the cell surface of haploid cells of Saccharomyces cerevisiae (X2180, mt^a and mt) were purified and analysed. The constitutive agglutinin from mt^a cells was extractable with 3 mM dithiothreitol. It was shown to be a glycoprotein (3% mannose) with an apparent Mr of 43,000 based on gel filtration, but in SDS-PAGE it behaved as a much smaller molecule (Mr between 18,000 and 26,000). About one in three amino acids was a hydroxyamino acid. Its biological activity was resistant to boiling for 1 h, but sensitive to pronase. Intact mt cells retained their agglutinability in the presence of dithiothreitol but limited trypsinizing released a biologically active agglutinin fragment. It had an apparent Mr of 320,000 (gel filtration). When analysed by SDS-PAGE, a single diffuse band with an apparent Mr of 225,000 was observed. The protein was 94% (w/w) mannose with a trace of N-acetyl glucosamine. Its biological activity was almost completely lost after boiling for 1 h. Both agglutinins behaved as monovalent molecules and specifically inhibited the biological activity of both noninduced and pheromone-induced cells. Pheromone treatment of mt^a cells resulted in an apparent 32-fold increase in agglutinin activity at the cell surface, whereas pheromone treatment of mt cells only doubled the apparent agglutinin activity.Abbreviations mt mating type - DTT dithiothreitol - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - YPG yeast-peptone-glucose - PAS periodic-acid-Schiff reagent     

Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase

The tri-functional enzyme of Saccharomyces cerevisiae dihydroneopterin aldolase (DHNA)-dihydropterin pyrophosphokinase (PPPK)-dihydropteroate synthase (DHPS) catalyzes three sequential steps in folate biosynthesis. A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been cloned. This bi-functional enzyme was expressed as a His(6) fusion protein in Escherichia coli and the protein was purified to apparent homogeneity. The purified protein possesses both PPPK and DHPS activities as measured by the incorporation of [(3)H]p-ABA into the appropriate substrate. The pH optimum of the DHPS activity was determined to be 8.5. Gel filtration measurement indicates that the protein exists as a dimer in solution. A robotic screening method was used to identify crystallization conditions. Bi-pyramidal crystals of the enzyme formed with the protein in the presence of a pterin substrate analog in phosphate buffer (pH 6.3) and these diffracted to 2.3A. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis.     

Functional and physical interactions of Faf1p, a Saccharomyces cerevisiae nucleolar protein

We report the discovery and characterisation of a novel nucleolar protein of Saccharomyces cerevisiae. We identified this protein encoded by ORF YIL019w, designated in SGD base as Faf1p, in a two hybrid interaction screen using the known nucleolar protein Krr1 as bait. The presented data indicate that depletion of the Faf1 protein has an impact on the 40S ribosomal subunit biogenesis resulting from a decrease in the production of 18S rRNA. The primary defect is apparently due to inefficient processing of 35S rRNA at the A(0), A(1), and A(2) cleavage sites.     

Saccharomyces cerevisiae Coq9 polypeptide is a subunit of the mitochondrial coenzyme Q biosynthetic complex

Coenzyme Q (Q) is a redox active lipid that is an essential component of the electron transport chain. Here, we show that steady state levels of Coq3, Coq4, Coq6, Coq7 and Coq9 polypeptides in yeast mitochondria are dependent on the expression of each of the other COQ genes. Submitochondrial localization studies indicate Coq9p is a peripheral membrane protein on the matrix side of the mitochondrial inner membrane. To investigate whether Coq9p is a component of a complex of Q-biosynthetic proteins, the native molecular mass of Coq9p was determined by Blue Native-PAGE. Coq9p was found to co-migrate with Coq3p and Coq4p at a molecular mass of approximately 1 MDa. A direct physical interaction was shown by the immunoprecipitation of HA-tagged Coq9 polypeptide with Coq4p, Coq5p, Coq6p and Coq7p. These findings, together with other work identifying Coq3p and Coq4p interactions, identify at least six Coq polypeptides in a multi-subunit Q biosynthetic complex.     

Purification and characterization of human caseinomacropeptide produced by a recombinant Saccharomyces cerevisiae

Caseinomacropeptide (CMP) is a biologically active polypeptide derived from the C-terminal of milk kappa-casein. CMP is heterogeneous since it is modified differently by glycosylation and phosphorylation after translation. Recently, recombinant human CMP (hCMP) has been produced as a secretory product in yeast. The present study aimed at the purification and characterization of recombinant hCMP. By sequential molecular cut-off ultrafiltration and anion-exchange chromatography, the recombinant hCMP in the culture broth could be purified to an HPLC purity over 94%. The authenticity of the purified hCMP was confirmed by sequence analysis of N-terminal amino acids. The recombinant hCMP was estimated to be 7.0kDa by SDS-PAGE, and showed a lower glycosylation than the natural bovine CMP.     

The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae

Summary The induction of mitotic gene conversion and crossing-over inSaccharomyces cerevisiae diploid cells homozygous for thepso4-1 mutation was examined in comparison to the corresponding wild-type strain. Thepso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth. Concerning the lethal effect, diploids homozygous for thepso4-1 mutation are more sensitive to all agents tested in any growth phase. However, this effect is more pronounced in the G2 phase of the cell cycle. These results imply that the ploidy effect and the resistance of budding cells are under the control of thePSO4 gene. On the other hand, thepso4-1 mutant is mutationally defective for all agents used. Therefore, thepso4-1 mutant has a generalized block in both recombination and mutation ability. This indicates that thePSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between thepso4-1 mutation and theRecA orLexA mutation ofEscherichia coli.     

Arachidonic and oleic acids stimulate zygote formation in the presence of mating pheromone in the yeast,Saccharomyces cerevisiae

Effect of exogenous fatty acids on zygote formation in Saccharomyces cerevisiae was studied. Arachidonic and oleic acids considerably stimulated zygote formation, but other fatty acids tested, linoleic, linolenic, stearic and palmitic acids, did not. Pretreatment experiments with arachidonic acid showed that the stimulation of zygote formation by the fatty acid required the presence of mating pheromone.Abbreviations YPD yeast-peptone-dextrose medium - A₅₃₀ absorbance at 530 nm     

Cloning of the LEU2 gene of Saccharomyces cerevisiae by in vivo recombination

We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites. A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene. In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene. In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.     

Metabolic changes induced during adaptation of Saccharomyces cerevisiae to a water stress

When exponentially growing Saccharomyces cerevisiae was transferred from a normal high water activity growth medium (a_w 0.997) to a medium containing 8% NaCl low water activity growth medium (a_w 0.955), glycerol accumulation during the first eight hours of the adaptation was both retarded and greatly diminished in magnitude. Investigation of the underlying reasons for the slow onset of glycerol accumulation revealed that not only was overall glycerol production reduced by salt transfer, but also the rates of ethanol production and glucose consumption were reduced. Measurement of glycolytic intermediates revealed an accumulation of glucose-6-phosphate, fructose-6-phosphate, fructose 1,6 bisphosphate and phosphoenolpyruvate in S. cerevisiae 3 to 4 h after transfer to salt, suggesting that one or more glycolytic enzymes were inhibited. Potassium ions accumulated in S. cerevisiae after salt transfer and reached a maximum about 6 h after transfer, whereas the sodium ion content increased progressively during the adaptation period. The trehalose content also increased in adapting cells. It is suggested that inhibition of glycerol production during the initial period of adaptation could be due to either the inhibition of glycerol-3-phosphate dehydrogenase by increased cation content or the inhibitin of glycolysis, glycerol being produced glycolytically in S. cerevisiae. The increased accumulation of glycerol towards the end of the 8-h period suggests that the osmoregulatory response of S. cerevisiae involves complex sets of adjustments in which inhibition of glycerol-3-phosphate dehydrogenase must be relieved before glycerol functions as a major osmoregulator.     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.