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Saccharomyces Cerevisiae (saccharomyces + cerevisiae)
Kinds of Saccharomyces Cerevisiae Terms modified by Saccharomyces Cerevisiae Selected AbstractsINFLUENCE OF PULSED ELECTRIC FIELD ON SELENOCYSTEINE CONTENT IN SACCHAROMYCES CEREVISIAEJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2008URSZULA PANKIEWICZ ABSTRACT Culture of Saccharomyces cerevisiae with sodium selenite addition in medium was treated by pulsed electric fields (PEFs). Amino acids from yeast hydrolysates were separated by means of ion-exchange chromatography on amino acid analyzer according to previously established procedure. Selenocysteine was determined in a form of complex with ninhydrin, applying photometric technique. PEF treatment of S. cerevisiae cells resulted in about threefold content increase of selenium bonded within selenocysteine. PRACTICAL APPLICATIONS Se yeast is an attractive source of Se because of its low cost and its ability to act as a precursor for selenoprotein synthesis. Se yeast can be consumed as such and as a nutritional supplement. Another possibility is to use selenized yeast instead of conventional yeast for baking bread. Bread is generally low in Se, and hence the use of selenized yeast for this purpose could result in higher Se intakes because bread is a common product consumed by many individuals (Dumont et al. 2006). The presented way to enrich the baking yeast in selenium, namely selenomethionine, may be successfully applied in yeast production, because the studied method is a relatively simple, nontoxic and cheap technique for introducing macrocompounds into the yeast cells. Such enriched selenium yeast may be a valuable and safe source of selenium at diet supplementation. [source] PARTIAL PURIFICATION AND CHARACTERIZATION OF NEUTRAL TREHALASE FROM COMMERCIAL BAKER'S YEAST, SACCHAROMYCES CEREVISIAEJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2000SANIYE YARAR ABSTRACT The neutral trehalase of a commercial baker's yeast (S. cerevisiae) strain has been partially purified using ammonium sidfate fractionation and DEAE-cellulose column chromatography techniques. Trehalase was precipitated between 35,50% ammonium sulfate saturation and approximately 5,8 fold purification was achieved. The yeast cAMP-dependent protein kinase was also precipitated in the same fraction and these two proteins were separated by DEAE-cellulose column chromatography. Trehalase became totally inactive after ion exchange chromatography, "cryptic trehalase" (tre-c), but was later activated with the addition of partially purified protem kinase together with cAMP and ATP. A 215 fold purification was obtained after DEAE-ceUulose column chromatography. One mM EDTA caused complete inhibition of the enzyme in crude extract, however the inhibition levels in ammonium sulfate and DEAE-cellulose fractions were 73.5% and 50%, respectively. Optimal pH range and temperature of the enzyme were determined as pH 6,6.8 and 30C, respectively. The kinetic parameters, Km and Vmax, were estimated as 11.78 mM trehalose and 12.47 ,mole glucose/min-mg protein, respectively. [source] Deletion of mdmB impairs mitochondrial distribution and morphology in Aspergillus nidulansCYTOSKELETON, Issue 2 2003Katrin V. Koch Abstract Mitochondria form a dynamic network of interconnected tubes in the cells of Saccharomyces cerevisiae or filamentous fungi such as Aspergillus nidulans,Neurospora crassa, or Podospora anserina. The dynamics depends on the separation of mitochondrial fragments, their movement throughout the cell, and their subsequent fusion with the other parts of the organelle. Interestingly, the microtubule network is required for the distribution in N. crassa and S. pombe, while S. cerevisiae and A. nidulans appear to use the actin cytoskeleton. We studied a homologue of S. cerevisiae Mdm10 in A. nidulans, and named it MdmB. The open reading frame is disrupted by two introns, one of which is conserved in mdm10 of P. anserina. The MdmB protein consists of 428 amino acids with a predicted molecular mass of 46.5 kDa. MdmB shares 26% identical amino acids to Mdm10 from S. cerevisiae, 35% to N. crassa, and 32% to the P. anserina homologue. A MdmB-GFP fusion protein co-localized evenly distributed along mitochondria. Extraction of the protein was only possible after treatment with a non-ionic and an ionic detergent (1% Triton X-100; 0.5% SDS) suggesting that MdmB was tightly bound to the mitochondrial membrane fraction. Deletion of the gene in A. nidulans affected mitochondrial morphology and distribution at 20°C but not at 37°C. mdmB deletion cells contained two populations of mitochondria at lower temperature, the normal tubular network plus some giant, non-motile mitochondria. Cell Motil. Cytoskeleton 55:114,124, 2003. © 2003 Wiley-Liss, Inc. [source] Mutagenesis of ,-tubulin cysteine residues in Saccharomyces cerevisiae: Mutation of cysteine 354 results in cold-stable microtubulesCYTOSKELETON, Issue 2 2001Mohan L. Gupta Jr. Abstract Cysteine residues play important roles in the control of tubulin function. To determine which of the six cysteine residues in ,-tubulin are critical to tubulin function, we mutated the cysteines in Saccharomyces cerevisiae ,-tubulin individually to alanine and serine residues. Of the twelve mutations, only three produced significant effects: C12S, C354A, and C354S. The C12S mutation was lethal in the haploid, but the C12A mutation had no observable phenotype. Based on interactive views of the electron crystallographic structure of tubulin, we suggest that substitution of serine for cysteine at this position has a destabilizing effect on the interaction of tubulin with the exchangeable GTP. The two C354 mutations, although not lethal, produced dramatic effects on microtubules and cellular processes that require microtubules. The C354 mutant cells had decreased growth rates, a slowed mitosis, increased resistance to benomyl, and impaired nuclear migration and spindle assembly. The C354A mutation produced a more severe phenotype than the C354S mutation: the haploid cells had chromosome segregation defects, only 50% of cells in a culture were viable, and a significant percentage of the cells were misshapened. Cytoplasmic microtubules in the C354S and C354A cells were longer than in the control strain and spindle structures appeared shorter and thicker. Both cytoplasmic and spindle microtubules in the two C354 mutants were extremely stable to cold temperature. After 24 h at 4°C, the microtubules were still present and, in fact, very long and thick tubulin polymers had formed. Evidence exists to indicate that the C354 residue in mammalian tubulin is near the colchicine binding site and the electron crystal structure of tubulin places the residue at the interface between the ,- and ,-subunits. The sulfhydryl group is situated in a polar environment, which may explain why the alanine mutation is more severe than the serine mutation. When the C12S and the two C354 mutations were made in a diploid strain, the mutated tubulin was incorporated into microtubules and the resulting heterozygotes had phenotypes that were intermediate between those of the mutated haploids and the wild-type strains. The results suggest that the C12 and C354 residues play important roles in the structure and function of tubulin. Cell Motil. Cytoskeleton 49:67,77, 2001. © 2001 Wiley-Liss, Inc. [source] The Mediated Electrochemical Method for Rapid Fermentation Ability AssessmentELECTROANALYSIS, Issue 14 2008Jinsheng Zhao Abstract The mediated electrochemical method characterized by the combinational utilization of two double mediator systems was proposed to assess the fermentation efficiency of three yeasts. The mediator systems selected were the menadione/ferricyanide system and the 2,6-dichlorophenolindophenol (DCPIP)/ferricyanide system. Both the electrochemical responses and succinate dehydrogenase assay suggested that the menadione/ferricyanide system made the yeasts switch to anaerobic respiration and, the DCPIP/ferricyanide system let the yeasts remain in theirs fermentative mode. The ethanol yields (g ethanol/g glucose) and the RD/RM (RD and RM refer to the electrochemical response from the menadione/ferricyanide system and the DCPIP/ferricyanide system, respectively) values are 0.47 and 0.38, 0.24 and 0.75, 0.23 and 0.81, respectively, for Saccharomyces cerevisiae, Pachysolen tannophilus and Pichia stipitis. The results showed that there was a negative correlation between the ethanol yields and the RD/RM values of the three yeasts, which showed the feasibility of the mediated electrochemical method in rapid fermentation ability assay. [source] Improved 2-DE of microorganisms after acidic extractionELECTROPHORESIS, Issue 8 2006Ben R. Herbert Professor Abstract 2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100,kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH,3 by 80,mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains. [source] Enhancement of the NAD(P)(H) Pool in Saccharomyces cerevisiaeENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2008A. Knepper Abstract Asymmetric biosyntheses allow for an efficient production of chiral building blocks. The application of whole cells as biocatalysts for asymmetric syntheses is advantageous because they already contain the essential coenzymes NAD(H) or NADP(H), which additionally can be regenerated in the cells. Unfortunately, reduced catalytic activity compared to the oxidoreductase activity is observed in many cases during whole-cell biotransformation. This may be caused by low intracellular coenzyme pool sizes and/or a decline in intracellular coenzyme concentrations. To enhance the intracellular coenzyme pool sizes, the effects of the precursor metabolites adenine and nicotinic acid on the intracellular accumulation of NAD(H) and NADP(H) were studied in Saccharomyces cerevisiae. Based on the results of simple batch experiments with different precursor additions, fed-batch processes for the production of yeast cells with enhanced NAD(H) or enhanced NADP(H) pool sizes were developed. Supplementation of the feed medium with 95,mM adenine and 9.5,mM nicotinic acid resulted in an increase of the intracellular NAD(H) concentration by a factor of 10 at the end of the fed-batch process compared to the reference process. The final NAD(H) concentration remains unchanged if the feed medium was solely supplemented with 95,mM adenine, but intracellular NADP(H) was increased by a factor of 4. The effects of NADP(H) pool sizes on the asymmetric reduction of ethyl-4-chloro acetoacetate (CAAE) to the corresponding (S)-4-chloro-3-hydroxybutanoate (S-CHBE) was evaluated with S.,cerevisiae,FasB,His6 as an example. An intracellular threshold concentration above 0.07,mM NADP(H) was sufficient to increase the biocatalytic S-CHBE productivity by 25,% compared to lower intracellular NADP(H) concentrations. [source] Exploring the Phospholipid Biosynthetic Pathways of Aspergillus fumigatus by Computational Genome AnalysisENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 6 2005H. Do Abstract Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systematic diseases (invasive aspergillosis) with high mortality rates. In recent years, considerable progress in the genome sequencing of this fungus has been made by an international consortium, which includes the Wellcome Trust Sanger Institute (UK) and the Institute for Genome Research (USA). A tenfold whole genome shotgun sequence assembly of A. fumigatus has been made publicly available. In this study, it was attempted to identify the genes related to the phospholipid biosynthesis from the A. fumigatus genome by a gene prediction program (GlimmerM) and to reconstruct the metabolic pathway for phospholipids of A. fumigatus. Fifteen genes related to phospholipid pathway were identified in the A. fumigatus genomic sequence. The open reading frames predicted by GlimmerM showed a high amino acid sequence similarity with the other fungal phospholipid biosynthetic genes and well-conserved functional domains. The obtained results also demonstrated that the reconstructed pathway of A. fumigatus in phospholipid biosynthesis was very similar to that of other fungi such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, and Neurospora crassa. Therefore it is postulated that the antifungal drugs targeted for the biosynthesis of phospholipids could also be effective against A. fumigatus. [source] The Composition of Jerusalem Artichoke (Helianthus tuberosus L.) Spirits Obtained from Fermentation with Bacteria and YeastsENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2005K. Szambelan Abstract The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains,3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains,Bc16a and D2 and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K.,fragilis. The highest ethanol yield was achieved when Z.,mobilis,3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94,% of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by-products compared to the distillates resulting from the single species process. Ester production of 0.30,2.93,g/L, responsible for the aromatic quality of the spirits, was noticed when K.,fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04,g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z.,mobilis, were lower than those achieved for yeasts. [source] Structure,activity relationships for gene activation oestrogenicity: Evaluation of a diverse set of aromatic chemicalsENVIRONMENTAL TOXICOLOGY, Issue 1 2002T. Wayne Schultz Abstract Structure,activity relationships for oestrogenicity were developed based on 120 aromatic chemicals evaluated in the Saccharomyces cerevisiae -based Lac -Z reporter assay. Relative gene activation was compared to 17,-estradiol and varied over eight orders of magnitude. Analysis of the data compared to 17,-estradiol identified three structural criteria that were related to xenoestrogen activity and potency: (1) the hydrogen-bonding ability of the phenolic ring mimicking the A-ring, (2) a hydrophobic centre similar in size and shape to the B- and C-rings, and (3) a hydrogen-bond donor mimicking the 17,-hydroxyl moiety of the D-ring, especially with an oxygen-to-oxygen distance similar to that between the 3- and 17,-hydroxyl groups of 17,-estradiol. Binding data were segregated into activity clusters including strong, moderate, weak, and detectable gene expression, and those compounds that were inactive. The hydrogen-bonding ability of hydroxy group in the 3-position on 17,-estradiol was observed to be essential for gene activation. Compounds with a 4-hydroxyl substituted benzene ring and a hydrophobic moiety of size and shape equivalent to the B-ring of 17,-estradiol were generally observed to be weakly active compounds. Moderately active compounds have a 4-hydroxyl substituted benzene ring with a hydrophobic moiety equivalent in size and shape to the B- and C-ring of 17,-estradiol, or have a high hydrogen-bond donor capacity owing to the presence of halogens on a nonphenolic ring. Strongly active compounds, similar to 4,4,-diethylethylene bisphenol (DES), possess the same hydrophobic ring structure as described for moderately active compounds and an additional hydroxyl group with an oxygen-to-oxygen distance close to that exhibited by the 3- and 17-hydroxyl groups of 17,-estradiol. © 2002 by Wiley Periodicals, Inc. Environ Toxicol 17: 14,23, 2002 [source] Xenoestrogenic gene exression: Structural features of active polycyclic aromatic hydrocarbonsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2002T. Wayne Schultz Abstract Estrogenicity was assessed using the Saccharomyces cerevisiae -based Lac-Z reporter assay and was reported as the logarithm of the inverse of the 50% molar ,-galactosidase activity (log[EC50,1]). In an effort to quantify the relationship between molecular structure of polycyclic aromatic hydrocarbons (PAHs) and estrogenic gene expression, a series of PAHs were evaluated. With noted exceptions, the results of these studies indicate that the initial two-dimensional structural warning for estrogenicity, the superpositioning of a hydroxylated aromatic system on the phenolic A-ring of 17-,-estradiol, can be extended to the PAHs. This two-dimensional-alignment criterion correctly identified estrogenicity of 22 of the 29 PAHs evaluated. Moreover, the estrogenic potency of these compounds was directly related to the size of the hydrophobic backbone. The seven compounds classified incorrectly by this structural feature were either dihydroxylated naphthalenes or aromatic nitrogen-heterocyclic compounds; all such compounds were false positives. Results with dihydroxylated naphthalenes reveal derivatives that were nonestrogenic when superimposed on the phenolic A-ring of 17-,-estradiol had the second hydroxyl group in the position of the C-ring or were catechol-like in structure. Structural alerts for nitrogen-heterocyclic compounds must take into account the position of the hydroxyl group and the in-ring nitrogen atom; compounds with the hydroxyl group and nitrogen atom involved with the same ring were observed to be nonactive. [source] Potential multidrug resistance gene POHL: An ecologically relevant indicator in marine spongesENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2001Anatoli Krasko Abstract Sponges are sessile filter feeders found in all aquatic habitats from the tropics to the arctic. Against potential environmental hazards, they are provided with efficient defense systems, e.g., protecting chaperones and/or the P-170/multidrug resistance pump system. Here we report on a further multidrug resistance pathway that is related to the pad one homologue (POH1) mechanism recently identified in humans. It is suggested that proteolysis is involved in the inactivation of xenobiotics by the POH1 system. Two cDNAs were cloned, one from the demosponge Geodia cydoniumand a second from the hexactinellid sponge Aphrocallistes vastus. The cDNA from G. cydonium, termed GCPOHL, encodes a deduced polypeptide with a size of 34,591 Da and that from A. vastus, AVPOHL, a protein of a calculated Mr of 34,282. The two sponge cDNAs are highly similar to each other as well as to the known sequences from fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae) and other Metazoa (from Schistosoma mansoni to humans). Under controlled laboratory conditions, the expression of the potential multidrug resistance gene POHL is, in G. cydonium, strongly upregulated in response to the toxins staurosporin (20 ,M) or taxol (50 ,M); the first detectable transcripts appear after 1 d and reach a maximum after 3 to 5 d of incubation. The relevance of the expression pattern of the G. cydonium gene POHL for the assessment of pollution in the field was determined at differently polluted sites in the area around Rovinj (Croatia; Mediterranean Sea, Adriatic Sea). The load of the selected sites was assessed by measuring the potency of XAD-7 concentrates of water samples taken from those places to induce the level of benzo[a]pyrene monooxygenase (BaPMO) in fish and to impair the multidrug resistance (MDR)/P-170 extrusion pump in clams. These field experiments revealed that the levels of inducible BaPMO activity in fish and of the MDR potential by the water concentrates are highly correlated with the level of expression of the potential multidrug resistance gene POHL in G. cydonium. This report demonstrates that the detoxification POH pathway, here mediated by the G. cydonium GCPOHL gene, is an additional marker for the assessment of the environmental load in a given marine area. [source] DENSITY-DEPENDENT EFFECTS ON ALLELOPATHIC INTERACTIONS IN YEASTEVOLUTION, Issue 3 2008Duncan Greig The ability of rare types to invade populations is important for the maintenance of diversity and spread of beneficial variants. Spatial structure promotes strategies of interference competition by limiting diffusion of interference toxins and resources, often allowing interference competitors to invade when rare. Consistent with previous results in other microbial systems, toxin production by Saccharomyces cerevisiae is advantageous in spatially structured, high-density environments, but not in unstructured environments. However, at low density and at low frequency, rare toxin producers cannot invade populations of common, sensitive, toxin nonproducers. This is because the likelihood of interaction between toxin producers and sensitives depends upon the density and frequency of both competitors. [source] A NEGATIVE RELATIONSHIP BETWEEN MUTATION PLEIOTROPY AND FITNESS EFFECT IN YEASTEVOLUTION, Issue 6 2007Tim F. Cooper It is generally thought that random mutations will, on average, reduce an organism's fitness because resulting phenotypic changes are likely to be maladaptive. This relationship leads to the prediction that mutations that alter more phenotypic traits, that is, are more pleiotropic, will impose larger fitness costs than mutations that affect fewer traits. Here we present a systems approach to test this expectation. Previous studies have independently estimated fitness and morphological effects of deleting all nonessential genes in Saccharomyces cerevisiae. Using datasets generated by these studies, we examined the relationship between the pleiotropic effect of each deletion mutation, measured as the number of morphological traits differing from the parental strain, and its effect on fitness. Pleiotropy explained ,18% of variation in fitness among the mutants even once we controlled for correlations between morphological traits. This relationship was robust to consideration of other explanatory factors, including the number of protein,protein interactions and the network position of the deleted genes. These results are consistent with pleiotropy having a direct role in affecting fitness. [source] Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltoseFEBS JOURNAL, Issue 20 2010Keizo Yamamoto The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 Å, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (,/,)8 -barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo-1,6-glucosidase is isomaltotriose; other, longer-chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310,315, and because the isomaltase pocket is shallower than that of other oligo-1,6-glucosidases. These features of the isomaltase active site pocket prevent isomalto-oligosaccharides from binding to the active site effectively. [source] Experimental and steady-state analysis of the GAL regulatory system in Kluyveromyces lactisFEBS JOURNAL, Issue 14 2010Venkat R. Pannala The galactose uptake mechanism in yeast is a well-studied regulatory network. The regulatory players in the galactose regulatory mechanism (GAL system) are conserved in Saccharomyces cerevisiae and Kluyveromyces lactis, but the molecular mechanisms that occur as a result of the molecular interactions between them are different. The key differences in the GAL system of K. lactis relative to that of S. cerevisiae are: (a) the autoregulation of KlGAL4; (b) the dual role of KlGal1p as a metabolizing enzyme as well as a galactose-sensing protein; (c) the shuttling of KlGal1p between nucleus and cytoplasm; and (d) the nuclear confinement of KlGal80p. A steady-state model was used to elucidate the roles of these molecular mechanisms in the transcriptional response of the GAL system. The steady-state results were validated experimentally using measurements of ,-galactosidase to represent the expression for genes having two binding sites. The results showed that the autoregulation of the synthesis of activator KlGal4p is responsible for the leaky expression of GAL genes, even at high glucose concentrations. Furthermore, GAL gene expression in K. lactis shows low expression levels because of the limiting function of the bifunctional protein KlGal1p towards the induction process in order to cope with the need for the metabolism of lactose/galactose. The steady-state model of the GAL system of K. lactis provides an opportunity to compare with the design prevailing in S. cerevisiae. The comparison indicates that the existence of a protein, Gal3p, dedicated to the sensing of galactose in S. cerevisiae as a result of genome duplication has resulted in a system which metabolizes galactose efficiently. [source] Analysis of DNA-binding sites on Mhr1, a yeast mitochondrial ATP-independent homologous pairing proteinFEBS JOURNAL, Issue 6 2010Tokiha Masuda The Mhr1 protein is necessary for mtDNA homologous recombination in Saccharomyces cerevisiae. Homologous pairing (HP) is an essential reaction during homologous recombination, and is generally catalyzed by the RecA/Rad51 family of proteins in an ATP-dependent manner. Mhr1 catalyzes HP through a mechanism similar, at the DNA level, to that of the RecA/Rad51 proteins, but without utilizing ATP. However, it has no sequence homology with the RecA/Rad51 family proteins or with other ATP-independent HP proteins, and exhibits different requirements for DNA topology. We are interested in the structural features of the functional domains of Mhr1. In this study, we employed the native fluorescence of Mhr1's Trp residues to examine the energy transfer from the Trp residues to etheno-modified ssDNA bound to Mhr1. Our results showed that two of the seven Trp residues (Trp71 and Trp165) are spatially close to the bound DNA. A systematic analysis of mutant Mhr1 proteins revealed that Asp69 is involved in Mg2+ -dependent DNA binding, and that multiple Lys and Arg residues located around Trp71 and Trp165 are involved in the DNA-binding activity of Mhr1. In addition, in vivo complementation analyses showed that a region around Trp165 is important for the maintenance of mtDNA. On the basis of these results, we discuss the function of the region surrounding Trp165. [source] Evolutionary divergence of valosin-containing protein/cell division cycle protein 48 binding interactions among endoplasmic reticulum-associated degradation proteinsFEBS JOURNAL, Issue 5 2009Giacomo Morreale Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cell-autonomous process that eliminates large quantities of misfolded, newly synthesized protein, and is thus essential for the survival of any basic eukaryotic cell. Accordingly, the proteins involved and their interaction partners are well conserved from yeast to mammals, and Saccharomyces cerevisiae is widely used as a model system with which to investigate this fundamental cellular process. For example, valosin-containing protein (VCP) and its yeast homologue cell division cycle protein 48 (Cdc48p), which help to direct polyubiquitinated proteins for proteasome-mediated degradation, interact with an equivalent group of ubiquitin ligases in mouse and in S. cerevisiae. A conserved structural motif for cofactor binding would therefore be expected. We report a VCP-binding motif (VBM) shared by mammalian ubiquitin ligase E4b (Ube4b),ubiquitin fusion degradation protein 2a (Ufd2a), hydroxymethylglutaryl reductase degradation protein 1 (Hrd1),synoviolin and ataxin 3, and a related sequence in Mr 78 000 glycoprotein,Amfr with slightly different binding properties, and show that Ube4b and Hrd1 compete for binding to the N-terminal domain of VCP. Each of these proteins is involved in ERAD, but none has an S. cerevisiae homologue containing the VBM. Some other invertebrate model organisms also lack the VBM in one or more of these proteins, in contrast to vertebrates, where the VBM is widely conserved. Thus, consistent with their importance in ERAD, evolution has developed at least two ways to bring these proteins together with VCP,Cdc48p. However, the differing molecular architecture of VCP,Cdc48p complexes indicates a key point of divergence in the molecular details of ERAD mechanisms. [source] Quantitative modeling of triacylglycerol homeostasis in yeast , metabolic requirement for lipolysis to promote membrane lipid synthesis and cellular growthFEBS JOURNAL, Issue 22 2008Jürgen Zanghellini Triacylglycerol metabolism in Saccharomyces cerevisiae was analyzed quantitatively using a systems biological approach. Cellular growth, glucose uptake and ethanol secretion were measured as a function of time and used as input for a dynamic flux-balance model. By combining dynamic mass balances for key metabolites with a detailed steady-state analysis, we trained a model network and simulated the time-dependent degradation of cellular triacylglycerol and its interaction with fatty acid and membrane lipid synthesis. This approach described precisely, both qualitatively and quantitatively, the time evolution of various key metabolites in a consistent and self-contained manner, and the predictions were found to be in excellent agreement with experimental data. We showed that, during pre-logarithmic growth, lipolysis of triacylglycerol allows for the rapid synthesis of membrane lipids, whereas de novo fatty acid synthesis plays only a minor role during this growth phase. Progress in triacylglycerol hydrolysis directly correlates with an increase in cell size, demonstrating the importance of lipolysis for supporting efficient growth initiation. [source] Requirements for chromatin reassembly during transcriptional downregulation of a heat shock gene in Saccharomyces cerevisiaeFEBS JOURNAL, Issue 11 2008Mette M. Jensen Heat shock genes respond to moderate heat stress by a wave of transcription. The induction phase is accompanied by the massive eviction of histones, which later reassemble with DNA during the ensuing phase of transcription downregulation. In this article, we identify determinants of this reassembly throughout the heat shock protein 104 gene (HSP104) transcription unit. The results show that, although histone H3 lacking amino acids 4,30 of its N-terminal tail (H3,4,30) is normally deposited, reassembly of H3,4,40 is obliterated with an accompanying sustained transcription. On mutation of the histone chaperones Spt6p and Spt16p, but not Asf1p, reassociation of H3 with DNA is compromised. However, despite a lasting open chromatin structure, transcription ceases normally in the spt6 mutant. Thus, transcriptional downregulation can be uncoupled from histone redeposition and ongoing transcription is not required to prevent chromatin reassembly. [source] Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1 , a molecular link between recombination and chromatin assembly during meiosisFEBS JOURNAL, Issue 9 2008Satomi Ishii In eukaryotes, meiosis leads to genetically variable gametes through recombination between homologous chromosomes of maternal and paternal origin. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes. However, the mechanism of chromatin organization after meiotic recombination is unknown. In this study we report that the meiosis-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the C. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharomyces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo. ,-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15. Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells. Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1. Taken together, these results suggest a novel role of the CAF-1,PCNA complex in meiotic events. We propose that the CAF-1,PCNA complex modulates chromatin assembly following meiotic recombination. [source] Sulfide : quinone oxidoreductase (SQR) from the lugworm Arenicola marina shows cyanide- and thioredoxin-dependent activityFEBS JOURNAL, Issue 6 2008Ursula Theissen The lugworm Arenicola marina inhabits marine sediments in which sulfide concentrations can reach up to 2 mm. Although sulfide is a potent toxin for humans and most animals, because it inhibits mitochondrial cytochrome c oxidase at micromolar concentrations, A. marina can use electrons from sulfide for mitochondrial ATP production. In bacteria, electron transfer from sulfide to quinone is catalyzed by the membrane-bound flavoprotein sulfide : quinone oxidoreductase (SQR). A cDNA from A. marina was isolated and expressed in Saccharomyces cerevisiae, which lacks endogenous SQR. The heterologous enzyme was active in mitochondrial membranes. After affinity purification, Arenicola SQR isolated from yeast mitochondria reduced decyl-ubiquinone (Km = 6.4 ,m) after the addition of sulfide (Km = 23 ,m) only in the presence of cyanide (Km = 2.6 mm). The end product of the reaction was thiocyanate. When cyanide was substituted by Escherichia coli thioredoxin and sulfite, SQR exhibited one-tenth of the cyanide-dependent activity. Six amino acids known to be essential for bacterial SQR were exchanged by site-directed mutagenesis. None of the mutant enzymes was active after expression in yeast, implicating these amino acids in the catalytic mechanism of the eukaryotic enzyme. [source] Leishmania infantum LeIF protein is an ATP-dependent RNA helicase and an eIF4A-like factor that inhibits translation in yeastFEBS JOURNAL, Issue 22 2006Mourad Barhoumi LeIF, a Leishmania protein similar to the eukaryotic initiation factor eIF4A, which is a prototype of the DEAD box protein family, was originally described as a Th1-type natural adjuvant and as an antigen that induces an IL12-mediated Th1 response in the peripheral blood mononuclear cells of leishmaniasis patients. This study aims to characterize this protein by comparative biochemical and genetic analysis with eIF4A in order to assess its potential as a target for drug development. We show that a His-tagged, recombinant, LeIF protein of Leishmania infantum, which was purified from Escherichia coli, is both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described previously for other members of the DEAD box helicase protein family. In vivo experiments show that the LeIF gene cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. In contrast, expression of LeIF inhibits yeast growth when endogenous eIF4A is expressed off only one of its two encoding genes. Furthermore, in vitro binding assays show that LeIF interacts with yeast eIF4G. These results show an unproductive interaction of LeIF with translation initiation factors in yeast. Furthermore, the 25 amino terminal residues were shown to enhance the ability of LeIF to interfere with the translation machinery in yeast. [source] The crystal structure of pyruvate decarboxylase from Kluyveromyces lactisFEBS JOURNAL, Issue 18 2006Implications for the substrate activation mechanism of this enzyme The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 Å resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106,113 and 292,301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105,113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105,113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase. [source] Three mammalian cytochromes b561 are ascorbate-dependent ferrireductasesFEBS JOURNAL, Issue 16 2006Dan Su Cytochromes b561 are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cytochromes b561 (chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochrome b) were expressed in a Saccharomyces cerevisiae,fre1,fre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity, to study their ability to reduce ferric iron (Fe3+). The expression of each of these cytochromes b561 was able to rescue the growth defect of the ,fre1,fre2 mutant cells in iron-deficient conditions, suggesting their involvement in iron metabolism. Plasma membrane ferrireductase activities were measured using intact yeast cells. Each cytochrome b561 showed significant FeCN and Fe3+ -EDTA reductase activities that were dependent on the presence of intracellular ascorbate. Site-directed mutagenesis of lysosomal cytochrome b was conducted to identify amino acids that are indispensable for its activity. Among more than 20 conserved or partially conserved amino acids that were investigated, mutations of four His residues (H47, H83, H117 and H156), one Tyr (Y66) and one Arg (R67) completely abrogated the FeCN reductase activity, whereas mutations of Arg (R149), Phe (F44), Ser (S115), Trp (W119), Glu (E196), and Gln (Q131) affected the ferrireductase activity to some degree. These mutations may affect the heme coordination, ascorbate binding, and/or ferric substrate binding. Possible roles of these residues in lysosomal cytochrome b are discussed. This study demonstrates the ascorbate-dependent transmembrane ferrireductase activities of members of the mammalian cytochrome b561 family of proteins. [source] A steady-state modeling approach to validate an in vivo mechanism of the GAL regulatory network in Saccharomyces cerevisiaeFEBS JOURNAL, Issue 20 2004Malkhey Verma Cellular regulation is a result of complex interactions arising from DNA,protein and protein,protein binding, autoregulation, and compartmentalization and shuttling of regulatory proteins. Experiments in molecular biology have identified these mechanisms recruited by a regulatory network. Mathematical models may be used to complement the knowledge-base provided by in vitro experimental methods. Interactions identified by in vitro experiments can lead to the hypothesis of multiple candidate models explaining the in vivo mechanism. The equilibrium dissociation constants for the various interactions and the total component concentration constitute constraints on the candidate models. In this work, we identify the most plausible in vivo network by comparing the output response to the experimental data. We demonstrate the methodology using the GAL system of Saccharomyces cerevisiae for which the steady-state analysis reveals that Gal3p neither dimerizes nor shuttles between the cytoplasm and the nucleus. [source] Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome bFEBS JOURNAL, Issue 11 2004Nicholas Fisher Saccharomyces cerevisiae has been used as a model system to characterize the effect of cytochrome b mutations found in fungal and oomycete plant pathogens resistant to Qo inhibitors (QoIs), including the strobilurins, now widely employed in agriculture to control such diseases. Specific residues in the Qo site of yeast cytochrome b were modified to obtain four new forms mimicking the Qo binding site of Erysiphe graminis, Venturia inaequalis, Sphaerotheca fuliginea and Phytophthora megasperma. These modified versions of cytochrome b were then used to study the impact of the introduction of the G143A mutation on bc1 complex activity. In addition, the effects of two other mutations F129L and L275F, which also confer levels of QoI insensitivity, were also studied. The G143A mutation caused a high level of resistance to QoI compounds such as myxothiazol, axoxystrobin and pyraclostrobin, but not to stigmatellin. The pattern of resistance conferred by F129L and L275F was different. Interestingly G143A had a slightly deleterious effect on the bc1 function in V. inaequalis, S. fuliginea and P. megasperma Qo site mimics but not in that for E. graminis. Thus small variations in the Qo site seem to affect the impact of the G143A mutation on bc1 activity. Based on this observation in the yeast model, it might be anticipated that the G143A mutation might affect the fitness of pathogens differentially. If so, this could contribute to observed differences in the rates of evolution of QoI resistance in fungal and oomycete pathogens. [source] The transporters Pdr5p and Snq2p mediate diazaborine resistance and are under the control of the gain-of-function allele PDR1-12FEBS JOURNAL, Issue 6 2004Eva Wehrschütz-Sigl The spontaneous acquisition of resistance to a variety of unrelated cytotoxic compounds has important implications in medical treatment of infectious diseases and anticancer therapy. In the yeast Saccharomyces cerevisiae this phenomenon is caused by overexpression of membrane efflux pumps and is called pleiotropic drug resistance. We have found that allelic forms of the genes for the transcription activators Pdr1p and Pdr3p, designated PDR1-12 and PDR3-33, respectively, mediate resistance to diazaborine. Here we demonstrate that the transporters Pdr5p and Snq2p are involved in diazaborine detoxification. We report that in the PDR3-33 mutant diazaborine resistance is exerted mainly via overexpression of the PDR5 and SNQ2 genes, while in the PDR1-12 mutant, additional genes, i.e. the Yap1p target genes FLR1 and YCF1, are also involved in diazaborine detoxification. In addition, we show that in the presence of cycloheximide or diazaborine PDR5 can be activated by additional transcription factors beside Pdr1p and Pdr3p. [source] Characterization of the products of the genes SNO1 and SNZ1 involved in pyridoxine synthesis in Saccharomyces cerevisiaeFEBS JOURNAL, Issue 4 2004Yi-Xin Dong Genes SNO1 and SNZ1 are Saccharomyces cerevisiae homologues of PDX2 and PDX1 which participate in pyridoxine synthesis in the fungus Cercospora nicotianae. In order to clarify their function, the two genes SNO1 and SNZ1 were expressed in Escherichia coli either individually or simultaneously and with or without a His-tag. When expressed simultaneously, the two protein products formed a complex and showed glutaminase activity. When purified to homogeneity, the complex exhibited a specific activity of 480 nmol·mg,1·min,1 as glutaminase, with a Km of 3.4 mm for glutamine. These values are comparable to those for other glutamine amidotransferases. In addition, the glutaminase activity was impaired by 6-diazo-5-oxo- l -norleucine in a time- and dose-dependent manner and the enzyme was protected from deactivation by glutamine. These data suggest strongly that the complex of Sno1p and Snz1p is a glutamine amidotransferase with the former serving as the glutaminase, although the activity was barely detectable with Sno1p alone. The function of Snz1p and the amido acceptor for ammonia remain to be identified. [source] Ycf1p-dependent Hg(II) detoxification in Saccharomyces cerevisiaeFEBS JOURNAL, Issue 11 2003Olivier Gueldry In Saccharomyces cerevisiae, disruption of the YCF1 gene increases the sensitivity of cell growth to mercury. Transformation of the resulting ycf1 null mutant with a plasmid harbouring YCF1 under the control of the GAL promoter largely restores the wild-type resistance to the metal ion. The protective effect of Ycf1p against the toxicity of mercury is especially pronounced when yeast cells are grown in rich medium or in minimal medium supplemented with glutathione. Secretory vesicles from S. cerevisiae cells overproducing Ycf1p are shown to exhibit ATP-dependent transport of bis(glutathionato)mercury. Moreover, using ,-galactosidase as a reporter protein, a relationship between mercury addition and the activity of the YCF1 promoter can be shown. Altogether, these observations indicate a defence mechanism involving an induction of the expression of Ycf1p and transport by this protein of mercury,glutathione adducts into the vacuole. Finally, possible coparticipation in mercury tolerance of other ABC proteins sharing close homology with Ycf1p was investigated. Gene disruption experiments enable us to conclude that neither Bpt1p, Yor1p, Ybt1p nor YHL035p plays a major role in the detoxification of mercury. [source] | |||||||||||||||||||||||||||||||||||||