Showing papers on "Yeast published in 1994"

Vesicle fusion from yeast to man.

Abstract: Membrane budding and fusion occur in all eukaryotic cells. Their underlying mechanisms have been studied in mammalian neurons and in yeast, a simple eukaryote. The differences between these two systems would suggest that fusion events in yeast and the neuron would operate by different mechanisms, but recent advances indicate that this is not true.

RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex.

Abstract: Rapamycin is a potent immunosuppressant that blocks the G1/S transition in antigen-activated T cells and in yeast. The similar effects of rapamycin in animal cells and yeast suggest that the biochemical steps affected by rapamycin are conserved. Using a two-hybrid system we isolated mammalian clones that interact with the human FK506/rapamycin-binding protein (FKBP12) in the presence of rapamycin. Specific interactors, designated RAPT1, encode overlapping sequences homologous to yeast Tor, a putative novel phosphatidylinositol 3-kinase. A region of 133 amino acids of RAPT1 is sufficient for binding to the FKBP12/rapamycin complex. The corresponding region in yeast Tor contains the serine residue that when mutated to arginine confers resistance to rapamycin. Introduction of this mutation into RAPT1 abolishes its interaction with the FKBP12/rapamycin complex.

A multisubunit complex containing the SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products isolated from yeast.

Abstract: A complex containing the products of the SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 genes and four additional polypeptides has been purified from extracts of the yeast Saccharomyces cerevisiae. Physical association of these proteins was demonstrated by copurification and coimmunoprecipitation. A potent DNA-dependent ATPase copurified with the complex, and this activity was evidently associated with SWI2/SNF2.

The role of trehalose synthesis for the acquisition of thermotolerance in yeast. II. Physiological concentrations of trehalose increase the thermal stability of proteins in vitro.

Abstract: In baker's yeast (Saccharomyces cerevisiae), accumulation of the non-reducing disaccharide, trehalose, is triggered by stimuli that activate the heat-shock response. Previously, trehalose levels have been shown to be closely correlated with thermotolerance, suggesting a protective function of this substance. Genetic evidence in support of this view is presented in an accompanying paper [De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 179-186]. In this study, we have examined the effect of trehalose on the thermal stability of proteins, a parameter thought to be a major determinant of thermotolerance. Physiological concentrations of trehalose (up to 0.5 M) were found to efficiently protect enzymes of yeast (glucose-6P-dehydrogenase, phosphoglucose-isomerase) as well as enzymes of non-yeast origin (bovine glutamic dehydrogenase, EcoRI) against heat inactivation in vitro. Trehalose also reduced the heat-induced formation of protein aggregates. The disaccharide proved to be a compatible solute, as even at very high concentrations (up to 1 M) it did not significantly interfere with the activity of test enzymes. Trehalose was at least as good or better a protein stabilizer than any of a number of other compatible solutes (including sugars, polyalcohols and amino acids), while the structurally related trehalose-6P was devoid of any protective effect. Thermoprotection of enzymes by trehalose was evident even in solutions containing high concentrations of yeast protein or substrate. The data indicate that trehalose accumulation may increase the thermotolerance of yeast by enhancing protein stability in intact cells.

Identification of a high affinity NH4+ transporter from plants.

Abstract: Despite the important role of the ammonium ion in metabolism, i.e. as a form of nitrogen that is taken up from the soil by microorganisms and plants, little is known at the molecular level about its transport across biomembranes. Biphasic uptake kinetics have been observed in roots of several plant species. To study such transport processes, a mutant yeast strain that is deficient in two NH4+ uptake systems was used to identify a plant NH4+ transporter. Expression of an Arabidopsis cDNA in the yeast mutant complemented the uptake deficiency. The cDNA AMT1 contains an open reading frame of 501 amino acids and encodes a highly hydrophobic protein with 9-12 putative membrane spanning regions. Direct uptake measurements show that mutant yeast cells expressing the protein are able to take up [14C]methylamine. Methylamine uptake can be efficiently competed by NH4+ but not by K+. The methylamine uptake is optimal at pH 7 with a Km of 65 microM and a Ki for NH4+ of approximately 10 microM, is energy-dependent and can be inhibited by protonophores. The plant protein is highly related to an NH4+ transporter from yeast (Marini et al., accompanying manuscript). Sequence homologies to genes of bacterial and animal origin indicate that this type of transporter is conserved over a broad range of organisms. Taken together, the data provide strong evidence that a gene for the plant high affinity NH4+ uptake has been identified.

SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein

Abstract: Summary An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H+ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. The KM-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors. The VMAX but not the KM-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.

Method of screening for cDNA encoding novel secreted mammalian proteins in yeast

Abstract: A novel method of screening for novel secreted mammalian proteins is described in which mammalian secretory leader sequences are detected using the yeast invertase gene as a reporter system.

Requirement for the yeast gene LON in intramitochondrial proteolysis and maintenance of respiration

Abstract: The role of protein degradation in mitochondrial homeostasis was explored by cloning of a gene from Saccharomyces cerevisiae that encodes a protein resembling the adenosine triphosphate (ATP)-dependent bacterial protease Lon. The predicted yeast protein has a typical mitochondrial matrix-targeting sequence at its amino terminus. Yeast cells lacking a functional LON gene contained a nonfunctional mitochondrial genome, were respiratory-deficient, and lacked an ATP-dependent proteolytic activity present in the mitochondria of Lon+ cells. Lon- cells were also impaired in their ability to catalyze the energy-dependent degradation of several mitochondrial matrix proteins and they accumulated electron-dense inclusions in their mitochondrial matrix.

Characterization of lipid particles of the yeast, Saccharomyces cerevisiae.

Abstract: Lipid particles of the yeast, Saccharomyces cerevisiae, were isolated to high purity and their components were analysed. The hydrophobic core of this organelle consists of triacylglycerols and steryl esters, which are almost exclusively located to that compartment. Lipid particles are stabilized by a surface membrane consisting of phospholipids and proteins. Electron microscopy confirmed the purity of the preparations and the proposed structure deduced from biochemical experiments. Major proteins of lipid particles have molecular weights of 72, 52, 43 and 34 kDa, respectively. The 43 kDa protein reacts with an antiserum against human apolipoprotein AII. In lipid particles of the yeast mutant strain S. cerevisiae erg6, which is deficient in sterol delta 24-methyltransferase, this protein is missing thereby identifying the protein and confirming our previous finding (Zinser et al., 1993) that sterol delta 24-methylation is associated with lipid particles. A possible involvement of surface proteins of lipid particles in the interaction with other organelles is discussed with respect to sterol translocation in yeast.

Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene.

Abstract: The ATF1 gene, which encodes alcohol acetyltransferase (AATase), was cloned from Saccharomyces cerevisiae and brewery lager yeast (Saccharomyces uvarum). The nucleotide sequence of the ATF1 gene isolated from S. cerevisiae was determined. The structural gene consists of a 1,575-bp open reading frame that encodes 525 amino acids with a calculated molecular weight of 61,059. Although the yeast AATase is considered a membrane-bound enzyme, the results of a hydrophobicity analysis suggested that this gene product does not have a membrane-spanning region that is significantly hydrophobic. A Southern analysis of the yeast genomes in which the ATF1 gene was used as a probe revealed that S. cerevisiae has one ATF1 gene, while brewery lager yeast has one ATF1 gene and another, homologous gene (Lg-ATF1). Transformants carrying multiple copies of the ATF1 gene or the Lg-ATF1 gene exhibited high AATase activity in static cultures and produced greater concentrations of acetate esters than the control.

Characterization of β‐glucosidase activity in yeasts of oenological origin

Abstract: I. ROSI, M. VINELLA AND P. DOMIZIO. 1994. Three hundred and seventeen strains representing 20 species of yeasts were screened for the presence of β-glucosidase activity. All of the strains of the species Debaryomyces castellii, Deb. hansenii, Deb. polymorphus, Kloeckera apiculata and Hansenula anomala showed β-glucosidase activity, but only one of 153 strains of Saccharomyces cerevisiae. The other species behaved differently, depending upon the strain. The strains that hydrolysed arbutin were checked to localize the β-glucosidase activity. A strain of Deb. hansenii exhibited the highest exocellular activity and some wall-bound and intracellular activity. The β-glucosidase synthesis from this yeast was enhanced by aerobic conditions of growth, was repressed by high glucose concentration (9%) and occurred during exponential growth. The optimum conditions for enzymatic preparations of Deb. hansenii were between pH 4.0 and 5.0 and 40dC. A high concentration of ethanol and glucose did not reduce the ezymatic activity. The enzymatic preparations of Deb. hansenii released monoterpenols and other alcohols from a grape glycoside extract.

A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast.

Abstract: Summary A method is presented for the isolation of genes encoding hydrolytic enzymes without any knowledge of the corresponding proteins. cDNA made from the organism of interest is cloned into a yeast vector to construct an expression library in the yeast Saccharomyces cerevisiae. Colonies producing hydrolytic enzymes are screened by activity plate assays. In this work, we constructed a yeast expression library from the filamentous fungus Trichoderma reesel and isolated a new β-1,4-endoglucanase gene on plates containing β-glucan. This gene, eg15, codes for a previously unknown small protein of 242 amino acids. Despite its small size, the protein contains two conservative domains found in Trichoderma cellulases, namely the cellulose-binding domain (CBD) and the iinker region that connects the CBD to the catalytic core domain. Molecular modelling of the EGV CBD revealed some interesting structural differences compared to the CBD of the major celluiase CBHI from T. reesei. The catalytic core of EGV is unusually small for a ceiiulase and represents a new family of ceilulases (Family K) and of glycosyl hydrolases (Famlly 45) together with the endoglucanase B of Pseudomonas fluorescens and the endoglucanase V of Humicola insolens on the basis of hydrophobic ciuster anaiysis.

The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of carbohydrates

Abstract: Interactions betweenLactobacillus brevis subsp.lindneri CB1,L. plantarum DC400,Saccharomyces cerevisiae 141 andS.exiguus M14 from sourdoughs were studied in a co-culture model system using a synthetic medium. The lack of competition for maltose whenS.exiguus M14 was present in co-culture with each of the lactic acid bacteria (LAB) enhanced the bacterial cell yield and lactic and acetic acid production.L.brevis subsp.lindneri CB1 resting cells hydrolysed maltose and accumulated glucose in the medium, allowing the growth of maltose negative yeast.S.cerevisiae 141 competed greatly with each of the LAB for glucose and only withL.plantarum DC400 for fructose, causing a decrease in the bacterial cell number and in acid production. As a result of the glucose and fructose availability after the invertase activity of both yeasts,L.plantarum DC400 grew optimally in the presence of sucrose as a carbon source. All of the interactions indicated were confirmed by studying the behaviour of the co-cultures in wheat flour hydrolysate.

An Arabidopsis heat shock protein complements a thermotolerance defect in yeast.

Abstract: The heat shock protein Hsp104 of the yeast Saccharomyces cerevisiae plays a key role in promoting survival at extreme temperatures. We found that when diverse higher plant species are exposed to high temperatures they accumulate proteins that are antigenically related to Hsp104. We isolated a cDNA corresponding to one of these proteins from Arabidopsis. The protein, AtHSP101, is 43% identical to yeast Hsp104. DNA gel blot analysis indicated that AtHSP101 is encoded by a single- or low-copy number gene. AtHsp101 mRNA was undetectable in the absence of stress but accumulated to high levels during exposure to high temperatures. When AtHSP101 was expressed in yeast, it complemented the thermotolerance defect caused by a deletion of the HSP104 gene. The ability of AtHSP101 to protect yeast from severe heat stress strongly suggests that this HSP plays an important role in thermotolerance in higher plants.

Molecular genetics of yeast :a practical approach

Abstract: 1: Procedures for isolating yeast DNA for different purposes. 2: Construction of cloning and expression vectors. 3: Cosmid cloning of yeast DNA. 4: The construction and use of cDNA libraries for genetic selections. 5: Pulsed field gel electrophoresis. 6: Plasmid shuffling and mutant isolation. 7: Ty insertional mutagenesis. 8: High efficiency transformation with lithium acetate. 9: Measurement of transcription. 10: Measurement of mRNA stability. 11: Production of foreign proteins at high level. 12: Cell-free translation of natural and synthetic mRNAs. 13: Virus-like particles: Ty retrotransposons. 14: The K1 killer toxin: molecular and genetic applications to secretion and cell surface assembly. 15: Immuno-electron microscopy. 16: Industrial Saccharomyces yeasts

Metabolic Engineering for Production of β-Carotene and Lycopene in Saccharomyces cerevisiae

Abstract: We have engineered a conventional yeast, Saccharomyces cerevisiae, to confer a novel biosynthetic pathway for the production of β-carotene and lycopene by introducing the bacterial carotenoid biosynthesis genes, which are individually surrounded by the promoters and terminators derived from S. cerevisiae. β-Carotene and lycopene accumulated in the cells of this yeast, which was considered to be a result of the carbon flow for the ergosterol biosynthetic pathway being partially directed to the pathway for the carotenoid production.

Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast. Kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway.

Abstract: Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Saccharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S. cerevisiae strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H. When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced iron/carbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4-hydroxylase activity upon coupling with the different P450 reductases and NADPH. Some CA4H protein was found also addressed to the yeast mitochondria but as a low-activity form. The spectral and kinetic characterizations of the yeast-produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constituted by the overexpressed yeast P450 reductase and CA4H is characterized by a 1:1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH-dependent P450 reaction. Based on spectral perturbation and inhibition studies, coumarate appeared to have no detectable affinity for the enzyme. A possible geometry of the substrate recognition pocket is discussed in the light of these data.