Nuclear localization of aspartate transcabamoylase in Saccharomyces cerevisiae.

M Nagy; J Laporte; B Penverne; G Hervé

doi:10.1083/jcb.92.3.790

Nuclear localization of aspartate transcabamoylase in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.92.3.790 ◽

1982 ◽

Vol 92 (3) ◽

pp. 790-794 ◽

Cited By ~ 25

Author(s):

M Nagy ◽

J Laporte ◽

B Penverne ◽

G Hervé

Keyword(s):

Saccharomyces Cerevisiae ◽

Feedback Inhibition ◽

Nuclear Accumulation ◽

Lead Phosphate ◽

Carbamoylphosphate Synthetase ◽

Aspartate Carbamoyltransferase ◽

Encoding Gene ◽

Atcase Activity ◽

Cytochemical Technique

The cytochemical technique using the in situ precipitation of orthophosphate ions liberated specifically by the aspartate carbamoyltransferase (ATCase) (EC 2.1.3.2) reaction indicated that in Saccharomyces cerevisiae this enzyme is confined to the nucleus. This observation is in accordance with the result reported by Bernhardt and Davis (1972), Proc. Natl. Acad. Sci. U. S. A. 69:1868-1872) on Neurospora crassa. The nuclear compartmentation was also observed in a mutant strain lacking proteinase B activity. This finding indicates that this proteinase is not involved in the nuclear accumulation of ATCase, and that the activity observed in the nucleus corresponds to the multifunctional form associated with the uracil path-specific carbamoylphosphate synthetase and sensitive to feedback inhibition by UTP. In a ura2 strain transformed by nonintegrated pFL1 plasmids bearing the URA2-ATCase activity encoding gene, the lead phosphate precipitate was observed predominantly in the cytoplasm. This finding enhances the reliability of the technique used by eliminating the possibility of an artifactual displacement of an originally cytoplasmic reaction product during the preparation of the material for electron microscopy. On the other hand, nuclei isolated under hypoosmotic conditions do not exhibit the ATCase activity that is recovered in the cytosolic fractions after differential centrifugation of the lysate in Percoll gradient. A release of the protein from the nuclei during the lysis step, consistent with its nucleoplasmic localization, is postulated.

Download Full-text

Improvement of 2-phenylethanol production in Saccharomyces cerevisiae by evolutionary and rational metabolic engineering

PLoS ONE ◽

10.1371/journal.pone.0258180 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258180

Author(s):

Linghuan Zhu ◽

Sha Xu ◽

Youran Li ◽

Guiyang Shi

Keyword(s):

Saccharomyces Cerevisiae ◽

Pyruvate Decarboxylase ◽

Widespread Application ◽

Innovative Strategies ◽

In Situ Product Recovery ◽

Glutamate Oxidase ◽

Encoding Gene ◽

Food Exploitation ◽

First Time

2-Phenylethanol (2-PE) is a valuable aromatic compound with favorable flavors and good properties, resulting in its widespread application in the cosmetic, food and medical industries. In this study, a mutant strain, AD032, was first obtained by adaptive evolution under 2-PE stress. Then, a fusion protein from the Ehrlich pathway, composed of tyrB from Escherichia coli, kdcA from Lactococcus lactis and ADH2 from Saccharomyces cerevisiae, was constructed and expressed. As a result, 3.14 g/L 2-PE was achieved using L-phenylalanine as a precursor. To further increase 2-PE production, L-glutamate oxidase from Streptomyces overexpression was applied for the first time in our research to improve the supply of α-ketoglutarate in the transamination of 2-PE synthesis. Furthermore, we found that the disruption of the pyruvate decarboxylase encoding gene PDC5 caused an increase in 2-PE production, which has not yet been reported. Finally, assembly of the efficient metabolic modules and process optimization resulted in the strain RM27, which reached 4.02 g/L 2-PE production from 6.7 g/L L-phenylalanine without in situ product recovery. The strain RM27 produced 2-PE (0.8 mol/mol) with L-phenylalanine as a precursor, which was considerably high, and displayed manufacturing potential regarding food safety and process simplification aspects. This study suggests that innovative strategies regarding metabolic modularization provide improved prospects for 2-PE production in food exploitation.

Download Full-text

Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6875 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6875-6883 ◽

Cited By ~ 165

Author(s):

D J Katzmann ◽

T C Hallstrom ◽

M Voet ◽

W Wysock ◽

J Golin ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Gene ◽

Sequence Similarity ◽

Yeast Cells ◽

Atp Binding ◽

Zinc Cluster ◽

Atp Binding Cassette Transporter ◽

Transporter Family ◽

Encoding Gene ◽

Atp Binding Cassette

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

Download Full-text

The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2719 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2719-2727 ◽

Cited By ~ 58

Author(s):

S Silve ◽

P Leplatois ◽

A Josse ◽

P H Dupuy ◽

C Lanau ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Proliferation ◽

Gene Product ◽

In Vitro Assays ◽

Gene Products ◽

Yeast Saccharomyces Cerevisiae ◽

Isomerase Activity ◽

Encoding Gene ◽

Resistant Mutants

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

Download Full-text

Automated cryo-lamella preparation for high-throughput in-situ structural biology

10.1101/797506 ◽

2019 ◽

Author(s):

Genevieve Buckley ◽

Gediminas Gervinskas ◽

Cyntia Taveneau ◽

Hari Venugopal ◽

James C. Whisstock ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Structural Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Automated Method ◽

Cellular Environment ◽

Transmission Electron

AbstractCryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae and demonstrate that this method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

Download Full-text

Cloning and sequencing of the GMP synthetase-encoding gene of Saccharomyces cerevisiae

Gene ◽

10.1016/0378-1119(94)90535-5 ◽

1994 ◽

Vol 139 (1) ◽

pp. 127-132 ◽

Cited By ~ 16

Author(s):

Geneviève Dujardin ◽

Michèle Kermorgant ◽

Piotr P. Slonimski ◽

Hélian Boucherie

Keyword(s):

Saccharomyces Cerevisiae ◽

Cloning And Sequencing ◽

Encoding Gene ◽

Gmp Synthetase

Download Full-text

Effects of Live Yeast Saccharomyces cerevisiae on Fermentation Parameters and Microbial Populations of Rumen, Total Tract Digestibility of Diet Nutrients and on the in situ Degradability of Alfalfa Hay in Iranian Chall Sheep

Pakistan Journal of Biological Sciences ◽

10.3923/pjbs.2007.590.597 ◽

2007 ◽

Vol 10 (4) ◽

pp. 590-597 ◽

Cited By ~ 11

Author(s):

A. A. Khadem ◽

M. Pahlavan ◽

A. Afzalzadeh ◽

M. Rezaeian

Keyword(s):

Saccharomyces Cerevisiae ◽

Microbial Populations ◽

Yeast Saccharomyces Cerevisiae ◽

Alfalfa Hay ◽

Fermentation Parameters ◽

Live Yeast

Download Full-text

Metabolic Engineering of Saccharomyces cerevisiae for Conversion of d-Glucose to Xylitol and Other Five-Carbon Sugars and Sugar Alcohols

Applied and Environmental Microbiology ◽

10.1128/aem.02707-06 ◽

2007 ◽

Vol 73 (17) ◽

pp. 5471-5476 ◽

Cited By ~ 23

Author(s):

Mervi H. Toivari ◽

Laura Ruohonen ◽

Andrei N. Miasnikov ◽

Peter Richard ◽

Merja Penttilä

Keyword(s):

Saccharomyces Cerevisiae ◽

Metabolic Engineering ◽

Growth Medium ◽

Fold Increase ◽

Pichia Stipitis ◽

Xylitol Dehydrogenase ◽

Sugar Alcohols ◽

Pentose Sugar ◽

Encoding Gene ◽

Pentose Sugars

ABSTRACT Recombinant Saccharomyces cerevisiae strains that produce the sugar alcohols xylitol and ribitol and the pentose sugar d-ribose from d-glucose in a single fermentation step are described. A transketolase-deficient S. cerevisiae strain accumulated d-xylulose 5-phosphate intracellularly and released ribitol and pentose sugars (d-ribose, d-ribulose, and d-xylulose) into the growth medium. Expression of the xylitol dehydrogenase-encoding gene XYL2 of Pichia stipitis in the transketolase-deficient strain resulted in an 8.5-fold enhancement of the total amount of the excreted sugar alcohols ribitol and xylitol. The additional introduction of the 2-deoxy-glucose 6-phosphate phosphatase-encoding gene DOG1 into the transketolase-deficient strain expressing the XYL2 gene resulted in a further 1.6-fold increase in ribitol production. Finally, deletion of the endogenous xylulokinase-encoding gene XKS1 was necessary to increase the amount of xylitol to 50% of the 5-carbon sugar alcohols excreted.

Download Full-text

Effect of valine and the herbicide sulfometuron methyl on acetolactate synthase activity in nuclear and plasmid-borne sulphometuron methyl resistant Saccharomyces cerevisiae strains

Canadian Journal of Microbiology ◽

10.1139/m88-112 ◽

1988 ◽

Vol 34 (5) ◽

pp. 680-685 ◽

Cited By ~ 5

Author(s):

S. N. Maiti ◽

M. W. Zink ◽

G. H. Rank

Keyword(s):

Saccharomyces Cerevisiae ◽

Copy Number ◽

Specific Activity ◽

Feedback Inhibition ◽

Acetolactate Synthase ◽

Wild Type ◽

Sulfometuron Methyl ◽

Isogenic Lines

Acetolactate synthase (ALS) specific activity was evaluated in isogenic lines of Saccharomyces cerevisiae carrying the wild-type ILV2 gene or mutations in this gene for resistance to the herbicide sulfometuron methyl (SM). Statistical comparisons were made between two nuclear alleles and among five alleles borne on a YE chimaeric plasmid transformed into a strain carrying a 1.5-kilobase deletion in the nuclear ILV2 gene. Decreased ALS activity of plasmid-borne SM-resistant mutations was shown not to be caused by copy number effects. ALS-specific activity in strains carrying the wild-type ILV2 allele exhibited strong feedback inhibition by valine and was sensitive to SM. All nuclear and plasmid-borne SM-resistance alleles resulted in ALS-specific activity highly resistant to SM and resistant to valine feedback inhibition.

Download Full-text

Gastrointestinal expression and partial cDNA cloning of murine Muc2

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.1.g115 ◽

1999 ◽

Vol 276 (1) ◽

pp. G115-G124 ◽

Cited By ~ 16

Author(s):

B. Jan-Willem Van Klinken ◽

Alexandra W. C. Einerhand ◽

Louise A. Duits ◽

Mireille K. Makkink ◽

Kristien M. A. J. Tytgat ◽

...

Keyword(s):

Small Intestine ◽

Cdna Probe ◽

Northern Blotting ◽

Western Blots ◽

Peptide Epitopes ◽

Colonic Mucin ◽

Partial Cdna ◽

Encoding Gene

To help us investigate the role of mucin in the protection of the colonic epithelium in the mouse, we aimed to identify the murine colonic mucin (MCM) and its encoding gene. We isolated MCM, raised an anti-MCM antiserum, and studied the biosynthesis of MCM in the gastrointestinal tract. Isolated MCM resembled other mucins in physicochemical properties. Anti-MCM recognized MCM as well as rat and human MUC2 on Western blots, interacting primarily with peptide epitopes, indicating that MCM was identical to murine Muc2. Using anti-MCM and previously characterized anti-human and anti-rat MUC2 antibodies, we identified a murine Muc2 precursor in the colon of ∼600 kDa, which appeared similar in size to rat and human MUC2 precursors. Western blotting, immunoprecipitation of metabolically labeled mucins, and immunohistochemistry showed that murine Muc2 was expressed in the colon and the small intestine but was absent in the stomach. To independently identify murine Muc2, we cloned a cDNA fragment from murine colonic mRNA, encoding the 302 NH2-terminal amino acids of murine Muc2. The NH2terminus of murine Muc2 showed 86 and 75% identity to the corresponding rat and human MUC2 peptide sequences, respectively. Northern blotting with a murine Muc2 cDNA probe showed hybridization to a very large mRNA, which was expressed highly in the colon and to some extend in the small intestine but was absent in the stomach. In situ hybridization showed that the murine Muc2 mRNA was confined to intestinal goblet cells. In conclusion, by two independent sets of experiments we identified murine Muc2, which appears homologous to rat and human MUC2. Because Muc2 is prominently expressed in the colon, it is most likely to be the predominant mucin in the colonic mucus layer.

Download Full-text