Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles.

We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine. The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates. It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants. The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4). The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T. Donahue and S. Henry, J. Biol. Chem. 256:7077-7085, 1981). In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine. The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.

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Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

Enzyme Research ◽

10.4061/2011/157294 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Alexsandro Sobreira Galdino ◽

Roberto Nascimento Silva ◽

Muriele Taborda Lottermann ◽

Alice Cunha Morales Álvares ◽

Lídia Maria Pepe de Moraes ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Soluble Starch ◽

Alpha Amylase ◽

Enzyme Specificity ◽

Wild Type ◽

Spectra Analysis ◽

Exclusion Chromatography ◽

The Difference ◽

Thermal Stability Of

An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH4+ and inhibited by Cu+2 and Hg+2. Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to Km values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1w, points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.

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Characterization of Hailey-Hailey Disease-mutants in presence and absence of wild type SPCA1 using Saccharomyces cerevisiae as model organism

Scientific Reports ◽

10.1038/s41598-019-48866-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Daniel Muncanovic ◽

Mette Heberg Justesen ◽

Sarah Spruce Preisler ◽

Per Amstrup Pedersen

Keyword(s):

Saccharomyces Cerevisiae ◽

Model Organism ◽

Wild Type ◽

Presence And Absence

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Nucleotide sequence and characterization of a Gene conferring resistance to ethionine in yeast Saccharomyces cerevisiae

Journal of Fermentation and Bioengineering ◽

10.1016/0922-338x(91)90269-m ◽

1991 ◽

Vol 71 (4) ◽

pp. 211-215 ◽

Cited By ~ 28

Author(s):

Naofumi Shiomi ◽

Hideki Fukuda ◽

Yasuki Fukuda ◽

Kousaku Murata ◽

Akira Kimura

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Sequence ◽

Yeast Saccharomyces Cerevisiae

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Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.

The Journal of Cell Biology ◽

10.1083/jcb.130.3.567 ◽

1995 ◽

Vol 130 (3) ◽

pp. 567-577 ◽

Cited By ~ 58

Author(s):

D Karaoglu ◽

D J Kelleher ◽

R Gilmore

Keyword(s):

Saccharomyces Cerevisiae ◽

Functional Characterization ◽

Null Mutant ◽

Wild Type ◽

Membrane Glycoproteins ◽

En Bloc ◽

Oligosaccharide Moiety

Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides. The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD. Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase. Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast. Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature. Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay. Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane-bound glycoproteins in vivo. Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins. The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates.

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The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2479-2485.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2479-2485

Author(s):

B S Loewy ◽

S A Henry

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Regulatory Genes ◽

Wild Type ◽

Inositol 1 ◽

Cytoplasmic Enzyme ◽

Pleiotropic Phenotype ◽

Reduced Rate ◽

Phosphate Synthase

We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine. The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates. It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants. The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4). The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T. Donahue and S. Henry, J. Biol. Chem. 256:7077-7085, 1981). In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine. The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.

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