Identification of the Aldo-Keto Reductase Responsible for d-galacturonic Acid Conversion to l-galactonate in Saccharomyces cerevisiae

d-galacturonic acid (d-GalUA) is the main constituent of pectin, a complex polysaccharide abundant in several agro-industrial by-products such as sugar beet pulp or citrus peel. During several attempts to valorise d-GalUA by engineering the popular cell factory Saccharomyces cerevisiae, it became obvious that d-GalUA is, to a certain degree, converted to l-galactonate (l-GalA) by an endogenous enzymatic activity. The goal of the current work was to clarify the identity of the responsible enzyme(s). A protein homology search identified three NADPH-dependent unspecific aldo-keto reductases in baker’s yeast (encoded by GCY1, YPR1 and GRE3) that show sequence similarities to known d-GalUA reductases from filamentous fungi. Characterization of the respective deletion mutants and an in vitro enzyme assay with a Gcy1 overproducing strain verified that Gcy1 is mainly responsible for the detectable reduction of d-GalUA to l-GalA.

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Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

Eukaryotic Cell ◽

10.1128/ec.00284-06 ◽

2007 ◽

Vol 6 (12) ◽

pp. 2214-2221 ◽

Cited By ~ 53

Author(s):

Lois M. Douglas ◽

Li Li ◽

Yang Yang ◽

A. M. Dranginis

Keyword(s):

Saccharomyces Cerevisiae ◽

Biofilm Formation ◽

In Vitro System ◽

Glycosylphosphatidylinositol Anchor ◽

Fungal Adhesion ◽

Very High ◽

Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

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Partial characterization of an RNA component that copurifies with Saccharomyces cerevisiae RNase P

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2536-2543.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2536-2543

Author(s):

J Y Lee ◽

D R Engelke

Keyword(s):

Saccharomyces Cerevisiae ◽

Secondary Structure ◽

Schizosaccharomyces Pombe ◽

Rnase P ◽

Partial Characterization ◽

Rna Sequences ◽

Extensive Sequence ◽

Sequence Similarities

Saccharomyces cerevisiae cellular RNase P is composed of both protein and RNA components that are essential for activity. The isolated holoenzyme contains a highly structured RNA of 369 nucleotides that has extensive sequence similarities to the 286-nucleotide RNA associated with Schizosaccharomyces pombe RNase P but bears little resemblance to the analogous RNA sequences in procaryotes or S. cerevisiae mitochondria. Even so, the predicted secondary structure of S. cerevisiae RNA is strikingly similar to the bacterial phylogenetic consensus rather than to previously predicted structures of other eucaryotic RNase P RNAs.

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In Vitro Characterization of Lactobacillus Strains Isolated from Fruit Processing By-Products as Potential Probiotics

Probiotics and Antimicrobial Proteins ◽

10.1007/s12602-017-9318-2 ◽

2017 ◽

Vol 10 (4) ◽

pp. 704-716 ◽

Cited By ~ 17

Author(s):

Thatyane Mariano Rodrigues de Albuquerque ◽

Estefânia Fernandes Garcia ◽

Amanda de Oliveira Araújo ◽

Marciane Magnani ◽

Maria Saarela ◽

...

Keyword(s):

In Vitro Characterization ◽

Fruit Processing ◽

By Products

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Molecular Characterization of Saccharomyces cerevisiae TFIID

Molecular and Cellular Biology ◽

10.1128/mcb.22.16.6000-6013.2002 ◽

2002 ◽

Vol 22 (16) ◽

pp. 6000-6013 ◽

Cited By ~ 78

Author(s):

Steven L. Sanders ◽

Krassimira A. Garbett ◽

P. Anthony Weil

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Molecular Mass ◽

Molecular Characterization ◽

Biochemical Characterization ◽

Gel Filtration ◽

Calculated Molecular Mass ◽

General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

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Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2396-2405.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2396-2405

Author(s):

R L Last ◽

J B Stavenhagen ◽

J L Woolford

Keyword(s):

Saccharomyces Cerevisiae ◽

Single Copy ◽

Yeast Genome ◽

Temperature Sensitive ◽

In Vitro Mutagenesis ◽

Mrna Accumulation ◽

Isolation And Characterization ◽

Polyadenylated Rna

Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains. The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy. RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).

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The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8282 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8282-8291 ◽

Cited By ~ 137

Author(s):

B T Roberts ◽

K A Farr ◽

M A Hoyt

Keyword(s):

Saccharomyces Cerevisiae ◽

Normal Cell ◽

Kinase Activity ◽

Cell Multiplication ◽

In Vitro Experiments ◽

Microtubule Inhibitor ◽

Cycle Progression ◽

Novel Protein

Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.

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Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.177 ◽

1987 ◽

Vol 7 (1) ◽

pp. 177-184 ◽

Cited By ~ 74

Author(s):

M E Dihanich ◽

D Najarian ◽

R Clark ◽

E C Gillman ◽

N C Martin ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Yeast Cells ◽

Transferase Activity ◽

Isopentenyl Transferase ◽

Nuclear Mutation ◽

Isolation And Characterization ◽

Isopentenyl Adenosine ◽

Cellular Compartments

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

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