Overcoming glucose repression in mixed sugar fermentation by co-expressing a cellobiose transporter and a β-glucosidase in Saccharomyces cerevisiae

Abstract An ssu2 mutation in Sacccharomyces cermisiae, previously shown to cause sulfite sensitivity, was found to be allelic to GRR1, a gene previously implicated in glucose repression. The suppressor rgt1, which suppresses the growth defects of grr1 strains on glucose, did not fully suppress the sensitivity on glucose or nonglucose carbon sources, indicating that it is not strictly linked to a defect in glucose metabolism. Because the Cln1 protein was previously shown to be elevated in grr1 mutants, the effect of CLN1 overexpression on sulfite sensitivity was investigated. Overexpression in GRR1 cells resulted in sulfite sensitivity, suggesting a connection between CLN1 and sulfite metabolism. Multicopy FZF1, a putative transcription factor, was found to suppress the sulfite sensitive phenotype of grr1 strains, but not the glucose derepression or aberrant cell morphology. Multicopy FZF1 was also found to suppress the sensitivity of a number of other unrelated sulfite-sensitive mutants, but not that of ssu1 or met20, implying that FZF1 may act through Ssulp and Met20p. Disruption of FZF1 resulted in sulfite sensitivity when the construct was introduced in single copy at the FZF1 locus in a GRR1 strain, providing evidence that FZF1 is involved in sulfite metabolism.

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Interaction of the Repressors Nrg1 and Nrg2 With the Snf1 Protein Kinase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/158.2.563 ◽

2001 ◽

Vol 158 (2) ◽

pp. 563-572 ◽

Cited By ~ 1

Author(s):

Valmik K Vyas ◽

Sergei Kuchin ◽

Marian Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Hybrid System ◽

Fluorescent Protein ◽

Zinc Finger Protein ◽

Glucose Repression ◽

Snf1 Kinase ◽

Cell Extracts ◽

Green Fluorescent ◽

Two Hybrid

Abstract The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae. We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system. Nrg2 is a C2H2 zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene. Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts. A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor. An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source. Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene. These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

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Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4757-4769.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4757-4769

Author(s):

J S Flick ◽

M Johnston

Keyword(s):

Saccharomyces Cerevisiae ◽

Base Pair ◽

Glucose Repression ◽

Gene Products ◽

Gal1 Promoter ◽

Base Pair Fragment ◽

Common Signal ◽

Multiple Sequences ◽

Pair Fragment ◽

Gal1 Gene

Expression of the GAL1 gene in Saccharomyces cerevisiae is strongly repressed by growth on glucose. We show that two sites within the GAL1 promoter mediate glucose repression. First, glucose inhibits transcription activation by GAL4 protein through UASG. Second, a promoter element, termed URSG, confers glucose repression independently of GAL4. We have localized the URSG sequences responsible for glucose repression to an 87-base-pair fragment located between UASG and the TATA box. Promoters deleted for small (20-base-pair) segments that span this sequence are still subject to glucose repression, suggesting that there are multiple sequences within this region that confer repression. Extended deletions across this region confirm that it contains at least two and possibly three URSG elements. To identify the gene products that confer repression upon UASG and URSG, we have analyzed glucose repression mutants and found that the GAL83, REG1, GRR1, and SSN6 genes are required for repression mediated by both UASG and URSG. In contrast, GAL82 and HXK2 are required only for UASG repression. A mutation designated urr1-1 (URSG repression resistant) was identified that specifically relieves URSG repression without affecting UASG repression. In addition, we observed that the SNF1-encoded protein kinase is essential for derepression of both UASG and URSG. We propose that repression of UASG and URSG is mediated by two independent pathways that respond to a common signal generated by growth on glucose.

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Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.2941-2948.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 2941-2948

Author(s):

A Lombardo ◽

G P Cereghino ◽

I E Scheffler

Keyword(s):

Saccharomyces Cerevisiae ◽

Succinate Dehydrogenase ◽

Half Life ◽

Glucose Repression ◽

Promoter Sequence ◽

Regulatory Elements ◽

Mrna Levels ◽

Protein Subunit ◽

Gene Encoding ◽

Regulatory Complex

We have examined the expression of the gene encoding the iron-protein subunit (Ip) of succinate dehydrogenase in Saccharomyces cerevisiae. The gene had been cloned by us and shown to be subject to glucose regulation (A. Lombardo, K. Carine, and I. E. Scheffler, J. Biol. Chem. 265:10419-10423, 1990). We discovered that a significant part of the regulation of the Ip mRNA levels by glucose involves the regulation of the turnover rate of this mRNA. In the presence of glucose, the half-life appears to be less than 5 min, while in glycerol medium, the half-life is greater than 60 min. The gene is also regulated transcriptionally by glucose. The upstream promoter sequence appeared to have four regulatory elements with consensus sequences shown to be responsible for the interaction with the HAP2/3/4 regulatory complex. A deletion analysis has shown that the two distal elements are redundant. These measurements were carried out by Northern (RNA) analyses of Ip mRNA transcripts as well as by assays of beta-galactosidase activity in cells carrying constructs of the Ip promoter linked to the lacZ coding sequence. These observations on the regulation of mRNA stability were also extended to the mRNA of the flavoprotein subunit of succinate dehydrogenase and in some experiments of iso-1-cytochrome c.

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