Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae.

We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.

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ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4197-4208.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4197-4208

Author(s):

S Silve ◽

P R Rhode ◽

B Coll ◽

J Campbell ◽

R O Poyton

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Glucose Repression ◽

Carbon Sources ◽

Growth Conditions ◽

Source Control ◽

Specific Target Gene ◽

Phosphorylation Pattern ◽

Nonfermentable Carbon Source ◽

In Cells

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

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ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4197 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4197-4208 ◽

Cited By ~ 35

Author(s):

S Silve ◽

P R Rhode ◽

B Coll ◽

J Campbell ◽

R O Poyton

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Glucose Repression ◽

Carbon Sources ◽

Growth Conditions ◽

Source Control ◽

Specific Target Gene ◽

Phosphorylation Pattern ◽

Nonfermentable Carbon Source ◽

In Cells

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

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Role of Tos3, a Snf1 Protein Kinase Kinase, during Growth of Saccharomyces cerevisiae on Nonfermentable Carbon Sources

Eukaryotic Cell ◽

10.1128/ec.4.5.861-866.2005 ◽

2005 ◽

Vol 4 (5) ◽

pp. 861-866 ◽

Cited By ~ 18

Author(s):

Myoung-Dong Kim ◽

Seung-Pyo Hong ◽

Marian Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Catalytic Activity ◽

Protein Kinase ◽

Carbon Source ◽

Carbon Sources ◽

Growth Conditions ◽

Single Mutation ◽

Glucose Depletion ◽

Nonfermentable Carbon Source ◽

Responsive Element

ABSTRACT In Saccharomyces cerevisiae, Snf1 protein kinase of the Snf1/AMP-activated protein kinase family is required for growth on nonfermentable carbon sources and nonpreferred sugars. Three kinases, Pak1, Elm1, and Tos3, activate Snf1 by phosphorylation of its activation-loop threonine, and the absence of all three causes the Snf− phenotype. No phenotype has previously been reported for the tos3Δ single mutation. We show here that, when cells are grown on glycerol-ethanol, tos3Δ reduces growth rate, Snf1 catalytic activity, and activation of the Snf1-dependent carbon source-responsive element (CSRE) in the promoters of gluconeogenic genes. In contrast, tos3Δ did not significantly affect Snf1 catalytic activity or CSRE function during abrupt glucose depletion, indicating that Tos3 has a more substantial role in activating Snf1 protein kinase during growth on a nonfermentable carbon source than during acute carbon stress. We also report that Tos3 is localized in the cytosol during growth in either glucose or glycerol-ethanol. These findings lend support to the idea that the Snf1 protein kinase kinases make different contributions to cellular regulation under different growth conditions.

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Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.6229 ◽

1991 ◽

Vol 11 (12) ◽

pp. 6229-6247 ◽

Cited By ~ 32

Author(s):

S M Miller ◽

B Magasanik

Keyword(s):

Saccharomyces Cerevisiae ◽

Glutamine Synthetase ◽

Nitrogen Source ◽

Glutamate Dehydrogenase ◽

Regulatory System ◽

Sole Source ◽

Lacz Fusion ◽

Upstream Region ◽

In Cells

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

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The Function and Properties of the Azf1 Transcriptional Regulator Change with Growth Conditions in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.5.2.313-320.2006 ◽

2006 ◽

Vol 5 (2) ◽

pp. 313-320 ◽

Cited By ~ 24

Author(s):

Matthew G. Slattery ◽

Dritan Liko ◽

Warren Heideman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Quaternary Structure ◽

Protein A ◽

Sodium Dodecyl ◽

Carbon Sources ◽

Extraction Procedure ◽

Growth Conditions ◽

Growth Defect ◽

Calcofluor White

ABSTRACT Azf1 activates CLN3 transcription in Saccharomyces cerevisiae cells growing in glucose. Paradoxically, other studies have shown Azf1 to be almost undetectable in glucose-grown cells. Microarray experiments showed that Azf1 activates nonoverlapping gene sets in different carbon sources: in glucose, Azf1 activates carbon and energy metabolism genes, and in glycerol-lactate, Azf1 activates genes needed for cell wall maintenance. Consistent with the decreased expression of cell wall maintenance genes observed with azf1Δ mutants, we observed a marked growth defect in the azf1Δ cells at 37°C in nonfermentable medium. Cell wall integrity assays, such as sensitivity to calcofluor white, sodium dodecyl sulfate, or caffeine, confirmed cell wall defects in azf1Δ mutants in nonfermentable medium. Gel shift experiments show that Azf1 binds to DNA elements with the sequence AAAAGAAA (A4GA3), a motif enriched in the promoters of Azf1-sensitive genes and predicted by whole-genome studies. This suggests that many of the Azf1-dependent transcripts may be regulated directly by Azf1 binding. We found that the levels of Azf1 protein in glucose-grown cells were comparable to Azf1 levels in cells grown in glycerol-lactate; however, this could only be demonstrated with a cell extraction procedure that minimizes proteolysis. Glucose produces conditions that destabilize the Azf1 protein, a finding that may reflect a glucose-induced change in Azf1 tertiary or quaternary structure.

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Derepression of citrate synthase in Saccharomyces cerevisiae may occur at the level of transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.4.2.247 ◽

1984 ◽

Vol 4 (2) ◽

pp. 247-253 ◽

Cited By ~ 7

Author(s):

M A Hoosein ◽

A S Lewin

Keyword(s):

Saccharomyces Cerevisiae ◽

Citrate Synthase ◽

Carbon Sources ◽

Stationary Growth Phase ◽

Stationary Growth ◽

Whole Cells ◽

Reactive Protein ◽

Nonfermentable Carbon Source ◽

Cell Free Protein Synthesis

Pulse-chase labeling in whole cells and cell-free protein synthesis were used to establish that the mitochondrial enzyme citrate synthase is made as a larger precursor in Saccharomyces cerevisiae. A 54,000 Mr precursor form appeared to be a primary translation product since it could be labeled with N-[35S]formylmethionine in vitro. The induction of citrate synthase was monitored in S. cerevisiae cells grown on fermentable (glucose) and nonfermentable (ethanol and glycerol) carbon sources. The amount of citrate synthase activity and immune-reactive protein increased more than 15-fold as S. cerevisiae cells entered the stationary growth phase on glucose-containing medium. This increase was paralleled by an increase in translatable RNA for the enzyme. When cells were grown on a nonfermentable carbon source, no increase in either citrate synthase or its mRNA was detected. The results suggest that the release of citrate synthase from catabolite repression may occur at the level of transcription.

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The Defect in Transcription-Coupled Repair Displayed by a Saccharomyces cerevisiae rad26 Mutant Is Dependent on Carbon Source and Is Not Associated With a Lack of Transcription

Genetics ◽

10.1093/genetics/158.3.989 ◽

2001 ◽

Vol 158 (3) ◽

pp. 989-997

Author(s):

Miriam Bucheli ◽

Lori Lommel ◽

Kevin Sweder

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Carbon Source ◽

Excision Repair ◽

Carbon Sources ◽

Repair Process ◽

Growth Conditions ◽

Nucleotide Excision ◽

Evolutionarily Conserved ◽

Transcription Activators

Abstract Nucleotide excision repair (NER) is an evolutionarily conserved pathway that removes DNA damage induced by ultraviolet irradiation and various chemical agents that cause bulky adducts. Two subpathways within NER remove damage from the genome overall or the transcribed strands of transcribing genes (TCR). TCR is a faster repair process than overall genomic repair and has been thought to require the RAD26 gene in Saccharomyces cerevisiae. Rad26 is a member of the SWI/SNF family of proteins that either disrupt chromatin or facilitate interactions between the RNA Pol II and transcription activators. SWI/SNF proteins are required for the expression or repression of a diverse set of genes, many of which are differentially transcribed in response to particular carbon sources. The remodeling of chromatin by Rad26 could affect transcription and/or TCR following formation of DNA damage and other stress-inducing conditions. We speculate that another factor(s) can substitute for Rad26 under particular growth conditions. We therefore measured the level of repair and transcription in two different carbon sources and found that the defect in the rad26 mutant for TCR was dependent on the type of carbon source. Furthermore, TCR did not correlate with transcription rate, suggesting that disruption of RAD26 leads to a specific defect in DNA repair and not transcription.

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Saccharomyces cerevisiae exhibits a sporulation-specific temporal pattern of transcript accumulation.

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.751 ◽

1985 ◽

Vol 5 (4) ◽

pp. 751-761 ◽

Cited By ~ 36

Author(s):

D B Kaback ◽

L R Feldberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Specific Pattern ◽

Transcript Accumulation ◽

Yeast Saccharomyces Cerevisiae ◽

Vegetative Cells ◽

Activation Of Transcription ◽

Nonfermentable Carbon Source ◽

General Activation ◽

Diploid Cells

Cultures of the yeast Saccharomyces cerevisiae that are heterozygous for the mating type (MATa/MAT alpha) undergo synchronous meiosis and spore formation when starved for nitrogen and supplied with a nonfermentable carbon source such as acetate. Haploid and homozygous MAT alpha/MAT alpha and MATa/MATa diploid cells incubated under the same conditions fail to undergo meiosis and are asporogenous. It has not yet been firmly established that gene expression during sporulation is controlled at the level of transcript accumulation. To examine this question, we used cloned genes that encode a variety of "housekeeping" functions to probe Northern blots to assay the appearance of specific transcripts in both sporulating and asporogenous S. cerevisiae. In sporulating cells, each transcript showed a characteristic pattern of accumulation, reaching a maximum relative abundance at one of several different periods. In contrast, in both asporogenous haploid MATa and diploid MAT alpha/MAT alpha cells, all transcripts accumulated with similar kinetics. These results suggest a sporulation-specific pattern for transcript appearance. During these studies, high levels of several different transcripts were observed at unexpected times in sporulating cells. Histone (H)2A and (H)2B1 transcripts, although most abundant during premeiotic DNA synthesis, remained at one-third to one-half maximal levels after its end and were found in mature ascospores. Their appearance at this time is in sharp contrast to vegetative cells in which these histone transcripts are only found just before and during the period of DNA synthesis. Furthermore, transcripts from GAL10 and CDC10 genes, which are believed to be dispensable for sporulation, were much more abundant in sporulating cells than in asporogenous cells and vegetative cells grown on glucose or acetate. The presence of these transcripts did not appear to be due to a general activation of transcription because each accumulated with different kinetics. In addition, the transcript for at least one gene, HO, that is also dispensable for sporulation was not detected. The increased abundance of transcripts from some genes not required for sporulation leads us to propose that genes preferentially expressed during sporulation need not be essential for this differentiation.

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EFFECT OF DIFFERENT CULTURE CONDITION ON ANTIOXIDANT SECONDARY METABOLITES FROM ENDOPHYTIC FUNGI ISOLATED FROM TURMERIC ROOT

Majalah Obat Tradisional ◽

10.22146/tradmedj.24308 ◽

2017 ◽

Vol 22 (1) ◽

pp. 31 ◽

Cited By ~ 1

Author(s):

Eris Septiana ◽

Partomuan Simanjuntak

Keyword(s):

Antioxidant Activity ◽

Nitrogen Source ◽

Yeast Extract ◽

Carbon Sources ◽

Basal Medium ◽

Nitrogen Sources ◽

Growth Conditions ◽

Carbon And Nitrogen ◽

Antioxidant Compound ◽

Initial Ph

Antioxidant is an interesting topic due to their capability to inhibit free radical and prevent damage because of oxidative processes. Endophyt fungi is one of antioxidant compound resources in nature. The low yield to gain antioxidant compound from fungi challenges to look for the composition of media and optimalization of growth conditions. This research aimed to know the effect of medium condition in different carbon and nitrogen sources as well as initial pH towards antioxidant activity of endophyt fungi Bo.Ci.Cl.A3. Shaker fermentation was used on 120 rpm at room temperature for 14 days. The carbon sources were glucose, sucrose, and starch and nitrogen sources were NaNO3, NH4NO3, and yeast extract with initial pH at 5, 7, and 9. Ethyl acetate was used as extractor. The results showed that endophyt fungi can produce secondary metabolite as antioxidant at all variation of fermented media. The nitrogen source of yeast extract could increase antioxidant activity of endophyt fungi Bo.Ci.Cl.A3, while other sources such as nitrogen source, carbon sources, and different initial pH on the basal medium that were used did not give increasing antioxidant activity. The conclusion of this research was the substitution of nitrogen source with yeast extract (3 g/L) on the basal medium Czapek Dox’s Broth could increase antioxidant activity of endophyt fungi Bo.Ci.Cl.A3.

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