Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (4) ◽  
pp. 1659-1666 ◽  
Author(s):  
P K Hwang ◽  
S Tugendreich ◽  
R J Fletterick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glycogen Phosphorylase ◽  
Glucose Repression ◽  
Essential Gene ◽  
Yeast Cells ◽  
Exponential Growth Phase ◽  
Phosphorylase Activity ◽  
Glycogen Accumulation ◽  
Multiple Copies ◽  
Diploid Cells

In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme. We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae. To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPH1 gene or containing multiple copies of the gene were constructed. GPH1 was found not to be an essential gene in yeast cells. Haploid cells disrupted in GPH1 lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells. Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores. Absence of functional GPH1 did not impair cells from synthesizing and storing trehalose. Increases in phosphorylase activity of 10- to 40-fold were detected in cells carrying multiple copies of GPH1-containing 2 microns plasmid. Northern (RNA) analysis indicated that GPH1 transcription was induced at the late exponential growth phase, almost simultaneous with the onset of intracellular glycogen accumulation. Thus, the low level of glycogen in exponential cells was not primarily maintained through regulating the phosphorylation state of a constitutive amount of phosphorylase. GPH1 did not appear to be under formal glucose repression, since transcriptional induction occurred well in advance of glucose depletion from the medium.

scholarly journals Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (4) ◽  
pp. 1659-1666 ◽  
Author(s):  
P K Hwang ◽  
S Tugendreich ◽  
R J Fletterick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glycogen Phosphorylase ◽  
Glucose Repression ◽  
Essential Gene ◽  
Yeast Cells ◽  
Exponential Growth Phase ◽  
Phosphorylase Activity ◽  
Glycogen Accumulation ◽  
Multiple Copies ◽  
Diploid Cells

In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme. We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae. To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPH1 gene or containing multiple copies of the gene were constructed. GPH1 was found not to be an essential gene in yeast cells. Haploid cells disrupted in GPH1 lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells. Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores. Absence of functional GPH1 did not impair cells from synthesizing and storing trehalose. Increases in phosphorylase activity of 10- to 40-fold were detected in cells carrying multiple copies of GPH1-containing 2 microns plasmid. Northern (RNA) analysis indicated that GPH1 transcription was induced at the late exponential growth phase, almost simultaneous with the onset of intracellular glycogen accumulation. Thus, the low level of glycogen in exponential cells was not primarily maintained through regulating the phosphorylation state of a constitutive amount of phosphorylase. GPH1 did not appear to be under formal glucose repression, since transcriptional induction occurred well in advance of glucose depletion from the medium.

Temporal patterns of tissue glycogen, glucose, and glycogen phosphorylase activity prior to hibernation in freeze-tolerant chorus frogs, Pseudacris triseriata

2008 ◽  
Vol 86 (10) ◽  
pp. 1095-1100 ◽  
Author(s):  
Steve C. Dinsmore ◽  
David L. Swanson
Keyword(s):  
Glycogen Phosphorylase ◽  
Threshold Level ◽  
Liver Glycogen ◽  
Critical Threshold ◽  
Hepatic Glycogen ◽  
Phosphorylase Activity ◽  
Continuous Increase ◽  
Glycogen Accumulation ◽  
Chorus Frogs ◽  
Glycogen Stores

Freezing survival may differ among winters in chorus frogs ( Pseudacris triseriata (Wied-Neuwied, 1838)), and low freezing survival is associated with low hepatic glycogen stores. The pattern of prehibernation liver glycogen accumulation in chorus frogs is unknown. Frogs might accumulate hepatic glycogen stores until a threshold level sufficient for winter survival is attained, after which frogs enter hibernation (critical threshold hypothesis). According to this model, frogs active late in the season should only be those with low hepatic glycogen stores. Alternatively, hepatic glycogen levels might continue to increase throughout the fall as long as frogs remain active (continuous increase hypothesis). We tested these hypotheses by measuring liver and leg muscle glycogen, glucose, and glycogen phosphorylase activities in chorus frogs throughout the fall prehibernation period in southeastern South Dakota. Hepatic glycogen levels were significantly related to date and increased throughout the fall period, consistent with the continuous increase hypothesis. This suggests that hepatic glycogen levels do not serve as a cue for entrance into hibernation. Liver phosphorylase activity did not vary significantly with progression of the fall season and activity was lower than in winter, suggesting that the winter increment of phosphorylase activity requires some stimulus during hibernation (e.g., low temperatures).

GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats

1991 ◽  
Vol 11 (10) ◽  
pp. 5101-5112
Author(s):  
J S Flick ◽  
M Johnston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Interactions ◽  
Tandem Repeats ◽  
Glucose Repression ◽  
Molecular Data ◽  
Primary Response ◽  
Yeast Cells ◽  
Cell Fractionation ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae

Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene. GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression.

scholarly journals The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (10) ◽  
pp. 6789-6796 ◽  
Author(s):  
J Tu ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Genetic Analysis ◽  
Protein Phosphatase ◽  
Glucose Repression ◽  
Regulatory Mechanisms ◽  
Genetic Studies ◽  
Glycogen Accumulation ◽  
Regulatory Response

We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.

Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae

1989 ◽  
Vol 9 (9) ◽  
pp. 3992-3998
Author(s):  
A M Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Yeast Cells ◽  
Specific Gene ◽  
Activity Levels ◽  
Sporulation Medium ◽  
Mrna Levels ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Diploid Cells

STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts. It has been suggested that STA1 is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S. cerevisiae. A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.

scholarly journals Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase.

1989 ◽  
Vol 9 (11) ◽  
pp. 5045-5054 ◽  
Author(s):  
J L Celenza ◽  
F J Eng ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Glucose Repression ◽  
Gene Dosage ◽  
Essential Gene ◽  
Glucose Deprivation ◽  
Protein Kinase Activity ◽  
Lacz Gene ◽  
Physical Association

The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in Saccharomyces cerevisiae. Previous evidence suggested that SNF4 is functionally related to SNF1, another essential gene in this global regulatory system that encodes a protein kinase. Increased SNF1 gene dosage partially compensates for a mutation in SNF4, and the SNF4 function is required for maximal SNF1 protein kinase activity in vitro. We have cloned SNF4 and identified its 1.2-kilobase RNA, which is not regulated by glucose repression. A 36-kilodalton SNF4 protein is predicted from the nucleotide sequence. Disruption of the chromosomal SNF4 locus revealed that the requirement for SNF4 function is less stringent at low temperature (23 degrees C). A bifunctional SNF4-lacZ gene fusion that includes almost the entire SNF4 coding sequence was constructed. The fusion protein was shown by immunofluorescence microscopy to be distributed throughout the cell, with partial localization to the nucleus. The SNF4-beta-galactosidase protein coimmunoprecipitated with the SNF1 protein kinase, thus providing evidence for the physical association of the two proteins.

scholarly journals A common bacterial metabolite elicits prion-based bypass of glucose repression

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
David M Garcia ◽  
David Dietrich ◽  
Jon Clardy ◽  
Daniel F Jarosz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Glucose Metabolism ◽  
Carbon Source ◽  
Glucose Repression ◽  
Carbon Sources ◽  
Yeast Cells ◽  
Genetic Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
The Common

Robust preference for fermentative glucose metabolism has motivated domestication of the budding yeast Saccharomyces cerevisiae. This program can be circumvented by a protein-based genetic element, the [GAR+] prion, permitting simultaneous metabolism of glucose and other carbon sources. Diverse bacteria can elicit yeast cells to acquire [GAR+], although the molecular details of this interaction remain unknown. Here we identify the common bacterial metabolite lactic acid as a strong [GAR+] inducer. Transient exposure to lactic acid caused yeast cells to heritably circumvent glucose repression. This trait had the defining genetic properties of [GAR+], and did not require utilization of lactic acid as a carbon source. Lactic acid also induced [GAR+]-like epigenetic states in fungi that diverged from S. cerevisiae ~200 million years ago, and in which glucose repression evolved independently. To our knowledge, this is the first study to uncover a bacterial metabolite with the capacity to potently induce a prion.

Extramitochondrial citrate synthase activity in bakers' yeast

1986 ◽  
Vol 6 (2) ◽  
pp. 488-493
Author(s):  
T M Rickey ◽  
A S Lewin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Citrate Synthase ◽  
Glucose Repression ◽  
Yeast Cells ◽  
Mitochondrial Enzyme ◽  
Wild Type ◽  
Reading Frame ◽  
Leu2 Gene ◽  
Growth Requirements ◽  
Wild Type Yeast

We isolated the gene for citrate synthase (citrate oxaloacetate lyase; EC 4.1.3.7) from Saccharomyces cerevisiae and ablated it by inserting the yeast LEU2 gene within its reading frame. This revealed a second, nonmitochondrial citrate synthase. Like the mitochondrial enzyme, this enzyme was sensitive to glucose repression. It did not react with antibodies against mitochondrial citrate synthase. Haploid cells lacking a gene for mitochondrial citrate synthase grew somewhat slower than wild-type yeast cells, but exhibited no auxotrophic growth requirements.

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (12) ◽  
pp. 5643-5649
Author(s):  
H Ma ◽  
L M Bloom ◽  
C T Walsh ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Point Mutations ◽  
Inverse Correlation ◽  
Invertase Activity ◽  
Yeast Cells ◽  
Hexokinase Ii ◽  
Mutant Proteins ◽  
Hexokinase Activity

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.

The Saccharomyces cerevisiae ADR1 gene is a positive regulator of transcription of genes encoding peroxisomal proteins

1991 ◽  
Vol 11 (2) ◽  
pp. 699-704 ◽  
Author(s):  
M Simon ◽  
G Adam ◽  
W Rapatz ◽  
W Spevak ◽  
H Ruis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Northern Hybridization ◽  
Multicopy Plasmid ◽  
Beta Oxidation ◽  
Positive Regulator ◽  
Ethanol Medium ◽  
Genes Encoding ◽  
Multiple Copies ◽  
Regulatory Functions

Expression of the CTA1 gene of Saccharomyces cerevisiae, encoding catalase A, the peroxisomal catalase of this yeast, is sensitive to glucose repression. A DNA fragment cloned as a multicopy plasmid suppressing the glucose repression of CTA1 transcription was demonstrated to contain the ADR1 gene. Multiple copies of ADR1 increased catalase A formation not only on 10% glucose, but also on ethanol medium and in the presence of oleic acid, an inducer of peroxisome proliferation. Compared with wild-type cells, adr1 null mutants produced by disruption of the gene exhibit reduced CTA1 expression. This demonstrates that ADR1 is a true positive regulator of CTA1. Further experiments showed that it acts directly on CTA1. Alcohol dehydrogenase II, which is under ADR1 control, was excluded as a mediator of the effect on CTA1; deletion of bases -123 to -168 of CTA1 reduces expression and eliminates the response to the ADR1 multicopy plasmid without eliminating fatty acid induction; and gel retardation experiments demonstrated that ADR1 binds to a CTA1 upstream fragment (-156 to -184) with limited similarity to the ADR1 binding site of ADH2. Northern hybridization experiments further demonstrated that expression of two genes encoding enzymes of peroxisomal beta-oxidation (beta-ketothiolase, trifunctional enzyme) and of a gene involved in peroxisome assembly (PAS1) is also negatively affected by the adr1 null mutation. These findings demonstrate that the ADR1 protein has much broader regulatory functions than previously recognized.

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