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

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.

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The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2922 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2922-2931 ◽

Cited By ~ 47

Author(s):

D L Frederick ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Protein Phosphatase ◽

Slow Growth ◽

Glucose Repression ◽

Growth Defect ◽

Loss Of Function ◽

Cell Extracts

The GLC7 gene of Saccharomyces cerevisiae encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is essential for cell growth. We have isolated a previously uncharacterized gene, REG2, on the basis of its ability to interact with Glc7p in the two-hybrid system. Reg2p interacts with Glc7p in vivo, and epitope-tagged derivatives of Reg2p and Glc7p coimmunoprecipitate from cell extracts. The predicted protein product of the REG2 gene is similar to Reg1p, a protein believed to direct PP1 activity in the glucose repression pathway. Mutants with a deletion of reg1 display a mild slow-growth defect, while reg2 mutants exhibit a wild-type phenotype. However, mutants with deletions of both reg1 and reg2 exhibit a severe growth defect. Overexpression of REG2 complements the slow-growth defect of a reg1 mutant but does not complement defects in glycogen accumulation or glucose repression, two traits also associated with a reg1 deletion. These results indicate that REG1 has a unique role in the glucose repression pathway but acts together with REG2 to regulate some as yet uncharacterized function important for growth. The growth defect of a reg1 reg2 double mutant is alleviated by a loss-of-function mutation in the SNF1-encoded protein kinase. The snf1 mutation also suppresses the glucose repression defects of reg1. Together, our data are consistent with a model in which Reg1p and Reg2p control the activity of PP1 toward substrates that are phosphorylated by the Snf1p kinase.

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REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae.

The EMBO Journal ◽

10.1002/j.1460-2075.1995.tb00282.x ◽

1995 ◽

Vol 14 (23) ◽

pp. 5939-5946 ◽

Cited By ~ 148

Author(s):

J. Tu ◽

M. Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Phosphatase ◽

Glucose Repression ◽

Protein Phosphatase Type 1 ◽

Protein Phosphatase Type

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Genetic Interactions Between REG1/HEX2 and GLC7, the Gene Encoding the Protein Phosphatase Type 1 Catalytic Subunit in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/143.1.119 ◽

1996 ◽

Vol 143 (1) ◽

pp. 119-127 ◽

Cited By ~ 7

Author(s):

Dongqing Huang ◽

Kristin T Chun ◽

Mark G Goebl ◽

Peter J Roach

Keyword(s):

Protein Phosphatase ◽

Glucose Repression ◽

Glycogen Synthesis ◽

Catalytic Subunit ◽

Loss Of Function ◽

Gene Encoding ◽

Glycogen Accumulation ◽

Protein Phosphatase Type 1 ◽

Protein Phosphatase Type

Abstract Mutations in GLC7, the gene encoding the type 1 protein phosphatase catalytic subunit, cause a variety of abberrant phenotypes in yeast, such as impaired glycogen synthesis and relief of glucose repression of the expression of some genes. Loss of function of the REG1/HEX2 gene, necessary for glucose repression of several genes, was found to suppress the glycogendeficient phenotype of the glc7-1 allele. Deletion of REG1 in a wild-type background led to overaccumulation of glycogen as well as slow growth and an enlarged cell size. However, loss of REG1 did not suppress other phenotypes associated with GLC7 mutations, such as inability to sporulate or, in cells bearing the glc7Y-170 allele, lack of growth at 14°. The effect of REG1 deletion on glycogen accumulation is not simply due to derepression of glucose-repressed genes, although it does require the presence of SAF1, which encodes a protein kinase essential for expression of glucose-repressed genes and for glycogen accumulation. We propose that REG1 has a role in controlling glycogen accumulation.

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The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.896-905.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 896-905

Author(s):

J S Stuart ◽

D L Frederick ◽

C M Varner ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Phosphatase ◽

Glycogen Synthase ◽

Regulatory Subunit ◽

Loss Of Function ◽

Mutant Type ◽

Glycogen Accumulation

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

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The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.896 ◽

1994 ◽

Vol 14 (2) ◽

pp. 896-905 ◽

Cited By ~ 65

Author(s):

J S Stuart ◽

D L Frederick ◽

C M Varner ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Phosphatase ◽

Glycogen Synthase ◽

Regulatory Subunit ◽

Loss Of Function ◽

Mutant Type ◽

Glycogen Accumulation

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

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Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4357 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4357-4365 ◽

Cited By ~ 64

Author(s):

D Huang ◽

I Farkas ◽

P J Roach

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Kinase Activity ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Mutant Gene ◽

Glycogen Synthase Kinase ◽

Partial Purification ◽

Glycogen Accumulation ◽

Dependent Protein

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.

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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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Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2502 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2502-2510 ◽

Cited By ~ 83

Author(s):

F Randez-Gil ◽

N Bojunga ◽

M Proft ◽

K D Entian

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Transcriptional Activation ◽

Phosphoenolpyruvate Carboxykinase ◽

Glucose Repression ◽

Sodium Dodecyl ◽

Binding Motif ◽

Gluconeogenic Enzymes ◽

Zinc Cluster ◽

Activating Function

The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.

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