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

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 563-572 ◽  
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.

scholarly journals Sip5 Interacts With Both the Reg1/Glc7 Protein Phosphatase and the Snf1 Protein Kinase of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 99-107
Author(s):  
Pascual Sanz ◽  
Katja Ludin ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Glucose Starvation ◽  
Glucose Limitation ◽  
Snf1 Kinase ◽  
Cell Extracts ◽  
Two Hybrid ◽  
New Protein

Abstract The Snf1 protein kinase is an essential component of the glucose starvation signalling pathway in Saccharomyces cerevisiae. We have used the two-hybrid system to identify a new protein, Sip5, that interacts with the Snf1 kinase complex in response to glucose limitation. Coimmunoprecipitation studies confirmed the association of Sip5 and Snf1 in cell extracts. We found that Sip5 also interacts strongly with Reg1, the regulatory subunit of the Reg1/Glc7 protein phosphatase 1 complex, in both two-hybrid and coimmunoprecipitation assays. Previous work showed that Reg1/Glc7 interacts with the Snf1 kinase under glucose-limiting conditions and negatively regulates its activity. Sip5 is the first protein that has been shown to interact with both Snf1 and Reg1/Glc7. Genetic analysis showed that the two-hybrid interaction between Reg1 and Snf1 is reduced threefold in a sip5Δ mutant. These findings suggest that Sip5 facilitates the interaction between the Reg1/Glc7 phosphatase and the Snf1 kinase.

scholarly journals Protein phosphatase type 1 interacts with proteins required for meiosis and other cellular processes in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4199-4206 ◽  
Author(s):  
J Tu ◽  
W Song ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Specific Substrate ◽  
Type I ◽  
Cellular Processes ◽  
Cell Extracts ◽  
Two Hybrid ◽  
Protein Phosphatase Type

Protein phosphatase type I (PP1) is involved in diverse cellular processes, and its activity toward specific substrates is thought to be controlled by different regulatory or targeting subunits. To identify regulatory subunits and substrates of the Saccharomyces cerevisiae PP1, encoded by GLC7, we used the two-hybrid system to detect interacting proteins. Among the many proteins identified were Gac1, a known glycogen regulatory subunit, and a protein with homology to Gac1. We also characterized a new gene designated GIP1, for Glc7-interacting protein. We show that a Gip1 fusion protein coimmunoprecipitates with PP1 from cell extracts. Molecular and genetic analyses indicate that GIP1 is expressed specifically during meiosis, affects transcription of late meiotic genes, and is essential for sporulation. Thus, the Gip1 protein is a candidate for a meiosis-specific substrate or regulator of PP1. Finally, we recovered two genes, RED1 and SCD5, with roles in meiosis and the vesicular secretory pathway, respectively. These results provide strong evidence implicating PP1 function in meiosis. In addition, this study indicates that the two-hybrid system offers a promising approach to understanding the multiple roles and interactions of PP1 in cellular regulation.

scholarly journals Interaction of a Swi3 homolog with Sth1 provides evidence for a Swi/Snf-related complex with an essential function in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (4) ◽  
pp. 1768-1775 ◽  
Author(s):  
I Treich ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Leucine Zipper Motif ◽  
Cell Extracts ◽  
Essential Function ◽  
Self Association ◽  
Two Hybrid

The Saccharomyces cerevisiae Swi/Snf complex has a role in remodeling chromatin structure to facilitate transcriptional activation. The complex has 11 components, including Swi1/Adr6, Swi2/Snf2, Swi3, Snf5, Snf6, Snf11, Swp73/Snf12, and Tfg3. Mammalian homologs of these proteins have been shown to form multiple Swi/Snf-related complexes. Here we characterize an S. cerevisiae Swi3 homolog (Swh3) and present evidence that it associates in a complex with a Snf2 homolog, Sthl. We identified Swh3 as a protein that interacts with the N terminus of Snf2 in the two-hybrid system. Swh3 and Swi3 are functionally distinct, and overexpression of one does not compensate for loss of the other. Swh3 is essential for viability and does not activate transcription of reporters. The Snf2 sequence that interacts with Swh3 was mapped to a region conserved in Sth1. We show that Swh3 and Sth1 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. We also map interactions between Swh3 and Sth1 and examine the role of a leucine zipper motif in self-association of Swh3. These findings, together with previous analysis of Sth1, indicate that Swh3 and Sth1 are associated in a complex that is functionally distinct from the Swi/Snf complex and essential for viability.

scholarly journals Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (3) ◽  
pp. 1972-1978 ◽  
Author(s):  
E J Hubbard ◽  
R Jiang ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Glucose Repression ◽  
Binding Studies ◽  
Glucose Limitation ◽  
Snf1 Kinase ◽  
Multicopy Suppressor ◽  
C Terminus ◽  
In The Wild

The SNF1 protein kinase of Saccharomyces cerevisiae is required to relieve glucose repression of transcription. To identify components of the SNF1 pathway, we isolated multicopy suppressors of defects caused by loss of SNF4, an activator of the SNF1 kinase. Increased dosage of the MSN3 gene restored invertase expression in snf4 mutants and also relieved glucose repression in the wild type. Deletion of MSN3 caused no substantial phenotype, and we identified a homolog, MTH1, encoding a protein 61% identical to MSN3. Both are also homologous to chicken fimbrin, human plastin, and yeast SAC6 over a 43-residue region. Deletion of MSN3 and MTH1 together impaired derepression of invertase in response to glucose limitation. Finally, MSN3 physically interacts with the SNF1 protein kinase, as assayed by a two-hybrid system and by in vitro binding studies. MSN3 is the same gene as STD1, a multicopy suppressor of defects caused by overexpression of the C terminus of TATA-binding protein (R. W. Ganster, W. Shen, and M. C. Schmidt, Mol. Cell. Biol. 13:3650-3659, 1993). Taken together, these data suggest that MSN3 modulates the regulatory response to glucose and may couple the SNF1 pathway to transcription.

scholarly journals 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.

1996 ◽  
Vol 16 (6) ◽  
pp. 2922-2931 ◽  
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.

scholarly journals Effect of the Pheromone-Responsive Gα and Phosphatase Proteins of Saccharomyces cerevisiae on the Subcellular Localization of the Fus3 Mitogen-Activated Protein Kinase

2003 ◽  
Vol 23 (4) ◽  
pp. 1135-1150 ◽  
Author(s):  
Ernest Blackwell ◽  
Izabel M. Halatek ◽  
Hye-Jin N. Kim ◽  
Alexis T. Ellicott ◽  
Andrey A. Obukhov ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Nucleocytoplasmic Transport ◽  
Mitogen Activated Protein Kinase ◽  
Mutant Form ◽  
Relative Level ◽  
Mitogen Activated Protein ◽  
Mating Signal ◽  
Green Fluorescent

ABSTRACT The mating-specific Gα protein of Saccharomyces cerevisiae, Gpa1, stimulates adaptation to pheromone by a mechanism independent of Gβγ sequestration. Genetic evidence suggests that Gpa1 targets the Fus3 mitogen-activated protein kinase, and it has recently been shown that the two proteins interact in cells responding to pheromone. To test the possibility that Gpa1 downregulates the mating signal by affecting the localization of Fus3, we created a Fus3-green fluorescent protein (GFP) fusion protein. In vegetative cells, Fus3-GFP was found in both the cytoplasm and the nucleus. Pheromone stimulated a measurable increase in the ratio of nuclear to cytoplasmic Fus3-GFP. In contrast, the relative level of nuclear Fus3-GFP decreased as cells recovered from pheromone arrest and did not increase when cells adapted to chronic stimulus were challenged again. Accumulation of Fus3-GFP in the nuclei of stimulated cells was also inhibited by overexpression of either wild-type Gpa1, the E364K hyperadaptive mutant form of Gpa1, or the Msg5 dually specific phosphatase. The effects of Gpa1 and Msg5 on Fus3 are partially interdependent. In a genetic screen for adaptive defective mutants, a nonsense allele of the nucleocytoplasmic transport receptor, Kap104, was identified. Truncation of the Kap104 cargo-binding domain blocked the effect of both Gpa1E364K and Msg5 on Fus3-GFP localization. Based on these results, we propose that Gpa1 and Msg5 work in concert to downregulate the mating signal and that they do so by inhibiting the pheromone-induced increase of Fus3 in the nucleus. Kap104 is required for the Gα/phosphatase-mediated effect on Fus3 localization.

A Split Enhanced Green Fluorescent Protein-Based Reporter in Yeast Two-Hybrid System

2007 ◽  
Vol 26 (2) ◽  
pp. 107-116 ◽  
Author(s):  
Kyoungsook Park ◽  
So Yeon Yi ◽  
Chang-Soo Lee ◽  
Kyoon Eon Kim ◽  
Hyun-Sook Pai ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Hybrid System ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Green Fluorescent ◽  
Two Hybrid

scholarly journals Stimulation of yeast meiotic gene expression by the glucose-repressible protein kinase Rim15p.

1997 ◽  
Vol 17 (5) ◽  
pp. 2688-2697 ◽  
Author(s):  
S Vidan ◽  
A P Mitchell
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Glucose Repression ◽  
Glucose Inhibition ◽  
Two Hybrid ◽  
Residue Polypeptide ◽  
Regulatory Sites ◽  
Stimulation Of

The Saccharomyces cerevisiae RIM15 gene was identified previously through a mutation that caused reduced ability to undergo meiosis. We report here an analysis of the cloned RIM15 gene, which specifies a 1,770-residue polypeptide with homology to serine/threonine protein kinases. Rim15p is most closely related to Schizosaccharomyces pombe cek1+. Analysis of epitope-tagged derivatives indicates that Rim15p has autophosphorylation activity. Deletion of RIM15 causes reduced expression of several early meiotic genes (IME2, SPO13, and HOP1) and of IME1, which specifies an activator of early meiotic genes. However, overexpression of IME1 does not permit full expression of early meiotic genes in a rim15delta mutant. Ime1p activates early meiotic genes through its interaction with Ume6p, and analysis of Rim15p-dependent regulatory sites at the IME2 promoter indicates that activation through Ume6p is defective. Two-hybrid interaction assays suggest that Ime1p-Ume6p interaction is diminished in a rim15 mutant. Glucose inhibits Ime1p-Ume6p interaction, and we find that Rim15p accumulation is repressed in glucose-grown cells. Thus, glucose repression of Rim15p may be responsible for glucose inhibition of Ime1p-Ume6p interaction.

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