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.

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 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 Springing into Action: Reg2 Negatively Regulates Snf1 Protein Kinase and Facilitates Recovery from Prolonged Glucose Starvation in Saccharomyces cerevisiae

2016 ◽  
Vol 82 (13) ◽  
pp. 3875-3885 ◽  
Author(s):  
Marcin Maziarz ◽  
Aishwarya Shevade ◽  
LaKisha Barrett ◽  
Sergei Kuchin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Carbon Source ◽  
Glucose Deprivation ◽  
Regulatory Subunit ◽  
Glucose Starvation ◽  
Wild Type ◽  
Glucose Limitation ◽  
Content Type ◽  
Glucose Depletion

ABSTRACTGlucose is the preferred carbon source for the yeastSaccharomyces cerevisiae. Glucose limitation activates Snf1 protein kinase, a key regulator of energy homeostasis that promotes utilization of alternative carbon sources and enforces energy conservation. Snf1 activation requires phosphorylation of its T-loop threonine (Thr210) by upstream kinases. When glucose is abundant, Snf1 is inhibited by Thr210 dephosphorylation. This involves the function of the type 1 protein phosphatase Glc7, which is targeted to Snf1 by a regulatory subunit, Reg1. Thereg1mutation causes increased Snf1 activity and mimics various aspects of glucose limitation, including slower growth. Reg2 is another Glc7 regulatory subunit encoded by a paralogous gene,REG2. Previous evidence indicated that thereg2mutation exacerbates the Snf1-dependent slow-growth phenotype caused byreg1, suggesting a link between Reg2 and Snf1. Here, we explore this link in more detail and present evidence that Reg2 contributes to Snf1 Thr210 dephosphorylation. Consistent with this role, Reg2 interacts with wild-type Snf1 but not with nonphosphorylatable Snf1-T210A. Reg2 accumulation increases in a Snf1-dependent manner during prolonged glucose deprivation, and glucose-starved cells lacking Reg2 exhibit delayed Snf1 Thr210 dephosphorylation and slower growth recovery upon glucose replenishment. Accordingly, cells lacking Reg2 are outcompeted by wild-type cells in the course of several glucose starvation/replenishment cycles. Collectively, our results support a model in which Reg2-Glc7 contributes to the negative control of Snf1 in response to glucose refeeding after prolonged starvation. The competitive growth advantage provided by Reg2 underscores the evolutionary significance of this paralog forS. cerevisiae.IMPORTANCEThe ability of microorganisms to respond to stress is essential for their survival. However, rapid recovery from stress could be equally crucial in competitive environments. Therefore, a wise stress response program should prepare cells for quick recovery upon reexposure to favorable conditions. Glucose is the preferred carbon source for the yeastS. cerevisiae. Glucose depletion activates the stress response protein kinase Snf1, which functions to limit energy-consuming processes, such as growth. We show that prolonged glucose deprivation also leads to Snf1-dependent accumulation of Reg2 and that this protein helps to inhibit Snf1 and to accelerate growth recovery upon glucose replenishment. Cells lacking Reg2 are readily outcompeted by wild-type cells during glucose depletion/replenishment cycles. Thus, while prolonged glucose deprivation might seem to put yeast cells “on their knees,” concomitant accumulation of Reg2 helps configure the cells into a “sprinter's crouch start position” to spring into action once glucose becomes available.

scholarly journals Regulatory Interactions between the Reg1-Glc7 Protein Phosphatase and the Snf1 Protein Kinase

2000 ◽  
Vol 20 (4) ◽  
pp. 1321-1328 ◽  
Author(s):  
Pascual Sanz ◽  
Geoffrey R. Alms ◽  
Timothy A. J. Haystead ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Protein Phosphatase ◽  
Glucose Repression ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Binding Motif ◽  
Glucose Limitation ◽  
Snf1 Kinase ◽  
Regulatory Interactions ◽  
Functional Relationships

ABSTRACT Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression inSaccharomyces cerevisiae. Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose. Here, we explore the functional relationships between Reg1, Glc7, and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1. We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex. Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1. We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex.

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.

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 Snf1 Protein Kinase Regulates Phosphorylation of the Mig1 Repressor in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (11) ◽  
pp. 6273-6280 ◽  
Author(s):  
Michelle A. Treitel ◽  
Sergei Kuchin ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Immunoblot Analysis ◽  
Dna Binding Protein ◽  
Glucose Inhibition ◽  
Cell Extracts ◽  
Differential Phosphorylation ◽  
Two Hybrid

ABSTRACT In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeastSaccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 andreg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.

Sign in / Sign up

Export Citation Format

Share Document