Snf1 Kinases with Different β-Subunit Isoforms Play Distinct Roles in Regulating Haploid Invasive Growth

ABSTRACT The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion. Cells contain three forms of the Snf1 kinase, each with a different β-subunit isoform, either Gal83, Sip1, or Sip2. We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation. The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation. Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1- and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene. In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11. Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms.

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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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Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1

Eukaryotic Cell ◽

10.1128/ec.00291-10 ◽

2011 ◽

Vol 10 (3) ◽

pp. 313-319 ◽

Cited By ~ 19

Author(s):

Yang Liu ◽

Xinjing Xu ◽

Marian Carlson

Keyword(s):

Protein Kinase ◽

Kinase Domain ◽

Catalytic Subunit ◽

Glucose Limitation ◽

Content Type ◽

Snf1 Kinase ◽

Phosphorylation State ◽

Glucose Depletion ◽

C Terminus ◽

Amp Activated Protein Kinase

ABSTRACT The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1972 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1972-1978 ◽

Cited By ~ 30

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.

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The RA Domain of Ste50 Adaptor Protein Is Required for Delivery of Ste11 to the Plasma Membrane in the Filamentous Growth Signaling Pathway of the Yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.912-928.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 912-928 ◽

Cited By ~ 62

Author(s):

Dagmar M. Truckses ◽

Joshua E. Bloomekatz ◽

Jeremy Thorner

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Protein Kinase ◽

Adaptor Protein ◽

Mitogen Activated Protein Kinase ◽

Invasive Growth ◽

Pheromone Response ◽

Yeast Saccharomyces Cerevisiae ◽

Mapk Kinase

ABSTRACT In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (high-osmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called “Ras association” (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00154-16 ◽

2016 ◽

Vol 82 (13) ◽

pp. 3875-3885 ◽

Cited By ~ 6

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.

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Snf1 Protein Kinase and the Repressors Nrg1 and Nrg2 Regulate FLO11, Haploid Invasive Growth, and Diploid Pseudohyphal Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.3994-4000.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 3994-4000 ◽

Cited By ~ 141

Author(s):

Sergei Kuchin ◽

Valmik K. Vyas ◽

Marian Carlson

Keyword(s):

Protein Kinase ◽

Surface Glycoprotein ◽

Invasive Growth ◽

Glucose Limitation ◽

Cell Surface Glycoprotein ◽

Snf1 Kinase ◽

Repressor Proteins ◽

A Cell ◽

Pseudohyphal Differentiation

ABSTRACT The Snf1 protein kinase of Saccharomyces cerevisiae is important for many cellular responses to glucose limitation, including haploid invasive growth. We show here that Snf1 regulates transcription of FLO11, which encodes a cell surface glycoprotein required for invasive growth. We further show that Nrg1 and Nrg2, two repressor proteins that interact with Snf1, function as negative regulators of invasive growth and as repressors of FLO11. We also examined the role of Snf1, Nrg1, and Nrg2 in two other Flo11-dependent processes. Mutations affected the initiation of biofilm formation, which is glucose sensitive, but also affected diploid pseudohyphal differentiation, thereby unexpectedly implicating Snf1 in a response to nitrogen limitation. Deletion of the NRG1 and NRG2 genes suppressed the defects of a snf1 mutant in all of these processes. These findings suggest a model in which the Snf1 kinase positively regulates Flo11-dependent developmental events by antagonizing Nrg-mediated repression of the FLO11 gene.

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

Genetics ◽

10.1093/genetics/154.1.99 ◽

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.

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Role of the yeast Snf1 protein kinase in invasive growth

Biochemical Society Transactions ◽

10.1042/bst0310175 ◽

2003 ◽

Vol 31 (1) ◽

pp. 175-177 ◽

Cited By ~ 32

Author(s):

S. Kuchin ◽

V.K. Vyas ◽

M. Carlson

Keyword(s):

Protein Kinase ◽

Nitrogen Limitation ◽

Invasive Growth ◽

Gene Encoding ◽

Glucose Limitation ◽

Snf1 Kinase ◽

A Cell ◽

Developmental Responses ◽

Pseudohyphal Differentiation

The sucrose non-fermenting 1 (Snf1) protein kinase of Saccharomyces cerevisiae is important for transcriptional, metabolic and developmental responses to glucose limitation. Here we discuss the role of the Snf1 kinase in regulating filamentous invasive growth. Haploid invasive growth occurs in response to glucose limitation and requires FLO11, a gene encoding a cell-surface adhesin. Snf1 regulates transcription of FLO11 by antagonizing the function of two repressors, Nrg1 and Nrg2. Snf1 and the Nrg repressors also affect diploid pseudohyphal differentiation, which is a response to nitrogen limitation, suggesting an unexpected signalling role for the Snf1 kinase.

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