Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH

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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The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4744-4756.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4744-4756

Author(s):

J Schultz ◽

L Marshall-Carlson ◽

M Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Function Analysis ◽

Normal Growth ◽

Negative Regulator ◽

Tetratricopeptide Repeat ◽

Functional Domain ◽

Snf1 Kinase ◽

C Terminus ◽

Protein Functions

The SSN6 protein functions as a negative regulator of a variety of genes in Saccharomyces cerevisiae and is required for normal growth, mating, and sporulation. It is a member of a family defined by a repeated amino acid sequence, the TPR (tetratricopeptide repeat) motif. Here, we have used specific antibody to identify and characterize the SSN6 protein. Both SSN6 and a bifunctional SSN6-beta-galactosidase fusion protein were localized in the nucleus by immunofluorescence staining. The N-terminal one-third of the protein containing the TPR units was identified as the region that is important for SSN6 function. Analysis of four nonsense alleles, isolated as intragenic suppressors of an ssn6::URA3 insertion, revealed that polypeptides truncated after TPR unit 7 provide SSN6 function. Deletion analysis suggested that TPR units are required but that 4 of the 10 TPR units are sufficient. In addition, deletion studies indicated that three very long, homogeneous tracts of polyglutamine and poly(glutamine-alanine) are dispensable. Previous genetic evidence suggested the SSN6 protein as a possible target of the SNF1 protein kinase. Here, we show that the C terminus of SSN6 is phosphorylated in vivo and that the SNF1 kinase is not responsible for most of the phosphorylation. Finally, SSN6 has a modest effect on the maintenance of minichromosomes.

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Regulation of Gluconeogenesis in Saccharomyces cerevisiae Is Mediated by Activator and Repressor Functions of Rds2

Molecular and Cellular Biology ◽

10.1128/mcb.01055-07 ◽

2007 ◽

Vol 27 (22) ◽

pp. 7895-7905 ◽

Cited By ~ 46

Author(s):

Nitnipa Soontorngun ◽

Marc Larochelle ◽

Simon Drouin ◽

François Robert ◽

Bernard Turcotte

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Target Genes ◽

Tricarboxylic Acid Cycle ◽

Glyoxylate Cycle ◽

Dual Function ◽

Snf1 Kinase ◽

Zinc Cluster ◽

Chip Technology

ABSTRACT In Saccharomyces cerevisiae, RDS2 encodes a zinc cluster transcription factor with unknown function. Here, we unravel a key function of Rds2 in gluconeogenesis using chromatin immunoprecipitation-chip technology. While we observed that Rds2 binds to only a few promoters in glucose-containing medium, it binds many additional genes when the medium is shifted to ethanol, a nonfermentable carbon source. Interestingly, many of these genes are involved in gluconeogenesis, the tricarboxylic acid cycle, and the glyoxylate cycle. Importantly, we show that Rds2 has a dual function: it directly activates the expression of gluconeogenic structural genes while it represses the expression of negative regulators of this pathway. We also show that the purified DNA binding domain of Rds2 binds in vitro to carbon source response elements found in the promoters of target genes. Finally, we show that upon a shift to ethanol, Rds2 activation is correlated with its hyperphosphorylation by the Snf1 kinase. In summary, we have characterized Rds2 as a novel major regulator of gluconeogenesis.

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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 SNF1 kinase complex from Saccharomyces cerevisiae phosphorylates the transcriptional repressor protein Mig1p in vitro at four sites within or near regulatory domain 1

FEBS Letters ◽

10.1016/s0014-5793(99)00725-5 ◽

1999 ◽

Vol 453 (1-2) ◽

pp. 219-223 ◽

Cited By ~ 71

Author(s):

Fiona C. Smith ◽

Stephen P. Davies ◽

Wayne A. Wilson ◽

David Carling ◽

D.Grahame Hardie

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Repressor ◽

Regulatory Domain ◽

Repressor Protein ◽

Snf1 Kinase

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The role of the Snf1 kinase in the adaptive response of Saccharomyces cerevisiae to alkaline pH stress

Biochemical Journal ◽

10.1042/bj20112099 ◽

2012 ◽

Vol 444 (1) ◽

pp. 39-49 ◽

Cited By ~ 30

Author(s):

Antonio Casamayor ◽

Raquel Serrano ◽

María Platara ◽

Carlos Casado ◽

Amparo Ruiz ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Glucose Sensing ◽

Limiting Factors ◽

Alkaline Ph ◽

Short Term ◽

Triple Mutant ◽

Snf1 Kinase ◽

High Ph ◽

Ph Stress ◽

Alkaline Ph Stress

Alkaline pH stress invokes a potent and fast transcriptional response in Saccharomyces cerevisiae that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and -response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. In the present study we show that the addition of glucose to the medium improves the growth of wild-type cells at high pH, fully abolishes the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr210. Lack of Elm1, one of the three upstream Snf1 kinases (Tos3, Elm1 and Sak1), markedly increases alkali sensitivity, whereas the phenotype of the triple mutant tos3 elm1 sak1 is even more pronounced than that of snf1 cells and is poorly rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of the genes induced in the short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those encoding high-affinity hexose transporters and phosphorylating enzymes. The induction of genes encoding enzymes involved in glycogen, but not trehalose, metabolism is largely dependent of the presence of Snf1. Therefore the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium resulted in an additive effect and allows near-normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment.

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Std1p (Msn3p) Positively Regulates the Snf1 Kinase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/163.2.507 ◽

2003 ◽

Vol 163 (2) ◽

pp. 507-514 ◽

Cited By ~ 1

Author(s):

Sergei Kuchin ◽

Valmik K Vyas ◽

Ellen Kanter ◽

Seung-Pyo Hong ◽

Marian Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Kinase Activity ◽

Catalytic Domain ◽

Snf1 Kinase ◽

Glucose Signaling ◽

Catalytic Domains ◽

Upstream Kinase ◽

Two Hybrid

Abstract The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.

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Rod1, an arrestin-related protein, is phosphorylated by Snf1-kinase in Saccharomyces cerevisiae

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2007.09.134 ◽

2007 ◽

Vol 364 (2) ◽

pp. 258-263 ◽

Cited By ~ 17

Author(s):

Junro Shinoda ◽

Yoshiko Kikuchi

Keyword(s):

Saccharomyces Cerevisiae ◽

Related Protein ◽

Snf1 Kinase

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Gis4, a New Component of the Ion Homeostasis System in the Yeast Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00215-06 ◽

2006 ◽

Vol 5 (10) ◽

pp. 1611-1621 ◽

Cited By ~ 14

Author(s):

Tian Ye ◽

Raúl García-Salcedo ◽

José Ramos ◽

Stefan Hohmann

Keyword(s):

Saccharomyces Cerevisiae ◽

Salt Tolerance ◽

Cell Surface ◽

Ion Homeostasis ◽

Yeast Saccharomyces Cerevisiae ◽

Snf1 Kinase ◽

C Terminus ◽

Surface Localization ◽

As Stress

ABSTRACT Gis4 is a new component of the system required for acquisition of salt tolerance in Saccharomyces cerevisiae. The gis4Δ mutant is sensitive to Na+ and Li+ ions but not to osmotic stress. Genetic evidence suggests that Gis4 mediates its function in salt tolerance, at least partly, together with the Snf1 protein kinase and in parallel with the calcineurin protein phosphatase. When exposed to salt stress, mutants lacking gis4Δ display a defect in maintaining low intracellular levels of Na+ and Li+ ions and exporting those ions from the cell. This defect is due to diminished expression of the ENA1 gene, which encodes the Na+ and Li+ export pump. The protein sequence of Gis4 is poorly conserved and does not reveal any hints to its molecular function. Gis4 is enriched at the cell surface, probably due to C-terminal farnesylation. The CAAX box at the C terminus is required for cell surface localization but does not seem to be strictly essential for the function of Gis4 in salt tolerance. Gis4 and Snf1 seem to share functions in the control of ion homeostasis and ENA1 expression but not in glucose derepression, the best known role of Snf1. Together with additional evidence that links Gis4 genetically and physically to Snf1, it appears that Gis4 may function in a pathway in which Snf1 plays a specific role in controlling ion homeostasis. Hence, it appears that the conserved Snf1 kinase plays roles in different pathways controlling nutrient as well as stress response.

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Growth Inhibition by Amino Acids in Saccharomyces cerevisiae

Microorganisms ◽

10.3390/microorganisms9010007 ◽

2020 ◽

Vol 9 (1) ◽

pp. 7

Author(s):

Stephanie J. Ruiz ◽

Joury S. van ’t Klooster ◽

Frans Bianchi ◽

Bert Poolman

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Growth Defect ◽

Amino Acid Transporters ◽

Amino Acid Permease ◽

Cytosolic Ph ◽

Acid Sensitivity ◽

Growth Inhibitory

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced downregulation of amino acid transporters in Saccharomyces cerevisiae is thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4, or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5–5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory to S. cerevisiae when transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on the in vivo activity of transporters and has allowed us to identify new transporter-specific substrates.

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