Depression of Saccharomyces cerevisiae invasive growth on non-glucose carbon sources requires the Snf1 kinase

Haploid Saccharomyces cerevisiae cells growing on media lacking glucose but containing high concentrations of carbon sources such as fructose, galactose, raffinose, and ethanol exhibit enhanced agar invasion. These carbon sources also promote diploid filamentous growth in response to nitrogen starvation. The enhanced invasive and filamentous growth phenotypes are suppressed by the addition of glucose to the media and require the Snf1 kinase. Mutations in the PGI1 and GND1 genes encoding carbon source utilization enzymes confer enhanced invasive growth that is unaffected by glucose but requires active Snf1. Carbon source does not modulate FLO11 flocculin expression, but enhanced polarized bud site selection is necessary for invasion on certain carbon sources. Interestingly, deletion of SNF1 blocks invasion without affecting bud site selection. Snf1 is also required for formation of spokes and hubs in multicellular mats. To examine glucose repression of invasive growth more broadly, we performed genome-wide microarray expression analysis in wild-type cells growing on glucose and galactose, and snf1 Delta cells on galactose. SNF1 probably mediates glucose repression of multiple genes potentially involved in invasive and filamentous growth. FLO11-independent cell-cell attachment, cell wall integrity, and/or polarized growth are affected by carbon source metabolism. In addition, derepression of cell cycle genes and signalling via the cAMP-PKA pathway appears to depend upon SNF1 activity during growth on galactose.     

Nitrogen availability and TOR regulate the Snf1 protein kinase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the Snf1 protein kinase of the Snf1/AMP-activated protein kinase (AMPK) family regulates a wide range of responses to stress caused by glucose deprivation. The stress signal is relayed via upregulation of Snf1, which depends on phosphorylation of its activation loop Thr210 residue by upstream kinases. Although Snf1 is also required for coping with various stresses unrelated to glucose deprivation, some evidence suggests a role for low-level basal activity of unphosphorylated Snf1, rather than a specific signaling function. We previously found that Snf1 is required for diploid pseudohyphal differentiation, a developmental response to nitrogen limitation. Here, we present evidence that Snf1 is directly involved in nitrogen signaling. First, genetic analyses suggest that pseudohyphal differentiation depends on the stimulatory phosphorylation of Snf1 at Thr210. Second, immunochemical data indicate that nitrogen limitation improves Thr210 phosphorylation. Analyses of pseudohyphal differentiation in cells with catalytically inactive and hyperactive Snf1 support the role of Snf1 activity. Finally, we show that Snf1 is negatively regulated by the rapamycin-sensitive TOR kinase which plays essential roles in signaling nitrogen and amino acid availability. This and other evidence implicate Snf1 in the integration of signals regarding nitrogen and carbon stress. TOR and Snf1/AMPK are highly conserved in evolution, and their novel functional interaction in yeast suggests similar mechanisms in other eukaryotes.     

Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae

Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.     

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

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.     

Std1p (Msn3p) positively regulates the Snf1 kinase in Saccharomyces cerevisiae

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.     

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

Metazoan arrestin proteins bind to seven-transmembrane proteins, mediate their internalization and play central roles in the subsequent signal transduction pathway. In Saccharomyces cerevisiae, there are several arrestin-related proteins. One of those proteins, Rod1, has been identified to have the ability to confer resistance to o-dinitrobenzene. We found that Rod1 interacted with Snf4, a subunit of Snf1-kinase complex. Both snf4 and snf1 mutants were also sensitive to the drug and the kinase activity of Snf1 was required for the drug tolerance. In immunoblotting analysis, the Rod1 protein was phosphorylated in an Snf1-dependent manner in vivo, and the phosphorylation of the serine residue 447 of Rod1 was responsible for the band-shift. Furthermore, the Rod1 protein was directly phosphorylated by Snf1-kinase in vitro. The substitution of the serine residue 447 to alanine slightly enhanced the resistance to the drug. We discuss possible functions of Rod1.     

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.

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.     

Changes in the enzyme activities of Saccharomyces cerevisiae during aerobic growth on different carbon sources

1. The activities of the enzymes of the citric acid cycle, the glyoxylate by-pass and some other enzymes acting on the substrates of these cycles have been measured at the pH of the yeast cell during the aerobic growth of yeast on different carbon sources and in different growth media. 2. Sugars induced an anaerobic type of metabolism as measured by ethanol production. Glucose was much more effective in inducing the anaerobic pathways than was galactose. The production of ethanol by cells grown on pyruvate was very small. 3. Glucose was also a more effective repressor than was galactose of the citric acid-cycle enzymes but both were equally effective in repressing almost completely the enzymes of the glyoxylate by-pass. 4. Disappearance of the sugars from the growth medium resulted in an increase in the activities of the enzymes of the citric acid cycle and in the appearance of substantial activities of the enzymes of the glyoxylate cycle. By contrast, the activities of purely biosynthetic enzymes (glutamate-oxaloacetate transaminase, NADP(+)-linked glutamate dehydrogenase) and of pyruvate decarboxylase were decreased. 5. The 2-oxoglutarate-oxidase system was found to be the least active enzyme of the citric acid cycle. 6. The regulatory control at the levels of pyruvate and acetaldehyde and the control of the citric acid cycle are discussed.     

Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae

The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.     

Sip5 interacts with both the Reg1/Glc7 protein phosphatase and the Snf1 protein kinase of Saccharomyces cerevisiae

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 sip5Delta mutant. These findings suggest that Sip5 facilitates the interaction between the Reg1/Glc7 phosphatase and the Snf1 kinase.     

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

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pH_c) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pH_c by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pH_c causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pH_c in regulating the metabolic program of yeast cells.     

New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1

We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation.     

Mapping the interaction of Snf1 with TORC1 in Saccharomyces cerevisiae

Nutrient sensing and coordination of metabolic pathways are crucial functions for all living cells, but details of the coordination under different environmental conditions remain elusive. We therefore undertook a systems biology approach to investigate the interactions between the Snf1 and the target of rapamycin complex 1 (TORC1) in Saccharomyces cerevisiae. We show that Snf1 regulates a much broader range of biological processes compared with TORC1 under both glucose‐ and ammonium‐limited conditions. We also find that Snf1 has a role in upregulating the NADP⁺‐dependent glutamate dehydrogenase (encoded by GDH3) under derepressing condition, and therefore may also have a role in ammonium assimilation and amino‐acid biosynthesis, which can be considered as a convergence of Snf1 and TORC1 pathways. In addition to the accepted role of Snf1 in regulating fatty acid (FA) metabolism, we show that TORC1 also regulates FA metabolism, likely through modulating the peroxisome and β‐oxidation. Finally, we conclude that direct interactions between Snf1 and TORC1 pathways are unlikely under nutrient‐limited conditions and propose that TORC1 is repressed in a manner that is independent of Snf1.