PP1 phosphatase-binding motif in Reg1 protein of Saccharomyces cerevisiae is required for interaction with both the PP1 phosphatase Glc7 and the Snf1 protein kinase

In Saccharomyces cerevisiae, Snf1 kinase, the ortholog of the mammalian AMP-activated protein kinase, is activated by an increase in the phosphorylation of the conserved threonine residue in its activation loop. The phosphorylation status of this key site is determined by changes in the rate of dephosphorylation catalyzed by the yeast PP1 phosphatase Glc7 in a complex with the Reg1 protein. Reg1 and many PP1 phosphatase regulatory subunits utilize some variation of the conserved RVxF motif for interaction with PP1. In the Snf1 pathway, the exact role of the Reg1 protein is uncertain since it binds to both the Glc7 phosphatase and to Snf1, the Glc7 substrate. In this study we sought to clarify the role of Reg1 by separating the Snf1- and Glc7-binding functions. We generated a series of Reg1 proteins, some with deletions of conserved domains and one with two amino acid changes in the RVxF motif. The ability of Reg1 to bind Snf1 and Glc7 required the same domains of Reg1. Further, the RVxF motif that is essential for Reg1 binding to Glc7 is also required for binding to Snf1. Our data suggest that the regulation of Snf1 dephosphorylation is imparted through a dynamic competition between the Glc7 phosphatase and the Snf1 kinase for binding to the PP1 regulatory subunit Reg1.     

Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase

Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression in Saccharomyces 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.     

Glc7/protein phosphatase 1 regulatory subunits can oppose the Ipl1/aurora protein kinase by redistributing Glc7

Faithful chromosome segregation depends on the opposing activities of the budding yeast Glc7/PP1 protein phosphatase and Ipl1/Aurora protein kinase. We explored the relationship between Glc7 and Ipl1 and found that the phosphorylation of the Ipl1 substrate, Dam1, was altered by decreased Glc7 activity, whereas Ipl1 levels, localization, and kinase activity were not. These data strongly suggest that Glc7 ensures accurate chromosome segregation by dephosphorylating Ipl1 targets rather than regulating the Ipl1 kinase. To identify potential Glc7 and Ipl1 substrates, we isolated ipl1-321 dosage suppressors. Seven genes (SDS22, BUD14, GIP3, GIP4, SOL1, SOL2, and PEX31) encode newly identified ipl1 dosage suppressors, and all 10 suppressors encode proteins that physically interact with Glc7. The overexpression of the Gip3 and Gip4 suppressors altered Glc7 localization, indicating they are previously unidentified Glc7 regulatory subunits. In addition, the overexpression of Gip3 and Gip4 from the galactose promoter restored Dam1 phosphorylation in ipl1-321 mutant cells and caused wild-type cells to arrest in metaphase with unsegregated chromosomes, suggesting that Gip3 and Gip4 overexpression impairs Glc7's mitotic functions. We therefore propose that the overexpression of Glc7 regulatory subunits can titrate Glc7 away from relevant Ipl1 targets and thereby suppress ipl1-321 cells by restoring the balance of phosphatase/kinase activity.     

Genetic interaction between the Ras-CAMP pathway and the Dis2s1/Glc7 protein phosphatase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1 ^ts mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene.     

Saccharomyces cerevisiae Afr1 protein is a protein phosphatase 1/Glc7-targeting subunit that regulates the septin cytoskeleton during mating

Glc7, the type1 serine/threonine phosphatase in the yeast Saccharomyces cerevisiae, is targeted by auxiliary subunits to numerous locations in the cell, where it regulates a range of physiological pathways. We show here that the accumulation of Glc7 at mating projections requires Afr1, a protein required for the formation of normal projections. AFR1-null mutants fail to target Glc7 to projections, and an Afr1 variant specifically defective in binding to Glc7 [Afr1(V546A F548A)] forms aberrant projections. The septin filaments in mating projections of AFR1 mutants initiate normally but then rearrange asymmetrically as the projection develops, suggesting that the Afr1-Glc7 holoenzyme may regulate the maintenance of septin complexes during mating. These results demonstrate a previously unknown role for Afr1 in targeting Glc7 to mating projections and in regulating the septin architecture during mating.     

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.     

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.     

The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex.

The Snf1 protein kinase plays a central role in the response to glucose starvation in the yeast Saccharomyces cerevisiae. Previously, we showed that two-hybrid interaction between Snf1 and its activating subunit, Snf4, is inhibited by high levels of glucose. These findings, together with biochemical evidence that Snf1 and Snf4 remain associated in cells grown in glucose, suggested that another protein (or proteins) anchors Snf1 and Snf4 into a complex. Here, we examine the possibility that a family of proteins, comprising Sip1, Sip2, and Gal83, serves this purpose. We first show that the fraction of cellular Snf4 protein that is complexed with Snf1 is reduced in a sip1delta sip2delta gal83delta triple mutant. We then present evidence that Sip1, Sip2, and Gal83 each interact independently with both Snf1 and Snf4 via distinct domains. A conserved internal region binds to the Snf1 regulatory domain, and the conserved C-terminal ASC domain binds to Snf4. Interactions were mapped by using the two-hybrid system and were confirmed by in vitro binding studies. These findings indicate that the Sip1/Sip2/Gal83 family anchors Snf1 and Snf4 into a complex. Finally, the interaction of the yeast Sip2 protein with a plant Snf1 homolog suggests that this function is conserved in plants.     

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.     

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.     

The Glc7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M.

We isolated a mutant carrying a conditional mutation in the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by selection of suppressors that restored the growth defect of cdc24 mutants at high temperature and simultaneously conferred cold-sensitive growth. This cold sensitivity for growth is caused by a single mutation (glc7Y-170) at position 170 of the Glc7 protein, resulting in replacement of cysteine with tyrosine. Genetic analysis suggested that the glc7Y-170 allele is associated with a recessive negative phenotype, reducing the activity of Glc7 in the cell. The glc7Y-170 mutant missegregated chromosome III at the permissive temperature, arrested growth as large-budded cells at the restrictive temperature, exhibited a significant increase in the number of nuclei at or in the neck, and had a short spindle. Furthermore, the glc7Y-170 mutant exhibited a high level of CDC28-dependent protein kinase activity when incubated at the restrictive temperature. These findings suggest that the glc7Y-170 mutation is defective in the G2/M phase of the cell cycle. Thus, type 1 protein phosphatase in Saccharomyces cerevisiae is essential for the G2/M transition.     

Sds22p is a subunit of a stable isolatable form of protein phosphatase 1 (Glc7p) from Saccharomyces cerevisiae

Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. PP1 activity is believed to be controlled by the interaction of PP1 catalytic subunit with various regulatory subunits. The essential gene GLC7 encodes the PP1 catalytic subunit in Saccharomyces cerevisiae. In this study, full-length GLC7(1-312), C-terminal deletion mutants, and C-terminally poly-his tagged mutants were constructed and expressed in a GLC7 knockout strain of S. cerevisiae. Viability studies of the GLC7 knockout strains carrying the plasmids expressing GLC7 C-terminal deletion mutants and their tagged forms showed that the mutants 1-295 and 1-304 were functional, whereas the mutant 1-245 was not. The C-terminally poly-his tagged Glc7p with and without an N-terminal hemagglutinin (HA) tag was partially purified by immobilized Ni(2+) affinity chromatography and further analyzed by gel filtration and ion exchange chromatography. Phosphatase activity assays, SDS-PAGE, and Western blot analyses of the chromatographic fractions suggested that the Glc7p associated with regulatory subunits in vivo. A 40-kDa protein was copurified with tagged Glc7p through several chromatographic procedures. Monoclonal antibody against the HA tag coimmunoprecipitated the tagged Glc7p and the 40-kDa protein. This protein was further purified by a reverse phase HPLC column. Analysis by CNBr digestion, peptide sequencing, and electrospray mass spectrometry showed that this 40-kDa protein is Sds22p, one of the proteins proposed to be a regulatory subunit of Glc7. These results demonstrate that Sds22p forms a complex with Glc7p and that Sds22p:Glc7p is a stable isolatable form of yeast PP1.     

Interaction Maps of the Saccharomyces cerevisiae ESCRT-III Protein Snf7

The Saccharomyces cerevisiae ESCRT-III protein Snf7 is part of an intricate interaction network at the endosomal membrane. Interaction maps of Snf7 were established by measuring the degree of binding of individual binding partners to putative binding motifs along the Snf7 sequence by glutathione S-transferase (GST) pulldown. For each interaction partner, distinct binding profiles were obtained. The following observations were made. The ESCRT-III subunits Vps20 and Vps24 showed a complementary binding pattern, suggesting a model for the series of events in the ESCRT-III functional cycle. Vps4 bound to individual Snf7 motifs but not to full-length Snf7. This suggests that Vps4 does not bind to the closed conformation of Snf7. We also demonstrate for the first time that the ALIX/Bro1 homologue Rim20 binds to the α6 helix of Snf7. Analysis of a Snf7 α6 deletion mutant showed that the α6 helix is crucial for binding of Bro1 and Rim20 in vivo and is indispensable for the multivesicular body (MVB)-sorting and Rim-signaling functions of Snf7. The Snf7Δα6 protein still appeared to be incorporated into ESCRT-III complexes at the endosomal membrane, but disassembly of the complex seemed to be defective. In summary, our study argues against the view that the ESCRT cycle is governed by single one-to-one interactions between individual components and emphasizes the network character of the ESCRT interactions.     

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.     

Pho85 phosphorylates the Glc7 protein phosphatase regulator Glc8 in vivo

The budding yeast Glc7 serine/threonine protein phosphatase-1 is regulated by Glc8, the yeast ortholog of mammalian phosphatase inhibitor-2. In this work, we demonstrated that similarly to inhibitor-2, Glc8 function is regulated by phosphorylation. The cyclin-dependent protein kinase, Pho85, in conjunction with the related cyclins Pcl6 and Pcl7 comprise the major Glc8 kinase in vivo and in vitro. Several glc7 mutations are dependent on the presence of Glc8 for viability. For example, glc7 alleles R121K, R142H, and R198D are lethal in combination with a glc8 deletion. We found that glc7-R121K is lethal in combination with a pho85 deletion. This finding indicates that Pho85 is the sole Glc8 kinase in vivo. Furthermore, glc7-R121K is also lethal when combined with deletions of pcl6, plc7, pcl8, and pcl10, indicating that these related cyclins redundantly activate Pho85 for Glc8 phosphorylation in vivo. In vitro kinase assays and genetic results indicate that Pho85 cyclins Pcl6 and Pcl7 comprise the predominant Glc8 kinase.     

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.