Journal articles: 'Saccharomyces cerevisiae, cell cycle, Snf1'

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Newcomb, Laura L., Jasper A. Diderich, Matthew G. Slattery, and Warren Heideman. "Glucose Regulation of Saccharomyces cerevisiae Cell Cycle Genes." Eukaryotic Cell 2, no. 1 (February 2003): 143–49. http://dx.doi.org/10.1128/ec.2.1.143-149.2003.

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ABSTRACT Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G1.

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Soontorngun, Nitnipa, Marc Larochelle, Simon Drouin, François Robert, and Bernard Turcotte. "Regulation of Gluconeogenesis in Saccharomyces cerevisiae Is Mediated by Activator and Repressor Functions of Rds2." Molecular and Cellular Biology 27, no. 22 (September 17, 2007): 7895–905. http://dx.doi.org/10.1128/mcb.01055-07.

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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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Timblin, Barbara K., Kelly Tatchell, and Lawrence W. Bergman. "Deletion of the Gene Encoding the Cyclin-Dependent Protein Kinase Pho85 Alters Glycogen Metabolism in Saccharomyces cerevisiae." Genetics 143, no. 1 (May 1, 1996): 57–66. http://dx.doi.org/10.1093/genetics/143.1.57.

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Abstract Pho85, a protein kinase with significant homology to the cyclin-dependent kinase, Cdc28, has been shown to function in repression of transcription of acid phosphatase (APase, encoded by PHO5) in high phosphate (Pi) medium, as well as in regulation of the cell cycle at G1/S. We describe several unique phenotypes associated with the deletion of the PHO85 gene including growth defects on a variety of carbon sources and hyperaccumulation of glycogen in rich medium high in Pi. Hyperaccumulation of glycogen in the pho85 strains is independent of other APase regulatory molecules and is not signaled through Snf1 kinase. However, constitutive activation of cAPK suppresses the hyperaccumulation of glycogen in a pho85 mutant. Mutation of the type-1 protein phosphatase encoded by GLC7 only partially suppresses the glycogen phenotype of the pho85 mutant. Additionally, strains containing a deletion of the PHO85 gene show an increase in expression of GSY2. This work provides evidence that Pho85 has functions in addition to transcriptional regulation of APase and cell-cycle progression including the regulation of glycogen levels in the cell and may provide a link between the nutritional state of the cell and these growth related responses.

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Gray, Joseph V., Gregory A. Petsko, Gerald C. Johnston, Dagmar Ringe, Richard A. Singer, and Margaret Werner-Washburne. "“Sleeping Beauty”: Quiescence in Saccharomyces cerevisiae." Microbiology and Molecular Biology Reviews 68, no. 2 (June 2004): 187–206. http://dx.doi.org/10.1128/mmbr.68.2.187-206.2004.

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SUMMARY The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one “model” quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.

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Miura, Natsuko, Masahiro Shinohara, Yohei Tatsukami, Yasuhiko Sato, Hironobu Morisaka, Kouichi Kuroda, and Mitsuyoshi Ueda. "Spatial Reorganization of Saccharomyces cerevisiae Enolase To Alter Carbon Metabolism under Hypoxia." Eukaryotic Cell 12, no. 8 (June 7, 2013): 1106–19. http://dx.doi.org/10.1128/ec.00093-13.

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ABSTRACTHypoxia has critical effects on the physiology of organisms. In the yeastSaccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by anSNF1knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-13C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia,S. cerevisiaecells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol viade novoprotein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.

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Dombek, Kenneth M., Valentina Voronkova, Alexa Raney, and Elton T. Young. "Functional Analysis of the Yeast Glc7-Binding Protein Reg1 Identifies a Protein Phosphatase Type 1-Binding Motif as Essential for Repression of ADH2 Expression." Molecular and Cellular Biology 19, no. 9 (September 1, 1999): 6029–40. http://dx.doi.org/10.1128/mcb.19.9.6029.

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ABSTRACT In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose. Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2. ConstitutiveADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells. To further elucidate the functional role of Reg1 in repressingADH2 expression, deletions scanning the entire length of the protein were analyzed. Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression. Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional. Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses. Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex. Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source. When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression. These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression. This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm.

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Huang, Dongqing, Jason Moffat, Wayne A. Wilson, Lynda Moore, Christine Cheng, Peter J. Roach, and Brenda Andrews. "Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10." Molecular and Cellular Biology 18, no. 6 (June 1, 1998): 3289–99. http://dx.doi.org/10.1128/mcb.18.6.3289.

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ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlikepho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation ofPHO85 suppressed the glycogen storage deficiency ofsnf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

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Miller, M. E., B. R. Cairns, R. S. Levinson, K. R. Yamamoto, D. A. Engel, and M. M. Smith. "Adenovirus E1A specifically blocks SWI/SNF-dependent transcriptional activation." Molecular and Cellular Biology 16, no. 10 (October 1996): 5737–43. http://dx.doi.org/10.1128/mcb.16.10.5737.

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Expression of the adenovirus E1A243 oncoprotein in Saccharomyces cerevisiae produces a slow-growth phenotype with accumulation of cells in the G1 phase of the cell cycle. This effect is due to the N-terminal and CR1 domains of E1A243, which in rodent cells are involved in triggering cellular transformation and also in binding to the cellular transcriptional coactivator p300. A genetic screen was undertaken to identify genes required for the function of E1A243 in S. cerevisiae. This screen identified SNF12, a gene encoding the 73-kDa subunit of the SWI/SNF transcriptional regulatory complex. Mutation of genes encoding known members of the SWI/SNF complex also led to loss of E1A function, suggesting that the SWI/SNF complex is a target of E1A243. Moreover, expression of E1A in wild-type cells specifically blocked transcriptional activation of the INO1 and SUC2 genes, whose activation pathways are distinct but have a common requirement for the SWI/SNF complex. These data demonstrate a specific functional interaction between E1A and the SWI/SNF complex and suggest that a similar interaction takes place in rodent and human cells.

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Neef, Daniel W., and Michael P. Kladde. "Polyphosphate Loss Promotes SNF/SWI- and Gcn5-Dependent Mitotic Induction of PHO5." Molecular and Cellular Biology 23, no. 11 (June 1, 2003): 3788–97. http://dx.doi.org/10.1128/mcb.23.11.3788-3797.2003.

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ABSTRACT Approximately 800 transcripts in Saccharomyces cerevisiae are cell cycle regulated. The oscillation of ∼40% of these genes, including a prominent subclass involved in nutrient acquisition, is not understood. To address this problem, we focus on the mitosis-specific activation of the phosphate-responsive promoter, PHO5. We show that the unexpected mitotic induction of the PHO5 acid phosphatase in rich medium requires the transcriptional activators Pho4 and Pho2, the cyclin-dependent kinase inhibitor Pho81, and the chromatin-associated enzymes Gcn5 and Snf2/Swi2. PHO5 mitotic activation is repressed by addition of orthophosphate, which significantly increases cellular polyphosphate. Polyphosphate levels also fluctuate inversely with PHO5 mRNA during the cell cycle, further substantiating an antagonistic link between this phosphate polymer and PHO5 mitotic regulation. Moreover, deletion of PHM3, required for polyphosphate accumulation, leads to premature onset of PHO5 expression, as well as an increased rate, magnitude, and duration of PHO5 activation. Orthophosphate addition, however, represses mitotic PHO5 expression in a phm3Δ strain. Thus, polyphosphate per se is not necessary to repress PHO transcription but, when present, replenishes cellular phosphate during nutrient depletion. These results demonstrate a dynamic mechanism of mitotic transcriptional regulation that operates mostly independently of factors that drive progression through the cell cycle.

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Du, Jian, Irem Nasir, Benjamin K. Benton, Michael P. Kladde, and Brehon C. Laurent. "Sth1p, a Saccharomyces cerevisiae Snf2p/Swi2p Homolog, Is an Essential ATPase in RSC and Differs From Snf/Swi in Its Interactions With Histones and Chromatin-Associated Proteins." Genetics 150, no. 3 (November 1, 1998): 987–1005. http://dx.doi.org/10.1093/genetics/150.3.987.

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Abstract The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo. The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p. The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function. The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways. Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi. These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle.

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Barber, Louise J., Thomas A. Ward, John A. Hartley, and Peter J. McHugh. "DNA Interstrand Cross-Link Repair in the Saccharomyces cerevisiae Cell Cycle: Overlapping Roles for PSO2 (SNM1) with MutS Factors and EXO1 during S Phase." Molecular and Cellular Biology 25, no. 6 (March 15, 2005): 2297–309. http://dx.doi.org/10.1128/mcb.25.6.2297-2309.2005.

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ABSTRACT Pso2/Snm1 is a member of the β-CASP metallo-β-lactamase family of proteins that include the V(D)J recombination factor Artemis. Saccharomyces cerevisiae pso2 mutants are specifically sensitive to agents that induce DNA interstrand cross-links (ICLs). Here we establish a novel overlapping function for PSO2 with MutS mismatch repair factors and the 5′-3′ exonuclease Exo1 in the repair of DNA ICLs, which is confined to S phase. Our data demonstrate a requirement for NER and Pso2, or Exo1 and MutS factors, in the processing of ICLs, and this is required prior to the repair of ICL-induced DNA double-strand breaks (DSBs) that form during replication. Using a chromosomally integrated inverted-repeat substrate, we also show that loss of both pso2 and exo1/msh2 reduces spontaneous homologous recombination rates. Therefore, PSO2, EXO1, and MSH2 also appear to have overlapping roles in the processing of some forms of endogenous DNA damage that occur at an irreversibly collapsed replication fork. Significantly, our analysis of ICL repair in cells synchronized for each cell cycle phase has revealed that homologous recombination does not play a major role in the direct repair of ICLs, even in G2, when a suitable template is readily available. Rather, we propose that recombination is primarily involved in the repair of DSBs that arise from the collapse of replication forks at ICLs. These findings have led to considerable clarification of the complex genetic relationship between various ICL repair pathways.

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Ferreira, Monica E., Kacie Flaherty, and Philippe Prochasson. "The Saccharomyces cerevisiae Histone Chaperone Rtt106 Mediates the Cell Cycle Recruitment of SWI/SNF and RSC to the HIR-Dependent Histone Genes." PLoS ONE 6, no. 6 (June 15, 2011): e21113. http://dx.doi.org/10.1371/journal.pone.0021113.

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Lambert, Sarah, Sarah J. Mason, Louise J. Barber, John A. Hartley, Jackie A. Pearce, Anthony M. Carr, and Peter J. McHugh. "Schizosaccharomyces pombe Checkpoint Response to DNA Interstrand Cross-Links." Molecular and Cellular Biology 23, no. 13 (July 1, 2003): 4728–37. http://dx.doi.org/10.1128/mcb.23.13.4728-4737.2003.

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ABSTRACT Drugs that produce covalent interstrand cross-links (ICLs) in DNA remain central to the treatment of cancer, but the cell cycle checkpoints activated by ICLs have received little attention. We have used the fission yeast, Schizosaccharomyces pombe, to elucidate the checkpoint responses to the ICL-inducing anticancer drugs nitrogen mustard and mitomycin C. First we confirmed that the repair pathways acting on ICLs in this yeast are similar to those in the main organisms studied to date (Escherichia coli, budding yeast, and mammalian cells), principally nucleotide excision repair and homologous recombination. We also identified and disrupted the S. pombe homologue of the Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this activity is required for normal resistance to cross-linking agents, but not other forms of DNA damage. Survival and biochemical analysis indicated a key role for the “checkpoint Rad” family acting through the chk1-dependent DNA damage checkpoint in the ICL response. Rhp9-dependent phosphorylation of Chk1 correlates with G2 arrest following ICL induction. In cells able to bypass the G2 block, a second-cycle (S-phase) arrest was observed. Only a transient activation of the Cds1 DNA replication checkpoint factor occurs following ICL formation in wild-type cells, but this is increased and persists in G2 arrest-deficient mutants. This likely reflects the fraction of cells escaping the G2 damage checkpoint and arresting in the subsequent S phase due to ICL replication blocks. Disruption of cds1 confers increased resistance to ICLs, suggesting that this second-cycle S-phase arrest might be a lethal event.

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Honigberg, Saul M., and Rita H. Lee. "Snf1 Kinase Connects Nutritional Pathways Controlling Meiosis in Saccharomyces cerevisiae." Molecular and Cellular Biology 18, no. 8 (August 1, 1998): 4548–55. http://dx.doi.org/10.1128/mcb.18.8.4548.

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ABSTRACT Glucose inhibits meiosis in Saccharomyces cerevisiae at three different steps (IME1 transcription, IME2transcription, and entry into late stages of meiosis). Because many of the regulatory effects of glucose in yeast are mediated through the inhibition of Snf1 kinase, a component of the glucose repression pathway, we determined the role of SNF1 in regulating meiosis. Deleting SNF1 repressed meiosis at the same three steps that were inhibited by glucose, suggesting that glucose blocks meiosis by inhibiting Snf1. For example, the snf1Δ mutant completely failed to induce IME1 transcripts in sporulation medium. Furthermore, even when this block was bypassed by expression ofIME1 from a multicopy plasmid, IME2transcription and meiotic initiation occurred at only 10 to 20% of the levels seen in wild-type cells. The addition of glucose did not further inhibit IME2 transcription, suggesting that Snf1 is the primary mediator of glucose controls on IME2 expression. Finally, in snf1Δ cells in which both blocks on meiotic initiation were bypassed, early stages of meiosis (DNA replication and commitment to recombination) occurred, but later stages (chromosome segregation and spore formation) did not, suggesting that Snf1 controls later stages of meiosis independently from the two controls on meiotic initiation. Because Snf1 is known to activate the expression of genes required for acetate metabolism, it may also serve to connect glucose and acetate controls on meiotic differentiation.

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Treitel, Michelle A., Sergei Kuchin, and Marian Carlson. "Snf1 Protein Kinase Regulates Phosphorylation of the Mig1 Repressor in Saccharomyces cerevisiae." Molecular and Cellular Biology 18, no. 11 (November 1, 1998): 6273–80. http://dx.doi.org/10.1128/mcb.18.11.6273.

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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.

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Elbing, Karin, Rhonda R. McCartney, and Martin C. Schmidt. "Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae." Biochemical Journal 393, no. 3 (January 13, 2006): 797–805. http://dx.doi.org/10.1042/bj20051213.

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Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of β and γ subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its β and γ subunits.

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Hubbard, E. J., R. Jiang, and M. Carlson. "Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae." Molecular and Cellular Biology 14, no. 3 (March 1994): 1972–78. http://dx.doi.org/10.1128/mcb.14.3.1972-1978.1994.

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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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Hubbard, E. J., R. Jiang, and M. Carlson. "Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae." Molecular and Cellular Biology 14, no. 3 (March 1994): 1972–78. http://dx.doi.org/10.1128/mcb.14.3.1972.

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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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Ye, Tian, Raúl García-Salcedo, José Ramos, and Stefan Hohmann. "Gis4, a New Component of the Ion Homeostasis System in the Yeast Saccharomyces cerevisiae." Eukaryotic Cell 5, no. 10 (October 2006): 1611–21. http://dx.doi.org/10.1128/ec.00215-06.

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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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Vyas, Valmik K., Sergei Kuchin, Cristin D. Berkey, and Marian Carlson. "Snf1 Kinases with Different β-Subunit Isoforms Play Distinct Roles in Regulating Haploid Invasive Growth." Molecular and Cellular Biology 23, no. 4 (February 15, 2003): 1341–48. http://dx.doi.org/10.1128/mcb.23.4.1341-1348.2003.

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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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Celenza, J. L., and M. Carlson. "Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein." Molecular and Cellular Biology 9, no. 11 (November 1989): 5034–44. http://dx.doi.org/10.1128/mcb.9.11.5034-5044.1989.

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The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.

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Casamayor, Antonio, Raquel Serrano, María Platara, Carlos Casado, Amparo Ruiz, and Joaquín Ariño. "The role of the Snf1 kinase in the adaptive response of Saccharomyces cerevisiae to alkaline pH stress." Biochemical Journal 444, no. 1 (April 26, 2012): 39–49. http://dx.doi.org/10.1042/bj20112099.

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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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Kuchin, S., V. K. Vyas, and M. Carlson. "Role of the yeast Snf1 protein kinase in invasive growth." Biochemical Society Transactions 31, no. 1 (February 1, 2003): 175–77. http://dx.doi.org/10.1042/bst0310175.

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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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Vyas, Valmik K., Sergei Kuchin, and Marian Carlson. "Interaction of the Repressors Nrg1 and Nrg2 With the Snf1 Protein Kinase in Saccharomyces cerevisiae." Genetics 158, no. 2 (June 1, 2001): 563–72. http://dx.doi.org/10.1093/genetics/158.2.563.

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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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Sanz, Pascual, Katja Ludin, and Marian Carlson. "Sip5 Interacts With Both the Reg1/Glc7 Protein Phosphatase and the Snf1 Protein Kinase of Saccharomyces cerevisiae." Genetics 154, no. 1 (January 1, 2000): 99–107. http://dx.doi.org/10.1093/genetics/154.1.99.

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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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Wright, R. M., and R. O. Poyton. "Release of two Saccharomyces cerevisiae cytochrome genes, COX6 and CYC1, from glucose repression requires the SNF1 and SSN6 gene products." Molecular and Cellular Biology 10, no. 3 (March 1990): 1297–300. http://dx.doi.org/10.1128/mcb.10.3.1297-1300.1990.

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We show here that SNF1 and SSN6 are required for derepression of the glucose-repressible yeast genes COX6 and CYC1, which encode the mitochondrial proteins cytochrome c oxidase subunit VI and iso-1-cytochrome c, respectively. In an snf1 mutant genetic background, the transcription of both COX6 and CYC1 continued to be repressed after cells were shifted into derepressing media. In an ssn6 mutant genetic background, both COX6 and CYC1 were expressed constitutively at high levels in repressing media. SSN6 acted epistatically to SNF1 in the regulation of both cytochrome genes. These findings are similar to previous findings on the effects of SNF1 and SSN6 on SUC2 expression in Saccharomyces cerevisiae and are consistent with a model proposing that SNF1 exerts its effect through SSN6 on COX6 and CYC1.

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Kuchin, Sergei, Valmik K. Vyas, and Marian Carlson. "Snf1 Protein Kinase and the Repressors Nrg1 and Nrg2 Regulate FLO11, Haploid Invasive Growth, and Diploid Pseudohyphal Differentiation." Molecular and Cellular Biology 22, no. 12 (June 15, 2002): 3994–4000. http://dx.doi.org/10.1128/mcb.22.12.3994-4000.2002.

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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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Muranaka, T., H. Banno, and Y. Machida. "Characterization of tobacco protein kinase NPK5, a homolog of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae." Molecular and Cellular Biology 14, no. 5 (May 1994): 2958–65. http://dx.doi.org/10.1128/mcb.14.5.2958-2965.1994.

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We have isolated a cDNA (cNPK5) that encodes a protein kinase of 511 amino acids from suspension cultures of tobacco cells. The predicted kinase domain of NPK5 is 65% identical in terms of amino acid sequence to that of the SNF1 serine/threonine protein kinase of Saccharomyces cerevisiae, which plays a central role in catabolite repression in yeast cells. SNF1 positively regulates transcription of various glucose-repressible genes of the yeast, such as the SUC2 gene for a secreted invertase, in response to glucose deprivation: snf1 mutants cannot utilize sucrose as a carbon source. Expression of cNPK5 in yeast cells allowed the snf1 mutant cells to utilize sucrose for growth and caused constitutive expression of the SUC2 gene in wild-type cells even in the presence of glucose, an indication that the NPK5 protein is present in a constitutively active form in S. cerevisiae. On the other hand, expression of cNPK5 failed to suppress the growth defect of the snf4 mutant cells in the presence of sucrose and to induce expression of the SUC2 gene. These results indicate that SNF4 is required for the induction of SUC2 expression by NPK5, as by SNF1, even if NPK5 is constitutively active in S. cerevisiae. The recombinant NPK5 protein is capable of autophosphorylation in vitro in a reaction that requires Mn2+ rather than Mg2+ ions but is inhibited by Ca2+ ions. Both dicotyledonous and monocotyledonous plants have several copies of the NPK5-related gene, which probably constitute a small gene family. NPK5-related genes were found to be expressed in the roots, leaves, and stems of tobacco plants. The high degree of structural conservation and the functional similarity of NPK5 to SNF1 lead us to speculate that NPK5 (or a related kinase) also plays a role in sugar metabolism in higher plants.

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Hedbacker, Kristina, Robert Townley, and Marian Carlson. "Cyclic AMP-Dependent Protein Kinase Regulates the Subcellular Localization of Snf1-Sip1 Protein Kinase." Molecular and Cellular Biology 24, no. 5 (March 1, 2004): 1836–43. http://dx.doi.org/10.1128/mcb.24.5.1836-1843.2004.

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ABSTRACT The Snf1/AMP-activated protein kinase family has diverse roles in cellular responses to metabolic stress. In Saccharomyces cerevisiae, Snf1 protein kinase has three isoforms of the β subunit that confer versatility on the kinase and that exhibit distinct patterns of subcellular localization. The Sip1 β subunit resides in the cytosol in glucose-grown cells and relocalizes to the vacuolar membrane in response to carbon stress. We show that translation of Sip1 initiates at the second ATG of the open reading frame, yielding a potential site for N myristoylation, and that mutation of the critical glycine abolishes relocalization. We further show that the cyclic AMP-dependent protein kinase (protein kinase A [PKA]) pathway maintains the cytoplasmic localization of Sip1 in glucose-grown cells. The Snf1 catalytic subunit also exhibits aberrant localization to the vacuolar membrane in PKA-deficient cells, indicating that PKA regulates the localization of Snf1-Sip1 protein kinase. These findings establish a novel mechanism of regulation of Snf1 protein kinase by the PKA pathway.

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Hedbacker, Kristina, Seung-Pyo Hong, and Marian Carlson. "Pak1 Protein Kinase Regulates Activation and Nuclear Localization of Snf1-Gal83 Protein Kinase." Molecular and Cellular Biology 24, no. 18 (September 15, 2004): 8255–63. http://dx.doi.org/10.1128/mcb.24.18.8255-8263.2004.

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ABSTRACT Three kinases, Pak1, Tos3, and Elm1, activate Snf1 protein kinase in Saccharomyces cerevisiae. This cascade is conserved in mammals, where LKB1 activates AMP-activated protein kinase. We address the specificity of the activating kinases for the three forms of Snf1 protein kinase containing the β-subunit isoforms Gal83, Sip1, and Sip2. Pak1 is the most important kinase for activating Snf1-Gal83 in response to glucose limitation, but Elm1 also has a significant role; moreover, both Pak1 and Elm1 affect Snf1-Sip2. These findings exclude the possibility of a one-to-one correspondence between the activating kinases and the Snf1 complexes. We further identify a second, unexpected role for Pak1 in regulating Snf1-Gal83: the catalytic activity of Pak1 is required for the nuclear enrichment of Snf1-Gal83 in response to carbon stress. The nuclear enrichment of Snf1 fused to green fluorescent protein (GFP) depends on both Gal83 and Pak1 and is abolished by a mutation of the activation loop threonine; in contrast, the nuclear enrichment of Gal83-GFP occurs in a snf1Δ mutant and depends on Pak1 only when Snf1 is present. Snf1-Gal83 is the only form of the kinase that localizes to the nucleus. These findings, that Pak1 both activates Snf1-Gal83 and controls its nuclear localization, implicate Pak1 in regulating nuclear Snf1 protein kinase activity.

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Madrigal-Perez, Luis Alberto, Gerardo M. Nava, Juan Carlos González-Hernández, and Minerva Ramos-Gomez. "Resveratrol increases glycolytic flux in Saccharomyces cerevisiae via a SNF1-dependet mechanism." Journal of Bioenergetics and Biomembranes 47, no. 4 (June 20, 2015): 331–36. http://dx.doi.org/10.1007/s10863-015-9615-y.

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Shirra, Margaret K., Jana Patton-Vogt, Andreas Ulrich, Oksana Liuta-Tehlivets, Sepp D. Kohlwein, Susan A. Henry, and Karen M. Arndt. "Inhibition of Acetyl Coenzyme A Carboxylase Activity Restores Expression of the INO1 Gene in a snf1Mutant Strain of Saccharomyces cerevisiae." Molecular and Cellular Biology 21, no. 17 (September 1, 2001): 5710–22. http://dx.doi.org/10.1128/mcb.21.17.5710-5722.2001.

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ABSTRACT Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy ofsnf1Δ strains. We identified mutations inACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the β subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show thatsnf1Δ strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying theACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression ofACC1, suppresses the inositol auxotrophy ofsnf1Δ strains. Together, these findings indicate that the inositol auxotrophy of snf1Δ strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important forINO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.

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Hong, Seung-Pyo, Milica Momcilovic, and Marian Carlson. "Function of Mammalian LKB1 and Ca2+/Calmodulin-dependent Protein Kinase Kinase α as Snf1-activating Kinases in Yeast." Journal of Biological Chemistry 280, no. 23 (April 14, 2005): 21804–9. http://dx.doi.org/10.1074/jbc.m501887200.

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The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation in response to stress. In the yeast Saccharomyces cerevisiae, the Snf1 kinase cascade comprises three Snf1-activating kinases, Pak1, Tos3, and Elm1. The only established mammalian AMPK kinase is LKB1. We show that LKB1 functions heterologously in yeast. In pak1Δ tos3Δ elm1Δ cells, LKB1 activated Snf1 catalytic activity and conferred a Snf+ growth phenotype. Coexpression of STRADα and MO25α, which form a complex with LKB1, enhanced LKB1 function. Thus, the Snf1/AMPK kinase cascade is functionally conserved between yeast and mammals. Ca2+/calmodulin-dependent kinase kinase (CaMKK) shows more sequence similarity to Pak1, Tos3, and Elm1 than does LKB1. When expressed in pak1Δ tos3Δ elm1Δ cells, CaMKKα activated Snf1 catalytic activity, restored the Snf+ phenotype, and also phosphorylated the activation loop threonine of Snf1 in vitro. These findings indicate that CaMKKα is a functional member of the Snf1/AMPK kinase family and support CaMKKα as a likely candidate for an AMPK kinase in mammalian cells. Analysis of the function of these heterologous kinases in yeast provided insight into the regulation of Snf1. When activated by LKB1 or CaMKKα, Snf1 activity was significantly inhibited by glucose, suggesting that a mechanism independent of the activating kinases can mediate glucose signaling in yeast. Finally, this analysis provided evidence that Pak1 functions in another capacity, besides activating Snf1, to regulate the nuclear enrichment of Snf1 protein kinase in response to carbon stress.

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O'Donnell, Allyson F., Rhonda R. McCartney, Dakshayini G. Chandrashekarappa, Bob B. Zhang, Jeremy Thorner, and Martin C. Schmidt. "2-Deoxyglucose Impairs Saccharomyces cerevisiae Growth by Stimulating Snf1-Regulated and α-Arrestin-Mediated Trafficking of Hexose Transporters 1 and 3." Molecular and Cellular Biology 35, no. 6 (December 29, 2014): 939–55. http://dx.doi.org/10.1128/mcb.01183-14.

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The glucose analog 2-deoxyglucose (2DG) inhibits the growth ofSaccharomyces cerevisiaeand human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity ofsnf1Δ cells. The addition of 2DG or the loss of Snf1 reducesHXT1andHXT3expression levels and stimulates transporter endocytosis and degradation in the vacuole. 2DG-stimulated trafficking of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/Art7, two members of the α-arrestin trafficking adaptor family. Mutations inROD1andROG3that block binding to the ubiquitin ligase Rsp5 eliminate Rod1- and Rog3-mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3, but via different mechanisms. Snf1 activated by 2DG phosphorylates Rod1 but fails to phosphorylate other known targets, such as the transcriptional repressor Mig1. We propose a novel mechanism for 2DG-induced toxicity whereby 2DG stimulates the modification of α-arrestins, which promote glucose transporter internalization and degradation, causing glucose starvation even when cells are in a glucose-rich environment.

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Ahuatzi, Deifilia, Alberto Riera, Rafael Peláez, Pilar Herrero, and Fernando Moreno. "Hxk2 Regulates the Phosphorylation State of Mig1 and Therefore Its Nucleocytoplasmic Distribution." Journal of Biological Chemistry 282, no. 7 (December 18, 2006): 4485–93. http://dx.doi.org/10.1074/jbc.m606854200.

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Mig1 and Hxk2 are two major mediators of glucose repression in Saccharomyces cerevisiae. However, the mechanism by which Hxk2 participates in the glucose repression signaling pathway is not completely understood. Recently, it has been demonstrated that Hxk2 interacts with Mig1 to generate a repressor complex located in the nucleus of S. cerevisiae. However, the mechanism by which Mig1 favors the presence of Hxk2 in the nucleus is not clear, and the function of Hxk2 at the nuclear repressor complex level is still unknown. Here, we report that serine 311 of Mig1 is a critical residue for interaction with Hxk2 and that this interaction is regulated by glucose. Our findings suggest that Snf1 interacts constitutively with the Hxk2 component of the repressor complex at high and low glucose conditions. Furthermore, we show that Snf1 binds to Mig1 under low glucose conditions and that binding is largely abolished after a shift to high glucose medium. We found that phosphorylation of serine 311 of Mig1 by Snf1 kinase is essential for Mig1 protein nuclear export and derepression of the SUC2 gene in glucose-limited cells. These results allow postulating that the Hxk2 operates by interacting both with Mig1 and Snf1 to inhibit the Mig1 phosphorylation at serine 311 during high glucose grown.

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Celenza, J. L., F. J. Eng, and M. Carlson. "Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase." Molecular and Cellular Biology 9, no. 11 (November 1989): 5045–54. http://dx.doi.org/10.1128/mcb.9.11.5045-5054.1989.

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The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in Saccharomyces cerevisiae. Previous evidence suggested that SNF4 is functionally related to SNF1, another essential gene in this global regulatory system that encodes a protein kinase. Increased SNF1 gene dosage partially compensates for a mutation in SNF4, and the SNF4 function is required for maximal SNF1 protein kinase activity in vitro. We have cloned SNF4 and identified its 1.2-kilobase RNA, which is not regulated by glucose repression. A 36-kilodalton SNF4 protein is predicted from the nucleotide sequence. Disruption of the chromosomal SNF4 locus revealed that the requirement for SNF4 function is less stringent at low temperature (23 degrees C). A bifunctional SNF4-lacZ gene fusion that includes almost the entire SNF4 coding sequence was constructed. The fusion protein was shown by immunofluorescence microscopy to be distributed throughout the cell, with partial localization to the nucleus. The SNF4-beta-galactosidase protein coimmunoprecipitated with the SNF1 protein kinase, thus providing evidence for the physical association of the two proteins.

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Thompson-Jaeger, S., J. François, J. P. Gaughran, and K. Tatchell. "Deletion of SNF1 affects the nutrient response of yeast and resembles mutations which activate the adenylate cyclase pathway." Genetics 129, no. 3 (November 1, 1991): 697–706. http://dx.doi.org/10.1093/genetics/129.3.697.

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Abstract We have isolated a snf1/ccr1 mutant of Saccharomyces cerevisiae which loses viability upon starvation and fails to accumulate glycogen in response to abrupt depletion of phosphate or glucose. A snf1 null mutant is sensitive to heat stress and starvation and fails to accumulate glycogen during growth in rich medium. The phenotypes of the snf1 mutants are those commonly associated with an overactivation of the adenylate cyclase pathway. Mutations in adenylate cyclase or RAS2 which decrease the level of cAMP in the cell moderate the snf1 phenotype. In contrast, a mutation in RAS2 (RAS2val19) which increases the level of cAMP or a mutation in the regulatory subunit (BCY1) of cAMP-dependent protein kinase which results in unregulated cAMP-dependent protein kinase activity accentuates the snf1 phenotype. However, the action of SNF1 in the stress response appears at least partly independent of cAMP-dependent protein kinase because a snf1 phenotype is observed in a strain that lacks all three of the genes that encode the catalytic subunits of cAMP-dependent protein kinase. SNF1 therefore acts at least in part through a cAMP-independent pathway.

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Ferrer-Dalmau, Jofre, Francisca Randez-Gil, Maribel Marquina, José A. Prieto, and Antonio Casamayor. "Protein kinase Snf1 is involved in the proper regulation of the unfolded protein response in Saccharomyces cerevisiae." Biochemical Journal 468, no. 1 (May 5, 2015): 33–47. http://dx.doi.org/10.1042/bj20140734.

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We show that Snf1, the yeast AMPK, affects the Ire1-RNase activity during the recovery phase of the UPR. Hence, our study suggests a connection between glucose availability and the UPR, identifying new targets for its regulation.

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Vincent, Olivier, Sergei Kuchin, Seung-Pyo Hong, Robert Townley, Valmik K. Vyas, and Marian Carlson. "Interaction of the Srb10 Kinase with Sip4, a Transcriptional Activator of Gluconeogenic Genes in Saccharomyces cerevisiae." Molecular and Cellular Biology 21, no. 17 (September 1, 2001): 5790–96. http://dx.doi.org/10.1128/mcb.21.17.5790-5796.2001.

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ABSTRACT Sip4 is a Zn2Cys6 transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptional repression but also positively regulates Gal4. We show that Srb10 is required for phosphorylation of Sip4 during growth in nonfermentable carbon sources and that the catalytic activity of Srb10 stimulates the ability of LexA-Sip4 to activate transcription of a reporter. Srb10 and Sip4 coimmunoprecipitate from cell extracts and interact in two-hybrid assays, suggesting that Srb10 regulates Sip4 directly. We also present evidence that the Srb10 and Snf1 kinases interact with different regions of Sip4. These findings support the view that the Srb10 kinase not only plays negative roles in transcriptional control but also has broad positive roles during growth in carbon sources other than glucose.

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Charbon, Godefroid, Karin D. Breunig, Ruddy Wattiez, Jean Vandenhaute, and Isabelle Noël-Georis. "Key Role of Ser562/661 in Snf1-Dependent Regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis." Molecular and Cellular Biology 24, no. 10 (May 15, 2004): 4083–91. http://dx.doi.org/10.1128/mcb.24.10.4083-4091.2004.

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ABSTRACT Utilization of nonfermentable carbon sources by Kluyveromyces lactis and Saccharomyces cerevisiae requires the Snf1p kinase and the Cat8p transcriptional activator, which binds to carbon source-responsive elements of target genes. We demonstrate that KlSnf1p and KlCat8p from K. lactis interact in a two-hybrid system and that the interaction is stronger with a kinase-dead mutant form of KlSnf1p. Of two putative phosphorylation sites in the KlCat8p sequence, serine 661 was identified as a key residue governing KlCat8p regulation. Serine 661 is located in the middle homology region, a regulatory domain conserved among zinc cluster transcription factors, and is part of an Snf1p consensus phosphorylation site. Single mutations at this site are sufficient to completely change the carbon source regulation of the KlCat8p transactivation activity observed. A serine-to-glutamate mutant form mimicking constitutive phosphorylation results in a nearly constitutively active form of KlCat8p, while a serine-to-alanine mutation has the reverse effect. Furthermore, it is shown that KlCat8p phosphorylation depends on KlSNF1. The Snf1-Cat8 connection is evolutionarily conserved: mutation of corresponding serine 562 of ScCat8p gave similar results in S. cerevisiae. The enhanced capacity of ScCat8S562E to suppress the phenotype caused by snf1 strengthens the hypothesis of direct phosphorylation of Cat8p by Snf1p. Unlike that of S. cerevisiae ScCAT8, KlCAT8 transcription is not carbon source regulated, illustrating the prominent role of posttranscriptional regulation of Cat8p in K. lactis.

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Salsaa, Michael, Kerestin Aziz, Pablo Lazcano, Michael W. Schmidtke, Maureen Tarsio, Maik Hüttemann, Christian A. Reynolds, Patricia M. Kane, and Miriam L. Greenberg. "Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH." Journal of Biological Chemistry 297, no. 4 (October 2021): 101110. http://dx.doi.org/10.1016/j.jbc.2021.101110.

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42

Celenza, J. L., F. J. Eng, and M. Carlson. "Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase." Molecular and Cellular Biology 9, no. 11 (November 1989): 5045–54. http://dx.doi.org/10.1128/mcb.9.11.5045.

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The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in Saccharomyces cerevisiae. Previous evidence suggested that SNF4 is functionally related to SNF1, another essential gene in this global regulatory system that encodes a protein kinase. Increased SNF1 gene dosage partially compensates for a mutation in SNF4, and the SNF4 function is required for maximal SNF1 protein kinase activity in vitro. We have cloned SNF4 and identified its 1.2-kilobase RNA, which is not regulated by glucose repression. A 36-kilodalton SNF4 protein is predicted from the nucleotide sequence. Disruption of the chromosomal SNF4 locus revealed that the requirement for SNF4 function is less stringent at low temperature (23 degrees C). A bifunctional SNF4-lacZ gene fusion that includes almost the entire SNF4 coding sequence was constructed. The fusion protein was shown by immunofluorescence microscopy to be distributed throughout the cell, with partial localization to the nucleus. The SNF4-beta-galactosidase protein coimmunoprecipitated with the SNF1 protein kinase, thus providing evidence for the physical association of the two proteins.

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43

Iida, H., S. Sakaguchi, Y. Yagawa, and Y. Anraku. "Cell cycle control by Ca2+ in Saccharomyces cerevisiae." Journal of Biological Chemistry 265, no. 34 (December 1990): 21216–22. http://dx.doi.org/10.1016/s0021-9258(17)45348-8.

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Duboc, P. "Physiology of Saccharomyces cerevisiae during cell cycle oscillations." Journal of Biotechnology 51, no. 1 (October 18, 1996): 57–72. http://dx.doi.org/10.1016/0168-1656(96)01566-0.

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SRIENC, FRIEDRICH, and BRUCE S. DIEN. "Kinetics of the Cell Cycle of Saccharomyces cerevisiae." Annals of the New York Academy of Sciences 665, no. 1 Biochemical E (October 1992): 59–71. http://dx.doi.org/10.1111/j.1749-6632.1992.tb42574.x.

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Bogomolnaya, Lydia M., Ritu Pathak, Jinbai Guo, Roxhana Cham, Rodolfo Aramayo, and Michael Polymenis. "Hym1p affects cell cycle progression in Saccharomyces cerevisiae." Current Genetics 46, no. 4 (September 10, 2004): 183–92. http://dx.doi.org/10.1007/s00294-004-0527-3.

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Shinoda, Junro, and Yoshiko Kikuchi. "Rod1, an arrestin-related protein, is phosphorylated by Snf1-kinase in Saccharomyces cerevisiae." Biochemical and Biophysical Research Communications 364, no. 2 (December 2007): 258–63. http://dx.doi.org/10.1016/j.bbrc.2007.09.134.

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Nadal, Marina, Maria D. Garcia-Pedrajas, and Scott E. Gold. "The snf1 Gene of Ustilago maydis Acts as a Dual Regulator of Cell Wall Degrading Enzymes." Phytopathology® 100, no. 12 (December 2010): 1364–72. http://dx.doi.org/10.1094/phyto-01-10-0011.

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Abstract:

Many fungal plant pathogens are known to produce extracellular enzymes that degrade cell wall elements required for host penetration and infection. Due to gene redundancy, single gene deletions generally do not address the importance of these enzymes in pathogenicity. Cell wall degrading enzymes (CWDEs) in fungi are often subject to carbon catabolite repression at the transcriptional level such that, when glucose is available, CWDE-encoding genes, along with many other genes, are repressed. In Saccharomyces cerevisiae, one of the main players controlling this process is SNF1, which encodes a protein kinase. In this yeast, Snf1p is required to release glucose repression when this sugar is depleted from the growth medium. We have employed a reverse genetic approach to explore the role of the SNF1 ortholog as a potential regulator of CWDE gene expression in Ustilago maydis. We identified U. maydis snf1 and deleted it from the fungal genome. Consistent with our hypothesis, the relative expression of an endoglucanase and a pectinase was higher in the wild type than in the Δsnf1 mutant strain when glucose was depleted from the growth medium. However, when cells were grown in derepressive conditions, the relative expression of two xylanase genes was unexpectedly higher in the Δsnf1 strain than in the wild type, indicating that, in this case, snf1 negatively regulated the expression of these genes. Additionally, we found that, contrary to several other fungal species, U. maydis Snf1 was not required for utilization of alternative carbon sources. Also, unlike in ascomycete plant pathogens, deletion of snf1 did not profoundly affect virulence in U. maydis.

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Kaniak, Aneta, Zhixiong Xue, Daniel Macool, Jeong-Ho Kim, and Mark Johnston. "Regulatory Network Connecting Two Glucose Signal Transduction Pathways in Saccharomyces cerevisiae." Eukaryotic Cell 3, no. 1 (February 2004): 221–31. http://dx.doi.org/10.1128/ec.3.1.221-231.2004.

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ABSTRACT The yeast Saccharomyces cerevisiae senses glucose, its preferred carbon source, through multiple signal transduction pathways. In one pathway, glucose represses the expression of many genes through the Mig1 transcriptional repressor, which is regulated by the Snf1 protein kinase. In another pathway, glucose induces the expression of HXT genes encoding glucose transporters through two glucose sensors on the cell surface that generate an intracellular signal that affects function of the Rgt1 transcription factor. We profiled the yeast transcriptome to determine the range of genes targeted by this second pathway. Candidate target genes were verified by testing for Rgt1 binding to their promoters by chromatin immunoprecipitation and by measuring the regulation of the expression of promoter lacZ fusions. Relatively few genes could be validated as targets of this pathway, suggesting that this pathway is primarily dedicated to regulating the expression of HXT genes. Among the genes regulated by this glucose signaling pathway are several genes involved in the glucose induction and glucose repression pathways. The Snf3/Rgt2-Rgt1 glucose induction pathway contributes to glucose repression by inducing the transcription of MIG2, which encodes a repressor of glucose-repressed genes, and regulates itself by inducing the expression of STD1, which encodes a regulator of the Rgt1 transcription factor. The Snf1-Mig1 glucose repression pathway contributes to glucose induction by repressing the expression of SNF3 and MTH1, which encodes another regulator of Rgt1, and also regulates itself by repressing the transcription of MIG1. Thus, these two glucose signaling pathways are intertwined in a regulatory network that serves to integrate the different glucose signals operating in these two pathways.

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Schultz, J., L. Marshall-Carlson, and M. Carlson. "The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae." Molecular and Cellular Biology 10, no. 9 (September 1990): 4744–56. http://dx.doi.org/10.1128/mcb.10.9.4744-4756.1990.

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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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Journal articles on the topic 'Saccharomyces cerevisiae, cell cycle, Snf1'

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