Schizosaccharomyces pombe Git7p, a member of the Saccharomyces cerevisiae Sgtlp family, is required for glucose and cyclic AMP signaling, cell wall integrity, and septation.
ABSTRACT: The Schizosaccharomyces pombe fbp1 gene, encoding fructose-1,6-bisphosphatase, is transcriptionally repressed by glucose. Mutations that confer constitutive fbp1 transcription identify git (glucose-insensitive transcription) genes that encode components of a cyclic AMP (cAMP) signaling pathway required for adenylate cyclase activation. Four of these genes encode the three subunits of a heterotrimeric G protein (gpa2, git5, and git11) and a G protein-coupled receptor (git3). Three additional genes, git1, git7, and git10, act in parallel to or downstream from the G protein genes. Here, we describe the cloning and characterization of the git7 gene. The Git7p protein is a member of the Saccharomyces cerevisiae Sgtlp protein family. In budding yeast, Sgtlp associates with Skplp and plays an essential role in kinetochore assembly, while in Arabidopsis, a pair of SGT1 proteins have been found to be involved in plant disease resistance through an interaction with RAR1. Like S. cerevisiae Sgtlp, Git7p is essential, but this requirement appears to be due to roles in septation and cell wall integrity, which are unrelated to cAMP signaling, as S. pombe cells lacking either adenylate cyclase or protein kinase A are viable. In addition, git7 mutants are sensitive to the microtubule-destabilizing drug benomyl, although they do not display a chromosome stability defect. Two alleles of git7 that are functional for cell growth and septation but defective for glucose-triggered cAMP signaling encode proteins that are altered in the highly conserved carboxy terminus. The S. cerevisiae and human SGT1 genes both suppress git7-93 but not git7-235 for glucose repression of fbp1 transcription and benomyl sensitivity. This allele-specific suppression indicates that the Git7p/Sgtlp proteins may act as multimers, such that Git7-93p but not Git7-235p can deliver the orthologous proteins to species-specific targets. Our studies suggest that members of the Git7p/Sgt1p protein family may play a conserved role in the regulation of adenylate cyclase activation in S. pombe, S. cerevisiae, and humans.
Project description:In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription. The git2 gene, also known as cyr1, encodes adenylate cyclase. Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways. The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway. In mammalian cells, adenylate cyclase enzymatic activity is regulated by heterotrimeric guanine nucleotide-binding proteins (G proteins). In the budding yeast Saccharomyces cerevisiae, adenylate cyclase enzymatic activity is regulated by monomeric, guanine nucleotide-binding Ras proteins. We show here that git8 is identical to the gpa2 gene that encodes a protein homologous to the alpha subunit of a G protein. Mutations in two additional genes, git3 and git5 are suppressed by gpa2+ in high copy number. Furthermore, a mutation in either git3 or git5 has an additive effect in strains deleted for gpa2 (git8), as it significantly increases expression of an fbp1-lacZ reporter gene. Therefore, git3 and git5 appear to act either in concert with or independently from gpa2 (git8) to regulate adenylate cyclase activity.
Project description:The fission yeast Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway. Elevated cAMP levels activate protein kinase A (PKA) to inhibit transcription of genes involved in sexual development and gluconeogenesis, including the fbp1(+) gene, which encodes fructose-1,6-bisphosphatase. Glucose-mediated activation of PKA requires the function of nine glucose-insensitive transcription (git) genes, encoding adenylate cyclase, the PKA catalytic subunit, and seven "upstream" proteins required for glucose-triggered adenylate cyclase activation. We describe the cloning and characterization of the git10(+) gene, which is identical to swo1(+) and encodes the S. pombe Hsp90 chaperone protein. Glucose repression of fbp1(+) transcription is impaired by both git10(-) and swo1(-) mutant alleles of the hsp90(+) gene, as well as by chemical inhibition of Hsp90 activity and temperature stress to wild-type cells. Unlike the swo1(-) mutant alleles, the git10-201 allele supports cell growth at 37 degrees , while severely reducing glucose repression of an fbp1-lacZ reporter, suggesting a separation-of-function defect. Sequence analyses of three swo1(-) alleles and the one git10(-) allele indicate that swo1(-) mutations alter core functional domains of Hsp90, while the git10(-) mutation affects the Hsp90 central domain involved in client protein binding. These results suggest that Hsp90 plays a specific role in the S. pombe glucose/cAMP pathway.
Project description:The fission yeast Schizosaccharomyces pombe responds to environmental glucose by activating adenylate cyclase. The resulting cAMP signal activates protein kinase A (PKA). PKA inhibits glucose starvation-induced processes, such as conjugation and meiosis, and the transcription of the fbp1 gene that encodes the gluconeogenic enzyme fructose-1,6-bisphosphatase. We previously identified a collection of git genes required for glucose repression of fbp1 transcription, including pka1/git6, encoding the PKA catalytic subunit, git2/cyr1, encoding adenylate cyclase, and six "upstream" genes required for adenylate cyclase activation. The git8 gene, identical to gpa2, encodes the alpha subunit of a heterotrimeric guanine-nucleotide binding protein (Galpha) while git5 encodes a Gbeta subunit. Multicopy suppression studies with gpa2(+) previously indicated that S. pombe adenylate cyclase activation may resemble that of the mammalian type II enzyme with sequential activation by Galpha followed by Gbetagamma. We show here that an activated allele of gpa2 (gpa2(R176H), carrying a mutation in the coding region for the GTPase domain) fully suppresses mutations in git3 and git5, leading to a refinement in our model. We describe the cloning of git3 and show that it encodes a putative seven-transmembrane G protein-coupled receptor. A git3 deletion confers the same phenotypes as deletions of other components of the PKA pathway, including a germination delay, constitutive fbp1 transcription, and starvation-independent conjugation. Since the git3 deletion is fully suppressed by the gpa2(R176H) allele with respect to fbp1 transcription, git3 appears to encode a G protein-coupled glucose receptor responsible for adenylate cyclase activation in S. pombe.
Project description:Schizosaccharomyces pombe regulates intracellular cAMP levels, and thus cAMP-dependent protein kinase (PKA) activity, in response to changes in nutrient conditions. Mutations in any of eight git genes inhibit glucose repression of fbp1 transcription, alter the cell morphology, and cause a reduction in the growth rate. The eight git genes encode components of an adenylate cyclase activation pathway, adenylate cyclase itself, and the catalytic subunit of PKA. Three of these genes have been identified in other studies as regulators of meiosis. Here we show that the sck1 gene, cloned as a high copy number suppressor of a mutation in git3, is able to suppress the defects conferred by a mutation in any of these git genes. Sequence analysis suggests that sck1 encodes a protein most closely related to the Saccharomyces cerevisiae SCH9 protein kinase that had previously been identified as a high copy number suppressor of mutations in S. cerevisiae that reduce or eliminate PKA activity. Disruption of the sck1 gene causes a significant delay in exit from stationary phase when combined with a disruption of the pka1 (git6) gene encoding the catalytic subunit of PKA. However, the sck1 disruption by itself has little or no effect upon fbp1 transcription, meiosis, or exit from stationary phase, and does not enhance the constitutive fbp1 transcription observed in a pka1 mutant. Therefore, sck1 appears to function in a redundant fashion to pka1, but to varying degrees, in the pathways regulated by pka1.
Project description:Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway, activating cAMP-dependent protein kinase A (PKA). This requires nine git (glucose insensitive transcription) genes that encode adenylate cyclase, the PKA catalytic subunit, and seven "upstream" proteins required for glucose-triggered adenylate cyclase activation, including three heterotrimeric G-protein subunits and its associated receptor. We describe here the cloning and characterization of the git1+ gene. Git1 is distantly related to a small group of uncharacterized fungal proteins, including a second S. pombe protein that is not functionally redundant with Git1, as well as to members of the UNC-13/Munc13 protein family. Mutations in git1+ demonstrate functional roles for the two most highly conserved regions of the protein, the C2 domain and the MHD2 Munc homology domain. Cells lacking Git1 are viable, but display phenotypes associated with cAMP-signaling defects, even in strains expressing a mutationally activated G alpha-subunit, which activates adenylate cyclase. These cells possess reduced basal cAMP levels and fail to mount a cAMP response to glucose. In addition, Git1 and adenylate cyclase physically interact and partially colocalize in the cell. Thus, Git1 is a critical component of the S. pombe glucose/cAMP pathway.
Project description:The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Galpha subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gbetagamma dimer to repress transcription of the glucose-regulated fbp1(+) gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2(+) chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Galpha function, thus extending our understanding of Galpha protein structure-function relationships.
Project description:A Schizosaccharomyces pombe gene encoding adenylate cyclase has been cloned by cross-hybridization with the Saccharomyces cerevisiae adenylate cyclase gene. The protein encoded consists of 1692 amino acids and has adenylate cyclase activity that cannot be activated by the Sa. cerevisiae RAS2 protein. Sc. pombe cyclase has a high degree of homology (approximately 60%) with the catalytic domain of Sa. cerevisiae cyclase precisely mapped by a gene-deletion analysis. A 25-40% identity is observed throughout the middle segments of approximately 1000 residues of both cyclases, large parts of which are composed of repetitions of a 23-amino acid motif similar to those found in human glycoproteins, Drosophila chaoptin, and Toll gene product. However, a segment corresponding to the NH2-terminal 620 residues of Sa. cerevisiae cyclase appears lost from Sc. pombe cyclase, and the COOH-terminal 140 residues are not well conserved between the two yeast species. Deletions involving the COOH-terminal residues of Sa. cerevisiae cyclase cause loss of activation by the RAS2 protein. These results suggest that Sc. pombe cyclase may have lost the ability to interact with RAS proteins by the loss of a regulatory site.
Project description:The Schizosaccharomyces pombe fbp1 gene is transcriptionally repressed by protein kinase A (PKA) that is activated by extracellular glucose via a cAMP-signaling pathway. We previously used an fbp1-ura4 reporter that places uracil biosynthesis under the control of the glucose-sensing pathway to identify mutations in genes of the cAMP pathway. More recently, this reporter has been used in high throughput screens for small molecule inhibitors of heterologously-expressed cyclic nucleotide phosphodiesterases (PDEs) that hydrolyse cAMP to 5' AMP. Here we show that strains lacking the adenylyl cyclase gene respond to either exogenous cAMP or cGMP to activate PKA, thus regulating fbp1-ura4 expression and other PKA-regulated processes such as conjugation and the nuclear export of an Rst2-GFP fusion protein. Expression of cGMP-specific PDEs or ones that hydrolyse both cAMP and cGMP increases the amount of exogenous cGMP required to activate PKA in order to repress fbp1-ura4 expression, creating conditions that allow detection of inhibitors of these PDEs. As proof of this concept, we screened a collection of compounds previously identified as inhibitors of cAMP-specific PDE4 or PDE7 enzymes for their ability to inhibit the mammalian cGMP-specific PDE5A enzyme. We identified compound BC76, which inhibits PDE5A in an in vitro enzyme assay with an IC(50) of 232nM. Further yeast-based assays show that BC76 inhibits PDE1, PDE4, PDE5, PDE8, PDE10 and PDE11, thus demonstrating the utility of this system for detecting and characterising inhibitors of either cAMP- or cGMP-metabolising PDEs.
Project description:Sgt1p is a highly conserved eucaryotic protein that is required for both SCF (Skp1p/Cdc53p-Cullin-F-box)-mediated ubiquitination and kinetochore function in yeast. We show here that Sgtlp is also involved in the cyclic AMP (cAMP) pathway in Saccharomyces cerevisiae. SGT1 is an allele-specific suppressor of cdc35-1, a thermosensitive mutation in the leucine-rich repeat domain of the adenylyl cyclase Cyrlp/Cdc35p. We demonstrate that Sgt1p and Cyrlp/Cdc35p physically interact and that the activity of the cAMP pathway is affected in an sgt1 conditional mutant. Sequence analysis suggests that Sgtlp has features of a cochaperone. Thus, Sgt1p is a novel activator of adenylyl cyclase in S. cerevisiae and may function in the assembly or the conformational activation of specific multiprotein complexes.
Project description:We describe regulation of the subcellular localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) regulatory (Cgs1p) and catalytic (Pka1p) subunits in the fission yeast Schizosaccharomyces pombe in response to physiological stresses and during sexual differentiation as determined by fluorescence microscopy of the Cgs1-green fluorescent protein (GFP) and Pka1-GFP fusion proteins, respectively. In wild-type S. pombe cells cultured to log phase under normal growth conditions, Cgs1p and Pka1p are concentrated in the nucleus and more diffusely present in the cytoplasm. Nuclear localization of both proteins is dependent on cAMP, since in cells lacking adenylate cyclase they are detectable only in the cytoplasm. In cells lacking Cgs1p or both Cgs1p and adenylate cyclase, Pka1p is concentrated in the nucleus, demonstrating a role for Cgs1p in the nuclear exclusion of Pka1p. Nuclear-cytoplasmic redistribution of Cgs1p and Pka1p is triggered by growth in glucose-limited or hyperosmotic media and in response to stationary-phase growth. In addition, both proteins are excluded from the nucleus in mating cells undergoing karyogamy and subsequently concentrated in postmeiotic spores. Cgs1p is required for subcellular redistribution of Pka1p induced by growth in glucose-limited and hyperosmotic media and during karyogamy but is not required for Pka1p redistribution triggered by stationary-phase growth or for the enrichment of Pka1p in spores. Our results demonstrate that PKA localization is regulated by cAMP and regulatory subunit-dependent and -independent mechanisms in S. pombe.