Loss of the TOR kinase Tor2 mimics nitrogen starvation and activates the sexual development pathway in fission yeast.
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ABSTRACT:
SUBMITTER: Matsuo T
PROVIDER: S-EPMC1899950 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.
| S-EPMC4612450 | biostudies-literature
Project description:Microorganisms naturally respond to changes in nutritional conditions by adjusting their morphology and physiology. The cellular response of the fission yeast S. pombe to nitrogen starvation has been extensively studied. Here, we report time course metabolomic analysis during one hour immediately after nitrogen starvation, prior to any visible changes in cell morphology except for a tiny increase of cell length per division cycle. We semi-quantitatively measured 75 distinct metabolites, 60% of which changed their level over 2-fold. The most significant changes occurred during the first 15 min, when trehalose, 2-oxoglutarate, and succinate increased, while purine biosynthesis intermediates rapidly diminished. At 30-60 min, free amino acids decreased, although several modified amino acids-including hercynylcysteine sulfoxide, a precursor to ergothioneine-accumulated. Most high-energy metabolites such as ATP, S-adenosyl-methionine or NAD+ remained stable during the whole time course. Very rapid metabolic changes such as the shut-off of purine biosynthesis and the rise of 2-oxoglutarate and succinate can be explained by the depletion of NH4Cl. The changes in the levels of key metabolites, particularly 2-oxoglutarate, might represent an important mechanistic step to trigger subsequent cellular regulations.
| S-EPMC3937841 | biostudies-literature
Project description:Gaf1 is the first GATA family zinc-finger transcription factor identified in Schizosaccharomyces pombe. Here, we report that Gaf1 functions as a negatively acting transcription factor of ste11(+), delaying the entrance of cells exposed to transient nitrogen starvation into the meiotic cycle. gaf1Δ strains exhibited accelerated G(1)-arrest upon nitrogen starvation. Moreover, gaf1Δ mutation caused increased mating and sporulation frequency under both nitrogen-starved and unstarved conditions, while overexpression of gaf1(+) led to a significant impairment of sporulation. By microarray analysis, we found that approximately 63% (116 genes) of the 183 genes up-regulated in unstarved gaf1Δ cells were nitrogen starvation-responsive genes, and furthermore that 25 genes among the genes up-regulated by gaf1Δ mutation are Ste11 targets (e.g., gpa1(+), ste4(+), spk1(+), ste11(+), and mei2(+)). The phenotype caused by gaf1Δ mutation was masked by ste11Δ mutation, indicating that ste11(+) is epistatic to gaf1(+) with respect to sporulation efficiency, and accordingly that gaf1(+) functions upstream of ste11(+) in the signaling pathway governing sexual development. gaf1Δ strains showed accelerated ste11(+) expression under nitrogen starvation and increased ste11(+) expression even under normal conditions. Electrophoretic mobility shift assay analysis demonstrated that Gaf1 specifically binds to the canonical GATA motif (5'-HGATAR-3') spanning from -371 to -366 in ste11(+) promoter. Consequently, Gaf1 provides the prime example for negative regulation of ste11(+) transcription through direct binding to a cis-acting motif of its promoter.
| S-EPMC3416868 | biostudies-literature
Project description:Rheb, a Ras-like small GTPase conserved from human to yeast, controls Tor kinase and plays a central role in the regulation of cell growth depending on extracellular conditions. Rhb1 (a fission yeast homolog of Rheb) regulates amino acid uptake as well as response to nitrogen starvation. In this study, we generated two mutants, rhb1-DA4 and rhb1-DA8, and characterized them genetically. The V17A mutation within the G1 box defined for the Ras-like GTPases was responsible for rhb1-DA4 and Q52R I76F within the switch II domain for rhb1-DA8. In fission yeast, two events--the induction of the meiosis-initiating gene mei2+ and cell division without cell growth--are a typical response to nitrogen starvation. Under nitrogen-rich conditions, Rheb stimulates Tor kinase, which, in turn, suppresses the response to nitrogen starvation. While amino acid uptake was prevented by both rhb1-DA4 and rhb1-DA8 in a dominant fashion, the response to nitrogen starvation was prevented only by rhb1-DA4. rhb1-DA8 thereby allowed genetic dissection of the Rheb-dependent signaling cascade. We postulate that the signaling cascade may branch below Rhb1 or Tor2 and regulate the amino acid uptake and response to nitrogen starvation independently.
| S-EPMC2766313 | biostudies-literature
Project description:From yeast to human, TOR (target of rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators.We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2-Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyperphosphorylation. The loss of functional Ryh1, TORC2, or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe, and therefore Rab6 may be a potential activator of TORC2 in mammals.In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2-Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe.
| S-EPMC3008323 | biostudies-literature
Project description:Amino acid transporters are located at specific subcellular compartments, and their localizations are regulated by the extracellular availability of amino acids. In yeast, target of rapamycin (TOR) activation induces the internalization of amino acid transporters located at the plasma membrane. However, whether and how TOR signaling regulates other amino acid transporters located at intracellular compartments remains unknown. Here, we demonstrate that in the fission yeast, the TOR inhibitor Torin-1 induces the transfer of several yellow fluorescent protein (YFP)-fused intracellular amino acid transporters, including Agp3, Isp5, Aat1, and Put4, from trans-Golgi/endosomes into the vacuoles. By contrast, the localizations of YFP-fused Can1, Fnx1, and Fnx2 transporter proteins were unaffected upon Torin-1 treatment. There are two TOR isoforms in fission yeast, Tor1 and Tor2. Whereas tor1 deletion did not affect the Torin-1-induced transfer of Agp3-YFP, Tor2 inhibition using a temperature-sensitive mutant induced the transfer of Agp3-YFP to the vacuolar lumen, similar to the effects of Torin-1 treatment. Tor2 inhibition also induced the transfer of the YFP-fused Isp5, Aat1, and Put4 transporter proteins to the vacuoles, although only partial transfer of the latter two transporters was observed. Under nitrogen depletion accompanied by reduced Tor2 activity, Agp3-YFP was transferred from the trans-Golgi/endosomes to the plasma membrane and then to the vacuoles, where it was degraded by the vacuolar proteases Isp6 and Psp3. Mutants with constitutively active Tor2 showed delayed transfer of Agp3-YFP to the plasma membrane upon nitrogen depletion. Cells lacking Tsc2, a negative regulator of Tor2, also showed a delay in this process in a Tor2-dependent manner. Taken together, these findings suggest that constitutive Tor2 activity is critical for the retention of amino acid transporters at trans-Golgi/endosomes. Moreover, nitrogen depletion suppresses Tor2 activity through Tsc2, thereby promoting the surface expression of these transporters.
| S-EPMC4598100 | biostudies-literature
Project description:Inositol is a precursor of numerous phospholipids and signalling molecules essential for the cell. Schizosaccharomyces pombe is naturally auxotroph for inositol as its genome does not have a homologue of the INO1 gene encoding inositol-1-phosphate synthase, the enzyme responsible for inositol biosynthesis. In this work, we demonstrate that inositol starvation in S. pombe causes cell death with apoptotic features. This apoptotic death is dependent on the metacaspase Pca1p and is affected by the UPR transducer Ire1p. Previously, we demonstrated that calnexin is involved in apoptosis induced by ER stress. Here, we show that cells expressing a lumenal version of calnexin exhibit a 2-fold increase in the levels of apoptosis provoked by inositol starvation. This increase is reversed by co-expression of a calnexin mutant spanning the transmembrane domain and C-terminal cytosolic tail. Coherently, calnexin is physiologically cleaved at the end of its lumenal domain, under normal growth conditions when cells approach stationary phase. This cleavage suggests that the two naturally produced calnexin fragments are needed to continue growth into stationary phase and to prevent cell death. Collectively, our observations indicate that calnexin takes part in at least two apoptotic pathways in S. pombe, and suggest that the cleavage of calnexin has regulatory roles in apoptotic processes involving calnexin.
| S-EPMC2705804 | biostudies-literature
Project description:Microorganisms naturally respond to changes in nutritional conditions by adjusting their morphology and physiology. The cellular response of the fission yeast S. pombe to nitrogen starvation has been extensively studied. Here, we report time course metabolomic analysis during one hour immediately after nitrogen starvation, prior to any visible changes in cell morphology except for a tiny increase of cell length per division cycle. We semi-quantitatively measured 75 distinct metabolites, 60% of which changed their level over 2-fold. The most significant changes occurred during the first 15 min, when trehalose, 2-oxoglutarate, and succinate increased, while purine biosynthesis intermediates rapidly diminished. At 30–60 min, free amino acids decreased, although several modified amino acids—including hercynylcysteine sulfoxide, a precursor to ergothioneine—accumulated. Most high-energy metabolites such as ATP, S-adenosyl-methionine or NAD+ remained stable during the whole time course. Very rapid metabolic changes such as the shut-off of purine biosynthesis and the rise of 2-oxoglutarate and succinate can be explained by the depletion of NH4Cl. The changes in the levels of key metabolites, particularly 2-oxoglutarate, might represent an important mechanistic step to trigger subsequent cellular regulations.
2013-11-26 | MTBLS67 | MetaboLights
Project description:In fission yeast, meiosis-specific transcripts are selectively eliminated during vegetative growth by the combined action of the YTH-family RNA-binding protein Mmi1 and the nuclear exosome. Upon nutritional starvation, the master regulator of meiosis Mei2 inactivates Mmi1, thereby allowing expression of the meiotic program. Here, we show that the E3 ubiquitin ligase subunit Not4/Mot2 of the evolutionarily conserved Ccr4-Not complex, which associates with Mmi1, promotes suppression of meiotic transcripts expression in mitotic cells. Our analyses suggest that Mot2 directs ubiquitination of Mei2 to preserve the activity of Mmi1 during vegetative growth. Importantly, Mot2 is not involved in the constitutive pathway of Mei2 turnover, but rather plays a regulatory role to limit its accumulation or inhibit its function. We propose that Mmi1 recruits the Ccr4-Not complex to counteract its own inhibitor Mei2, thereby locking the system in a stable state that ensures the repression of the meiotic program by Mmi1.
| S-EPMC5614563 | biostudies-literature
Project description:BackgroundChanges in gene expression are hallmarks of cellular differentiation. Sexual differentiation in fission yeast (Schizosaccharomyces pombe) provides a model system for gene expression programs accompanying and driving cellular specialization. The expression of hundreds of genes is modulated in successive waves during meiosis and sporulation in S. pombe, and several known transcription factors are critical for these processes.ResultsWe used DNA microarrays to investigate meiotic gene regulation by examining transcriptomes after genetic perturbations (gene deletion and/or overexpression) of rep1, mei4, atf21 and atf31, which encode known transcription factors controlling sexual differentiation. This analysis reveals target genes at a genome-wide scale and uncovers combinatorial control by Atf21p and Atf31p. We also studied two transcription factors not previously implicated in sexual differentiation whose meiotic induction depended on Mei4p: Rsv2p induces stress-related genes during spore formation, while Rsv1p represses glucose-metabolism genes. Our data further reveal negative feedback interactions: both Rep1p and Mei4p not only activate specific gene expression waves (early and middle genes, respectively) but are also required for repression of genes induced in the previous waves (Ste11p-dependent and early genes, respectively).ConclusionThese data give insight into regulatory principles controlling the extensive gene expression program driving sexual differentiation and highlight sophisticated interactions and combinatorial control among transcription factors. Besides triggering simultaneous expression of gene waves, transcription factors also repress genes in the previous wave and induce other factors that in turn regulate a subsequent wave. These dependencies ensure an ordered and timely succession of transcriptional waves during cellular differentiation.
| S-EPMC2246291 | biostudies-literature