TORC1 signaling is governed by two negative regulators in fission yeast.
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ABSTRACT:
SUBMITTER: Ma N
PROVIDER: S-EPMC3781973 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond.
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Project description:Mammalian target of rapamycin complex 1 (TORC1) is controlled by the GATOR complex composed of the GATOR1 subcomplex and its inhibitor, the GATOR2 subcomplex, sensitive to amino acid starvation. Previously, we identified fission yeast GATOR1 that prevents deregulated activation of TORC1 (Chia et al., 2017). Here, we report identification and characterization of GATOR2 in fission yeast. Unexpectedly, the GATOR2 subunit Sea3, an ortholog of mammalian WDR59, is physically and functionally proximal to GATOR1, rather than GATOR2, attenuating TORC1 activity. The fission yeast GATOR complex is dispensable for TORC1 regulation in response to amino acid starvation, which instead activates the Gcn2 pathway to inhibit TORC1 and induce autophagy. On the other hand, nitrogen starvation suppresses TORC1 through the combined actions of the GATOR1-Sea3 complex, the Gcn2 pathway, and the TSC complex, another conserved TORC1 inhibitor. Thus, multiple, parallel signaling pathways implement negative regulation of TORC1 to ensure proper cellular starvation responses.
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Project description:Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.
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Project description:Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD+ -dependent protein deacetylase, which regulates the expression of the ATP-dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho-mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)-like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2-Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.
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Project description:Compartment-specific Ras signaling is an emerging paradigm that may explain the multiplex outputs from a single GTPase. The fission yeast, Schizosaccharomyces pombe, affords a simple system in which to study Ras signaling because it has a single Ras protein, Ras1, that regulates two distinct pathways: one that controls mating through a Byr2-mitogen-activated protein kinase cascade and one that signals through Scd1-Cdc42 to maintain elongated cell morphology. We generated Ras1 mutants that are restricted to either the endomembrane or the plasma membrane. Protein binding studies showed that each could interact with the effectors of both pathways. However, when examined in ras1 null cells, endomembrane-restricted Ras1 supported morphology but not mating, and, conversely, plasma membrane-restricted Ras1 supported mating but did not signal to Scd1-Cdc42. These observations provide a striking demonstration of compartment-specific Ras signaling and indicate that spatial specificity in the Ras pathway is evolutionarily conserved.
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Project description:Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.
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Project description:Cellular systems are generally robust against fluctuations of intracellular parameters such as gene expression level. However, little is known about expression limits of genes required to halt cellular systems. In this study, using the fission yeast Schizosaccharomyces pombe, we developed a genetic 'tug-of-war' (gTOW) method to assess the overexpression limit of certain genes. Using gTOW, we determined copy number limits for 31 cell-cycle regulators; the limits varied from 1 to >100. Comparison with orthologs of the budding yeast Saccharomyces cerevisiae suggested the presence of a conserved fragile core in the eukaryotic cell cycle. Robustness profiles of networks regulating cytokinesis in both yeasts (septation-initiation network (SIN) and mitotic exit network (MEN)) were quite different, probably reflecting differences in their physiologic functions. Fragility in the regulation of GTPase spg1 was due to dosage imbalance against GTPase-activating protein (GAP) byr4. Using the gTOW data, we modified a mathematical model and successfully reproduced the robustness of the S. pombe cell cycle with the model.
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Project description:The target of Rapamycin complex1 (TORC1) senses and integrates several environmental signals, including amino acid (AA) availability, to regulate cell growth. Folliculin (FLCN) is a tumor suppressor (TS) protein in renal cell carcinoma, which paradoxically activates TORC1 in response to AA supplementation. Few tractable systems for modeling FLCN as a TS are available. Here, we characterize the FLCN-containing complex in Schizosaccharomyces pombe (called BFC) and show that BFC augments TORC1 repression and activation in response to AA starvation and supplementation, respectively. BFC co-immunoprecipitates V-ATPase, a TORC1 modulator, and regulates its activity in an AA-dependent manner. BFC genetic and proteomic networks identify the conserved peptide transmembrane transporter Ptr2 and the phosphoribosylformylglycinamidine synthase Ade3 as new AA-dependent regulators of TORC1. Overall, these data ascribe an additional repressive function to Folliculin in TORC1 regulation and reveal S. pombe as an excellent system for modeling the AA-dependent, FLCN-mediated repression of TORC1 in eukaryotes.
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Project description:Target of Rapamycin Complex 1 (TORC1) signaling promotes growth and aging. Inhibition of TORC1 leads to reduced protein translation, which promotes longevity. TORC1-dependent post-transcriptional regulation of protein translation has been well studied, while analogous transcriptional regulation is less understood. Here we screen fission yeast mutants for resistance to Torin1, which inhibits TORC1 and cell growth. Cells lacking the GATA factor Gaf1 (gaf1Δ) grow normally even in high doses of Torin1. The gaf1Δ mutation shortens the chronological lifespan of non-dividing cells and diminishes Torin1-mediated longevity. Expression profiling and genome-wide binding experiments show that upon TORC1 inhibition, Gaf1 directly upregulates genes for small-molecule metabolic pathways and indirectly represses genes for protein translation. Surprisingly, Gaf1 binds to and downregulates the tRNA genes, so it also functions as a transcription factor for RNA polymerase III. Thus, Gaf1 controls the transcription of both protein-coding and tRNA genes to inhibit translation and growth downstream of TORC1.
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