Project description:Rheb, a ras-like small GTPase conserved from human to yeast, controls Tor kinase and plays a central role in regulation of cell growth depending on extracellular conditions. Fission yeast Rheb regulates amino acid uptake as well as response to nitrogen starvation. In this study we generated two mutants of Rheb, rhb1-DA4 and rhb1-DA8, and characterized them genetically. 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, induction of a 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 Rheb in fission may have two downstream elements, Tor kinase for regulation of the response to nitrogen starvation and the other element for regulation of amino acid uptake.
Project description:Rheb, a ras-like small GTPase conserved from human to yeast, controls Tor kinase and plays a central role in regulation of cell growth depending on extracellular conditions. Fission yeast Rheb regulates amino acid uptake as well as response to nitrogen starvation. In this study we generated two mutants of Rheb, rhb1-DA4 and rhb1-DA8, and characterized them genetically. 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, induction of a 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 Rheb in fission may have two downstream elements, Tor kinase for regulation of the response to nitrogen starvation and the other element for regulation of amino acid uptake. Gene expression profile under nitrogen starvation in fission yeast. Type of experiment: Comparing between vegetatively-growing control cells and cells 3 hrs after nitrogen starvation. Experimental factor: Gene expression profile after nitrogen starvation in the rhb1-D4 or rhb1-D8 cells. Quality control steps taken: All experiments were repeated more than twice except for the tsc2D which was previously reported. Keywards: Nitrogen starvation, rhb1-D4, rhb1-D8
Project description:Cells must appropriately sense and integrate multiple metabolic resources to commit to proliferation. Here, we report that cells regulate nitrogen (amino acid) and carbon metabolic homeostasis through tRNA U34-thiolation. Despite amino acid sufficiency, tRNA-thiolation deficient cells appear amino acid starved. In these cells, carbon flux towards nucleotide synthesis decreases, and trehalose synthesis increases, resulting in metabolic a starvation-signature. Thiolation mutants have only minor translation defects. However, these cells exhibit strongly decreased expression of phosphate homeostasis genes, mimicking a phosphate-limited state. Reduced phosphate enforces a metabolic switch, where glucose-6-phosphate is routed towards storage carbohydrates. Notably, trehalose synthesis, which releases phosphate and thereby restores phosphate availability, is central to this metabolic rewiring. Thus, cells use thiolated tRNAs to perceive amino acid sufficiency, and balance amino acid and carbon metabolic flux to maintain metabolic homeostasis, by controlling phosphate availability. These results further biochemical explain how phosphate availability determines a switch to a ‘starvation-state’.
Project description:Two nutrient sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), control yeast growth and metabolism in response to changes in nutrient availability. Starvation for amino acids activates the GAAC pathway, involving Gcn2p phosphorylation of eIF2 and preferential translation of GCN4, a transcription activator of genes involved in amino acid metabolism. TOR senses nitrogen availability and regulates gene expression through transcription factors, such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome in response to amino acid starvation and rapamycin treatment. Of the ~2500 genes whose expression was changed by 2-fold or greater, Gcn4p and Gln3p were required for 542 and 657 genes, respectively. While Gcn4p activates a common core of 57 genes in response to amino acid starvation or rapamycin treatment, the different stress arrangements allow for variations in Gcn4p-directed transcription. With few exceptions, genes requiring Gcn2p eIF2 kinase for induced expression also required Gcn4p, emphasizing the role of Gcn2p as an upstream activator of Gcn4p-directed transcription. There is also significant coordination between the GAAC and TOR pathways, with Gcn4p being required for activation of more genes during rapamycin treatment than Gln3p. Importantly, TOR regulates the GAAC-directed transcription of genes required for assimilation of nitrogen sources, such as γ-amino-butyric acid. Therefore, yeast has integrated gene expression responses to amino acid abundance and nitrogen source quality through the control of Gcn2p phosphorylation of eIF2 and GCN4 translation. Keywords: gene expression In this study, we carried out microarray analyses in a collection of yeast strains deleted for GCN2, GCN4, and GLN3, individually or in combinations, to explore the importance of the TOR and GAAC pathways in directing the transcriptome in response to amino acid starvation and rapamycin treatment.