LC-MS analysis of yeast metabolome extracts under rapamycin treatment and nitrogen source downshift
Ontology highlight
ABSTRACT:
INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS
ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)
SUBMITTER: 
Duncan Holbrook-Smith
PROVIDER: MSV000089934 | MassIVE | Thu Jul 21 06:46:00 BST 2022
REPOSITORIES: MassIVE
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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
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Project description:We are interested in the study of the already reported sensitivity of yeast cells lacking the type 1 protein phosphatase Ptc1 to the drug rapamycin. In order to carry out our studies, we analyzed the changes in the transcription profile that a short-term incubation with rapamycin (200 ng/mL) has in both, wild type and ptc1 cells. It has been shown that inhibition of the TOR pathway by treatment with rapamycin has profound effects on the global transcriptional profile. We confirmed the previously described transcription changes induced by the drug in wild type cells. Under our working conditions rapamycin increased, in wild type cells, at least 2-fold the expression of 667 genes, whereas it decreased the mRNA level of 721 genes (13.8% and 14.9% of genes with measurable expression level, respectively). Gene ontology analysis shows that, as previously documented, many induced genes falls into the NCR, Msn2/Msn4 regulated stress response or Retrograde response categories, whereas genes encoding cytoplasmic, but not mitochondrial, ribosomal proteins and the so called RIBI regulon where largely repressed. Deletion of PTC1 decreases the number of genes induced or repressed at least 2-fold by rapamycin treatment. Remarkably, lack of Ptc1 seems to lead to a general attenuation of changes triggered by rapamycin.
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