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TOR inhibition drives accumulation of amino acids through transcriptional activation in algae

ABSTRACT: Cellular homeostasis is maintained by the balance between energy production and breakdown and is fundamental to all forms of life. The conserved, ancient target of rapamycin (TOR) kinase is a central metabolic regulator in eukaryotes that integrates carbon and nitrogen to maintain homeostasis and promote growth and development through protein synthesis. While TOR regulatory mechanisms of amino acid accumulation are well known in yeast and mammals, they remain unknown in photosynthetic organisms. Here, we developed the unicellular green alga Chromochloris zofingiensis as a simpler model system for understanding TOR function. Multiomics experiments showed that TOR inhibition leads to an increase in amino acid levels independent of hexokinase-mediated glucose signaling. We observed upregulation of selective amino acid biosynthesis pathways at the transcript and protein levels as potential mechanisms driving the increase in amino acids. Transcriptomics and proteomics experiments identified a basic helix-loop-helix (bHLH) transcription factor with rapid upregulation during TOR inhibition. DAP-seq analysis demonstrated that bHLH can bind directly to the promoters of amino acid biosynthesis genes, potentially regulating their transcription in response to TOR inhibition. We found high conservation of the bHLH-binding motif in the genomes of other green algae and plants, suggesting a conserved regulatory mechanism for amino acid biosynthesis across Viridiplantae. Phosphoproteomics experiments also revealed novel conserved targets that are not currently recognized as part of the TOR pathway. Altogether, our findings elucidate the transcriptional regulation of amino acid metabolism and explain how TOR regulates nitrogen metabolism to support growth and development in photosynthetic organisms.

ORGANISM(S): Chromochloris zofingiensis

PROVIDER: GSE309591 | GEO | 2025/10/03

REPOSITORIES: GEO

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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 Î³-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.

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TOR inhibition drives accumulation of amino acids through transcriptional activation in algae

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