Aminoacyl-tRNA quality control is required for efficient activation of the TOR pathway regulator Gln3p

Abstract

The aminoacylation status of the cellular tRNA pool regulates both general amino acid control (GAAC) and target of rapamycin (TOR) stress response pathways in yeast. Consequently, fidelity of translation at the level of aminoacyl-tRNA synthesis plays a central role in determining accuracy and sensitivity of stress responses. To investigate effects of translational quality control (QC) on cell physiology under stress conditions, phenotypic microarray analyses were used to identify changes in QC deficient cells. Nitrogen source growth assays showed QC deficient yeast grew differently compared to WT. The QC deficient strain was more tolerant to caffeine treatment than wild type through altered interactions with the TOR and GAAC pathways. Increased caffeine tolerance of the QC deficient strain was consistent with the observation that the activity of Gln3p, a transcription factor controlled by the TOR pathway, is decreased in the QC deficient strain compared to WT. GCN4 translation, which is typically repressed in the absence of nutritional stress, was enhanced in the QC deficient strain through TOR inhibition. QC did not impact cell cycle regulation; however, the chronological lifespan of QC deficient yeast strains decreased compared to wild type, likely due to translational errors and alteration of the TOR-associated regulon. These findings support the idea that changes in translational fidelity provide a mechanism of cellular adaptation by modulating TOR activity. This, in turn, supports a central role for aminoacyl-tRNA synthesis QC in the integrated stress response by maintaining the proper aa-tRNA pools necessary to coordinate the GAAC and TOR.

Keywords: Aminoacyl-tRNA; GAAC; TOR; nutritional stress; tRNA editing.

Figure 1.

Phenotypic analysis of WT and PheRS QC deficient yeast. (A) WT and QC deficient strains of yeast were used for phenotypic screening by Biolog Phenotypic Microarray (PM). PM conditions which facilitated growth different from wild type were mapped onto a gene interaction network (expanded shell by 10 connections) using stringDB. Genes linked to growth conditions conferring growth advantage are indicated by blue and red colored nodes for the WT (FRS1) and proofreading deficient (frs1-1) strains, respectively. See supplement for gene table. Green nodes indicate gene association with both WT and frs1-1 depending on growth condition. (B) PM data for growth in response to caffeine (included within the chemical sensitivity PM plate) was extracted from cumulative data set and plotted as a function of time. (C) Caffeine growth phenotypes observed from the PM were validated through independent growth experiments. WT and QC deficient yeast were grown in synthetic complete (SC) medium with varying amounts of caffeine supplementation. Growth was followed by monitoring the OD₆₀₀ over time. Results are representative of three independent replicates. Error bars colored blue for FRS1 and red for frs1-1 and indicate 1 S.D.

Figure 2.

Growth of WT and QC deficient strains with different nitrogen sources. WT and PheRS QC deficient strains of yeast were inoculated to an OD₆₀₀ of 0.01 and grown for 24 or 48 hours (A-C indicate maximum OD₆₀₀ values after incubation) on synthetic defined minimal media (SDMM) with varying sources of nitrogen. (A) Multiplexed growth assays were conducted in 96 well plates with yeast nitrogen base containing ammonium sulfate (YNB-NH₄) as the primary nitrogen source and supplemented with individual amino acids (2 mM). (B) Multiplexed growth assays were conducted in 96 well plates with yeast nitrogen base containing GABA (YNB-GABA) as the primary nitrogen source and supplemented with individual amino acids (2mM). (C) Multiplexed growth assays were conducted in 96 well plates with yeast nitrogen base supplemented with individual amino acids (YNB-2mM AA) as the primary nitrogen source. (D-F) WT and QC deficient cells were grown in YPD and serial dilutions (1 – 1 × 10⁻⁴) were spotted on solid SDMM with varying sources of nitrogen and grown for 24–48 hours. Growth assays were conducted in triplicate. Error bars are representative of 1 S.D.

Figure 3.

Regulation of Gln3p targets in response to diverse amino acid nitrogen sources. (A-B) WT (FRS1) and QC deficient (frs1-1) cells harboring plasmid pGATA:LacZ or pUGA1:LacZ were grown in the indicated medium. GABA was used to induce Gln3p expression. Significance was assessed using paired sample t-test where “*” represents p < 0.05, “**” represents p < 0.005 (C) WT and QC deficient cells harboring plasmid pGATA:LacZ were grown in synthetic defined minimal medium (SDMM) with ammonium sulfate as the primary nitrogen source and supplemented individually with each amino acid (1 mM). (D) WT and QC deficient cells harboring plasmid pGATA:LacZ were grown in synthetic defined minimal medium (SDMM) with each amino acid (1 mM) as the primary nitrogen source. Cells were analyzed for betagalactosidase activity using CPRG chromogenic substrate while monitoring the OD580 over time. Rate was determined in triplicate and normalized to OD600 values. Error bars reflect S.D. determined from at least three independent experiments.

Figure 4.

Translation of GCN4 during growth on ammonium. WT and QC deficient strains harboring a GCN4-GFP translation reporter plasmid (pGCN4Rep) were grown in either (A) synthetic complete (SC) or (B) synthetic defined minimal medium (SDMM) with ammonium as the primary nitrogen source (SC YNB-NH₄ and YNB-NH₄, respectively). GCN4 translation was followed by measuring GFP fluorescence over time and normalized to cellular concentration using OD₆₀₀ values. Results are the average of three independent replicates. Error bars represent 1 S.D.

Figure 5.

Impact of QC on TOR-dependent cellular physiology. (A) WT (FRS1) and QC deficient (frs1-1) cells were grown in minimal medium (SDMM) or minimal medium supplemented with 1.2 mM Tyrosine (SDMM + Tyr) to an OD600 of 0.8-1.0. The DNA content of cells stained with SYBR Green I was analyzed by flow cytometry. Univariate cell cycle analysis was conducted using a modified Watson Pragmatic algorithm (FlowJo). Coefficient of variation (CV) was determined using width at ½ G1 peak height from at least three independent experiments. (B) WT (FRS1) and QC deficient (frs1-1) cells harboring plasmid uORF-CLN3:LacZ were grown in the indicated medium. 3-aminotriazole (3-AT) was used to induce amino acid starvation. Cells were analyzed for betagalactosidase activity using CPRG chromogenic substrate while monitoring the OD₅₈₀ over time. Rate was determined in triplicate and normalized to OD₆₀₀ values. (C) WT (FRS1) and QC deficient (frs1-1) cells were grown in 50 mL of synthetic complete medium with ammonium as the primary nitrogen source. Percent survival was normalized for each culture to CFU/mL at day 5. Error bars reflect 1 S.D. determined from at least three independent experiments.

Figure 6.

Activation and cross-regulation of the GAAC and TOR pathways. Both the GAAC and TOR pathways respond to changes in amino acid availability. The TORC1 complex monitors the availability of nitrogenous compounds and in response to nitrogen limitation, mounts a stress response by freeing Sit4p from its complex. Sit4p then dephosphorylates Gln3p, facilitating dissociation from Ure2p and allowing for translocation of Gln3p to the nucleus to activate NCR gene transcription. Sit4p also dephosphorylates S577 on Gcn2p. Gcn2p is subsequently able to efficiently bind deacylated tRNA during periods of amino acid starvation. Activated Gcn2p phosphorylates eIF2α, decreasing levels of translation while increasing translation of Gcn4p. Gcn4p enters the nucleus and facilitates transcription of amino acid biosynthesis genes. Gcn4p and Gln3p co-regulate a subset of stress response genes which are common to both stress response pathways. The intersection of the aa-tRNA pool with the TOR pathway (blue arrows) occurs through Gcn2p, yet the exact mechanism of the interaction is unclear.