Selective control of amino acid metabolism by the GCN2 eIF2 kinase pathway in Saccharomyces cerevisiae

Abstract

Background: When eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. Activated Gcn2p up-regulates the general amino acid control pathway through phosphorylation of the translational initiation factor eIF2. In Saccharomyces cerevisiae, Gcn2p is the only kinase that phosphorylates eIF2 to regulate translation through this mechanism. We addressed changes in yeast growth and tRNA aminoacylation, or charging, during amino acid depletion in the presence and absence of GCN2. tRNA charging was measured using a microarray technique which simultaneously measures all cytosolic tRNAs. A fully prototrophic strain, and its isogenic gcn2 Delta counterpart, were used to study depletion for each of the 20 amino acids, with a focus on Trp, Arg, His and Leu, which are metabolically distinct and together provide a good overview on amino acid metabolism.

Results: While the wild-type strain had no observable phenotype upon depletion for any amino acid, the gcn2 Delta strain showed slow growth in media devoid of only Trp or Arg. Consistent with the growth phenotypes, profiles of genome-wide tRNA charging revealed significant decrease in cognate tRNA charging only in the gcn2 Delta strain upon depletion for Trp or Arg. In contrast, there was no change in tRNA charging during His and Leu depletion in either the wild-type or gcn2 Delta strains, consistent with the null effect on growth during loss of these amino acids. We determined that the growth phenotype of Trp depletion is derived from feedback inhibition of aromatic amino acid biosynthesis. By removing Phe and Tyr from the media in addition to Trp, regular growth was restored and tRNATrp charging no longer decreased. The growth phenotype of Arg depletion is derived from unbalanced nitrogen metabolism. By supplementing ornithine upon Arg depletion, both growth and tRNAArg charging were partially restored.

Conclusion: Under mild stress conditions the basal activity of Gcn2p is sufficient to allow for proper adaptation to amino acid depletion. This study highlights the importance of the GCN2 eIF2 kinase pathway for maintaining metabolic homeostasis, contributing to appropriate tRNA charging and growth adaptation in response to culture conditions deficient for the central amino acids, tryptophan and arginine.

Figure 1

Yeast growth upon single amino acid depletion shows recovery of Trp and Arg depletion dependent on GCN2. (A) Systematic depletion of amino acids from growth media in 96 well plates. Arrows indicate slow growth in Arg and Trp in the gcn2Δ strain. (B) Recovery of growth in flasks after amino acid depletion. Depletion occurred while cells were in stationary or mid log growth. (C) Measurements of the GCN4-lacZ reporter showed that the Gcn2p-dependent translational regulation of the GCN4 mRNA is significantly reduced in the gcn2Δ strain before and after Arg depletion. This result suggests that the level of the GCN4 protein is drastically lower in the gcn2Δ strain as expected. Blue arrows indicate time points at which tRNA charging profiles were measured.

Figure 2

Changes in tRNA charging profile correlate with growth phenotype. Cognate tRNAs for the amino acid depleted are indicated by red arrows. Non-cognate tRNAs whose charging also changes significantly are indicated by black arrows. (A) Trp depletion. Top: array spot for the Trp-tRNA probe; Middle: heatmap of the wild-type and isogenic gcn2Δ strains; Bottom: histogram of the relative charging level of tRNA^Trp before and after Trp depletion. (B) Comparison of the changes in tRNA charging (top) and tRNA abundance (bottom) before and after 60 min of Trp depletion of the gcn2Δ strain. Both tRNA^Trp and initiator tRNA_i^Met exhibited a large decrease in charging, but not in abundance. (C) Arg depletion. Top: array spot for the Arg-tRNA (anticodon ICG) probe; Middle: heatmap of the wild-type and isogenic gcn2Δ strains; Bottom: histogram of the relative charging level before and after 60 min of Arg depletion for the gcn2Δ strain. (D) Leu and His depletion showed no significant decrease in tRNA charging.

Figure 3

Partial Trp and Arg depletion of the gcn2Δ strain shows graded sensitivity of Trp and Arg availability to Gcn2p. The complete media contain 182 μg/ml Trp and 182 μg/ml Arg. (A) Trp. Growth at varying Trp concentrations (left), the tRNA^Trp charging levels 60 min after depletion (middle), time course of tRNA^Trp charging at various Trp concentration in the media (right). (B) Arg. Growth at varying Arg concentrations (left), the averaged charging levels of all four tRNA^Arg isoacceptors 60 min after depletion (middle), time course of the averaged charging levels of all four tRNA^Arg isoacceptors at various Arg concentration in the media (right).

Figure 4

GCN2-dependent effect upon Trp depletion is linked to aromatic amino acid biosynthesis. (A) Simplified schematic of aromatic amino acid anabolism indicating where excess amino acids inhibit biosynthesis according to reference [11]. The crucial step is the feedback inhibition of the first step (red lines). (B) Growth curve of gcn2Δ with depletion of Trp, Tyr and Phe showing growth recovery in complete media versus Trp depleted media. (C) Heatmap of the wild type and gcn2Δ strains depleted of all three aromatic amino acids. Red arrow indicates tRNA^Trp and black arrows indicate tRNA^Phe and tRNA^Tyr. (D) Histogram of the relative charging level before and 60 min after triple amino acid depletion for the gcn2Δ strain.

Figure 5

GCN2-dependent effect upon Arg depletion is linked to nitrogen metabolism. (A) Simplified schematic of nitrogen metabolism. Ornithine is not only necessary for completion of the urea cycle but is necessary for polyamine biosynthesis. The mitochondrion compartment is shown as a blue oval. (B) Growth curve of gcn2Δ strain in complete media, lacking arginine, and lacking arginine supplemented with ornithine, citrulline and/or polyamines. (C) Heatmap of the wild type and gcn2Δ strains after depletion of arginine and supplementation with 2 mg/ml ornithine. Arrows indicate tRNA^Arg isoacceptors. (D) Histogram of relative charging level before and after Arg-depletion/Orn supplementation for the gcn2Δ strain. Inset shows the charging levels of the tRNA^Arg isoacceptors in the absence and presence of ornithine.