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

Zaborske JM, Wu X, Wek RC, Pan T - BMC Biochem. (2010)

Bottom Line: 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 effect on growth during loss of these amino acids.By removing Phe and Tyr from the media in addition to Trp, regular growth was restored and tRNATrp charging no longer decreased.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.

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 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.

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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 tRNAArg 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 tRNAArg isoacceptors in the absence and presence of ornithine.
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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 tRNAArg 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 tRNAArg isoacceptors in the absence and presence of ornithine.

Mentions: In order to better understand why Arg depletion is deleterious to cells lacking GCN2, we focused on cellular utilization of arginine. Many genes in the arginine biosynthesis are transcriptionally regulated by GCN4. Arg is not only important for protein synthesis but is also a necessary precursor for the synthesis of other metabolites. For example, as a part of the urea cycle, Arg is the immediate precursor to ornithine, which is necessary to make citrulline and polyamines (Fig. 5A, [22]). To address the importance of these metabolic relationships in growth phenotype of the gcn2Δ cells in Arg-deficient medium, we depleted arginine from the SC media, but supplemented this medium with ornithine or citrulline (Fig. 5B). Growth was partially restored upon ornithine supplementation but not with citrulline. The addition of ornithine also partially alleviated the tRNAArg charging defect as compared to the absence of ornithine (Fig. 5C, D). Charging of all tRNAArg isoacceptors was essentially unchanged in the gcn2Δ cells after 15 minutes of Arg depletion, and showed a ~2-fold decrease only after 60 minutes, whereas the charging level of tRNAArg was reduced by 5-10-fold in the absence of ornithine.


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

Zaborske JM, Wu X, Wek RC, Pan T - BMC Biochem. (2010)

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 tRNAArg 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 tRNAArg isoacceptors in the absence and presence of ornithine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921344&req=5

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 tRNAArg 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 tRNAArg isoacceptors in the absence and presence of ornithine.
Mentions: In order to better understand why Arg depletion is deleterious to cells lacking GCN2, we focused on cellular utilization of arginine. Many genes in the arginine biosynthesis are transcriptionally regulated by GCN4. Arg is not only important for protein synthesis but is also a necessary precursor for the synthesis of other metabolites. For example, as a part of the urea cycle, Arg is the immediate precursor to ornithine, which is necessary to make citrulline and polyamines (Fig. 5A, [22]). To address the importance of these metabolic relationships in growth phenotype of the gcn2Δ cells in Arg-deficient medium, we depleted arginine from the SC media, but supplemented this medium with ornithine or citrulline (Fig. 5B). Growth was partially restored upon ornithine supplementation but not with citrulline. The addition of ornithine also partially alleviated the tRNAArg charging defect as compared to the absence of ornithine (Fig. 5C, D). Charging of all tRNAArg isoacceptors was essentially unchanged in the gcn2Δ cells after 15 minutes of Arg depletion, and showed a ~2-fold decrease only after 60 minutes, whereas the charging level of tRNAArg was reduced by 5-10-fold in the absence of ornithine.

Bottom Line: 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 effect on growth during loss of these amino acids.By removing Phe and Tyr from the media in addition to Trp, regular growth was restored and tRNATrp charging no longer decreased.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.

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 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.

Show MeSH
Related in: MedlinePlus