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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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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.
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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.

Mentions: Yeast can synthesize each of the twenty amino acids. To screen for conditions in which the GCN2 pathway is required for prototrophic yeast cells to recover from amino acid depletion, we systematically depleted one amino acid from the medium for each of the 20 amino acids. Yeast cells were first grown to saturation in SC medium supplemented with all 20 amino acids. Cells were then pelleted and resuspended to A600 ~ 0.1 in the same medium containing all 20 or just 19 amino acids-absent a selected depleted amino acid. Cell growth was monitored in microplates for 24 h (Fig. 1A). To determine the requirement for GCN2 during the nutrient shift, growth of a prototrophic strain containing an intact GAAC pathway was compared to an isogenic gcn2Δ strain. The wild-type GCN2 strain showed no significant growth differences during these drop-out media conditions. By comparison, the gcn2Δ strain showed no growth phenotype upon depletion for each of 18 amino acids; however, depletion of tryptophan or arginine showed a strong growth defect. To address whether depletion for Trp or Arg specifically blocked growth during the transition into early-log growth, we depleted these two amino acids in flasks after cells were grown to the mid-logarithmic phase (Fig. 1B). The same growth defect was observed, indicating that GCN2 is required in response to depletion for either Trp or Arg in SC medium. Using a GCN4-lacZ reporter, we show that the Gcn2p dependent translational regulation of the GCN4 mRNA is indeed drastically reduced in the gcn2Δ strain before and after depletion of Arg (Fig. 1C, [14]).


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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.
Mentions: Yeast can synthesize each of the twenty amino acids. To screen for conditions in which the GCN2 pathway is required for prototrophic yeast cells to recover from amino acid depletion, we systematically depleted one amino acid from the medium for each of the 20 amino acids. Yeast cells were first grown to saturation in SC medium supplemented with all 20 amino acids. Cells were then pelleted and resuspended to A600 ~ 0.1 in the same medium containing all 20 or just 19 amino acids-absent a selected depleted amino acid. Cell growth was monitored in microplates for 24 h (Fig. 1A). To determine the requirement for GCN2 during the nutrient shift, growth of a prototrophic strain containing an intact GAAC pathway was compared to an isogenic gcn2Δ strain. The wild-type GCN2 strain showed no significant growth differences during these drop-out media conditions. By comparison, the gcn2Δ strain showed no growth phenotype upon depletion for each of 18 amino acids; however, depletion of tryptophan or arginine showed a strong growth defect. To address whether depletion for Trp or Arg specifically blocked growth during the transition into early-log growth, we depleted these two amino acids in flasks after cells were grown to the mid-logarithmic phase (Fig. 1B). The same growth defect was observed, indicating that GCN2 is required in response to depletion for either Trp or Arg in SC medium. Using a GCN4-lacZ reporter, we show that the Gcn2p dependent translational regulation of the GCN4 mRNA is indeed drastically reduced in the gcn2Δ strain before and after depletion of Arg (Fig. 1C, [14]).

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