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

Abstract

BackgroundWhen 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 gcn2Δ 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.ResultsWhile the wild-type strain had no observable phenotype upon depletion for any amino acid, the gcn2Δ 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 gcn2Δ 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 gcn2Δ 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.ConclusionUnder 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.

Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state

“…Even the one component involved in the canonical starvation response that is conserved, the GCN2 ortholog PfeIK1, is seemingly dispensable, because its absence does not compromise the viability of parasites under isoleucine-limiting conditions, despite a well-documented ability to phosphorylate PfeIF2α during starvation. In prototrophic yeast, lack of GCN2 does not affect logarithmic growth in most single amino acid-dropout medium conditions because of compensatory crosstalk between other amino acid-regulatory pathways that function independently of GCN2-mediated signaling (42). However, there is no evidence to suggest that such metabolic complexity exists in P. falciparum, especially considering that the parasite is deficient in amino acid biosynthesis and is absolutely dependent on exogenous isoleucine.…”

Protein and amino acid restriction, aging and disease: from yeast to humans

“…The availability of strains lacking or overexpressing most of the genes in the nutrient signaling pathways combined with the use of biochemically defined media has allowed researchers to evaluate the impact of specific AAs on the pro-aging signaling described above and their subsequent role on macromolecular damage and lifespan [12–14]. Both glucose and AA restriction are sufficient to extend the yeast lifespan [13, 14].…”

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…As indicated in A, a variety of stimuli have been shown to result in increased levels of Gcn4 (Hinnebusch and Natarajan 2002). Some of these responses impinge directly on Gcn2 (Cherkasova et al 2010;Zaborske et al 2009Zaborske et al , 2010 and some function independently, apparently in parallel. Notably, Gcn4 stability is increased under amino acid starvation (Kornitzer et al 1994;Shemer et al 2002;Bomeke et al 2006;Aviram et al 2008;Streckfuss-Bomeke et al 2009).…”