Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells

GCN2 stimulates GCN4 translation in amino acid-starved cells by phosphorylating the alpha-subunit of translation initiation factor 2. GCN2 function in vivo requires the GCN1/GCN20 complex, which binds to the N-terminal domain of GCN2. A C-terminal segment of GCN1 (residues 2052-2428) was found to be necessary and sufficient for binding GCN2 in vivo and in vitro. Overexpression of this fragment in wild-type cells impaired association of GCN2 with native GCN1 and had a dominant Gcn(-) phenotype, dependent on Arg2259 in the GCN1 fragment. Substitution of Arg2259 with Ala in full-length GCN1 abolished complex formation with native GCN2 and destroyed GCN1 regulatory function. Consistently, the Gcn(-) phenotype of gcn1-R2259A, but not that of gcn1Delta, was suppressed by overexpressing GCN2. These findings prove that GCN2 binding to the C-terminal domain of GCN1, dependent on Arg2259, is required for high level GCN2 function in vivo. GCN1 expression conferred sensitivity to paromomycin in a manner dependent on its ribosome binding domain, supporting the idea that GCN1 binds near the ribosomal acceptor site to promote GCN2 activation by uncharged tRNA.     

Association of GCN1-GCN20 regulatory complex with the N-terminus of eIF2alpha kinase GCN2 is required for GCN2 activation

Stimulation of GCN4 mRNA translation due to phosphorylation of the alpha-subunit of initiation factor 2 (eIF2) by its specific kinase, GCN2, requires binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like domain in GCN2. GCN2 function in vivo also requires GCN1 and GCN20, but it was unknown whether these latter proteins act directly to promote the stimulation of GCN2 by uncharged tRNA. We found that the GCN1-GCN20 complex physically interacts with GCN2, binding to the N-terminus of the protein. Overexpression of N-terminal GCN2 segments had a dominant-negative phenotype that correlated with their ability to interact with GCN1-GCN20 and impede association between GCN1 and native GCN2. Consistently, this Gcn(-) phenotype was suppressed by overexpressing GCN2, GCN1-GCN20 or tRNA(His). The requirement for GCN1 was also reduced by overexpressing tRNA(His) in a gcn1Delta strain. We conclude that binding of GCN1-GCN20 to GCN2 is required for its activation by uncharged tRNA. The homologous N-terminus of Drosophila GCN2 interacted with yeast GCN1-GCN20 and had a dominant Gcn(-) phenotype, suggesting evolutionary conservation of this interaction.     

The GCN2 eIF2alpha kinase is required for adaptation to amino acid deprivation in mice

The GCN2 eIF2alpha kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2(-/-) knockout strain of mice. Gcn2(-/-) mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2(-/-) mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2(-/-) mice failed to show the normal induction of eIF2alpha phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2alpha and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2(-/-) mice.     

Polyribosome binding by GCN1 is required for full activation of eukaryotic translation initiation factor 2{alpha} kinase GCN2 during amino acid starvation

The protein kinase GCN2 mediates translational control of gene expression in amino acid-starved cells by phosphorylating eukaryotic translation initiation factor 2alpha. In Saccharomyces cerevisiae, activation of GCN2 by uncharged tRNAs in starved cells requires its direct interaction with both the GCN1.GCN20 regulatory complex and ribosomes. GCN1 also interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We describe point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1.GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN1.GCN20 and a stronger decrease in eukaryotic translation initiation factor 2alpha phosphorylation than did mutating in one segment alone. These findings provide strong evidence that ribosome binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain, related in sequence to translation elongation factor 3 (eEF3), decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells.     

GCN20, a novel ATP binding cassette protein, and GCN1 reside in a complex that mediates activation of the eIF-2 alpha kinase GCN2 in amino acid-starved cells.

GCN2 is a protein kinase that phosphorylates the alpha-subunit of translation initiation factor 2 (eIF-2) and thereby stimulates translation of GCN4 mRNA in amino acid-starved cells. We isolated a null mutation in a previously unidentified gene, GCN20, that suppresses the growth-inhibitory effect of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. The deletion of GCN20 in otherwise wild-type strains impairs derepression of GCN4 translation and reduces the level of eIF-2 alpha phosphorylation in vivo, showing that GCN20 is a positive effector of GCN2 kinase function. In accordance with this conclusion, GCN20 was co-immunoprecipitated from cell extracts with GCN1, another factor required to activate GCN2, and the two proteins interacted in the yeast two-hybrid system. We conclude that GCN1 and GCN20 are components of a protein complex that couples the kinase activity of GCN2 to the availability of amino acids. GCN20 is a member of the ATP binding cassette (ABC) family of proteins and is closely related to ABC proteins identified in Caenorhabditis elegans, rice and humans, suggesting that the function of GCN20 may be conserved among diverse eukaryotic organisms.     

The GCN2 kinase biases feeding behavior to maintain amino acid homeostasis in omnivores

To insure an adequate supply of nutrients, omnivores choose among available food sources. This process is exemplified by the well-characterized innate aversion of omnivores to otherwise nutritious foods of imbalanced amino acid content. We report that brain-specific inactivation of GCN2, a ubiquitously expressed protein kinase that phosphorylates translation initiation factor 2 alpha (eIF2alpha) in response to intracellular amino acid deficiency, impairs this aversive response. GCN2 inactivation also diminishes phosphorylated eIF2alpha levels in the mouse anterior piriform cortex following consumption of an imbalanced meal. An ancient intracellular signal transduction pathway responsive to amino acid deficiency thus affects feeding behavior by activating a neuronal circuit that biases consumption against imbalanced food sources.     

Microbial metabolism of 2-methyl amino acids

Eighty-nine microorganisms were isolated that were able to use 2-methyl amino acids and related compounds as the sole source of nitrogen. All of these cultures produced low levels of ammonia in culture supernatant solutions None was capable of fixing nitrogen gas. Whole-cell and cell-free-extract experiments showed that ammonia was not released directly from the 2-methyl amino acids. All of these strains except those isolated with 2-methylserine as a nitrogen source appeared to metabolize 2-methyl amino compounds by a single enzymatic reaction involving simultaneous decarboxylation and transamination. Pyruvate served as an acceptor for the transamination with the concomitant formation of alanine. The strains utilizing 2-methylserine produced a specific 2-methylserine transhydroxymethylase.     

Autophagy contributes to sulfonylurea herbicide tolerance via GCN2-independent regulation of amino acid homeostasis

Sulfonylurea (SU) herbicides inhibit branched-chain amino acid (BCAA) biosynthesis by targeting acetolactate synthase. Plants have evolved target-site resistance and metabolic tolerance to SU herbicides; the GCN2 (general control non-repressible 2) pathway is also involved in SU tolerance. Here, we report a novel SU tolerance mechanism, autophagy, which we call ‘homeostatic tolerance,’ is involved in amino acid signaling in Arabidopsis. The activation and reversion of autophagy and GCN2 by the SU herbicide tribenuron-methyl (TM) and exogenous BCAA, respectively, confirmed that TM-induced BCAA starvation is responsible for the activation of autophagy and GCN2. Genetic and biochemical analyses revealed a lower proportion of free BCAA and more sensitive phenotypes in atg5, atg7, and gcn2 single mutants than in wild-type seedlings after TM treatment; the lowest proportion of free BCAA and the most sensitive phenotypes were found in atg5 gcn2 and atg7 gcn2 double mutants. Immunoblotting and microscopy revealed that TM-induced activation of autophagy and GCN2 signaling do not depend on the presence of each other, and these 2 pathways may serve as mutually compensatory mechanisms against TM. TM inhibited the TOR (target of rapamycin), and activated autophagy in an estradiol-induced TOR RNAi line, suggesting that TM-induced BCAA starvation activates autophagy, probably via TOR inactivation. Autophagy and GCN2 were also activated, and independently contributed to TM tolerance in plants conferring metabolic tolerance. Together, these data suggest that autophagy is a proteolytic process for amino acid recycling and contributes to GCN2-independent SU tolerance, probably by its ability to replenish fresh BCAA.     

The GCN2 eIF2alpha kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid

Metabolic adaptation is required to cope with episodes of protein deprivation and malnutrition. GCN2 eIF2alpha kinase, a sensor of amino acid deficiency, plays a key role in yeast and mammals in modulating amino acid metabolism as part of adaptation to nutrient deprivation. The role of GCN2 in adaptation to long-term amino acid deprivation in mammals, however, is poorly understood. We found that expression of lipogenic genes and the activity of fatty acid synthase (FAS) in the liver are repressed and lipid stores in adipose tissue are mobilized in wild-type mice upon leucine deprivation. In contrast, GCN2-deficient mice developed liver steatosis and exhibited reduced lipid mobilization. Liver steatosis in Gcn2(-/-) mice was found to be caused by unrepressed expression of lipogenic genes, including Srebp-1c and Fas. Thus, our study identifies a novel function of GCN2 in regulating lipid metabolism during leucine deprivation in addition to regulating amino acid metabolism.     

Evaluation of the immunocytochemical method for amino acids

Free amino acids can be coupled to proteins by glutaraldehyde. Rabbits immunised with a bovine serum albumin-glutaraldehyde-amino acid conjugate form antibodies that recognise similar conjugates with brain proteins in glutaraldehyde-fixed tissue. Antisera raised against conjugated GABA (gamma-aminobutyrate), glutamate, aspartate, taurine, glutamine, or glycine were tested against a variety of small molecular compounds that had been fixed by glutaraldehyde to brain protein and immobilised on cellulose ester filters for processing together with the brain sections. This system permitted closely similar conditions for testing and immunocytochemistry. After removing antibodies against the carrier used for immunisation and against cross reacting amino acid conjugates the antisera showed a high specificity. The specific nature of the antisera was corroborated by solid phase adsorption to the homologous antigens and by inhibition experiments with free amino acids and amino acid-glutaraldehyde fixation complexes. After transection of the striatonigral pathway the ipsilateral substantia nigra was almost depleted of GABA-like immunoreactivity; this observation lends additional support to the selectivity of the GABA antiserum. A semiquantitative relation was established between the concentration of amino acid before fixation in a model system and the subsequent intensity of immunostaining. Similar model experiments suggested that the conjugation of an amino acid to brain protein with glutaraldehyde, and the immunoreactivity of the conjugates, may be significantly inhibited in the presence of high concentrations of other amino compounds.     

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-²H₃]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.