Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.

Damaging de novo missense variants in EEF1A2 lead to a developmental and degenerative epileptic-dyskinetic encephalopathy.

Heterozygous de novo variants in the eukaryotic elongation factor EEF1A2 have previously been described in association with intellectual disability and epilepsy but never functionally validated. Here we report 14 new individuals with heterozygous EEF1A2 variants. We functionally validate multiple variants as protein-damaging using heterologous expression and complementation analysis. Our findings allow us to confirm multiple variants as pathogenic and broaden the phenotypic spectrum to include dystonia/choreoathetosis, and in some cases a degenerative course with cerebral and cerebellar atrophy. Pathogenic variants appear to act via a haploinsufficiency mechanism, disrupting both the protein synthesis and integrated stress response functions of EEF1A2. Our studies provide evidence that EEF1A2 is highly intolerant to variation and that de novo pathogenic variants lead to an epileptic-dyskinetic encephalopathy with both neurodevelopmental and neurodegenerative features. Developmental features may be driven by impaired synaptic protein synthesis during early brain development while progressive symptoms may be linked to an impaired ability to handle cytotoxic stressors.

Persistent contamination of Salmonella, Campylobacter, Escherichia coli, and Staphylococcus aureus at a broiler farm in New Zealand.

Intensive poultry production due to public demand raises the risk of contamination, creating potential foodborne hazards to consumers. The prevalence and microbial load of the pathogens Campylobacter, Salmonella, Staphylococcus aureus, and Escherichia coli was determined by standard methods at the farm level. After disinfection, swab samples collected from wall crevices, drinkers, and vents were heavily contaminated, as accumulated organic matter and dust likely protected the pathogens from the disinfectants used. The annex floor also showed high microbial concentrations, suggesting the introduction of pathogens from external environments, highlighting the importance of erecting hygiene barriers at the entrance of the main shed. Therefore, pathogen control measures and proper application of disinfectants are recommended as intervention strategies. Additionally, quantitative polymerase chain reaction (qPCR) was evaluated as a quantification tool. qPCR showed limitations with samples containing low microbial counts because of the low detection limit of the method. Thus, bacterial pre-enrichment of test samples may be necessary to improve the detection of pathogens by qPCR.

Perturbations in actin dynamics reconfigure protein complexes that modulate GCN2 activity and promote an eIF2 response.

Genetic and pharmacological interventions in yeast and mammalian cells have suggested a cross-talk between the actin cytoskeleton and protein synthesis. Regulation of the activity of the translation initiation factor 2 (eIF2) is a paramount mechanism for cells to rapidly adjust the rate of protein synthesis and to trigger reprogramming of gene expression in response to internal and external cues. Here, we show that disruption of F-actin in mammalian cells inhibits translation in a GCN2-dependent manner, correlating with increased levels of uncharged tRNA. GCN2 activation increased phosphorylation of its substrate eIF2α and the induction of the integrated stress response master regulator, ATF4. GCN2 activation by latrunculin-B is dependent on GCN1 and inhibited by IMPACT. Our data suggest that GCN2 occurs in two different complexes, GCN2-eEF1A and GCN2-GCN1. Depolymerization of F-actin shifts GCN2 to favor the complex with GCN1, concomitant with GCN1 being released from its binding to IMPACT, which is sequestered by G-actin. These events might further contribute to GCN2 activation. Our findings indicate that GCN2 is an important sensor of the state of the actin cytoskeleton.

Imprinted and ancient gene: a potential mediator of cancer cell survival during tryptophan deprivation.

BACKGROUND: Depletion of tryptophan and the accumulation of tryptophan metabolites mediated by the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1), trigger immune cells to undergo apoptosis. However, cancer cells in the same microenvironment appear not to be affected. Mechanisms whereby cancer cells resist accelerated tryptophan degradation are not completely understood. We hypothesize that cancer cells co-opt IMPACT (the product of IMPrinted and AnCienT gene), to withstand periods of tryptophan deficiency. METHODS: A range of bioinformatic techniques including correlation and gene set variation analyses was applied to genomic datasets of cancer (The Cancer Genome Atlas) and normal (Genotype Tissue Expression Project) tissues to investigate IMPACT's role in cancer. Survival of IMPACT-overexpressing GL261 glioma cells and their wild type counterparts cultured in low tryptophan media was assessed using fluorescence microscopy and MTT bio-reduction assay. Expression of the Integrated Stress Response proteins was measured using Western blotting. RESULTS: We found IMPACT to be upregulated and frequently amplified in a broad range of clinical cancers relative to their non-malignant tissue counterparts. In a subset of clinical cancers, high IMPACT expression associated with decreased activity of pathways and genes involved in stress response and with increased activity of translational regulation such as the mTOR pathway. Experimental studies using the GL261 glioma line showed that cells engineered to overexpress IMPACT, gained a survival advantage over wild-type lines when cultured under limiting tryptophan concentrations. No significant difference in the expression of proteins in the Integrated Stress Response pathway was detected in tryptophan-deprived GL261 IMPACT-overexpressors compared to that in wild-type cells. IMPACT-overexpressing GL261 cells but not their wild-type counterparts, showed marked enlargement of their nuclei and cytoplasmic area when stressed by tryptophan deprivation. CONCLUSIONS: The bioinformatics data together with our laboratory studies, support the hypothesis that IMPACT mediates a protective mechanism allowing cancer cells to overcome microenvironmental stresses such as tryptophan deficiency.

Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2α.

The common method for liberating proteins from Saccharomyces cerevisiae cells involves mechanical cell disruption using glass beads and buffer containing inhibitors (protease, phosphatase and/or kinase inhibitors), followed by centrifugation to remove cell debris. This procedure requires the use of costly inhibitors and is laborious, in particular when many samples need to be processed. Also, enzymatic reactions can still occur during harvesting and cell breakage. As a result low-abundance and labile proteins may be degraded, and enzymes such as kinases and phosphatases may still modify proteins during and after cell lysis. We believe that our rapid sample preparation method helps overcome the above issues and offers the following advantages: (a) it is cost-effective, as no inhibitors and breaking buffer are needed; (b) cell breakage is fast (about 15 min) since it only involves a few steps; (c) the use of formaldehyde inactivates endogenous proteases prior to cell lysis, dramatically reducing the risk of protein degradation; (d) centrifugation steps only occur prior to cell lysis, circumventing the problem of losing protein complexes, in particular if cells were treated with formaldehyde intended to stabilize and capture large protein complexes; and (e) since formaldehyde has the potential to instantly terminate protein activity, this method also allows the study of enzymes in live cells, i.e. in their true physiological environment, such as the short-term effect of a drug on enzyme activity. Taken together, the rapid sample preparation procedure provides a more accurate snapshot of the cell's protein content at the time of harvesting. Copyright © 2017 John Wiley & Sons, Ltd.

Gcn1 contacts the small ribosomal protein Rps10, which is required for full activation of the protein kinase Gcn2.

In eukaryotes, amino acid deprivation leads to the accumulation of uncharged tRNAs that are detected by Gcn2 (general control non-derepressible 2), which in turn phosphorylates eIF2α (α-subunit of eukaryotic translation initiation factor 2), an essential process for overcoming starvation. In Saccharomyces cerevisiae, sensing amino acid shortages requires that Gcn2 binds directly to its effector protein Gcn1 and both must associate with the ribosome. Our hypothesis is that uncharged tRNAs occur in the ribosomal A-site and that Gcn1 is directly involved in transfer of this starvation signal to Gcn2. In the present paper, we provide evidence that Gcn1 directly contacts the small ribosomal protein S10 (Rps10). Gcn1 residues 1060-1777 showed a yeast two-hybrid (Y2H) interaction with Rps10A. In vitro, Rps10A or Rps10B co-precipitated Gcn1[1060-1777] in an RNA-independent manner. rps10AΔ or rps10BΔ strains showed reduced eIF2α phosphorylation under replete conditions and shortly after onset of starvation, suggesting that Gcn1-mediated Gcn2 activation was impaired. Overexpression of GST-tagged Rps10 reduced growth under amino acid starvation and this was exacerbated by the Gcn1-M7A mutation known to impair Gcn1-ribosome interaction and Gcn2 activity. Under amino acid starvation, eEF3 (eukaryotic translation elongation factor 3) overexpression, known to weaken Gcn1 function on the ribosome, exacerbated the growth defect of rps10AΔ or rps10BΔ strains. Taken together, these data support the idea that Gcn1 contacts ribosome-bound Rps10 to efficiently mediate Gcn2 activation.

Keeping the eIF2 alpha kinase Gcn2 in check.

The protein kinase Gcn2 is present in virtually all eukaryotes and is of increasing interest due to its involvement in a large array of crucial biological processes. Some of these are universally conserved from yeast to humans, such as coping with nutrient starvation and oxidative stress. In mammals, Gcn2 is important for e.g. long-term memory formation, feeding behaviour and immune system regulation. Gcn2 has been also implicated in diseases such as cancer and Alzheimer's disease. Studies on Gcn2 have been conducted most extensively in Saccharomyces cerevisiae, where the mechanism of its activation by amino acid starvation has been revealed in most detail. Uncharged tRNAs stimulate Gcn2 which subsequently phosphorylates its substrate, eIF2α, leading to reduced global protein synthesis and simultaneously to increased translation of specific mRNAs, e.g. those coding for Gcn4 in yeast and ATF4 in mammals. Both proteins are transcription factors that regulate the expression of a myriad of genes, thereby enabling the cell to initiate a survival response to the initial activating cue. Given that Gcn2 participates in many diverse processes, Gcn2 itself must be tightly controlled. Indeed, Gcn2 is regulated by a vast network of proteins and RNAs, the list of which is still growing. Deciphering molecular mechanisms underlying Gcn2 regulation by effectors and inhibitors is fundamental for understanding how the cell keeps Gcn2 in check ensuring normal organismal function, and how Gcn2-associated diseases may develop or may be treated. This review provides a critical evaluation of the current knowledge on mechanisms controlling Gcn2 activation or activity.

Evolutionarily conserved IMPACT impairs various stress responses that require GCN1 for activating the eIF2 kinase GCN2.

In response to a range of environmental stresses, phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2α) represses general protein synthesis coincident with increased translation of specific mRNAs, such as those encoding the transcription activators GCN4 and ATF4. The eIF2α kinase GCN2 is activated by amino acid starvation by a mechanism involving GCN2 binding to an activator protein GCN1, along with association with uncharged tRNA that accumulates during nutrient deprivation. We previously showed that mammalian IMPACT and its yeast ortholog YIH1 bind to GCN1, thereby preventing GCN1 association with GCN2 and stimulation of this eIF2α kinase during amino acid depletion. GCN2 activity is also enhanced by other stresses, including proteasome inhibition, UV irradiation and lack of glucose. Here, we provide evidence that IMPACT affects directly and specifically the activation of GCN2 under these stress conditions in mammalian cells. We show that activation of mammalian GCN2 requires its interaction with GCN1 and that IMPACT promotes the dissolution of the GCN2-GCN1 complex. To a similar extent as the overexpression of YIH1, overexpression of IMPACT in yeast cells inhibited growth under all stress conditions that require GCN2 and GCN1 for cell survival, including exposure to acetic acid, high levels of NaCl, H2O2 or benomyl. This study extends our understanding of the roles played by GCN1 in GCN2 activation induced by a variety of stress arrangements and suggests that IMPACT and YIH1 use similar mechanisms for regulating this eIF2α kinase.

Yeast studies reveal moonlighting functions of the ancient actin cytoskeleton.

Classic functions of the actin cytoskeleton include control of cell size and shape and the internal organization of cells. These functions are manifest in cellular processes of fundamental importance throughout biology such as the generation of cell polarity, cell migration, cell adhesion, and cell division. However, studies in the unicellular model eukaryote Saccharomyces cerevisiae (Baker's yeast) are giving insights into other functions in which the actin cytoskeleton plays a critical role. These include endocytosis, control of protein translation, and determination of protein 3-dimensional shape (especially conversion of normal cellular proteins into prions). Here, we present a concise overview of these new "moonlighting" roles for the actin cytoskeleton and how some of these roles might lie at the heart of important molecular switches. This is an exciting time for researchers interested in the actin cytoskeleton. We show here how studies of actin are leading us into many new and exciting realms at the interface of genetics, biochemistry, and cell biology. While many of the pioneering studies have been conducted using yeast, the conservation of the actin cytoskeleton and its component proteins throughout eukaryotes suggests that these new roles for the actin cytoskeleton may not be restricted to yeast cells but rather may reflect new roles for the actin cytoskeleton of all eukaryotes.

Semi-quantitative colony immunoassay for determining and optimizing protein expression in Saccharomyces cerevisiae and Escherichia coli.

This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl ß-D-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.

GCN2 in Viral Defence and the Subversive Tactics Employed by Viruses.

The recent SARS-CoV-2 pandemic and associated COVID19 disease illustrates the important role of viral defence mechanisms in ensuring survival and recovery of the host or patient. Viruses absolutely depend on the host's protein synthesis machinery to replicate, meaning that impeding translation is a powerful way to counteract viruses. One major approach used by cells to obstruct protein synthesis is to phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Mammals possess four different eIF2α-kinases: PKR, HRI, PEK/PERK, and GCN2. While PKR is currently considered the principal eIF2α-kinase involved in viral defence, the other eIF2α-kinases have also been found to play significant roles. Unsurprisingly, viruses have developed mechanisms to counteract the actions of eIF2α-kinases, or even to exploit them to their benefit. While some of these virulence factors are specific to one eIF2α-kinase, such as GCN2, others target all eIF2α-kinases. This review critically evaluates the current knowledge of viral mechanisms targeting the eIF2α-kinase GCN2. A detailed and in-depth understanding of the molecular mechanisms by which viruses evade host defence mechanisms will help to inform the development of powerful anti-viral measures.

Overexpression of eukaryotic translation elongation factor 3 impairs Gcn2 protein activation.

In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn(-) phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn(-) phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn(-) phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.

Rapid yeast-based screen for Functionally Relevant Amino Acids (RS-FRAA) in a protein.

Here, we describe a fast and cost-effective procedure to generate a large array of mutant proteins and immediately screen for those with altered protein function. This protocol is a modification from three existing approaches: fusion PCR, Saccharomyces cerevisiae in-yeast recombination, and semi-quantitative growth assays. We also describe a mating step to reduce the occurrence of false positive findings due to ectopic mutations. The only requirement is that the protein elicits a phenotype in Saccharomyces cerevisiae.

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity.

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.

Gcn1 and actin binding to Yih1: implications for activation of the eIF2 kinase GCN2.

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.

Enterotoxigenicity and genetic relatedness of Staphylococcus aureus in a commercial poultry plant and poultry farm.

Raw (fresh) and frozen poultry products are frequently associated with Staphylococcus aureus contamination. New Zealand is among the developed countries with high incidences of staphylococcal food poisoning. The study investigated the S. aureus isolates obtained from various stages of poultry production, to determine the primary source of contamination. Viable cell counts of S. aureus were enumerated using Petrifilm™ Staph Express Count Plates, and the isolates were confirmed by Gram-stain and coagulase-positive test. Sixty S. aureus isolates were further confirmed by PCR. The PCR analysis used primers that specifically amplifies a fragment of the femA gene, unique to S. aureus. The confirmed S. aureus strains were further examined for enterotoxigenicity by PCR. Multilocus Sequence Typing (MLST) was then used to identify sequence types (STs) of the sixty isolates of S. aureus. The relatedness of the sequence types was investigated by eBURST. In this study, it was observed that all samples from the processing plant and live chickens at the farm were contaminated by S. aureus. Fifty-nine (59) of the 60 isolates were enterotoxigenic carrying enterotoxin genes: seg, sei, seh, sek, sel, sem, sen, or seo. The sixty isolates were categorised into six different sequence types: ST5, ST2594, ST101, ST83, ST398, ST1; where ST5, ST83 and ST2594 belonged to the Clonal Complex (CC) 5 with ST5 being the clonal ancestor. The sources of S. aureus contamination in the final poultry products were linked to fresh mechanically separated meat, fresh skin, fresh skin-on-breast fillet, rubber fingers on mechanical pluckers, and live chickens at the farm. The skin of live chickens at the farm was most likely the origin of S. aureus contamination on equipment and final products. Not all identified S. aureus strains at the farm were observed in the final products. Therefore, further investigation on other potential contamination sources such as gloves and knives used at the processing plant, and feeders and drinkers at the farm level is recommended.

A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation.

The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.

Asp56 in actin is critical for the full activity of the amino acid starvation-responsive kinase Gcn2.

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.

Experimentally based structural model of Yih1 provides insight into its function in controlling the key translational regulator Gcn2.

Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1-Gcn2 interaction by binding to Gcn1, thus preventing Gcn2-mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.