A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling.

The integrated stress response (ISR) enables cells to survive a variety of acute stresses, but chronic activation of the ISR underlies age-related diseases. ISR signaling downregulates translation and activates expression of stress-responsive factors that promote return to homeostasis and is initiated by inhibition of the decameric guanine nucleotide exchange factor eIF2B. Conformational and assembly transitions regulate eIF2B activity, but the allosteric mechanisms controlling these dynamic transitions and mediating the therapeutic effects of the small-molecule ISR inhibitor ISRIB are unknown. Using hydrogen-deuterium exchange-mass spectrometry and cryo-electron microscopy, we identified a central α-helix whose orientation allosterically coordinates eIF2B conformation and assembly. Biochemical and cellular signaling assays show that this 'switch-helix' controls eIF2B activity and signaling. In sum, the switch-helix acts as a fulcrum of eIF2B conformational regulation and is a highly conserved actuator of ISR signal transduction. This work uncovers a conserved allosteric mechanism and unlocks new therapeutic possibilities for ISR-linked diseases.

Protection of eIF2B from inhibitory phosphorylated eIF2: A viral strategy to maintain mRNA translation during the PKR-triggered integrated stress response.

The integrated stress response (ISR) protects cells from a variety of insults. Once elicited (e.g., by virus infections), it eventually leads to the block of mRNA translation. Central to the ISR are the interactions between translation initiation factors eIF2 and eIF2B. Under normal conditions, eIF2 drives the initiation of protein synthesis through hydrolysis of GTP, which becomes replenished by the guanine nucleotide exchange factor eIF2B. The antiviral branch of the ISR is activated by the RNA-activated kinase PKR which phosphorylates eIF2, thereby converting it into an eIF2B inhibitor. Here, we describe the recently solved structures of eIF2B in complex with eIF2 and a novel escape strategy used by viruses. While unphosphorylated eIF2 interacts with eIF2B in its "productive" conformation, phosphorylated eIF2 [eIF2(αP)] engages a different binding cavity on eIF2B and forces it into the "nonproductive" conformation that prohibits guanine nucleotide exchange factor activity. It is well established that viruses express so-called PKR antagonists that interfere with double-strand RNA, PKR itself, or eIF2. However recently, three taxonomically unrelated viruses were reported to encode antagonists targeting eIF2B instead. For one antagonist, the S segment nonstructural protein of Sandfly fever Sicilian virus, atomic structures showed that it occupies the eIF2(αP)-binding cavity on eIF2B without imposing a switch to the nonproductive conformation. S segment nonstructural protein thus antagonizes the activity of PKR by protecting eIF2B from inhibition by eIF2(αP). As the ISR and specifically eIF2B are central to neuroprotection and a wide range of genetic and age-related diseases, these developments may open new possibilities for treatments.

Surviving and Adapting to Stress: Translational Control and the Integrated Stress Response.

Significance: Organisms adapt to changing environments by engaging cellular stress response pathways that serve to restore proteostasis and enhance survival. A primary adaptive mechanism is the integrated stress response (ISR), which features phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2). Four eIF2α kinases respond to different stresses, enabling cells to rapidly control translation to optimize management of resources and reprogram gene expression for stress adaptation. Phosphorylation of eIF2 blocks its guanine nucleotide exchange factor, eIF2B, thus lowering the levels of eIF2 bound to GTP that is required to deliver initiator transfer RNA (tRNA) to ribosomes. While bulk messenger RNA (mRNA) translation can be sharply lowered by heightened phosphorylation of eIF2α, there are other gene transcripts whose translation is unchanged or preferentially translated. Among the preferentially translated genes is ATF4, which directs transcription of adaptive genes in the ISR. Recent Advances and Critical Issues: This review focuses on how eIF2α kinases function as first responders of stress, the mechanisms by which eIF2α phosphorylation and other stress signals regulate the exchange activity of eIF2B, and the processes by which the ISR triggers differential mRNA translation. To illustrate the synergy between stress pathways, we describe the mechanisms and functional significance of communication between the ISR and another key regulator of translation, mammalian/mechanistic target of rapamycin complex 1 (mTORC1), during acute and chronic amino acid insufficiency. Finally, we discuss the pathological conditions that stem from aberrant regulation of the ISR, as well as therapeutic strategies targeting the ISR to alleviate disease. Future Directions: Important topics for future ISR research are strategies for modulating this stress pathway in disease conditions and drug development, molecular processes for differential translation and the coordinate regulation of GCN2 and other stress pathways during physiological and pathological conditions. Antioxid. Redox Signal. 39, 351-373.

The role of eIF2 phosphorylation in cell and organismal physiology: new roles for well-known actors.

Control of protein synthesis (mRNA translation) plays key roles in shaping the proteome and in many physiological, including homeostatic, responses. One long-known translational control mechanism involves phosphorylation of initiation factor, eIF2, which is catalysed by any one of four protein kinases, which are generally activated in response to stresses. They form a key arm of the integrated stress response (ISR). Phosphorylated eIF2 inhibits eIF2B (the protein that promotes exchange of eIF2-bound GDP for GTP) and thus impairs general protein synthesis. However, this mechanism actually promotes translation of certain mRNAs by virtue of specific features they possess. Recent work has uncovered many previously unknown features of this regulatory system. Several studies have yielded crucial insights into the structure and control of eIF2, including that eIF2B is regulated by several metabolites. Recent studies also reveal that control of eIF2 and the ISR helps determine organismal lifespan and surprising roles in sensing mitochondrial stresses and in controlling the mammalian target of rapamycin (mTOR). The latter effect involves an unexpected role for one of the eIF2 kinases, HRI. Phosphoproteomic analysis identified new substrates for another eIF2 kinase, Gcn2, which senses the availability of amino acids. Several genetic disorders arise from mutations in genes for eIF2α kinases or eIF2B (i.e. vanishing white matter disease, VWM and microcephaly, epileptic seizures, microcephaly, hypogenitalism, diabetes and obesity, MEHMO). Furthermore, the eIF2-mediated ISR plays roles in cognitive decline associated with Alzheimer's disease. New findings suggest potential therapeutic value in interfering with the ISR in certain settings, including VWM, for example by using compounds that promote eIF2B activity.

Stepwise assembly of the eukaryotic translation initiation factor 2 complex.

The eukaryotic translation initiation factor 2 (eIF2) has key functions in the initiation step of protein synthesis. eIF2 guides the initiator tRNA to the ribosome, participates in scanning of the mRNA molecule, supports selection of the start codon, and modulates the translation of mRNAs in response to stress. eIF2 comprises a heterotrimeric complex whose assembly depends on the ATP-grasp protein Cdc123. Mutations of the eIF2γ subunit that compromise eIF2 complex formation cause severe neurological disease in humans. To this date, however, details about the assembly mechanism, step order, and the individual functions of eIF2 subunits remain unclear. Here, we quantified assembly intermediates and studied the behavior of various binding site mutants in budding yeast. Based on these data, we present a model in which a Cdc123-mediated conformational change in eIF2γ exposes binding sites for eIF2α and eIF2ß subunits. Contrary to an earlier hypothesis, we found that the associations of eIF2α and eIF2ß with the γ-subunit are independent of each other, but the resulting heterodimers are nonfunctional and fail to bind the guanosine exchange factor eIF2B. In addition, levels of eIF2α influence the rate of eIF2 assembly. By binding to eIF2γ, eIF2α displaces Cdc123 and thereby completes the assembly process. Experiments in human cell culture indicate that the mechanism of eIF2 assembly is conserved between yeast and humans. This study sheds light on an essential step in eukaryotic translation initiation, the dysfunction of which is linked to human disease.

A C-term truncated EIF2Bγ protein encoded by an intronically polyadenylated isoform introduces unfavorable EIF2Bγ-EIF2γ interactions.

Eukaryotic translation initiates upon recruitment of the EIF2-GTP·Met-tRNAi ternary complex (TC) to the ribosomes. EIF2 (α, ß, γ subunits) is a GTPase. The GDP to GTP exchange within EIF2 is facilitated by the guanine nucleotide exchange factor EIF2B (α-ε subunits). During stress-induced conditions, phosphorylation of the α-subunit of EIF2 turns EIF2 into an inhibitor of EIF2B. In turn, inhibition of EIF2B decreases TC formation and triggers the internal stress response (ISR), which determines the cell fate. Deregulated ISR has been linked to neurodegenerative disorders and cancer, positioning EIF2B as a promising therapeutic target. Hence, a better understanding of the mechanisms/factors that regulate EIF2B activity is required. Here, combining transcript and protein level analyses, we describe an intronically polyadenylated (IPA) transcript of EIF2B's γ-subunit. We show that the IPA mRNA isoform is translated into a C-terminus truncated protein. Using structural modeling, we predict that the truncated EIF2Bγ protein has unfavorable interactions with EIF2γ, leading to a potential decrease in the stability of the nonproductive EIF2:EIF2B complex. While we discovered and confirmed the IPA mRNA isoform in breast cancer cells, the expression of this isoform is not cancer-specific and is widely present in normal tissues. Overall, our data show that a truncated EIF2Bγ protein co-exists with the canonical protein and is an additional player to regulate the equilibrium between productive and nonproductive states of the EIF2:EIF2B complex. These results may have implications in stress-induced translation control in normal and disease states. Our combinatorial approach demonstrates the need to study noncanonical mRNA and protein isoforms to understand protein interactions and intricate molecular mechanisms.

Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B.

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2's dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five 'aromatic fingers'. NSs binding preserves eIF2B activity by maintaining eIF2B's conformation in its active A-State.

eIF2B-capturing viral protein NSs suppresses the integrated stress response.

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

Sugar phosphate activation of the stress sensor eIF2B.

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.

Identification of a Missense Variant in the EIF2B3 Gene Causing Vanishing White Matter Disease with Antenatal-Onset but Mild Symptoms and Long-Term Survival.

Vanishing white matter disease (VWM) is a rare autosomal recessive leukodystrophy caused by a mutation in any of the five gene encoding subunits of the translation initiation factors eIF2B1 to eIF2B5. Whole-exome sequencing was performed on a 7-year-old boy with prenatal symptoms, including intrauterine-growth retardation, decreased movements, and oligohydramnios as well as mild intellectual disability, optic atrophy, macrocephaly, mild ataxia, and white matter lesions after birth. Analysis of WES data revealed a homozygous missense variant, c.C590T (p.Thr197Met) in the EIF2B3 gene (NM_0203650). The candidate variant was confirmed by Sanger sequencing and found to co-segregate with disease in family members. Pathogenicity analysis, 3D protein modeling, and stability assessment showed the deleterious effects of this nucleotide change. Previous studies suggest a direct relationship between the onset of symptoms and the progression rate and severity of the disease. All described cases of EIF2B deficiency with antenatal-onset led prenatal death; if they were born, they experienced clinical exacerbation, seizure, severe encephalopathy, and consequent infantile death (< 1 year). The patient of this study had never had seizure, which could be a potential explanation for the observed mild clinical picture, chronic state, and long-term survival until the age of seven. This study reported the first VWM due to EIF2B gene deficiency with antenatal-onset but mild symptoms and long-term survival. The result of this study showed that stressor factors, particularly seizure, could have a substantial role in poor prognosis and early neonatal death.

eIF2B conformation and assembly state regulate the integrated stress response.

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a twofold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-electron microscopy structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

Eif2b3 mutants recapitulate phenotypes of vanishing white matter disease and validate novel disease alleles in zebrafish.

Leukodystrophy with vanishing white matter (VWM), also called Childhood Ataxia with Central Nervous System Hypomyelination, is caused by mutations in the subunits of the eukaryotic translation initiation factor, EIF2B1, EIF2B2, EIF2B3, EIF2B4 or EIF2B5. However, little is known regarding the underlying pathogenetic mechanisms, and there is no curative treatment for VWM. In this study, we established the first EIF2B3 animal model for VWM disease in vertebrates by CRISPR mutagenesis of the highly conserved zebrafish ortholog eif2b3. Using CRISPR, we generated two mutant alleles in zebrafish eif2b3, 10- and 16-bp deletions, respectively. The eif2b3 mutants showed defects in myelin development and glial cell differentiation, and increased expression of genes in the induced stress response pathway. Interestingly, we also found ectopic angiogenesis and increased VEGF expression. Ectopic angiogenesis in the eif2b3 mutants was reduced by the administration of VEGF receptor inhibitor SU5416. Using the eif2b3 mutant zebrafish model together with in silico protein modeling analysis, we demonstrated the pathogenicity of 18 reported mutations in EIF2B3, as well as of a novel variant identified in a 19-month-old female patient: c.503 T > C (p.Leu168Pro). In summary, our zebrafish mutant model of eif2b3 provides novel insights into VWM pathogenesis and offers rapid functional analysis of human EIF2B3 gene variants.

ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B.

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

eIF2B and the Integrated Stress Response: A Structural and Mechanistic View.

The eukaryotic translation initiation factor eIF2 is a GTPase, which brings the initiator Met-tRNAi to the ribosome as the eIF2-GTP·Met-tRNAi ternary complex (TC). TC regeneration is catalyzed by the guanine nucleotide exchange factor (GEF) eIF2B. eIF2 phosphorylation by several stress-induced kinases converts it into a competitive inhibitor of eIF2B. Inhibition of eIF2B activity lowers cellular TC concentrations, which in turn triggers the integrated stress response (ISR). Depending on its degree of activation and duration, the ISR protects the cell from the stress or can itself induce apoptosis. ISR dysregulation is a causative factor in the pathology of multiple neurodegenerative disorders, while ISR inhibitors are neuroprotective. The realization that eIF2B is a promising therapeutic target has triggered significant interest in its structure and its mechanisms of action and regulation. Recently, four groups published the cryo-electron microscopy structures of eIF2B with its substrate eIF2 and/or its inhibitor, phosphorylated eIF2 [eIF2(α-P)]. While all three structures of the nonproductive eIF2B·eIF2(α-P) complex are similar to each other, there is a sharp disagreement between the published structures of the productive eIF2B·eIF2 complex. One group reports a structure similar to that of the nonproductive complex, whereas two others observe a vastly different eIF2B·eIF2 complex. Here, we discuss the recent reports on the structure, function, and regulation of eIF2B; the preclinical data on the use of ISR inhibitors for the treatment of neurodegenerative disorders; and how the new structural and biochemical information can inform and influence the use of eIF2B as a therapeutic target.

eIF2B-catalyzed nucleotide exchange and phosphoregulation by the integrated stress response.

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a guanosine triphosphatase that becomes activated by eIF2B, a two-fold symmetric and heterodecameric complex that functions as eIF2's dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from a substrate into an inhibitor of eIF2B. We report cryo-electron microscopy structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B's bipartite catalytic centers to catalyze nucleotide exchange. Phosphorylation refolds eIF2α, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequestering it into a nonproductive complex.

Structural basis for eIF2B inhibition in integrated stress response.

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.

RGS2 promotes the translation of stress-associated proteins ATF4 and CHOP via its eIF2B-inhibitory domain.

Regulator of G protein signaling 2 (RGS2) is upregulated by multiple forms of stress and can augment translational attenuation associated with the phosphorylation of the initiation factor eIF2, a hallmark of several stress-induced coping mechanisms. Under stress-induced translational inhibition, key factors, such as ATF4, are selectively expressed via alternative translation mechanisms. These factors are known to regulate molecular switches that control cell fate by regulating pro-survival and pro-apoptotic signals. The molecular mechanisms that balance these opposing responses to stresses are unclear. The present results suggest that RGS2 may be an important regulatory component in the cellular stress response through its translational control abilities. Previously, we have shown that RGS2 can interact with the translation initiation factor, eIF2B, and inhibit de novo protein synthesis. Here, we demonstrate that the expression of either full length RGS2 or its eIF2B-interacting domain (RGS2eb) significantly increases levels of ATF4 and CHOP, both of which are linked to stress-induced apoptosis. Furthermore, we show that these effects are translationally regulated and independent of eIF2 phosphorylation. The present results thus point to a novel function of RGS2 in the stress response directly related to its ability to reduce global protein synthesis.

Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule.

Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from a substrate to a competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, thereby inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation, and in rodents it enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by cryo-electron microscopy. Formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and ISRIB promoted this step by cross-bridging a central symmetry interface. Thus, regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.

Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B.

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the ß and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.

eIF2B Mechanisms of Action and Regulation: A Thermodynamic View.

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor of the GTPase eIF2, which brings the initiator Met-tRNAi to the ribosome in the form of the eIF2-GTP·Met-tRNAi ternary complex (TC). The activity of eIF2B is inhibited by phosphorylation of its substrate eIF2 by several stress-induced kinases, which triggers the integrated stress response (ISR). The ISR plays a central role in maintaining homeostasis in the cell under various stress conditions, and its dysregulation is a causative factor in the pathology of a number of neurodegenerative disorders. Over the past three decades, virtually every aspect of eIF2B function has been the subject of uncertainty or controversy: from the catalytic mechanism of nucleotide exchange, to whether eIF2B only catalyzes nucleotide exchange on eIF2 or also promotes binding of Met-tRNAi to eIF2-GTP to form the TC. Here, we provide the first complete thermodynamic analysis of the process of recycling of eIF2-GDP to the TC. The available evidence leads to the conclusion that eIF2 is channeled from the ribosome (as an eIF5·eIF2-GDP complex) to eIF2B, converted by eIF2B to the TC, which is then channeled back to eIF5 and the ribosome. The system has evolved to be regulated by multiple factors, including post-translational modifications of eIF2, eIF2B, and eIF5, as well as directly by the energy balance in the cell, through the GTP:GDP ratio.