Guanine Nucleotide Exchange Factor eIF2B Research Articles

Global Translational Responses to Oxidative Stress Impact upon Multiple Levels of Protein Synthesis

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H(2)O(2) in the yeast Saccharomyces cerevisiae. Our data show that H(2)O(2) causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the alpha-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H(2)O(2) in a gcn2 mutant. Polysome analysis indicated that H(2)O(2) causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H(2)O(2) stress. For some candidate mRNAs, we show that a low concentration of H(2)O(2) results in increased protein production. In contrast, a high concentration of H(2)O(2) promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress.

Leucine Activates Pancreatic Translational Machinery in Rats and Mice through mTOR Independently of CCK and Insulin1–3

Feeding stimulates pancreatic digestive enzyme synthesis at the translational level, and this is thought to be mediated by hormones and neurotransmitters. However, BCAAs, particularly leucine, stimulate protein synthesis in several tissues. We investigated whether BCAA stimulated the translational machinery in murine pancreas and whether their effects were independent of hormones. Rats and mice were administered (i.g. gavage) individual BCAA at 1.35 mg/g (body weight) and rat isolated pancreatic acini were incubated with BCAA under different conditions. Activation of translation initiation factors and total protein synthesis were analyzed. BCAA gavage stimulated the phosphorylation of the initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase (S6K), with leucine being the most effective. Leucine also increased the association of the initiation factors eIF4E and eIF4G, but did not affect the activity of the guanine nucleotide exchange factor eIF2B, nor total protein synthesis. BCAA acted independently of insulin signaling on isolated pancreatic acini from diabetic rats. The ability of leucine to promote phosphorylation of 4E-BP1 and S6K as well as enhance the assembly of the eIF4F complex was unimpaired in CCK-deficient mice. Finally, rapamycin (0.75 mg/kg) administered to rats 2 h before leucine gavage inhibited the phosphorylation of S6 and 4E-BP1 induced by leucine. We conclude that leucine may participate, as a signal as well as a substrate, in activating the translational machinery in pancreatic acinar cells independently of hormonal effects and that this action is through the mTOR pathway.

Direct Binding of Translation Initiation Factor eIF2γ-G Domain to Its GTPase-activating and GDP-GTP Exchange Factors eIF5 and eIF2Bϵ

The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the gamma subunit of the heterotrimeric eIF2; however, only eIF2beta, and not eIF2gamma, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2gamma, as well as its isolated G domain, binds directly to eIF5 and the epsilon subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50-100 of yeast eIF5 impairs the interaction with recombinant eIF2gamma-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2gamma-G domain function via eIF2beta, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2gamma-G domain.

Inhibition of mammalian translation initiation by volatile anesthetics

Volatile anesthetics are essential for modern medical practice, but sites and mechanisms of action for any of their numerous cellular effects remain largely unknown. Previous studies with yeast showed that volatile anesthetics induce nutrient-dependent inhibition of growth through mechanisms involving inhibition of mRNA translation. Studies herein show that the volatile anesthetic halothane inhibits protein synthesis in perfused rat liver at doses ranging from 2 to 6%. A marked disaggregation of polysomes occurs, indicating that inhibition of translation initiation plays a key role. Dose- and time-dependent alterations that decrease the function of a variety of translation initiation processes are observed. At 6% halothane, a rapid and persistent increase in phosphorylation of the alpha-subunit of eukaryotic translation initiation factor (eIF)2 occurs. This is accompanied by inhibition of activity of the guanine nucleotide exchange factor eIF2B that is responsible for GDP-GTP exchange on eIF2. At lower doses, neither eIF2alpha phosphorylation nor eIF2B activity is altered. After extended exposure to 6% halothane, alterations in two separate responses regulated by the target of rapamycin pathway occur: 1) redistribution of eIF4E from its translation-stimulatory association with eIF4G to its translation-inactive complex with eIF4E-binding protein-1; and 2) decreased phosphorylation of ribosomal protein S6 (rpS6) with a corresponding decrease in active forms of a kinase that phosphorylates rpS6 (p70(S6K1)). Changes in the association of eIF4E and eIF4G are observed only after extended exposure to low anesthetic doses. Thus dose- and time-dependent alterations in multiple processes permit liver cells to adapt translation to variable degrees and duration of stress imposed by anesthetic exposure.

Structural Basis for Autoinhibition and Mutational Activation of Eukaryotic Initiation Factor 2α Protein Kinase GCN2*[boxs

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Dominant form of vanishing white matter–like leukoencephalopathy

Leukoencephalopathy with vanishing white matter syndrome (childhood ataxia with central nervous system hypomyelination/vanishing white matter disease) is an autosomal recessive disorder characterized by the occurrence of acute episodes of deterioration after minor head trauma or infection, and symmetrical demyelination on magnetic resonance with cavitation aspects. Mutations in each of the five subunits of eIF2B have been identified. We report in an affected man and his mother an adult-onset form of childhood ataxia with central nervous system hypomyelination/vanishing white matter disease-like disorder with no mutations in the EIF2B genes and normal guanine nucleotide exchange factor eIF2B activity, suggesting a new dominant inheritance of this syndrome that may involve other genes.

Role of mTOR signalling in the control of translation initiation and elongation by nutrients.

Protein synthesis requires nutrients both as precursors (amino acids) and as a source of energy, since this process consumes a high proportion of cellular metabolic energy. Recent work has shown that both types of nutrients directly influence the activities of components of the translational machinery in mammalian cells. Amino acids positively regulate signalling through the mammalian target of the rapamycin (mTOR) pathway, although the degree of dependency on external amino acids varies between cell types. mTOR signalling modulates several key components involved in mRNA translation, in particular (via repressor proteins) the cap-binding initiation factor eIF4E, the ribosomal protein S6 kinases, and elongation factor eEF2. The branched-chain amino acid leucine is the most effective one in most cell types. It is currently unclear how mammalian cells sense prevailing amino acid levels, although this may involve intracellular amino acids. Cellular ATP levels can also influence mTOR activity. The activities of some translation factors are modulated by mTOR-independent mechanisms. Examples include the regulation of eEF2 by cellular energy levels, which may be controlled via the AMP-activated protein kinase, and the activity of the guanine nucleotide-exchange factor eIF2B, which is modulated by amino acids and metabolic fuels.

The Crystal Structure of the N-terminal Region of the Alpha Subunit of Translation Initiation Factor 2 (eIF2α) from Saccharomyces cerevisiae Provides a View of the Loop Containing Serine 51, the Target of the eIF2α-specific Kinases

The α subunit of translation initiation factor 2 (eIF2α) is the target of specific kinases that can phosphorylate a conserved serine residue as part of a mechanism for regulating protein expression at the translational level in eukaryotes. The structure of the 20 kDa N-terminal region of eIF2α from Saccharomyces cerevisiae was determined by X-ray crystallography at 2.5Å resolution. In most respects, the structure is similar to that of the recently solved human eIF2α; the rather elongated protein contains a five-stranded antiparallel β-barrel in its N-terminal region, followed by an almost entirely helical domain. The S.cerevisiae eIF2α lacks a disulfide bridge that is present in the homologous protein in humans and some of the other higher eukaryotes. Interestingly, a conserved loop consisting of residues 51–65 and containing serine 51, the putative phosphorylation site, is visible in the electron density maps of the S.cerevisiae eIF2α; most of this functionally important loop was not observed in the crystal structure of the human protein. This loop is relatively exposed to solvent, and contains two short 310 helices in addition to some extended structure. Serine 51 is located at the C-terminal end of one of the 310 helices and near several conserved positively charged residues. The side-chain of serine 51 is sufficiently exposed so that its phosphorylation would not necessitate a substantial change in the protein structure. The structures and relative positions of residues that have been implicated in kinase binding and in the interaction with guanine nucleotide exchange factor (eIF2B) are described.

Caerulein-induced acute pancreatitis inhibits protein synthesis through effects on eIF2B and eIF4F.

Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2alpha. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 microg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2alpha phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2alpha phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.

Regulation of mammalian translation factors by nutrients.

Protein synthesis requires both amino acids, as precursors, and a substantial amount of metabolic energy. It is well established that starvation or lack of nutrients impairs protein synthesis in mammalian cells and tissues. Branched chain amino acids are particularly effective in promoting protein synthesis. Recent work has revealed important new information about the mechanisms involved in these effects. A number of components of the translational machinery are regulated through signalling events that require the mammalian target of rapamycin, mTOR. These include translational repressor proteins (eukaryotic initiation factor 4E-binding proteins, 4E-BPs) and protein kinases that act upon the small ribosomal subunit (S6 kinases). Amino acids, especially leucine, positively regulate mTOR signalling thereby relieving inhibition of translation by 4E-BPs and activating the S6 kinases, which can also regulate translation elongation. However, the molecular mechanisms by which amino acids modulate mTOR signalling remain unclear. Protein synthesis requires a high proportion of the cell's metabolic energy, and recent work has revealed that metabolic energy, or fuels such as glucose, also regulate targets of the mTOR pathway. Amino acids and glucose modulate a further important regulatory step in translation initiation, the activity of the guanine nucleotide-exchange factor eIF2B. eIF2B controls the recruitment of the initiator methionyl-tRNA to the ribosome and is activated by insulin. However, in the absence of glucose or amino acids, insulin no longer activates eIF2B. Since control of eIF2B is independent of mTOR, these data indicate the operation of additional, and so far unknown, regulatory mechanisms that control eIF2B activity.

Translational control of protein synthesis in pancreatic acinar cells.

Translational control of protein synthesis in the pancreas is important in regulating growth and the synthesis of digestive enzymes. Regulation of translation is primarily directed at the steps in initiation and involves reversible phosphorylation of initiation factors (eIFs) and ribosomal proteins. Major sites include the assembly of the eIF4F mRNA cap binding complex, the activity of guanine nucleotide exchange factor eIF2B, and the activity of ribosomal S6 kinase. All of these involve phosphorylation by different regulatory pathways. Stimulation of protein synthesis in acinar cells is primarily mediated by the phosphatidylinositol 3-kinase-mTOR pathway and involves both release of eIF4E (the limiting component of eIF4F) from its binding protein and phosphorylation of ribosomal S6 protein by S6K. eIF4E is itself phosphorylated by a distinct pathway. Inhibition of acinar protein synthesis can be mediated by inhibition of eIF2B following phosphorylation of eIF2alpha.

Tight binding of the phosphorylated alpha subunit of initiation factor 2 (eIF2alpha) to the regulatory subunits of guanine nucleotide exchange factor eIF2B is required for inhibition of translation initiation.

Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNA(Met) to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its alpha subunit [eIF2(alphaP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the alpha subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2alpha (glutathione S-transferase [GST]-SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(alphaP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(alphaP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(alphaP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the beta and gamma subunits of eIF2 in the manner required for GDP-GTP exchange.

Interplay between insulin and nutrients in the regulation of translation factors.

Protein synthesis in mammalian cells is regulated through alterations in the states of phosphorylation of eukaryotic initiation factors and elongation factors (eIFs and eEFs respectively) and of other regulatory proteins. This modulates their activities or their abilities to interact with one another. Insulin activates several of these proteins including the following: the guanine-nucleotide exchange factor eIF2B; the eIF4F complex, which (through eIF4E) interacts with the cap of the mRNA; p70 S6 kinase; and elongation factor eEF2, which mediates the translocation step of elongation. Control of the last three of these is linked to mTOR (mammalian target of rapamycin). In Chinese hamster ovary cells, regulation of all these proteins by insulin is modulated by the presence of amino acids and/or glucose in the medium. For example, p70 S6 kinase activity declines in the absence of amino acids and cannot be stimulated by insulin under this condition. The readdition of amino acids, especially leucine, restores activity and sensitivity to insulin. With eIF2B and eEF2, both amino acids and glucose must be provided for insulin to regulate their activities. In contrast, insulin-stimulation of the formation of eIF4F complexes requires glucose but not amino acids. Glucose metabolism is required for this permissive effect. Our recent studies have also identified the mechanism by which mTOR signalling regulates the phosphorylation of eEF2. eEF2 kinase is phosphorylated by p70 S6 kinase at Ser-366; this results in the inactivation of eEF2 kinase, especially at low (micromolar) Ca concentrations.

Regulation of protein synthesis by insulin through IRS-1.

Most regulatory mechanisms for the overall rate of protein synthesis described to date involve the initiation phase, which is catalyzed by the initiation factors (eIF1; Merrick and Hershey 1996; Rhoads 1999). Protein synthesis occurs in a series of discrete steps. A ternary complex of eIF2•GTP•Met-tRNAi binds to a complex of the 40 S ribosomal subunit and eIF3 to form the 43 S initiation complex. The eIF4 factors plus the poly(A)-binding protein collectively recognize the 5′-terminal cap or 3′-terminal poly(A) tract of mRNA, unwind its secondary structure, and transfer it to the 43 S initiation complex, resulting in the 48 S initiation complex. Scanning for the first initiation codon in good sequence context requires ATP hydrolysis by eIF4A and the presence of eIF1 and eIF1A (Pestova et al. 1998). Then, eIF5 stimulates GTP hydrolysis by eIF2, after which the initiation factors are replaced by the 60 S subunit to form the 80 S initiation complex. The released eIF2-GDP is recycled to eIF2-GTP by the guanine nucleotide exchange factor eIF2B. The first elongator aminoacyl-tRNA is brought to the A-site by eEF1, followed by synthesis of the first peptide bond and translocation catalyzed by eEF2

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2.

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.

A Ribosomal Protein Is Required for Translational Regulation of GCN4 mRNA: EVIDENCE FOR INVOLVEMENT OF THE RIBOSOME IN eIF2 RECYCLING

In amino acid-starved yeast cells, inhibition of the guanine nucleotide exchange factor eIF2B by phosphorylated translation initiation factor 2 results in increased translation of GCN4 mRNA. We isolated a suppressor of a mutant eIF2B. The suppressor prevents efficient GCN4 mRNA translation due to inactivation of the small ribosomal subunit protein Rps31 and results in low amounts of mutant 40 S ribosomal subunits. Deletion of one of two genes encoding ribosomal protein Rps17 also reduces the amounts of 40 S subunits but does not suppress eIF2B mutations or prevent efficient GCN4 translation. Our findings show that Rps31-deficient ribosomes are altered in a way that decreases the eIF2B requirement and that the small ribosomal subunit mediates the effects of low eIF2B activity on cell viability and translational regulation in response to eIF2 phosphorylation.

Homologous segments in three subunits of the guanine nucleotide exchange factor eIF2B mediate translational regulation by phosphorylation of eIF2.

eIF2B is a five-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the alpha subunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded by GCD2, GCD7, and GCN3 that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its alpha subunit [eIF2(alphaP)]. Most, if not all, of the mutations decrease sensitivity to eIF2(alphaP) without excluding GCN3, the nonessential subunit, from eIF2B; thus, all three proteins are critical for regulation of eIF2B by eIF2(alphaP). The mutations are clustered at both ends of the homologous region of each subunit, within two segments each of approximately 70 amino acids in length. Several mutations alter residues at equivalent positions in two or all three subunits. These results imply that structurally similar segments in GCD2, GCD7, and GCN3 perform related functions in eIF2B regulation. We propose that these segments form a single domain in eIF2B that makes multiple contacts with the alpha subunit of eIF2, around the phosphorylation site, allowing eIF2B to detect and respond to phosphoserine at residue 51. Most of the eIF2 is phosphorylated in certain mutants, suggesting that these substitutions allow eIF2B to accept phosphorylated eIF2 as a substrate for nucleotide exchange.

Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit.

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.

Identification of a Regulatory Subcomplex in the Guanine Nucleotide Exchange Factor eIF2B That Mediates Inhibition by Phosphorylated eIF2

Eukaryotic translation initiation factor 2B (eIF2B) is a five-subunit complex that catalyzes guanine nucleotide exchange on eIF2. Phosphorylation of the alpha subunit of eIF2 [creating eIF2(alphaP]) converts eIF2 x GDP from a substrate to an inhibitor of eIF2B. We showed previously that the inhibitory effect of eIF2(alphaP) can be decreased by deletion of the eIF2B alpha subunit (encoded by GCN3) and by point mutations in the beta and delta subunits of eIF2B (encoded by GCD7 and GCD2, respectively). These findings, plus sequence similarities among GCD2, GCD7, and GCN3, led us to propose that these proteins comprise a regulatory domain that interacts with eIF2(alphaP) and mediates the inhibition of eIF2B activity. Supporting this hypothesis, we report here that overexpression of GCD2, GCD7, and GCN3 specifically reduced the inhibitory effect of eIF2(alphaP) on translation initiation in vivo. The excess GCD2, GCD7, and GCN3 were coimmunoprecipitated from cell extracts, providing physical evidence that these three proteins can form a stable subcomplex. Formation of this subcomplex did not compensate for a loss of eIF2B function by mutation and in fact lowered eIF2B activity in strains lacking eIF2(alphaP). These findings indicate that the trimeric subcomplex does not possess guanine nucleotide exchange activity; we propose, instead, that it interacts with eIF2(alphaP) and prevents the latter from inhibiting native eIF2B. Overexpressing only GCD2 and GCD7 also reduced eIF2(alphaP) toxicity, presumably by titrating GCN3 from eIF2B and producing the four-subunit form of eIF2B that is less sensitive to eIF2(alphaP). This interpretation is supported by the fact that overexpressing GCD2 and GCD7 did not reduce eIF2(alphaP) toxicity in a strain lacking GCN3; however, it did suppress the impairment of eIF2B caused by the gcn3c-R104K mutation. An N-terminally truncated GCD2 protein interacted with other eIF2B subunits only when GCD7 and GCN3 were overexpressed, in accordance with the idea that the portion of GCD2 homologous to GCD7 and GCN3 is sufficient for complex formation by these three proteins. Together, our results provide strong evidence that GCN3, GCD7, and the C-terminal half of GCD2 comprise the regulatory domain in eIF2B.

EIF2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2. Sequence conservation between the alpha, beta and delta subunits of eIF2B from mammals and yeast.

The guanine nucleotide-exchange factor eIF2B mediates the exchange of GDP bound to translation initiation factor eIF2 for GTP. This exchange process is a key regulatory step for the control of translation initiation in eukaryotic organisms. To improve our understanding of the structure, function and regulation of eIF2B, we have obtained and sequenced cDNA species encoding all of its five subunits. Here we report the sequences of eIF2B beta and delta from rat. This paper focuses on sequence similarities between the alpha, beta and delta subunits of mammalian eIF2B. Earlier work showed that the amino acid sequences of the corresponding subunits of eIF2B in the yeast Saccharomyces cerevisiae (GCN3, GCD7 and GCD2) exhibit considerable similarity. We demonstrate that this is also true for the mammalian subunits. Moreover, alignment of the eIF2B alpha, beta and delta sequences from mammals and yeast, along with the sequence of the putative eIF2B alpha subunit from Caenorhabditis elegans and eIF2B delta from Schizosaccharomyces pombe shows that a large number of residues are identical or conserved between the C-terminal regions of all these sequences. This strong sequence conservation points to the likely functional importance of these residues. The implications of this are discussed in the light of results concerning the functions of the subunits of eIF2B in yeast and mammals. Our results also indicate that the large apparent differences in mobility on SDS/PAGE between eIF2B beta and delta subunits from rat and rabbit are not due to differences in their lengths but reflect differences in amino acid composition. We have also examined the relative expression of mRNA species encoding the alpha, beta, delta and epsilon subunits of eIF2B in a range of rat tissues by Northern blot analysis. As might be expected for mRNA species encoding subunits of a heterotrimeric protein, the ratios of expression levels of these subunits to one another did not vary between the different rat tissues examined (with the possible exception of liver). This represents the first analysis of the levels of expression of mRNA species encoding the different subunits of eIF2B.