Homologous Segments in Three Subunits of the Guanine Nucleotide Exchange Factor eIF2B Mediate Translational Regulation by Phosphorylation of eIF2 (original) (raw)

The beta/Gcd7 subunit of eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor, is crucial for binding eIF2 in vivo

Molecular and cellular biology, 2010

The ␤/Gcd7 Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2B), a Guanine Nucleotide Exchange Factor, Is Crucial for Binding eIF2 In Vivo

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor (GEF) for eukaryotic translation initiation factor 2, which stimulates formation of the eIF2-GTP-Met-tRNA i Met ternary complex (TC) in a manner inhibited by phosphorylated eIF2 [eIF2(␣P)]. While eIF2B contains five subunits, the /Gcd6 subunit is sufficient for GEF activity in vitro. The ␦/Gcd2 and ␤/Gcd7 subunits function with ␣/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(␣P)-GDP, but the essential functions of ␦/Gcd2 and ␤/Gcd7 are not well understood. We show that the depletion of wild-type ␤/Gcd7, three lethal ␤/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in ␤/Gcd7 and eIF2␣ all impair eIF2 binding to eIF2B without reducing /Gcd6 abundance in the native eIF2B-eIF2 holocomplex. Additionally, ␤/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2␣ (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, ␤/Gcd7 can overcome the toxicity of eIF2(␣P) and rescue native eIF2B function when overexpressed with ␦/Gcd2 or ␥/Gcd1. In aggregate, these findings provide compelling evidence that ␤/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (␣P).

Clues to the mechanism of action of eIF2B, the guanine-nucleotide-exchange factor for translation initiation

Biochemical Society Transactions, 2008

A variety of cellular processes rely on G-proteins, which cycle through active GTP-bound and inactive GDPbound forms. The switch between these states is commonly regulated by GEFs (guanine-nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Although G-proteins have structural similarity, GEFs are very diverse proteins. A complex example of this system is seen in eukaryotic translation initiation between eIF (eukaryotic initiation factor) 2, a G-protein, its five-subunit GEF, eIF2B, and its GAP, eIF5. eIF2 delivers Met-tRNA i (initiator methionyl-tRNA) to the 40S ribosomal subunit before mRNA binding. Upon AUG recognition, eIF2 hydrolyses GTP, aided by eIF5. eIF2B then re-activates eIF2 by removing GDP, thereby promoting association of GTP. In the present article, we review data from studies of representative G-protein-GEF pairs and compare these with observations from our research on eIF2 and eIF2B to propose a model for how interactions between eIF2B and eIF2 promote guanine nucleotide exchange.

Identification of Domains and Residues within the ɛ Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2Bɛ) Required for Guanine Nucleotide Exchange Reveals a Novel Activation Function Promoted by eIF2B Complex Formation

Molecular and Cellular Biology, 2000

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation

The EMBO Journal, 2006

In eukaryotic translation initiation, the eIF2 . GTP/Met-tRNA i Met ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2 . GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Be catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA i Met , and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Be mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Be with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Be segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Be to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA i Met , suggesting that tRNA i Met is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.

Localization of the Translational Guanine Nucleotide Exchange Factor eIF2B: A Common Theme for GEFs?

Cell Cycle, 2006

The eukaryotic initiation factor 2B (eIF2B) serves an essential recycling function in protein synthesis. As the guanine nucleotide exchange factor for eIF2, it recycles eIF2 from a GDP to a GTP bound form that is competent for translation initiation. Stress-dependent controls target this eIF2B-recycling step allowing a reprogramming of the global gene expression profile. In addition, a human disease, leukoencephalopathy with vanishing white matter (VWM), is caused by mutations in the eIF2B subunit genes. Recently, we have found that the eIF2B guanine nucleotide exchange factor resides in a specific cytoplasmic focus in the yeast, Saccharmoyces cerevisiae. eIF2B is a resident feature of this focus, whereas eIF2 shuttles to and fro. Moreover, the in vivo rate of eIF2 shuttling correlates with changes in guanine nucleotide exchange activity implicating this large cytoplasmic focus as a site of guanine nucleotide exchange. In this perspective, we discuss these findings in the wider context of the assortment of guanine nucleotide exchange factors.

Identification of Domains and Residues within the varepsilon Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2Bvarepsilon ) Required for Guanine Nucleotide Exchange Reveals a Novel Activation Function Promoted by eIF2B Complex Formation

Molecular and Cellular Biology, 2000

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2B). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2B in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gene encoding eIF2B (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2B (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2B without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2B disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2B alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2B alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2B and the other eIF2B subunits. This suggests that the N-terminal region of eIF2B is an activation domain that responds to eIF2B complex formation.

Tight Binding of the Phosphorylated Subunit of Initiation Factor 2 (eIF2 ) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

Molecular and Cellular Biology, 2001

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 ␣ subunit [eIF2(␣P)] 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 ␣ subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2␣ (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(␣P) 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(␣P) 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(␣P). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the ␤ and ␥ subunits of eIF2 in the manner required for GDP-GTP exchange.

Homologous Segments in Three Subunits of the Guanine Nucleotide Exchange Factor eIF2B Mediate Translational Regulation by Phosphorylation of eIF2 (original) (raw)

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