scholarly journals Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3.

1993 ◽  
Vol 13 (8) ◽  
pp. 4618-4631 ◽  
Author(s):  
J L Bushman ◽  
M Foiani ◽  
A M Cigan ◽  
C J Paddon ◽  
A G Hinnebusch

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

1993 ◽  
Vol 13 (8) ◽  
pp. 4618-4631
Author(s):  
J L Bushman ◽  
M Foiani ◽  
A M Cigan ◽  
C J Paddon ◽  
A G Hinnebusch

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.


2005 ◽  
Vol 25 (8) ◽  
pp. 3063-3075 ◽  
Author(s):  
Madhusudan Dey ◽  
Bruce Trieselmann ◽  
Emily G. Locke ◽  
Jingfang Lu ◽  
Chune Cao ◽  
...  

ABSTRACT Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2α (eIF2α) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2α, including those at residues flanking Ser51 and around 20 Å away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2α. Thus, two structurally distinct effectors of eIF2 function, eIF2α kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2α.


2010 ◽  
Vol 30 (21) ◽  
pp. 5218-5233 ◽  
Author(s):  
Kamal Dev ◽  
Hongfang Qiu ◽  
Jinsheng Dong ◽  
Fan Zhang ◽  
Dominik Barthlme ◽  
...  

ABSTRACT 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(αP)]. While eIF2B contains five subunits, the ε/Gcd6 subunit is sufficient for GEF activity in vitro. The δ/Gcd2 and β/Gcd7 subunits function with α/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(αP)-GDP, but the essential functions of δ/Gcd2 and β/Gcd7 are not well understood. We show that the depletion of wild-type β/Gcd7, three lethal β/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in β/Gcd7 and eIF2α all impair eIF2 binding to eIF2B without reducing ε/Gcd6 abundance in the native eIF2B-eIF2 holocomplex. Additionally, β/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2α (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, β/Gcd7 can overcome the toxicity of eIF2(αP) and rescue native eIF2B function when overexpressed with δ/Gcd2 or γ/Gcd1. In aggregate, these findings provide compelling evidence that β/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (αP).


2001 ◽  
Vol 21 (15) ◽  
pp. 5018-5030 ◽  
Author(s):  
Thanuja Krishnamoorthy ◽  
Graham D. Pavitt ◽  
Fan Zhang ◽  
Thomas E. Dever ◽  
Alan G. Hinnebusch

ABSTRACT Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNAMet to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its α subunit [eIF2(αP)] 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 α subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2α (glutathioneS-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(αP) 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(αP) 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(αP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the β and γ subunits of eIF2 in the manner required for GDP-GTP exchange.


2005 ◽  
Vol 170 (6) ◽  
pp. 925-934 ◽  
Author(s):  
Susan G. Campbell ◽  
Nathaniel P. Hoyle ◽  
Mark P. Ashe

The eukaryotic translation initiation factor 2B (eIF2B) provides a fundamental controlled point in the pathway of protein synthesis. eIF2B is the heteropentameric guanine nucleotide exchange factor that converts eIF2, from an inactive guanosine diphosphate–bound complex to eIF2-guanosine triphosphate. This reaction is controlled in response to a variety of cellular stresses to allow the rapid reprogramming of cellular gene expression. Here we demonstrate that in contrast to other translation initiation factors, eIF2B and eIF2 colocalize to a specific cytoplasmic locus. The dynamic nature of this locus is revealed through fluorescence recovery after photobleaching analysis. Indeed eIF2 shuttles into these foci whereas eIF2B remains largely resident. Three different strategies to decrease the guanine nucleotide exchange function of eIF2B all inhibit eIF2 shuttling into the foci. These results implicate a defined cytoplasmic center of eIF2B in the exchange of guanine nucleotides on the eIF2 translation initiation factor. A focused core of eIF2B guanine nucleotide exchange might allow either greater activity or control of this elementary conserved step in the translation pathway.


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