scholarly journals eIF2B, the guanine nucleotide-exchange factor for eukaryotic initiation factor 2. Sequence conservation between the α, β and δ subunits of eIF2B from mammals and yeast

1996 ◽  
Vol 318 (2) ◽  
pp. 637-643 ◽  
Author(s):  
Nigel T. PRICE ◽  
Harry MELLOR ◽  
Bridget L. CRADDOCK ◽  
Kevin M. FLOWERS ◽  
Scot R. KIMBALL ◽  
...  
Keyword(s):  
Amino Acid ◽  
Translation Initiation ◽  
Guanine Nucleotide Exchange Factor ◽  
Initiation Factor ◽  
Rat Tissues ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Mrna Species ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

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 eIF2Bβ and Δ from rat. This paper focuses on sequence similarities between the α, β and Δ 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 eIF2Bα, β and Δ sequences from mammals and yeast, along with the sequence of the putative eIF2Bα subunit from Caenorhabditis elegans and eIF2BΔ 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 eIF2Bβ and Δ 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 α, β, Δ and ϵ 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.

scholarly journals Cloning and expression of cDNAs for the β subunit of eukaryotic initiation factor-2B, the guanine nucleotide exchange factor for eukaryotic initiation factor-2

1995 ◽  
Vol 309 (3) ◽  
pp. 1009-1014 ◽  
Author(s):  
B L Craddock ◽  
N T Price ◽  
C G Proud
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Mammalian Cells ◽  
Guanine Nucleotide Exchange Factor ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

A key control point in the initiation of protein synthesis in mammalian cells is the recycling of eukaryotic initiation factor (eIF)-2 by the guanine nucleotide exchange factor eIF-2B. In mammalian cells, eIF-2B is a complex of five different subunits termed epsilon, delta, gamma, beta and alpha. To clone cDNAs for the beta subunit of rabbit eIF-2B, amino acid sequence data was first obtained and used to design redundant oligonucleotide primers for use in PCR. PCR products were used to screen a rabbit liver cDNA library in lambda gt11 to obtain full-length cDNAs for eIF-2B beta. The cDNAs were sequenced completely on both strands and revealed an open reading frame encoding a predicted 351-amino acid polypeptide of 39.0 kDa. The molecular mass and pI (5.99) of the predicted protein agree well with the properties of eIF-2B beta purified from rabbit reticulocytes. In vitro transcription/-translation of the cDNAs gave rise to a product that migrated at a position indistinguishable from that of this subunit of the purified protein. The amino acid sequence shows a high degree of similarity to that of GCD7, a Saccharomyces cerevisiae protein thought to be equivalent to mammalian eIF-2B beta. Northern-blot analysis revealed a single major mRNA species for eIF-2B beta in each of the four rabbit tissues tested.

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
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Eukaryotic Translation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation

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.

scholarly journals Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation.

1994 ◽  
Vol 14 (5) ◽  
pp. 3208-3222 ◽  
Author(s):  
C R Vazquez de Aldana ◽  
A G Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Inhibitory Effects ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Suppressor Mutations ◽  
Nucleotide Exchange Factor ◽  
In Cells

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.

scholarly journals Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

2001 ◽  
Vol 21 (15) ◽  
pp. 5018-5030 ◽  
Author(s):  
Thanuja Krishnamoorthy ◽  
Graham D. Pavitt ◽  
Fan Zhang ◽  
Thomas E. Dever ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Tight Binding ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Regulatory Subunits ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor 2

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.

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
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Eukaryotic Translation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation

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.

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