Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells

Bandyopadhyay, Amitabha; Maitra, Umadas

doi:10.1093/nar/27.5.1331

Cited by 33 publications

(22 citation statements)

References 28 publications

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“…In agreement with this hypothesis, we observed that mammalian eIF5 forms a complex with mammalian eIF2 (7), a component of the 40S initiation complex, and that eIF5-eIF2 complex formation occurs through the ␤ subunit of eIF2 (11). Complex formation between eIF5 and Nip1p subunit of eIF3 has also been reported (1,2).…”

supporting

confidence: 83%

Mutational Analysis of Mammalian Translation Initiation Factor 5 (eIF5): Role of Interaction between the β Subunit of eIF2 and eIF5 in eIF5 Function In Vitro and In Vivo

Das

Maitra

2000

Molecular and Cellular Biology

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Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S-eIF3-AUG-Met-tRNA f -eIF2-GTP) to promote the hydrolysis of ribosome-bound GTP. eIF5 also forms a complex with eIF2 by interacting with the ␤ subunit of eIF2. In this work, we have used a mutational approach to investigate the importance of eIF5-eIF2␤ interaction in eIF5 function. Binding analyses with recombinant rat eIF5 deletion mutants identified the C terminus of eIF5 as the eIF2␤-binding region. Alanine substitution mutagenesis at sites within this region defined several conserved glutamic acid residues in a bipartite motif as critical for eIF5 function. The E346A,E347A and E384A,E385A double-point mutations each caused a severe defect in the binding of eIF5 to eIF2␤ but not to eIF3-Nip1p, while a eIF5 hexamutant (E345A,E346A,E347A,E384A,E385A,E386A) showed negligible binding to eIF2␤. These mutants were also severely defective in eIF5-dependent GTP hydrolysis, in 80S initiation complex formation, and in the ability to stimulate translation of mRNAs in an eIF5-dependent yeast cell-free translation system. Furthermore, unlike wild-type rat eIF5, which can functionally substitute for yeast eIF5 in complementing in vivo a genetic disruption of the chromosomal copy of the TIF5 gene, the eIF5 double-point mutants allowed only slow growth of this ⌬TIF5 yeast strain, while the eIF5 hexamutant was unable to support cell growth and viability of this strain. These findings suggest that eIF5-eIF2␤ interaction plays an essential role in eIF5 function in eukaryotic cells.Eukaryotic initiation factor 5 (eIF5), a monomeric protein of 49 kDa in mammals (9, 10, 21) and 46 kDa in the yeast Saccharomyces cerevisiae (5, 6), plays an essential role in the initiation of protein synthesis. Following scanning of mRNA by the 40S preinitiation complex (40S-eIF3-Met-tRNA f -eIF2-GTP) and positioning of the initiator Met-tRNA f at the AUG codon of the mRNA to form the 40S initiation complex (eIF3-40S-AUG-Met-tRNA f -eIF2-GTP), the initiation factor eIF5 interacts with the 40S initiation complex to effect the hydrolysis of ribosome-bound GTP. Hydrolysis of GTP causes the release of eIF2-GDP, P i , and eIF3 from the 40S initiation complex, which is essential for the subsequent joining of the 60S ribosomal subunit to the 40S complex to form a functional 80S initiation complex (80S-mRNA-Met-tRNA f ) that is active in peptidyl transfer (for reviews, see references 16, 18, and 19). eIF5-dependent GTP hydrolysis has also been shown to play an important role in the selection of the AUG start codon (15).An interesting feature of the derived amino acid sequence of mammalian (rat and human) and yeast eIF5 proteins (26) is the presence of sequence motifs at the N-terminal region of eIF5 that have weak homology to characteristic domains present in proteins belonging to the GTPase superfamily (3). However, unlike these proteins, eIF5 neither binds nor hydrolyzes free GTP or GTP bound to the (Met-tRNA f -eIF2-GTP) ternary complex in the absence of 40S r...

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confidence: 83%

Mutational Analysis of Mammalian Translation Initiation Factor 5 (eIF5): Role of Interaction between the β Subunit of eIF2 and eIF5 in eIF5 Function In Vitro and In Vivo

Das

Maitra

2000

Molecular and Cellular Biology

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“…The 13th subunit of eIF3 was identified as GA17, a protein of unknown function that contains a PCI domain like those found in eIF3a, eIF3c, and eIF3e subunits. eIF3 binds to many other initiation factors, including eIFs 1, 1A, 2, 4B, 4G, and 5 (Methot et al 1996;Bandyopadhyay and Maitra 1999;Fletcher et al 1999;Asano et al 2000;Válasek et al 2002;Olsen et al 2003) and participates in organization of higher order multifactor complexes on the ribosome. However, since all activities of eIF3 involve its interaction with the 40S subunit, it is the single most important of eIF3's partners.…”

Section: Introductionmentioning

confidence: 99%

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

Kolupaeva¹,

Unbehaun²,

Lomakin³

et al. 2005

RNA

115

131

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The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j−, respectively). Although unlike eIF3j+, eIF3j− does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2·GTP·Met-tRNA i Met ternary complex, eIF3j−/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j− nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.

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“…eIF3 is a high molecular weight complex of 11 or more protein subunits that plays an important role in the initiation pathway. The factor helps dissociate 80S ribosomes into ribosomal subunits; it promotes the binding of Met-tRNA i and mRNA to the 40S subunit; and it binds directly to eIF1 [14], eIF4B [15], eIF4G [16] and eIF5 [17], perhaps playing an organizational role on the surface of the ribosome. The eIF3 a-subunit (p170) is present at high levels in human mammary carcinomas and esophageal squamous cell carcinomas [18].…”

Section: Introductionmentioning

confidence: 99%

Malignant transformation by the eukaryotic translation initiation factor 3 subunit p48 (eIF3e)

Mayeur

Hershey

2002

FEBS Letters

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Several components of translation, e.g. eIF4E and PKR, are implicated in cancer. The e-subunit (p48) of mammalian initiation factor 3 is encoded by the Int6 gene, a common site for integration of the mouse mammary tumor virus genome, leading to the production of a truncated eukaryotic initiation factor-3e (eIF3e). Stable expression of a truncated eIF3e in NIH 3T3 cells causes malignant transformation by four criteria: foci formation; anchorage independent growth; accelerated growth; and lack of contact inhibition. Stable expression of full-length eIF3e does not cause transformation. The truncated eIF3e also inhibits the onset of apoptosis caused by serum starvation. ß

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Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells

Cited by 33 publications

References 28 publications

Mutational Analysis of Mammalian Translation Initiation Factor 5 (eIF5): Role of Interaction between the β Subunit of eIF2 and eIF5 in eIF5 Function In Vitro and In Vivo

Mutational Analysis of Mammalian Translation Initiation Factor 5 (eIF5): Role of Interaction between the β Subunit of eIF2 and eIF5 in eIF5 Function In Vitro and In Vivo

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

Malignant transformation by the eukaryotic translation initiation factor 3 subunit p48 (eIF3e)

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