Purification and Characterization of a Protein Factor that Reverses the Inhibition of Protein Synthesis by the Heme‐Regulated Translational Inhibitor in Rabbit Reticulocyte Lysates

Amesz, Hans; Goumans, Hans; Haubrich‐Morree, Thea; Voorma, Harry O.; Benne, Rob

doi:10.1111/j.1432-1033.1979.tb13212.x

Cited by 107 publications

(53 citation statements)

References 41 publications

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“…For example, in the original work describing the identification of seven initiation factors required for globin mRNA-directed translation initiation (10, 11), eIF4F was not identified as a separate translation initiation factor because the protein was present as a contaminant in purified preparations of eIF3 and eIF4B (45). Likewise, eIF2B forms a stable complex with eIF2 and can be isolated as a stable eIF2⅐eIF2B complex from cell extracts (46,47). These two proteins which have distinct functions in translation initiation can only be separated from each other by high salt glycerol gradient centrifugation (47).…”

Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of Mammalian Translation Initiation Factor 3 (eIF3)

Chaudhuri

Chakrabarti²,

Maitra

1997

Journal of Biological Chemistry

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Eukaryotic translation initiation factor 3 (eIF3), which plays an essential role in initiation of protein synthesis, was purified from rabbit reticulocyte lysates using an assay that specifically measures its ability to stimulate the binding of Met-tRNA f (as a Met-tRNA f ⅐eIF2⅐ GTP ternary complex) to 40 S ribosomal subunits. Purified eIF3 consisted of six major polypeptides of molecular masses 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa polypeptide reported to be a constituent of other eIF3 preparations. Characterization of purified eIF3 lacking the 170-kDa polypeptide showed that the eIF3-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. eIF3, which was originally bound to the 40 S initiation complex, was released from the 40 S subunit during the subunit joining reaction. Additionally, chicken antibodies raised against rabbit reticulocyte eIF3 were used to immunochemically characterize eIF3 subunits and to isolate a 3.1-kilobase pair human cDNA that encodes the p110 subunit of mammalian eIF3. The derived amino acid sequence (calculated M r 95,214) shows that the p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1p, a subunit of yeast eIF3.

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Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of Mammalian Translation Initiation Factor 3 (eIF3)

Chaudhuri

Chakrabarti²,

Maitra

1997

Journal of Biological Chemistry

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“…The exchange of GTP for GDP in the eIF-2 * GDP complex is catalyzed by a multimeric protein factor called eIF-2B (previously called reversing factor or GEF) (1,22,26,33,41). The guanine nucleotide exchange is a prerequisite for eIF-2 to enter another initiation cycle and bind the initiator tRNA, MettRNAi.…”

mentioning

confidence: 99%

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.

Konakanchi¹,

Davies²,

Chen³

et al. 1994

Mol. Cell. Biol.

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The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2a) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2a phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2at subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2ao kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2ot. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2ot 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2a, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2oa impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2a and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2ot inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.In the initiation step of protein synthesis in eukaryotic cells, the joining of the 48S preinitiation complex with the 60S subunit to form the 80S initiation complex is accompanied by the hydrolysis of GTP and the formation of a binary complex of eukaryotic initiation factor 2 (eIF-2) and GDP (eIF-2 GDP) (reviewed in references 15 and 29). The exchange of GTP for GDP in the eIF-2 * GDP complex is catalyzed by a multimeric protein factor called eIF-2B (previously called reversing factor or GEF) (1,22,26,33,41). The guanine nucleotide exchange is a prerequisite for eIF-2 to enter another initiation cycle and bind the initiator tRNA, MettRNAi. eIF-2 is a heterotrimer composed of the a, P, and y subunits. The activity of eIF-2 is an important control point in the regulation of polypeptide chain initiation and is regulated at least i...

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“…However, recently we have found that eIF-2 occurs as a complex with a protein, designated anti-HRI [99]. This complex is 5-6-fold more efficient during methionyl-puromycin formation than eIF-2 alone, resulting in the recycling of eIF-2.…”

Section: Eif4dmentioning

confidence: 99%

“…Since this discovery attempts were made to show that phosphorylated eIF-2 was inactive in initiation of protein synthesis, but no concluding evidence was obtained [24,98]. Phosphorylation of eIF-2 seemed to have neither an effect on its activity in model assay systems [24, 98,99], nor on its capacity to recycle [loo]. It was concluded that the phosphorylation of eIF-2 is not the sole cause of cessation of protein synthesis.…”

Section: Eif4dmentioning

confidence: 99%

Initiation of eukaryotic protein synthesis

1981

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Purification and Characterization of a Protein Factor that Reverses the Inhibition of Protein Synthesis by the Heme‐Regulated Translational Inhibitor in Rabbit Reticulocyte Lysates

Cited by 107 publications

References 41 publications

Biochemical Characterization of Mammalian Translation Initiation Factor 3 (eIF3)

Biochemical Characterization of Mammalian Translation Initiation Factor 3 (eIF3)

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.

Initiation of eukaryotic protein synthesis

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