Characterization of Mammalian eIF2A and Identification of the Yeast Homolog

Wendy L Zoll,Lynn E Horton,Anton A Komar,Jack O Hensold,William C Merrick

doi:10.1074/jbc.m207109200

Wendy L Zoll, Lynn E Horton + Show 3 more

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https://doi.org/10.1074/jbc.m207109200

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Abstract

To begin the physical characterization of eukaryotic initiation factor (eIF) 2A, a translation initiation factor that binds Met-tRNA(i), tryptic peptides from rabbit reticulocyte eIF2A were analyzed to obtain amino acid sequence information. Sequences for 8 peptides were matched to three different expressed sequence tag clones. The sequence predicted for eIF2A is 585 amino acids. Matching of the cDNA sequence to the human genome revealed that the eIF2A mRNA is made up of 15 or 16 exons, and the gene is contained on chromosome 3. A homolog in Saccharomyces cerevisiae was identified, YGR054W, which is a non-essential gene. Hemagglutinin-tagged yeast eIF2A localizes on both 40 S and 80 S ribosomes. A knockout of both eIF2A and eIF5B yielded a "synthetically sick" yeast strain with a severe slow growth phenotype. The phenotype of this double mutant and the biochemical localization suggest that eIF2A participates in translation initiation. eIF2A does not appear to participate in re-initiation as the DeltaeIF2A strain shows the same level of GCN4 induction with amino acid starvation as seen in wild type yeast. The lack of any apparent phenotype in the DeltaeIF2A strain suggests that eIF2A functions in a minor pathway, perhaps internal initiation or in the translation of a small number of specific mRNAs.

Highlights

To begin the physical characterization of eukaryotic initiation factor 2A, a translation initiation factor that binds Met-tRNAi, tryptic peptides from rabbit reticulocyte Eukaryotic initiation factor 2A (eIF2A) were analyzed to obtain amino acid sequence information
Unlike IF2 or eIF2A, eIF2 was unable to catalyze poly(U)directed polyphenylalanine synthesis, it could direct the formation of puromycin-sensitive 80 S preinitiation complexes with Met-tRNAi [2]. eIF2A and IF2 have been shown to bind initiator tRNA in a GTP-independent manner, but require the presence of the small ribosomal subunit and the trinucleotide start codon, AUG
Both eIF2 and eIF2A were active in the methionyl-puromycin synthesis assay and have the same complementary factor and nucleotide requirements. eIF2 was found to out-compete eIF2A in the methionyl-puromycin synthesis assay when both proteins were present [4]

Summary

Introduction

To begin the physical characterization of eukaryotic initiation factor (eIF) 2A, a translation initiation factor that binds Met-tRNAi, tryptic peptides from rabbit reticulocyte eIF2A were analyzed to obtain amino acid sequence information. The lack of any apparent phenotype in the ⌬eIF2A strain suggests that eIF2A functions in a minor pathway, perhaps internal initiation or in the translation of a small number of specific mRNAs. Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that catalyzes the formation of puromycin-sensitive 80 S preinitiation complexes and the poly(U)-directed synthesis of polyphenylalanine at low [Mg2ϩ] [1]. Unlike IF2 or eIF2A, eIF2 was unable to catalyze poly(U)directed polyphenylalanine synthesis, it could direct the formation of puromycin-sensitive 80 S preinitiation complexes with Met-tRNAi [2]. EIF2 formed a ternary complex composed of Met-tRNAi and GTP, a step that did not require AUG [3] Both eIF2 and eIF2A were active in the methionyl-puromycin synthesis assay and have the same complementary factor and nucleotide requirements (eIF1A, eIF5A, eIF5B, and GTP). Both eIF2 and eIF2A were active in the methionyl-puromycin synthesis assay and have the same complementary factor and nucleotide requirements (eIF1A, eIF5A, eIF5B, and GTP). eIF2 was found to out-compete eIF2A in the methionyl-puromycin synthesis assay when both proteins were present [4]

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