Mutational analysis of mammalian translation initiation factor 5 (eIF5): role of interaction between the beta subunit of eIF2 and eIF5 in eIF5 function in vitro and in vivo.
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 beta subunit of eIF2. In this work, we have used a mutational approach to investigate the importance of eIF5-eIF2beta interaction in eIF5 function. Binding analyses with recombinant rat eIF5 deletion mutants identified the C terminus of eIF5 as the eIF2beta-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 eIF2beta but not to eIF3-Nip1p, while a eIF5 hexamutant (E345A,E346A, E347A,E384A,E385A,E386A) showed negligible binding to eIF2beta. 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 DeltaTIF5 yeast strain, while the eIF5 hexamutant was unable to support cell growth and viability of this strain. These findings suggest that eIF5-eIF2beta interaction plays an essential role in eIF5 function in eukaryotic cells.
Das
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Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.
Maitra
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D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001156:Artemia; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5; D005656:Fungal Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008322:Mammals; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012269:Ribosomal Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins
D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5; D005656:Fungal Proteins; C077915:NIP1 protein, S cerevisiae; D009687:Nuclear Proteins; D010448:Peptide Initiation Factors; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C039913:ribosomal protein S80; D006160:Guanosine Triphosphate; D000409:Alanine