Eukaryotic translation initiation factor 5 (eIF5) functions as a GTPase activating protein (GAP)
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Translation initiation factor 5 (eIF5) plays an essential role in initiation of protein synthesis in eukaryotic cells. 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 (40S•eIF3•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, Pi and eIF3 from the 40S subunit which is essential for the subsequent joining of the 60S ribosomal subunit to the 40S complex to form a functional 80S initiation complex (40S•AUG•Met-tRNA f) that is active in peptidyl transfer. However, eIF5, by itself, neither binds nor hydrolyzes either free GTP or GTP bound to eIF2 as a Met-tRNA f•eIF2•GTP ternary complex.;The aim of this thesis work is to investigate the mechanism by which eIF5 promotes the hydrolysis of eIF2-bound GTP in the 40S initiation complex. We show that eIF5 forms a complex with eIF2 by interacting with polylysine stretches at the N-terminus of the beta subunit of eIF2. A mutational approach was then used to investigate the importance of eIF5•eIF2beta interaction in eIF5 function. Using binding analyses with recombinant rat eIF5 deletion mutants, we have identified a bipartite motif at 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, 80S initiation complex formation, as well as in their 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 in yeast cells, 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. Additionally, like other typical GTPase Activating Proteins (GAPs), an invariant arginine residue at the N-terminal region of eIF5 is essential for its function. Typical GAPs bind to their respective GTPases and through the invariant arginine residue present in an arginine-finger motif stabilize the transition state and thus allow the hydrolysis of GTP. We have observed that mutation of the invariant arginine residue in eIF5 to alanine or even to lysine causes a severe defect in the ability of eIF5 to promote GTP hydrolysis from the 40S initiation complex. These mutants were also defective in overall protein synthesis as well as in their ability to support cell growth of a DeltaTIF5 yeast strain. These findings suggest that eIF5•eIF2beta interaction plays an essential role in eIF5 function and that eIF5 acts as a GTPase activating protein (GAP) in translation initiation.