Molecular genetic analysis of translation initiation factor 5 (eIF5) from Saccharomyces cerevisiae

Abstract

Eukaryotic translation initiation factor 5 (eIF5), in conjunction with GTP and other initiation factors, plays an essential role in initiation of protein synthesis in eukaryotic cells. In vitro, eIF5 interacts with the 40S initiation complex (40S{dollar}\cdot{dollar}mRNA{dollar}\cdot{dollar}Met{dollar}\cdot{dollar}tRNA{dollar}\sb{lcub}\rm f{rcub}\cdot{dollar}eIF2{dollar}\cdot{dollar}GTP) to mediate the hydrolysis of ribosome-bound GTP, an event that is essential for the subsequent joining of the 60S ribosomal subunit to the 40S complex to form an elongation competent 80S initiation complex (80S{dollar}\cdot{dollar}mRNA{dollar}\cdot{dollar}Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{dollar}).;In Saccharomyces cerevisiae, eIF5 is encoded by a single copy essential gene, TIF5 that encodes a protein of 45,346 Daltons. In this work, the open reading frame of the TIF5 gene has been expressed in Escherichia coli and the recombinant protein purified to apparent electrophoretic homogeneity. The recombinant protein mimics natural eIF5 in size and known biochemical properties thus confirming that the TIF5 gene encodes yeast eIF5. To understand the function of eIF5 in vivo, we constructed a conditional mutant yeast strain in which a functional but a rapidly degradable form of eIF5 fusion protein was synthesized from the repressible GAL10 promoter. Depletion of eIF5 from this mutant yeast strain resulted in inhibition of both cell growth and the rate of in vivo protein synthesis. Analysis of the polysome profiles of eIF5-depleted cells showed greatly diminished polysomes with simultaneous increase in free ribosomes. Furthermore, lysates of cells depleted of eIF5 were dependent on exogenously added yeast eIF5 for efficient translation of mRNAs in vitro. Additionally, we have also isolated a conditional Ts- mutant by replacing the wild-type chromosomal copy of the gene with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis at the non-permissive temperature with concomitant loss of polyribosomes and accumulation of free 80S ribosomes. Furthermore, cell-free translation extracts of Ts- mutant cells, preincubated at 37{dollar}\sp\circ{dollar}C, were dependent on exogenously added eIF5 for translation of mRNAs in vitro. We also show that mammalian eIF5 can functionally substitute for yeast eIF5 in translation of mRNAs in vitro as well as in complementing in vivo a genetic disruption in the chromosomal copy of TIF5. Taken together, these results provide the first direct evidence that the TIF5 gene encodes a protein (eIF5) that has an essential role in initiation of protein synthesis in eukaryotic cells.;Experiments designed to understand the mechanism of action of eIF5 in translation initiation have been carried out. Mutational analysis of the sequence motifs present in eIF5 that are characteristic of proteins of the GTPase superfamily indicated that these putative domains do not play a critical role in the function of eIF5 in vivo. We postulate that eIF5 does not function as a GTPase protein. Rather, the protein functions by activating the intrinsic GTPase activity of eIF2 bound to the 40S ribosomal subunit.;We also show that yeast eIF5 is constitutively phosphorylated on multiple serine residues by casein kinase II. However, mutations of serine residues at the consensus casein kinase II sequences in TIF5 that abolish phosphorylation of eIF5 in yeast cells did not cause any apparent deleterious effects on cell growth and viability, and hence did not presumably affect eIF5 function in vivo in any significant way under normal growth conditions. Thus the functional significance of phosphorylation of eIF5 in yeast cells is presently unknown.