Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae

Translation initiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIFS1A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90 % sequence identity, and each is ca. 63 % identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIFS1A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIFS5A and TIFS1B disrupted are not viable, indicating that eIF-5A is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that fo