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

Abstract

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 for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.Eukaryotic initiation factor 5A (eIF-5A; previously named eIF-4D) (initiation factors are named according to the revised nomenclature recommended by the International Union of Biochemistry [37]) is one of a number of protein factors that stimulate the initiation phase of protein synthesis (29). The purified protein from mammalian cells is small (16 to 18 kDa) and acidic (pI = 5.4) and is one of the most abundant of the initiation factors (2, 21). eIF-5A is distinguished by possession of a unique residue, hypusine [N--(4-amino-2-hydroxybutyl)-lysine], formed posttranslationally by transfer of a butylamino group from spermidine to a specific lysine followed by a hydroxylation reaction (9, 33). The eIF-5A protein and its hypusine modification are highly conserved from yeasts to humans (12), suggesting an important role in protein synthesis, cellular metabolism, or both. eIF-5A appears to function in protein synthesis by promoting formation of the first peptide bond, a reaction usually studied in vitro by a ...

Polyamines and cancer: old molecules, new understanding

“…In the early 1980s, Myung Park and collaborators demonstrated that the polyamine spermidine was the essential substrate for the posttranslational modification of the putative eukaryotic translation inhibition factor 5A (eIF5A) 103 . Subsequent studies have shown that the Saccharomyces cerevisiae equivalent of eIF5A is an essential gene in this yeast 104 , but is not essential for general protein synthesis 105,106 . Rather, eIF5A seems to be involved in the processing of specific RNAs 107,108 .…”

Crystal structure of human eIF5A1: Insight into functional similarity of human eIF5A1 and eIF5A2

“…Knock-out of either the eif5a or dhs gene is fatal in yeast. 2,3,14 Two eIF5A isoforms, eIF5A1 and eIF5A2, occur in humans and have 84% identity in their amino acid sequence. [15][16][17][18] The eif5a1 gene is ubiquitously expressed, whereas eif5a2 is highly expressed in the testis and at moderate levels in the brain.…”

Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae

“…Inhibition of deoxyhypusine synthase activity by chemicals [4] or by limiting the level of the substrate spermidine [5] arrests the growth of eukaryotic cells, suggesting that this enzyme may play a pivotal role in cell growth. It has been shown by mutational analysis that preservation of the site of hypusination of eIF-5A (Lys 51 in the yeast precursor) is vital for the factor's function(s) [6]. Yeast contains two forms of 5A, designated eIF-5Aa and eIF-5Ab, derived from two distinct genes reciprocally regulated by oxygen [6].…”