Eukaryotic initiation factor 5A (eIF5A) is a putative translation initiation factor and is the only cellular protein that contains the unique modified Lys, hypusine [N e -(4-amino-2-hydroxybutyl)lysine] [1]. Hypusine is formed post-translationally at one specific Lys residue of the eIF5A precursor in two consecutive enzymatic reactions [2,3]. The first enzyme, deoxyhypusine synthase (DHS) [4,5], catalyzes the transfer of the

The evolutionarily conserved factor eIF5A is the only protein known to undergo hypusination, a unique posttranslational modi¢cation triggered by deoxyhypusine synthase (Dys1). Although eIF5A is essential for cell viability, the function of this putative translation initiation factor is still obscure. To identify eIF5A-binding proteins that could clarify its function, we screened a two-hybrid library and identi¢ed two eIF-5A partners in S. cerevisiae: Dys1 and the protein encoded by the gene YJR070C, named Lia1 (Ligand of eIF5A). The interactions were con¢rmed by GST pulldown. Mapping binding sites for these proteins revealed that both eIF5A domains can bind to Dys1, whereas the C-terminal domain is su⁄cient to bind Lia1. We demonstrate for the ¢rst time in vivo that the N-terminal K K-helix of Dys1 can modulate enzyme activity by inhibiting eIF5A interaction. We suggest that this inhibition be abrogated in the cell when hypusinated and functional eIF5A is required. ß

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site‐directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF‐5A protein‐folding defects. Our data provided evidence that the C‐terminal α‐helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N‐terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature‐sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature‐sensitive strains produced stable mutant eIF5A proteins – eIF5AK56A and eIF5AQ22H,L93F– and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.