TRANSLATION INITIATION FACTOR-EIF-5A, THE HYPUSINE-CONTAINING PROTEIN, IS PHOSPHORYLATED ON SERINE IN SACCHAROMYCES-CEREVISIAE

Translation initiation factor eIF-5A (formerly called eIF-4D) is a sma ll, highly conserved protein in eukaryotic cells that undergoes a uniq ue modification at one of its lysine residues to form hypusine. eIF-5A stimulates in vitro the synthesis of methionyl-puromycin, a model rea ction for formation of the first peptide bond. In Saccharomyces cerevi siae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF5 1B, and each gene gives rise to two hypusinated isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic). In order to study the structural and functional differences between the two isoforms, both i soelectric forms were purified from a yeast strain overexpressing TIF5 1A and were shown to stimulate identically the synthesis of methionyl- puromycin in a heterologous mammalian assay system. Pulse-chase labeli ng of yeast cells with [S-35]methionine showed that the basic form, eI F-5Ab, is a precursor form of the acidic form, eIF-5Aa. Immunoprecipit ation of P-32-labeled cell lysates with rabbit antibodies specific for yeast eIF-5A, phosphoprotein phosphatase treatment of eIF-5Aa, and ph osphoamino acid analysis demonstrated that eIF-5Aa is generated by pho sphorylation of eIF-5Ab on serine. Therefore eIF-5A undergoes two post -translational modifications, hypusination and phosphorylation, where the activity of the factor is dependent on the first but is not influe nced in vitro by the second.