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Abstract
Translation initiation factor eIF-5A (formerly called eIF-4D) is a small, highly conserved protein in eukaryotic cells that undergoes a unique modification at one of its lysine residues to form hypusine. eIF-5A stimulates in vitro the synthesis of methionyl-puromycin, a model reaction for formation of the first peptide bond. In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B, 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 isoelectric forms were purified from a yeast strain overexpressing TIF51A and were shown to stimulate identically the synthesis of methionyl-puromycin in a heterologous mammalian assay system. Pulse-chase labeling of yeast cells with [35S]methionine showed that the basic form, eIF-5Ab, is a precursor form of the acidic form, eIF-5Aa. Immunoprecipitation of 32P-labeled cell lysates with rabbit antibodies specific for yeast eIF-5A, phosphoprotein phosphatase treatment of eIF-5Aa, and phosphoamino acid analysis demonstrated that eIF-5Aa is generated by phosphorylation 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 influenced in vitro by the second.
Highlights
Translation initiation factor eIF-5A (formerly called unusualaminoacidresidue,hypusine(~-(4-amino-2-hydroxyeIF-4D) is a small, highly conserved protein in eukar- buty1)lysine) (5).Hypusine is formed post-translationally by yotic cells that undergoes a unique modification at one the transfer of an aminobutyl group from spermidine to the of its lysine residues to form hypusine. eIF-5A stimulates in vitro the synthesis of methionyl-puromycin, a model reaction for formation of the firstpeptide bond
In Saccharomyces cerevisiae eIF-SA is encodedby two highly homologous genes, TIF51A and TIF51B, and each gene givesrise to two hypusinated isoelectric
TIF51A and were shown to stimulate identically the mycin synthesis assay (8).we recently showed synthesis of methionyl-puromycin in a heterologous that eIF-5A and its hypusine modification are necessary for mammalianassay system
Summary
Of Buffer A plus 50 mMKC1 was injected intramuscularly with Two Isoelectric Variants of Yeast eIF-5A Stimulate Identi-. When the wild-type yeast strain S173-6B was grown aerobically and labeled with [3H]spermidine and its lysate was analyzed by two-dimensional IEF/SDS-PAGE, we had observed previously two 20-kDa proteins having slightly different isoelectric points (which correspond to eIF-5Aa and eIF5Ab) and a third hypusinated form of lower molecular mass (-18 kDa) having a more basic PI (previously called spot c (9)). The samples were fractionated by 15% SDS-PAGE and thenanalyzed by Western immunoblotting as described for A. cell lysate proteins with polyclonal rabbit antibodies raised Treatment of purified eIF-5A with the phosphatase results in against the yeasetIF-5A protein. Precipitates were fractionated by 15% SDS-PAGE, and an labeled cell lysates as described under “Material and Methautoradiograph of the gel (Fig. 5A) showed a “S-labeled ods.’’ The HCl hydrolysate of 32P-labeled eIF-5Aprotein was protein (lane 2 ) of 20 kDa which is the expectedsize of eIF- separated by two-dimensional electrophoresis on thin-layer.
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