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 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.
Kang
H A
HA
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Schwelberger
H G
HG
Hershey
J W
JW
D001483:Base Sequence; D015153:Blotting, Western; D004277:DNA, Single-Stranded; D005800:Genes, Fungal; D007525:Isoelectric Focusing; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism; D011691:Puromycin / Q000031:analogs & derivatives / Q000096:biosynthesis; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D000097574:Eukaryotic Translation Initiation Factor 5A
D004277:DNA, Single-Stranded; D008239:Lysine; D010448:Peptide Initiation Factors; D011498:Protein Precursors; D011691:Puromycin; D016601:RNA-Binding Proteins; D012694:Serine; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine; C024777:methionylpuromycin