The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells.
When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D007526:Isoelectric Point; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis; D010053:Ovary / Q000032:analysis; D010448:Peptide Initiation Factors / Q000096:biosynthesis*; D010449:Peptide Mapping; D011498:Protein Precursors / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins; D013095:Spermidine / Q000378:metabolism*; D000097574:Eukaryotic Translation Initiation Factor 5A
D008239:Lysine; D010448:Peptide Initiation Factors; D011498:Protein Precursors; D016601:RNA-Binding Proteins; D013095:Spermidine; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine; C034924:deoxyhypusine