The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D). Purification of eIF-4D and its precursors and comparison of their activities.
Eukaryotic translation initiation factor 4D (eIF-4D) is the only protein known to contain the amino acid, hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This unusual amino acid is formed post-translationally by modification of a single specific lysine residue in an eIF-4D precursor protein. Two separate eIF-4D precursors, each of which contains a lysine residue in place of the hypusine residue and each of which thereby serves as a protein substrate for the hypusine modification, were purified from DL-2-difluoromethylornithine-treated Chinese hamster ovary cells by means of a five-step procedure. These two precursors termed PI and PII both have apparent molecular masses of approximately 17 kDa, indistinguishable from that of eIF-4D, but exhibit more acidic isoelectric points (5.1 and 5.25 for PI and PII, respectively, compared with 5.37 for eIF-4D). These physical characteristics, together with other properties, indicate that eIF-4D differs from PII only in possessing the hypusine residue in place of a lysine residue, whereas an additional structural difference exists between PI and eIF-4D. eIF-4D from CHO cells provides a significant enhancement of methionyl-puromycin synthesis, a model assay for translation initiation. Neither PI nor PII stimulates this in vitro system. These findings are the first direct evidence that hypusine is essential for the biological activity of eIF-4D.
Park
M H
MH
Laboratory of Cellular Development and Oncology, National Institute of Dental Research, Bethesda, Maryland 20892.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D000518:Eflornithine / Q000494:pharmacology; D007526:Isoelectric Point; D008239:Lysine / Q000031:analogs & derivatives; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification* / Q000378:metabolism; D011498:Protein Precursors / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011700:Putrescine / Q000378:metabolism; D016601:RNA-Binding Proteins; D013095:Spermidine / Q000378:metabolism; D013329:Structure-Activity Relationship; D000097574:Eukaryotic Translation Initiation Factor 5A
D000518:Eflornithine; D008239:Lysine; D010448:Peptide Initiation Factors; D011498:Protein Precursors; D011700:Putrescine; D016601:RNA-Binding Proteins; D013095:Spermidine; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine