Protein synthesis initiation factor eIF-4D. Functional comparison of native and unhypusinated forms of the protein

• Published in: The Journal of biological chemistry 1989-11, Vol.264 (31), p.18527-18530
• Main Authors: SMIT-MCBRIDE, Z, SCHNIER, J, KAUFMAN, R. J, HERSHEY, J. W. B
• Format: Article
• Language: English
• Subjects: Arginine - genetics; Biological and medical sciences; Codon; DNA - genetics; Escherichia coli; Escherichia coli - genetics; Eukaryotic Translation Initiation Factor 5A; Fundamental and applied biological sciences. Psychology; Gene Expression; Humans; Lysine - genetics; Lysine - physiology; Molecular and cellular biology; Molecular genetics; Mutation; Nucleic Acid Hybridization; Peptide Initiation Factors - genetics; Peptide Initiation Factors - physiology; Plasmids; RNA-Binding Proteins; Structure-Activity Relationship; Transfection; Translation. Translation factors. Protein processing
• ISSN: 0021-9258; 1083-351X

Protein synthesis initiation factor eIF-4D is a relatively abundant protein in mammalian cells and possesses a unique amino acid residue, hypusine. The role of the hypusine modification in eIF-4D function was addressed by studying the function of eIF-4D variants lacking hypusine. The cloned human cDNA encoding eIF-4D was overexpressed in Escherichia coli and a precursor form lacking hypusine was purified. This protein fails to stimulate methionyl-puromycin synthesis in vitro, nor does it significantly inhibit the action of native eIF-4D. Mammalian expression vectors were constructed with the wild-type cDNA and a mutant form in which the codon for lysine-50 (the residue hypusinated) was altered by site-directed mutagenesis to that for arginine. Transient co-transfection of COS-1 cells with the eIF-4D vector and a vector expressing dihydrofolate reductase led to strong synthesis of both eIF-4D and dihydrofolate reductase. This indicates that normal cellular levels of eIF-4D are saturating in these cells and that excess levels of eIF-4D are not detrimental. Cotransfection with the eIF-4D arginine variant caused no effect on dihydrofolate reductase synthesis, in agreement with the in vitro experiments. The inability of the unhypusinated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vivo strongly suggests that the hypusine modification is required for eIF-4D activity and for its interaction with the 80 S initiation complex in protein synthesis.