Protein synthesis initiation factor eIF-4D. Functional comparison of native and unhypusinated forms of the protein.
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.
Smit-McBride
Z
Z
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Schnier
J
J
Kaufman
R J
RJ
Hershey
J W
JW
D001120:Arginine / Q000235:genetics; D003062:Codon; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000502:physiology* / Q000031:analogs & derivatives; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010448:Peptide Initiation Factors / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D016601:RNA-Binding Proteins; D013329:Structure-Activity Relationship; D014162:Transfection; D000097574:Eukaryotic Translation Initiation Factor 5A
D001120:Arginine; D003062:Codon; D004247:DNA; D008239:Lysine; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine