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

Zeljka McBride, J. Schnier, R. J. Kaufman, J. W.B. Hershey

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

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 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 unhypunisated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vitro 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.

Original languageEnglish (US)
Pages (from-to)18527-18530
Number of pages4
JournalJournal of Biological Chemistry
Volume264
Issue number31
StatePublished - Jan 1 1989

Fingerprint

Peptide Initiation Factors
Proteins
Tetrahydrofolate Dehydrogenase
Puromycin
eukaryotic translation initiation factor 5A
Arginine
Complementary DNA
Mutagenesis
COS Cells
Site-Directed Mutagenesis
Codon
Escherichia coli
Lysine
Transfection

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Protein synthesis initiation factor eIF-4D. Functional comparison of native and unhypusinated forms of the protein. / McBride, Zeljka; Schnier, J.; Kaufman, R. J.; Hershey, J. W.B.

In: Journal of Biological Chemistry, Vol. 264, No. 31, 01.01.1989, p. 18527-18530.

Research output: Contribution to journalArticle

@article{cde1544786a2402694979275f7391fbb,
title = "Protein synthesis initiation factor eIF-4D. Functional comparison of native and unhypusinated forms of the protein",
abstract = "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 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 unhypunisated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vitro 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.",
author = "Zeljka McBride and J. Schnier and Kaufman, {R. J.} and Hershey, {J. W.B.}",
year = "1989",
month = "1",
day = "1",
language = "English (US)",
volume = "264",
pages = "18527--18530",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "31",

}

TY - JOUR

T1 - Protein synthesis initiation factor eIF-4D. Functional comparison of native and unhypusinated forms of the protein

AU - McBride, Zeljka

AU - Schnier, J.

AU - Kaufman, R. J.

AU - Hershey, J. W.B.

PY - 1989/1/1

Y1 - 1989/1/1

N2 - 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 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 unhypunisated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vitro 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.

AB - 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 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 unhypunisated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vitro 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.

UR - http://www.scopus.com/inward/record.url?scp=0024428107&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024428107&partnerID=8YFLogxK

M3 - Article

VL - 264

SP - 18527

EP - 18530

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 31

ER -