Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein

Zeljka McBride, T. E. Dever, J. W.B. Hershey, W. C. Merrick

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

Protein synthesis initiation factor 4D (eIF-4D) from mammalian cells contains the post-translationally modified lysine derivative hypusine. A highly purified preparation of the protein from rabbit reticulocytes was subjeted to chemical and enzymatic cleavage, and a large number of overlapping peptides were resolved by high performance liquid chromatography and sequenced. Two mixed 14-base DNA probes were synthesized based on suitable amino acid sequences and were used to screen a human cDNA library in λgt11. A cDNA insert containing eIF-4D encoding sequences was identified and a 558-base pair EcoRI-PstI fragment was sequenced. Northern blot hybridization of HeLa cell RNA shows a single size class (1.2 kilobase) of mRNA. The DNA encodes a protein comprising 154 residues with a mass of 16,703 daltons. Human eIF-4D matches all of the rabbit peptides sequenced, extending from residue 9 to 154 except for Cys-129 which is Ser in the rabbit protein. The residue modified to hypusine is identified as Lys-50 and the amino terminus is blocked. eIF-4D possesses rather little secondary structure in the amino-terminal two-thirds of the protein, but the carboxye-terminal third is rich in α helices.

Original languageEnglish (US)
Pages (from-to)1578-1583
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number3
StatePublished - Jan 1 1989

Fingerprint

Cloning
Sequence Analysis
Organism Cloning
Complementary DNA
Rabbits
Proteins
Peptide Initiation Factors
Peptides
Reticulocytes
DNA Probes
High performance liquid chromatography
Gene Library
HeLa Cells
Base Pairing
Northern Blotting
Lysine
Amino Acid Sequence
High Pressure Liquid Chromatography
Cells
hypusine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein. / McBride, Zeljka; Dever, T. E.; Hershey, J. W.B.; Merrick, W. C.

In: Journal of Biological Chemistry, Vol. 264, No. 3, 01.01.1989, p. 1578-1583.

Research output: Contribution to journalArticle

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