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

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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

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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