In vitro synthesis, phosphorylation, and localization on 48 S initiation complexes of human protein synthesis initiation factor 4E

L. S. Hiremath, S. T. Hiremath, W. Rychlik, S. Joshi, L. L. Domier, R. E. Rhoads

Research output: Contribution to journalArticlepeer-review

Abstract

Complementary DNA for human eukaryotic initiation factor 4E (eIF-4E) was transcribed in vitro and the transcripts used to direct protein synthesis in a cell-free reticulocyte translation system. The predominant translation product was 25 kDa, was bound to a m7HTP-Sepharose affinity column, and was specifically eluted with m7GTP. Both phosphorylated (P) and unphosphorylated (U) forms of eIF-4E were synthesized, and the P/U ration increased as a function of incubation time in the reticulocyte lysate system. Both forms were quantitatively retained on m7GTP-Sepharose. When translation reactions were resolved on sucrose density gradients, the 35S-labeled eIF-4E sedimented predominantly at 3-4 S. However, in the presence of edeine or guanylyl imidodiphosphate, both of which cause accumulation of 48 S initiation complexes, eIF-4E was detected in the 48 S region. In the presence of sparsomycin, used to accumulate 80 S initiation complexes, no eIF-4E was observed in the 80 S region. No change in the eIF-4E distribution was caused by m7GTP. These results are consistent with a model whereby eIF-4E is transferred to the 43 S initiation complex together with mRNA and is released from the initiation complex when the 60 S ribosomal subunit joins.

Original languageEnglish (US)
Pages (from-to)1132-1138
Number of pages7
JournalJournal of Biological Chemistry
Volume264
Issue number2
StatePublished - 1989
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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