Subunit assembly and guanine nucleotide exchange activity of eukaryotic initiation factor-2B expressed in Sf9 cells

John R. Fabian, Scot R. Kimball, Nina K. Heinzinger, Leonard S. Jefferson

Research output: Contribution to journalArticle

56 Scopus citations

Abstract

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide exchange factor (GEF) that plays a key role in the regulation of protein synthesis. In this study, we have used the baculovirus-infected Sf9 insect cell system to express and characterize the five dissimilar subunits of rat eIF-2B. GEF activity was detected in extracts of Sf9 cells expressing the E- subunit alone and was greatly increased when all five subunits were coexpressed. In addition, high GEF activity was observed in extracts containing a four-subunit complex lacking the α-subunit. Assembly of an eIF- 2B holoprotein was confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF activity of the five-subunit eIF-2B; this inhibition required the elF-2B α-subunit. The results demonstrate that eIF-2Bα functions as a regulatory sub-unit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2Bε is necessary and is perhaps sufficient for GEF activity in vitro.

Original languageEnglish (US)
Pages (from-to)12359-12365
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number19
DOIs
StatePublished - May 9 1997

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Subunit assembly and guanine nucleotide exchange activity of eukaryotic initiation factor-2B expressed in Sf9 cells'. Together they form a unique fingerprint.

  • Cite this