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Volume 272, Number 19, Issue of May 9, 1997 pp. 12359-12365
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Subunit Assembly and Guanine Nucleotide Exchange Activity of Eukaryotic Initiation Factor-2B Expressed in Sf9 Cells

(Received for publication, January 3, 1997)

From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

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 -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 -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 eIF-2B -subunit. The results demonstrate that eIF-2B functions as a regulatory subunit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2B is necessary and is perhaps sufficient for GEF activity in vitro.

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