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

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...

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Bibliographic Details
Published in:The Journal of biological chemistry 1997-05, Vol.272 (19), p.12359-12365
Main Authors: Fabian, J R, Kimball, S R, Heinzinger, N K, Jefferson, L S
Format: Article
Language:English
Subjects:
Animals
Chromatography, Gel
Electrophoresis, Polyacrylamide Gel
Eukaryotic Initiation Factor-2 - chemistry
Eukaryotic Initiation Factor-2 - metabolism
Guanine Nucleotide Exchange Factors
Guanine Nucleotides - metabolism
Mice
Phosphorylation
Protein Conformation
Proteins - chemistry
Proteins - metabolism
Rats
Spodoptera
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Summary: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 ε-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 eIF-2B α-subunit. The results demonstrate that eIF-2Bα 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-2Bε is necessary and is perhaps sufficient for GEF activity in vitro .
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.272.19.12359