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 excha nge factor (GEF) that plays a key role in the regulation of protein sy nthesis, In this study, we have used the baculovirus-infected Sf9 inse ct cell system to express and characterize the five dissimilar subunit s of rat eIF-2B, GEF activity was detected in extracts of Sf9 cells ex pressing the epsilon-subunit alone and was greatly increased when all five subunits were coexpressed, In addition, high GEF activity was obs erved in extracts containing a four subunit complex lacking the alpha- subunit. Assembly of an eIF-2B holoprotein was confirmed by coimmunopr ecipitation of all five subunits, Gel filtration chromatography reveal ed that recombinant eIF-2B had the same molecular mass as eIF-2B purif ied from rat liver and that it did indeed possess GEF activity, Phosph orylation of the substrate eIF-2 inhibited the GEF activity of the fiv e-subunit eIF-2B; this inhibition required the eIF-2B alpha-subunit, T he results demonstrate that eIF-2B alpha functions as a regulatory sub unit that is not required for GEF activity, but instead mediates the r egulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2B epsilon is necessary and is perhaps sufficient for GEF activity in vit ro.