Identification of domains within the epsilon-subunit of the translation initiation factor eIF2B that are necessary for guanine nucleotide exchange activity and eIF2B holoprotein formation.
Eukaryotic translation initiation factor, eIF2B, is a guanine nucleotide exchange factor (GEF) composed of five dissimilar subunits. eIF2B is important for regenerating GTP-bound eIF2 during the initiation process. This event is obligatory for eIF2 to bind initiator methionyl-tRNA, forming the ternary initiation complex. In the current investigation, deletion mutants of the catalytic subunit, eIF2B epsilon, were constructed to identify regions that are necessary for eIF2B catalytic activity and formation of the holoprotein. We used the baculovirus expression system to coexpress wild-type and truncated forms of the epsilon-subunit of mammalian eIF2B (eIF2B epsilon) with the other four subunits (alpha, beta, gamma, delta) of the protein in Sf9 cells. Removal of either the N- or the C-terminal conserved domains of eIF2B epsilon resulted in a significant loss of GEF activity and reduced or abolished interaction with the alpha-, gamma- and delta-subunits of eIF2B. Removal of the C-terminal 552 amino acids of eIF2B epsilon markedly reduced its interaction with the beta-subunit of eIF2 whereas loss of the N-terminal 431 amino acids did not. The results suggest that intact eIF2B epsilon is required for full catalytic activity and formation of the eIF2B holoprotein. In contrast, the C-terminal domain of eIF2B epsilon is sufficient alone for binding the beta-subunit of its substrate, eIF2, in vitro.
Anthony
T G
TG
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
Fabian
J R
JR
Kimball
S R
SR
Jefferson
L S
LS
D000704:Analysis of Variance; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D007313:Insecta; D011487:Protein Conformation; D051381:Rats
D020717:Eukaryotic Initiation Factor-2B; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides