FIG. 10.

eIF4AII binding by deletion mutants of eIF4H_i. Deletion mutants of eIF4H_i were previously used to define the regions of eIF4H_i necessary to bind Vhs by the conventional yeast two-hybrid system and in GST pull-down experiments (18). These mutants were now used to examine the region of eIF4H_i required to bind full-length eIF4AII. Full-length wild-type [³⁵S]methionine-labeled eIF4AII was produced by in vitro translation and analyzed for the ability to bind various fusion proteins containing GST fused to full-length eIF4H_i (lane 5) or deletion mutant forms of eIF4H_i (lanes 1 to 4). Full-length eIF4AII bound by the various GST-eIF4H_i polypeptides and eluted with 10 mM glutathione is shown in the autoradiogram in panel A. The material that bound to just GST is shown in lane 6. The eIF4H_i amino acids that were present in the wild-type and various mutant forms of eIF4H_i are shown in parentheses at the top of each lane of the gel. A Coomassie-stained gel of the mutant and wild-type GST-eIF4H_i proteins used in the GST pull-down reactions is shown in panel B. The ability of the eIF4H_i polypeptides to bind Vhs was reported previously (18) and is summarized above panel A. ++, the mutant eIF4H_i polypeptide binds Vhs as well as wild-type eIF4H_i by both the yeast two-hybrid and GST pull-down assays; +, the mutant eIF4H_i polypeptide binds Vhs as well as wild-type eIF4H_i by the yeast two-hybrid assay but binds threefold-less Vhs in GST pull-down experiments; −, there was no detectable interaction between the mutant eIF4H_i and Vhs by either the yeast two-hybrid or GST pull-down assay.