MECHANISM OF ACTION OF DEVELOPMENTALLY-REGULATED SEA-URCHIN INHIBITOR OF EIF-4

The developmentally regulated inhibitor of elF-4 function found in unfertilized sea urchin eggs has been partially purified and its mechanism of action studied in vitro using purified recombinant elF-4 alpha and cell-free translation systems. The results demonstrate that although the phosphorylation of elf-4 alpha is necessary to promote protein synthesis, it is not sufficient to maintain all aspects of elF-4 function. The egg inhibitor does not change elF-4 alpha. phosphorylation state. During the blockage of initiation caused by the egg inhibitor, elF-4 alpha remains phosphorylated but accumulates in a 48S initiation intermediate. This suggests that the egg inhibitor functions by preventing the release of elF-4 alpha from the small ribosomal subunit. The characteristics of the inhibitor in a reticulocyte translation system demonstrate that elF-4 activity is inhibited within 3-6 min. However, the inhibitor's characteristics in a mRNA-dependent translation system contrast with this. Preincubation with the inhibitor for 5-25 min prior to the addition of mRNA does not prevent endogenous elF-4 from participating in translation but diminishes its ability to be reutilized, consistent with the accumulation of elf-4 alpha on the small ribosomal subunit. The ribosomal localization of the inhibitor suggests that it could prevent elF-4 alpha release by direct binding. The gradual inactivation of the inhibitor following fertilization indicates that it represents a component of a novel regulatory cascade that modulates elF-4 activity. (C) 1993 Wiley-Liss, Inc.