Mechanism of action of developmentally regulated sea urchin inhibitor of eIF-4.
The developmentally regulated inhibitor of eIF-4 function found in unfertilized sea urchin eggs has been partially purified and its mechanism of action studied in vitro using purified recombinant eIF-4 alpha and cell-free translation systems. The results demonstrate that although the phosphorylation of eIF-4 alpha is necessary to promote protein synthesis, it is not sufficient to maintain all aspects of eIF-4 function. The egg inhibitor does not change eIF-4 alpha phosphorylation state. During the blockage of initiation caused by the egg inhibitor, eIF-4 alpha remains phosphorylated but accumulates in a 48S initiation intermediate. This suggests that the egg inhibitor functions by preventing the release of eIF-4 alpha from the small ribosomal subunit. The characteristics of the inhibitor in a reticulocyte translation system demonstrate that eIF-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 eIF-4 from participating in translation but diminishes its ability to be reutilized, consistent with the accumulation of eIF-4 alpha on the small ribosomal subunit. The ribosomal localization of the inhibitor suggests that it could prevent eIF-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 eIF-4 activity.
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Center of Marine Biotechnology, Baltimore, MD 21202.
Huang
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Hiremath
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Stern
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Rhoads
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D000818:Animals; D005260:Female; D005306:Fertilization / Q000235:genetics / Q000502:physiology; D014644:Genetic Variation; D009865:Oocytes / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012617:Sea Urchins / Q000235:genetics / Q000378:metabolism
D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C075833:eIF-4