A REEVALUATION OF THE CAP-BINDING PROTEIN, EIF4E, AS A RATE-LIMITING FACTOR FOR INITIATION OF TRANSLATION IN RETICULOCYTE LYSATE

The cap-binding eukaryotic initiation factor, eIF4E, is a key target f or the regulation of translation in mammalian cells and is widely thou ght to be present at very low molar concentrations, Here we present ob servations with the reticulocyte lysate that challenge this view, When reticulocyte ribosomes are harvested by centrifugation, most (similar to 75%) of the eIF4E remains in the postribosomal supernatant (PRS), In a reconstituted translation system we find that the ribosome-associ ated eIF4E alone can sustain much of the overall activity, suggesting that much of the factor in the PRS is functionally redundant, Consiste nt with this, our estimates of eIF4E in the reticulocyte lysate reveal much higher concentrations than previously reported, The association of a small proportion of eIF4E with the ribosome fraction appears to b e functional and dependent on interaction with the factor eIF4G, This fraction of eIF4E is, as expected, more highly phosphorylated than tha t in the PRS; however, at least half the total phosphorylated eIF4E in reticulocyte lysate translation systems resides in the PRS fraction, suggesting that, while phosphorylation may enhance activity, it is not in itself sufficient to promote utilization of the factor. We also sh ow that the eIF4E-binding factor, eIF4E-BP1 or PHAS-I, which regulates eIF4E activity in insulin-responsive cells, is present in the reticul ocyte PRS at an approximately 1:1 molar ratio relative to eIF4E and de monstrate by coimmunoprecipitation studies that the binding of PHAS-I and eIF4G to eIF4E is mutually exclusive, These data are consistent wi th a potential regulatory role for PHAS-I in the reticulocyte lysate.