title: A reevaluation of the cap-binding protein, eIF4E, as a rate-limiting factor for initiation of translation in reticulocyte lysate.
abstract: The cap-binding eukaryotic initiation factor, eIF4E, is a key target for the regulation of translation in mammalian cells and is widely thought to be present at very low molar concentrations. Here we present observations with the reticulocyte lysate that challenge this view. When reticulocyte ribosomes are harvested by centrifugation, most (approximately 75%) of the eIF4E remains in the postribosomal supernatant (PRS). In a reconstituted translation system we find that the ribosome-associated eIF4E alone can sustain much of the overall activity, suggesting that much of the factor in the PRS is functionally redundant. Consistent 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 be functional and dependent on interaction with the factor eIF4G. This fraction of eIF4E is, as expected, more highly phosphorylated than that 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 show that the eIF4E-binding factor, eIF4E-BP1 or PHAS-I, which regulates eIF4E activity in insulin-responsive cells, is present in the reticulocyte PRS at an approximately 1:1 molar ratio relative to eIF4E and demonstrate by co-immunoprecipitation studies that the binding of PHAS-I and eIF4G to eIF4E is mutually exclusive. These data are consistent with a potential regulatory role for PHAS-I in the reticulocyte lysate.
authors.affiliation: Department of Biochemistry, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Rau
authors.forename: T
authors.initials: T
authors.lastname: Ohlmann
authors.forename: S J
authors.initials: SJ
authors.lastname: Morley
authors.forename: V M
authors.initials: VM
authors.lastname: Pain
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D002451:Cell Compartmentation; D002474:Cell-Free System; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D010750:Phosphoproteins / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D010448:Peptide Initiation Factors; D010455:Peptides; D010750:Phosphoproteins; D012315:RNA Caps