The 60 S ribosomal subunit as a carrier of eukaryotic initiation factor 2 and the site of reversing factor activity during protein synthesis.
Studies on the recycling of eukaryotic initiation factor 2 (eIF-2) during protein synthesis in normal and heme-deficient reticulocyte lysates indicate that eIF-2 binds physiologically to the 60 S ribosomal subunit. Several findings suggest that the 60 S subunit serves as a carrier for eIF-2 during protein synthesis. The addition of purified eIF-2 (beta-32P) to normal hemin-supplemented lysates results in its binding to polyribosomal 60 S subunits; the binding is temperature-dependent. In lysates inhibited by heme deficiency, phosphorylated eIF-2 alpha can be detected on polyribosomal 60 S subunits early in the initial linear phase of protein synthesis; after polyribosomal disaggregation and shut-off of protein synthesis, phosphorylated eIF-2 alpha accumulates on free 60 S ribosome subunits and on the 60 S subunits of 80 S ribosome couples. The phosphorylated eIF-2 alpha associated with the 60 S subunits in heme-deficient lysates appears to be present as the binary complex [eIF-2 (alpha P) X GDP]; the binding of this complex to the 60 S subunit is tight and is not affected by treatment with 25 mM EDTA or by sedimentation in sucrose gradients. Reversal of the inhibition of protein synthesis in heme-deficient lysates by the addition of reversing factor results in a rapid binding of reversing factor to the 60 S subunits and a concomitant dissociation of [eIF-2(alpha P) X GDP]. These findings suggest that the [eIF-2 X GDP] binary complex formed during the assembly of the 80 S initiation complex binds to the 60 S subunit of polyribosomes and is subsequently released by the action of reversing factor.
D002499:Centrifugation, Density Gradient; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006427:Hemin / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000494:pharmacology; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D012330:RNA, Double-Stranded; D006153:Guanosine Diphosphate; D006418:Heme; D006427:Hemin; D004492:Edetic Acid