Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits.
The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).
D000818:Animals; D001798:Blood Proteins; D002250:Carbon Radioisotopes; D015852:Eukaryotic Initiation Factor-2; D006150:Guanine Nucleotides / Q000097:blood*; D006153:Guanosine Diphosphate / Q000097:blood*; D006160:Guanosine Triphosphate / Q000097:blood; D007700:Kinetics; D010448:Peptide Initiation Factors / Q000037:antagonists & inhibitors / Q000097:blood*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011506:Proteins / Q000037:antagonists & inhibitors; D012346:RNA, Transfer, Amino Acyl / Q000097:blood; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D001798:Blood Proteins; D002250:Carbon Radioisotopes; D015852:Eukaryotic Initiation Factor-2; D006150:Guanine Nucleotides; D010448:Peptide Initiation Factors; D010761:Phosphorus Radioisotopes; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; C026464:tRNA(m)(Met), methionine-; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate