Evidence That the Primary Effect of Phosphorylation of Eukaryotic Initiation Factor 2 ( a ) in Rabbit Reticulocyte Lysate Is Inhibition of the Release of Eukaryotic Initiation Factor - aeGDP from 60 S Ribosomal

The phosphorylation of eukaryotic initiation factor (eIF) 2a that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(1 .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 h min and [‘“C] eIF-2 or [CI-~~PIGTP, 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 wg/ml poly(1-C), eIF-2 and GDP binding to 60 S subunits was increased 1.5to 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(1-C) is eIF-2(a-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-tRNAr to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF2-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 eIF2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF2.GDP but not eIF-a(a-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 eIF2a 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).