REGULATION OF PROTEIN SYNTHESIS INITIATION IN RABBIT RETICULOCYTES

Protein synthesis in rabbit reticulocytes involves participation of several protein factors which include peptide chain initiation factors and initiation inhibitors. An inhibitor, TDI (Ternary Complex Dissociation Inhibitor), has been isolated from reticulocyte supernatant. TDI inhibits one of the peptide chain initiation factor activity, TDF (Ternary Complex Dissociation Factor), and the transfer of initiator t-RNA (Met-tRNA(,f)) to 40S ribosomes. This inhibition is also observed when GTP is replaced by its non-hydrolyzable analog, GMPPNP. Further studies on TDI revealed that TDI is functionally analogous to the widely studied protein synthesis inhibitor, HRI (Heme Regulated Inhibitor). Like TDI, HRI also inhibits TDF activity and Met-tRNA(,f) transfer into 40S ribosomes. HRI, a c-AMP independent protein kinase, is activated in reticulocyte lysates in absence of hemin and shuts off protein synthesis after an initial lag period of a few minutes. HRI specifically phosphorylates the smallest (38,000 dalton) subunit of the key initiation factor, eIF-2, which forms a ternary complex Met-tRNA(,f) eIF-2 GTP as the first step of eukaryotic peptide chain initiation. TDF (also termed Co-eIF-2B) dissociates this ternary complex in the presence of high Mg(\u27++) (5 mM) and low temperature (0(DEGREES)C) and also catalyses the transfer of Met-tRNA(,f) to 40S ribosomes in presence of AUG codon. The interaction of eIF-2 and Co-eIF-2B is abolished by HRI in presence of ATP. Non-hydrolyzable analog of ATP, AMPPNP, does not substitute for ATP. HRI (in presence of ATP) inhibits the formation of ternary complex in partially purified eIF-2 preparations but not in purified eIF-2 preparations. With purified eIF-2 preparations, however, a strong inhibition of ternary complex formation was observed in presence of low Mg(\u27++) (concentrations (0.5 mM). A protein factor, Co-eIF-2C, was isolated from partially purified initiation factor preparations that relieved the Mg(\u27++)-inhibition of eIF-2 activity (measured in terms of its ability to form ternary complex in absence of Mg(\u27++)). Co-eIF-2C is a high molecular weight (approx. mol. wt. 350,000 daltons), heat labile protein. The inhibition of ternary complex formation by HRI in partially purified preparations was due to the loss of interaction of eIF-2 and Co-eIF-2C. To ascertain whether only HRI catalyzed phophorylation of eIF-2 or phosphorylation of other component(s) are involved in the loss of interaction of eIF-2 with Co-eIF-2B (TDF) and Co-eIF-2C, eIF-2(P) {phosphorylated eIF-2, HRI catalysed}, free of HRI, was isolated by phosphocellulose column chromatography. Whereas the ternary complex formed by eIF-2 was strongly dissociated by Co-eIF-2B (TDF) and Mg(\u27++)-inhibition of eIF-2 activity was reversed by Co-eIF-2C, the ternary complex formed by eIF-2(P) was not dissociated by Co-eIF-2B (TDF) and Mg(\u27++)-inhibition of ternary complex formation by eIF-2(P) was not reversed by Co-eIF-2C. Since these reactions required no further addition of ATP or HRI or both, the results suggest that HRI induced phosphorylation of eIF-2 results in its loss of interaction with two protein factors, Co-eIF-2B and Co-eIF-2C, thus inhibiting ternary complex formation and Met-tRNA(,f)(.)40S ribosome complex formation and consequently protein synthesis. Using preformed 40S initiation complex, HRI was found to have no effect on the formation of 80S initiation complex