Roles of an eukaryotic initiation factor 2 (eIF-2) associated 67 kDa protein (p(67)) in the regulation of protein synthesis initiation in animal cells

Protein synthesis rapidly shuts off in hemin deficient reticulocyte lysate. It is widely believed that reticulocyte lysate contains a protein synthesis inhibitor in latent form. Under hemin deficiency it becomes activated and phosphorylates the $\alpha$-subunit of eukaryotic initiation factor 2 (eIF-2), thus shutting off protein synthesis. This inhibition can be reversed by the addition of exogenous hemin. Contrary to previous reports, our laboratory has established that this inhibitor, HRI (heme regulated inhibitor), is always active in hemin deficient reticulocyte lysate. But, active HRI can not phosphorylate the eIF-2 $\alpha$-subunit since an eIF-2 associated 67 kDa glycoprotein (p$\sp{67}$) is also present in reticulocyte lysate. This p$\sp{67}$ protects eIF-2 $\alpha$-subunit from kinase catalyzed phosphorylation and thus promotes the protein synthesis in the presence of active eIF-2 kinases such as HRI and dsI (double stranded RNA activated Inhibitor). p$\sp{67}$ is a glycoprotein and contains multiple O-linked GlcNAc moieties. p$\sp{67}$ binds specifically to the eIF-2 $\gamma$-subunit then uses the glycosyl residues to protect the eIF-2 $\alpha$-subunit phosphorylation site from inhibitory phosphorylation by eIF-2 kinases. In hemin deficient reticulocyte lysate, p$\sp{67}$ is rapidly deglycosylated and deglycosylated p$\sp{67}$ can not protect eIF-2 $\alpha$-subunit from eIF-2 kinase catalyzed phosphorylation. The level of control in reticulocyte lysate is at the level of a p$\sp{67}$ deglycosylase present in latent form and activated during hemin deficiency. Another eIF-2 kinase, dsI (double stranded RNA activated inhibitor), plays an important role in protein synthesis regulation during different physiological conditions such as viral infection. eIF-2$\cdot$p$\sp{67}$ complex is resistant to dsI phosphorylation since dsI can not bind to this complex. Autophosphorylated dsI binds to three subunit eIF-2 and then transfers its phosphoryl residue(s) to eIF-2 and forms eIF-2 $\alpha$(P) in an irreversible reaction. dsI dissociates from eIF-2(P), accepts phosphoryl residue from ATP and the cycle continues. When p$\sp{67}$ is bound to three subunit eIF-2 this phosphorylation cycle and consequent dsI phosphorylation is blocked