Phosphorothioated binary complex of eukaryotic initiation factor eIF-2 and GDP inhibits protein synthesis in hemin-supplemented reticulocyte lysates

Abstract

The rabbit reticulocyte heme-regulated eIF-2α kinase (HRI) utilizes adenosine-5′-0-(3-thiotriphosphate) (ATP-γ-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [ eIF-2(α-[ 35S]P)·GDP ], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2·[ 3H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF-2(α-[ 35S]P)·GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2·GDP alone, through the ability of eIF-2(α-P)·GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate.