Protein synthesis in rabbit reticulocytes: characteristics of the protein factor RF that reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates.

Abstract

During heme deficiency in reticulocyte lysates, the heme-regulated translational inhibitor of protein synthesis (HRI) is activated and shuts off protein synthesis. In partial reactions, HRI phosphorylates the MW 38,000 subunit (.alpha. subunit) of eukaryotic initiation factor 2 (eIF-2), which forms a ternary complex, Met-tRNA.cntdot.eIF-2.cntdot.GTP. The eIF-2.alpha.(P) thus formed is not recognized by 2 eIF-2 ancillary factors, Co-eIF-2B (which promotes the dissociation of the ternary complex at high Mg2+) and Co-eIF-2C (which reverses the inhibition of ternary complex formation), and thus, is presumably inactive in peptide chain initiation. A protein factor, designated RF, which reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates, was purified from reticulocyte cell supernatant. RF is a high MW (MW .simeq. 450,000) protein complex composed of multiple polypeptides. An active RF preparation contains Co-eIF-2B and Co-eIF-2C activities, and these 2 activities in RF preparation are not inhibited by HRI and ATP, i.e. eIF-2.alpha.(P) is recognized. During purification, RF remains associated with eIF-2 activity (eIF-2.cntdot.RF) and can be freed of this eIF-2 activity by CM-Sephadex chromatography. Both eIF-2.cntdot.RF and RF contain a MW 38,000 polypeptide component that is indistinguishable from the Mr 38,000 subunit of eIF-2 by 2-dimensional gel electrophoresis. A significant part of this MW 38,000 polypeptide component in eIF-2.cntdot.RF and almost the entire MW 38,000 polypeptide component in RF remain unphosphorylated after prolonged incubation with HRI and ATP. A possible role of this free MW 38,000 polypeptide in RF action is discussed.