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During heme deficiency in reticulocyte lysates, the heme-regulated protein synthesis inhibitor, HRI, phosphorylates the a subunit of eukaryotic initiation factor 2 (eIF-2) and thus inhibits protein synthesis. Two factors, eIF-2 and a reticulocyte-lysate supernatant factor that we term RF, reverse this inhibition. We now report the following. (i) An active eIF-2 preparation contained, in addition to the three subunits (α,β, and γ), a 67-kDa polypeptide. Pretreatment of eIF-2 with polyclonal antibodies against either isolated a subunit or 67-kDa polypeptide almost completely inhibited the reversal activity. Upon further fractionation, three-subunit eIF-2 and the 67-kDa polypeptide were resolved. Neither the three-subunit eIF-2 nor the 67-kDa polypeptide alone was active in protein synthesis inhibition reversal. The activity was, however, restored by combining both the three-subunit eIF-2 and the 67-kDa polypeptide. (ii) Active RF preparations contained eIF-2 a (unphosphorylated) and (3 subunits and the 67-kDa polypeptide. As with eIF-2, prior treatment of the RF preparation with antibodies to either the a subunit or the 67-kDa polypeptide almost completely inhibited the reversal activity. The RF preparation devoid of eIF-2 y subunit did not form ternary complex (Met-tRNAfMet•eIF-2•GTP). The eIF-2 γ subunit in the free form was isolated, and addition of this isolated γ subunit to RF promoted significant ternary-complex formation. (iii) Purified HRI efficiently phosphorylated the a subunit in the three subunit eIF-2. However, the extent of such phosphorylation was significantly reduced when eIF-2 containing the 67-kDa polypeptide was used. The 67-kDa polypeptide apparently protected eIF-2 a subunit from HRI-catalyzed phosphorylation but did not inhibit HRI activity. Based on these results, we suggest that the protein synthesis inhibition reversal activity in both eIF-2 and RF is due to the same components-namely, eIF-2 a subunit and the 67-kDa polypeptide. The 67-kDa polypeptide protects eIF-2 a subunit from HRI-catalyzed phosphorylation and may also be a necessary component of the functioning eIF-2 molecule.