Protein synthesis in rabbit reticulocytes: A study of the mechanism of action of the protein factor RF that reverses protein synthesis inhibition in heme-deficient reticulocyte lysates

A eukaryotic initiation factor 2 (eIF-2)-ancillary protein factor Co-eIF-2 promotes displacement of GDP from eIF-2.GDP and facilitates ternary complex (met-tRNA(f)-eIF-2.GTP) formation in the presence of Mg²⁺. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the α-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF-2 does not displace GDP from eIF-2α(P).GDP. RF, a high molecular weight cell supernatant factor, reverses protein synthesis inhibition in heme-deficient reticulocyte lysates and also reverses HRI inhibition of ternary complex formation. RF contains Co-eIF-2 activity. In addition, an active RF preparation contains excess α-subunit of eIF-2 in the free and unphosphorylated form and this α-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess α-subunit of eIF-2 and this α-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by eIF-2.[³H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2.[³H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNA(f).40S initiation complex formation; (v) both the ternary complex and the Met-tRNA(f).40S complex formation in the presence of HRI and ATP are completely inhibited by prior incubation of RF with GDP; (vi) upon further fractionation of an active RF fraction, a preparation can be obtained that contains HRi-sensitive Co-eIF-2 activity. However, this preparation does not efficiently reverse protein synthesis inhibition in heme-deficient reticulocyte lysates and does not contain excess α-subunit of eIF-2. Based on the observations, we have suggested (a) RF provides the unphosphorylated α-subunit to eIF-2α(P).GDP and restores eIF-2 activity. This RF activity is inhibited as the α-subunit in the RF preparation becomes phosphorylated by HRI and ATP in the presence of GDP; (b) RF contains Co-eIF-2 activity, which has dual functions: (i) stimulation of ternary complex formation by eIF-2 and (ii) GDP displacemet from eIF-2.GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 still stimulates ternary complex formation by unphosphorylated eIF-2 but does not displace GDP from eIF-2α(P).GDP.