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 X GDP and facilitates ternary complex (Met-tRNAf X eIF-2 X GTP) formation in the presence of Mg2+. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the alpha-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF-2 does not displace GDP from eIF-2 alpha (P) X 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 alpha-subunit of eIF-2 in the free and unphosphorylated form and this alpha-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess alpha-subunit of eIF-2 and this alpha-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by eIF-2 X [3H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2 X [3H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNAf X 40S initiation complex formation; (v) both the ternary complex and the Met-tRNAf X 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 alpha-subunit of eIF-2. Based on these observations, we have suggested (a) RF provides the unphosphorylated alpha-subunit to eIF-2 alpha (P) X GDP and restores eIF-2 activity. This RF activity is inhibited as the alpha-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 displacement from eIF-2 X GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 but does not displace GDP from eIF-2 alpha(P) X GDP.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006418:Heme / Q000172:deficiency*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects; D011494:Protein Kinases / Q000502:physiology; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism*; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D006153:Guanosine Diphosphate; D006418:Heme; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D019892:eIF-2 Kinase; D008274:Magnesium