title: Protein synthesis in rabbit reticulocytes: mechanism of protein synthesis inhibition by heme-regulated inhibitor.
abstract: Partially purified Met-tRNAf binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography. Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNAf and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg2+ and dissociated by Co-eIF-2B at 5 mM Mg2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-e-IF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNAf binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNAf . 40S . AUG complex to the 60S ribosomal subunit to form Met-tRNAf-80S . AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006418:Heme / Q000502:physiology*; D007700:Kinetics; D008715:Methionine; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D010448:Peptide Initiation Factors; D006418:Heme; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D008715:Methionine