Purification of the eukaryotic initiation factor 2-eukaryotic initiation factor 2B complex and characterization of its guanine nucleotide exchange activity during protein synthesis initiation.

Abstract

AM, = 390,000 complex of eukaryotic initiation factor 2 (eIF-2; 3 subunits, M, = 38, 52, and 55 X lo3) and a factor required for catalytic recycling of eIF-2 (eIF-2B; 5 subunits, M, = 26, 39, 58,67, and 82 X lo3) has been purified from rabbit reticulocyte lysate.The eIF-2 eIF-2B complex can be dissociated into free eIF-2B and eIF-2 by high salt or phosphorylation of the M, = 38,000 eIF-2a subunit at physiologic salt concentrations.elF-2 elF-2B association promotes formation of the eIF-2 Met-tRNAi.GTP ternary compIex at physiologic concentrations of GTP and GDP.The basis of this activity appears to be an increase in the KOGDp of eIF-2 from 3.1 X to 1.8 X lo" M when associated with eIF-2B.elF-2B, itself, does not have any intrinsic Met-tRNAi binding activity and binding of the ternary eIF-2.Met-tRNAi*GTP complex to initiating 43 S ribosomal subunits appears to be preceded by eIF-2 * elF-2B dissociation.While the addition of free elF-2 to inhibited hemindeficient reticulocyte lysates restores the synthesis of an equivalent amount of globin, both free eIF-2B and eIF-2.eIF-2Bcatalyze the synthesis of 30-40 pmol of globin/pmol of factor.Alternatively, control rates of protein synthesis in hemin-deficient lysates can be immediately restored by increasing the Concentration of GTP to 1 mM.These data indicate that the inhibition of eIF-2 recycling in hemin-deficient lysates is the result of its altered interaction with eIF-2B which interferes with the GTP exchange required for eIF-2-GDP reactivation.Eukaryotic initiation factor 2 occupies a central role in the mechanism of translational regulation by hemin (1, see reviews in Refs.2-5), the oxidation/reduction state of pyridine nucleotides and thiols (6, 7), the energy charge of the adenine and guanine nucleotide pools (8), amino acid starvation (for review see Ref. 9), and viral infection (see reviews in Refs.2, 10, and 11).In hemin-deficient reticulocyte lysates, phosphorylation of eIF-2' was thought to be directly responsible for the inhibition of protein synthesis initiation (12-17, also see reviews in Refs. 2 and 3).A more complex mechanism is required, however, since phosphorylation of eIF-2a does not directly inhibit formation of the eIF-2 e Met-tRNA, .