Protein synthesis in rabbit reticulocytes: characteristics of Co-eIF-2 protein complex

Abstract

A high molecular weight reticulocyte protein factor, named Co-eIF-2, contains Co-eIF-2A, Co-eIF-2B, and Co-eIF-2C activities and stimulates Met-tRNAf binding to eIF-2 both in the presence and absence of Mg2+. Some characteristics of this stimulation in the absence of Mg2+ are: (1) Stimulation is most pronounced at low eIF-2 levels. (2) Stimulation is partially resistant to heat and NEM treatment, and thus appears to be due to the combined action of both heat and NEM-insensitive Co-eIF-2A, and heat and NEM-sensitive Co-eIF-2C activities. (3) [3H]GDP bound in eIF-2 . [3H]GDP complex is rapidly displaced by unlabelled GTP during ternary complex formation Co-eIF-2 stimulates Met-tRNAf binding to eIF-2 even when added after the [3H]GDP from eIF-2 . [3H]GDP has been completely displaced. This indicates that Co-eIF-2-stimulation is not due to GDP displacement from eIF-2 . GDP. We propose that eIF-2 molecules become inactive in the presence of Mg2+ and at high dilution, and Co-eIF-2 restores the inactive eIF-2 molecules into an active form.

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.

“…The sources of the materials used in these studies were the same as described (1,(14)(15)(16). Unlabeled HPLC-purified Abbreviations: eIF-2, eukaryotic initiation factor 2, which forms ternary complex, Met-tRNAf'eIF-2'GTP; Co-eIF-2, ancillary protein factor that stimulates ternary complex formation by eIF-2; RF, high molecular weight cell supernatant factor that reverses protein synthesis inhibition in heme-deficient reticulocyte lysates; HRI, hemeregulated translational inhibitor; eIF-2a(P), phosphorylated eIF-2 (HRI catalyzed).…”

“…The following mechanism has been proposed for the inhibition of ternary complex formation: A high molecular weight protein factor that we purify from reticulocyte ribosomal salt wash and term Co-eIF-2 (1) [also termed Co-eIF-2C (2), SF (3), eIF-2-RF (4)] is required for ternary complex formation by eIF-2 in the presence of Mg2+. The bulk of eIF-2 is present as eIF-2-GDP (5).…”

“…Co-eIF-2 promotes displacement of GDP from eIF-2-GDP and thus facilitates ternary complex formation (5-10). Co-eIF-2 activity does not displace GDP from eIF2a(P)-GDP (eIF-2 phosphorylated by HRI and ATP) and ternary complex formation is inhibited (1)(2)(3)(4)(5)(6)(7)(8)(9)(10).…”

“…Co-eIF-2 preparations were obtained-after the CM-Sephadex chromatographic step (1,16). eIF-24[3H]GDP was prepared by incubating purified eIF-2 with [3H]GDP as described (1), and the complex formed was further purified by using CMSephadex chromatography.…”

“…Standard Millipore filtration methods were used for assays of eIF-2 and RF activities (1,14,15). Met-tRNAf-40S initiation complex formation was assayed by using the density gradient centrifugation procedure (2).…”