Protein synthesis in rabbit reticulocytes: Characteristics of a postribosomal supernatant factor that reverses inhibition of protein synthesis in heme-deficient lysates and inhibition of ternary complex (Met-tRNA_f^Met•eIF-2•GTP) forma

During heme deficiency in reticulocyte lysates, a translational inhibitor (heme-regulated inhibitor, HRI) that blocks polypeptide chain initiation is activated. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAfMet binding factor, eIF-2. Phosphorylation of eIF-2 by HRI prevents its interaction with at least two additional factors, resulting in a net reduction in formation of ternary complex (Met-tRNAfMet eIF-2.GTP) and AUG-dependent transfer of Met-tRNAfMet to 40S ribosomal subunits. A factor isRF) that reverses protein synthesis inhibition in heme-deficient Iysates has been purified from reticulocyte postribosomal supernatant. sRF also reverses the inhibition of ternary complex formation by HRI in a fractionated system. The ternary complex inhibition reversal activity and the protein synthesis inhibition reversal activity cosediment at 12.5 S upon glycerol density gradient centrifugation, and both activities are sensitive to heat or N-ethylmaleirmide. Purified sRF does not dephosphorylate eIF-2 whose phosphorylation has been catalyzed by HRI, nor does the sRF prevent the phosphorylation of eIF-2 by HRI in a fractionated system. sRF stimulates ternary complex formation by both phosphorylated and nonphosphorylated eIF-2. These observations suggest that the sensitivity of protein synthesis to phosphorylation of eIF-2 by HRI may be modulated by the concentration and activity of sRF. During heme deficiency in reticulocyte lysates, protein synthesis shuts off abruptly due to activation of a latent protein synthesis inhibitor, the heme-regulated inhibitor (HRI) (9-19). HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAfMet (tRNAfMet will be abbreviated to tRNAf) binding factor, eukaryotic initiation factor 2 (eIF-2), producing eIF-2(P) (20-24). Phosphorylation of eIF-2 does not inhibit its activity per se but prevents its interaction with at least two factors: Co-eIF-2B (or ternary complex dissociation factor, TDF) (25, 26) and Co-eIF-2C (1, 7). Co-eIF-2B is required for AUG-dependent Met-tRNAf binding to 40S ribosomal subunits (4). Co-eIF-2C relieves the Mg2+ inhibition of ternary complex formation, resulting in an apparent stimulation (1, 7). Factors similar to Co-eIF-2C have been reported by deHaro et al. (27, 28) and Ranu and London (29). Phosphorylation of eIF-2 by HRI thus results in a net reduction of ternary complex formation and loss of efficient Met-tRNAf transfer to 40S ribosomal subunits. The inhibition of protein synthesis in heme-deficient lysates can be overcome by the addition of the ribosomal salt (0.5 M KCI) wash or a partially purified preparation of eIF-2 (30-33). We have reported (8) that this activity (protein synthesis inhibition reversal factor from ribosomes, rRF) is not due to eIF-2 nor is it correlated with eIF-2 activity or concentration. rRF was resolved from the bulk of eIF-2. Also, homogeneous eIF-2 was inactive in reversal of protein synthesis inhibition in hemedeficient lysates. Another factor that reverses protein synthesis inhibition in heme-deficient lysates has been isolated from reticulocyte postribosomal supernatant (34-36). In this paper, we describe further purification and characterization of this factor, protein synthesis inhibition reversal factor from supernatant (sRF). sRF efficiently restores the protein synthesis activity of heme-deficient lysates to the level observed in the presence of hemin and also reverses the inhibition of ternary complex formation by HRI in a fractionated system. The sRF activities for ternary complex inhibition reversal and for protein synthesis inhibition reversal cosediment at 12.5 S upon glycerol density gradient centrifugation, and both activities are sensitive to N-ethylmaleimide and heat. sRF neither dephosphorylates eIF-2(P) nor blocks phosphorylation of eIF-2 by HRI in a fractionated system. sRF stimulates ternary complex formation by both phosphorylated and nonphosphorylated eIF-2. MATERIALS AND METHODS Materials. The materials were obtained from the following sources: [14C]leucine [289-355 mCi/mmol (1 Ci = 3.7 X 1010 becquerels)] from New England Nuclear; [85S]methionine (900 Ci/mmol) from Amersham; [,y-32P]ATP (2.7-3.1 Ci/mmol) from ICN; PM-10 ultrafiltration membranes from Amicon, Lexington, MA; GTP-agarose from Sigma. The sources of most of the other materials used in these studies have been described (8). Preparations of Rabbit Reticulocyte Lysates and Assay of Protein Synthesis. The procedures for the preparation of reticulocytes and reticulocyte lysates and the incubation mixtures for protein synthesis have been described (14, 20). Protein synthesis was assayed by the incorporation of [14C]leucine into hot trichloroacetic acid-insoluble protein. The specific activity Abbreviations: The present nomenclature for the peptide chain eukaryotic initiation factors (eIFs) (1), their past nomenclature (shown in parentheses) (2) as used by our laboratory, and their characteristic functions are: eIF-2 (EIF-1), Met-tRNAfMet binding factor (3, 4); eIF-2(P), phosphorylated eIF-2 (phosphorylation catalyzed by heme-regulated translational inhibitor); Co-eIF-2A (Co-EIF-1), stimulates Met-tRNAfMet binding to eIF-2 (5, 6); Co-eIF-2B (EIF-2, TDF), promotes dissociation of ternary complex at high Mg2+ (4); Co-eIF-2C (EIF-2B), reverses Mg2+ inhibition of ternary complex formation (7); HRI, heme-regulated translational inhibitor; rRF, ribosomal salt wash factor that reverses protein synthesis inhibition in heme-deficient lysates (8); sRF, postribosomal supernatant factor that reverses protein synthesis inhibition in heme-deficient lysates. tRNAf, tRNAfMet; NaDodSO4, sodium dodecyl sulfate; MalNEt, N-ethylmaleimide. * This paper is no. 25 in a series. Paper no. 24 is ref. 1. 5490 The publication costs of this article were defrayed in part by page charge payment This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 76 (1979) 5491 of the ['4C]leucine used is shown in the figure legends. The KC1 concentration in the lysates was 80 mM in all cases. Incubation was at 30'C for 30 min unless otherwise indicated. Three different reaction volumes were used (12, 25, and 50,ul) for convenience in particular experiments. Specific activities of [14C]leucine and aliquot size were adjusted so that the amounts of [14C]leucine incorporated in the aliquots were comparable in all three systems. Sodium Dodecyl Sulfate (NaDodSO4) Gel Electrophoresis and Autoradiography. Electrophoresis of proteins in NaDodSO4 polyacrylamide gels was performed according to the method of Laemmli (37). The gels contained 10% acrylamide (10% acrylamide, 0.27% N,N'-methylenebisacrylamide). The separating gels were formed as 1.5 X 100 X 150-mm slabs, with 1.5-cm stacking gels (5% acrylamide). Electrophoresis was performed at 150 V for 31/2 hr in a Bio-Rad model 220 electrophoresis apparatus. The gels were stained with Coomassie brilliant blue. Autoradiography was performed as described with Kodak X-Omat R film (21). Protein Determination. Protein concentration was determined according to the method of Bradford (38) as modified by Spector (39) with bovine serum albumin as the standard. Preparation of sRF. The sRF was prepared as described by Gross (35), except that 10 mM Tris-HCI, pH 7.8 was used instead of Hepes, and the DEAE-cellulose column was eluted by using 0.1 M, 0.2 M, 0.3 M, and 0.5 M KCI. The bulk of the sRF activity eluted with the 0.2 M KCI wash. The active fractions were pooled and concentrated by ultrafiltration. The concentrated sRF (1.5 ml, 14 mg/ml) was loaded on a 1.2 X 4.8-cm GTP-agarose column equilibrated with buffer A (10 mM Tris-HCI, pH 7.8/1 mM dithiothreitol/0.1 mM EDTA/0.1 M KCI). The bulk of the sRF activity does not bind to the column and elutes in the void volume. The active fractions were pooled and concentrated by dialysis against 80-90% saturated ammonium sulfate as described by Schreier et al. (40). The concentrated sRF was dissolved in buffer A containing 10% (vol/ vol) glycerol at a protein concentration of 4-8 mg/ml and stored at -96°C.