Protein synthesis in rabbit reticulocytes: Characteristics of a ribosomal factor that reverses inhibition of protein synthesis

A ribosomal salt (0.5 M KCI) wash factor (RF) that reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates has been resolved from the bulk of MettRNAfmet-binding factor (EIF-1), Co-EIF-I, and EIF-2 (ternary complex dissociation factor, TDF). The purified RF restores protein synthesis activity of heme-deficient lysates to the level observed in the presence of hemin. No direct correlation exists between amount of EIF-i activity and ability to reverse inhibition of protein synthesis in heme-deficient lysates. Homogeneous preparations of EIF-i are completely inactive in reversal of protein synthesis inhibition in heme-deficient lysates. These findings suggest that RF activity is not due to EIF-i alone but may or may not require EIF-i as a component of a complex factor. Protein synthesis in reticulocyte lysates is regulated by heme (4-6). In heme deficiency, there is rapid activation of a translational inhibitor [HRI, heme-reversible inhibitor, also called HCR (7)] (7-9) that blocks protein chain initiation (5, 6, 8-13). The translational inhibitor HRI has now been extensively purified (14) and has been shown to be a cyclic-AMP-independent protein kinase that specifically phosphorylates the 38,000-dalton subunit of the peptide chain initiation factor EIF-1 (14-18). Recent reports indicate that HRI-phosphorylated EIF-I may not be recognized by the initiation factors EIF-2 (TDF) (19-20) and also possibly Co-EIF-1 (20, 21), and is inactive in the formation of the initiation complex of Met-tRNAfMet.40S ribosomal subunit (Met-tRNAf-40S) (19-22). An interaction between EIF-1, EIF-2, and Co-EIF-i is presumably necessary for the formation of 40S initiation complex (1, 23). Protein synthesis inhibition in heme-deficient lysates can be overcome by addition of the ribosomal salt (0.5 M KCI) wash or a partially purified preparation of EIF-1 (24-27). This observation and the recent reports of possible inactivation of EIF-1 by HRI-induced phosphorylation of the smallest subunit of EIF-1 (19-22) have led to the current belief that HRI inactivates EIF-1 by phosphorylation, thus inhibiting protein synthesis, and addition of exogeneous EIF-1 overcomes this inhibition. In this paper, we describe the purification of a factor (RF) from the 0.5 M KCl ribosomal salt wash that reverses protein synthesis inhibition in heme-deficient lysates. The RF activity has been resolved from the bulk of EIF-1 activity. Purified RF very efficiently restores protein synthesis in heme-deficient lysates. Equivalent amounts of EIF-1 activity at various stages of purification have little or no ability to reverse protein synthesis inhibition in heme-deficient lysates. Homogeneous EIF-1 preparations have no inhibition reversal activity at any concentration tested. MATERIALS AND METHODS Materials. The materials were obtained from the following sources: ['4C]leucine (294 Ci/mol) from New England Nuclear; [85S]methionine (970 Ci/mmol) from Amersham/Searle; DEAE-cellulose (DE-li and DE-52) and phosphocellulose (P-il) from Whatman. Preparation of Rabbit Reticulocyte Lysates and Protein Synthesis Assay. Rabbit reticulocyte lysates were prepared according to the method of Hunt et al. (9). Preparation of incubation mixtures and measurements of protein synthesis were performed according to the methods of Ranu and London (14). The concentration of exogeneous KC1 in the reaction mixture was adjusted to 136 mM unless otherwise stated. The KC1 optimum for our lysates is approximately 100 mM. The higher KC1 concentration (136 mM) was used in all cases because this is the concentration that exists in the presence of maximum addition of our factors. At 136mM KC1, protein synthesis in our lysates is inhibited only slightly (less than 10%) during the 40-min incubation period, compared to protein synthesis in the presence of 100 mM KC1. Several different lysates were used during the course of these experiments. These lysates varied in their abilities to incorporate ['4C]leucine in the absence of hemin. However, the characteristics of the effects of hemin and the various factors on protein synthesis in these lysates were essentially identical. Also, all of the comparisons between hemin and the various factors were made with a single lysate in a single experiment. Protein synthesis was assayed by the incorporation of [14C]leucine into protein in 5-,l aliquots at 30"C; 1.2 nmol of [14C]leucine (specific activity 220 cpm/pmol) was added to a standard 25-,4l reaction mixture. RF activity in fractions from various stages of purification was assayed by its stimulatory effect in a standard 25jdl reaction mixture without added hemin after 40 min of incubation. Assays for Peptide Chain Initiation Factors. EIF-1 activity was determined by using a Millipore filtration assay for formation of [-5S]Met-tRNAf-EIF-1-GTP ternary complex as described previously (28). Ternary complex dissociation factor (TDF) activity was determined by using a Millipore filtration assay as described (1). The standard incubation mixture (75 ,u) contained 5-10 pmol of [-5S]Met-tRNAf (specific activity 10,000-12,000 cpm/pmol). Preparation of EIF-1. The purification of EIF-1 has been Abbreviations: The nomenclature for the peptide chain initiation factors is according to Majumdar et al. (1). tRNAf, tRNAfMet; EIF-1, Met-tRNAf-binding factor, also called eIF-2 according to Anderson et al. (2); EIF-2 (TDF), ternary complex dissociation factor; Co-EIF-1, a factor stimulating Met-tRNAf binding to EIF-1; HRI, heme-regulated translational inhibitor; 40S (in complexes), 40S ribosomal subunit; RF, the reticulocyte ribosomal salt wash factor that reverses inhibition of protein synthesis in heme-deficient lysates; NaDodSO4, sodium dodecyl sulfate. * This is paper 23 in a series. Paper 22 is ref. 3. 4858 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. Biochemistry: Ralston et al. Proc. Natl. Acad. Sc. USA 75 (1978) 4859 described (29-31). The homogeneous EIF-1 preparation used in these experiments showed three protein bands upon sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis, corresponding to approximate molecular weights of 54,000,52,000, and 38,000. NaDodSO4 Gel Electrophoresis. Electrophoresis of proteins in NaDodSO4/polyacrylamide gels was performed according to the method of Laemmli (32). 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 3 hr in a Bio-Rad model 220 electrophoresis apparatus.