A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ ATP/GTP/Pi exchange system is described which has been used to maintain [y-32P]ATP and [.)-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf to 40-S subunits. Three phosphorylated proteins co-migrate with eIF-3, the factor facilitating entry of Met-tRNAf onto the 40-S subunit and promoting dissociation of 8 0 3 ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of the phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 4 0 3 ribosomal subunits, the other is an acidic protein.Recently considerable progress has been made toward the identification of initiation factors (eIF) involved in eukaryotic translation. eIF-2 is involved in the formation of a ternary complex with Met-tRNAf and GTP, and in the binding of this complex to 4 0 3 ribosomal subunits [l -91. eIF-2A directs the binding of Met-tRNAf in a codon-dependent reaction [lo-151. eIF-3, a multisubunit complex, is found on native 40-S ribosomes [16-181 and facilitates entry of the ternary complex onto 40-S subunits [19,20]. Two factors are involved in binding of mRNA to the 4 0 3 initiation complex, eIF-4A 23]. eIF-4C [22,24] and eIF-4D [24] are required for maximal globin synthesis, and eIF-5 has a ribosome-dependent GTPase activity and is involved in the formation of the 80-S initiaton complex [9,22,25].Initiation is of particular interest as a site of regulation of protein synthesis, since binding of MetAhhrn.irrtioris. eIF, eukaryotic initiation factor M ; 32Pi. inorganic phosphate substituted with 32P; cyclic AMP, adenosine 3': 5'-monophosphate.-~ -~ tRNAf to 40-S subunits, has been shown to be specifically inhibited in reticulocyte lysates under conditions of hemin deficiency [26,27] [36,41], and eIF-5 [38,41] have also been reported ; however, these latter modification events have not been linked with a regulatory function. In order to examine regulation of initiation of protein synthesis, we have developed a two-dimensional gel electrophoresis system for rapid identification of the initiation factors in the l...

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The effects of N6,O2-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) and sodium fluoride on the phosphorylation of ribosomal proteins S6 and on protein synthesis were examined. Rabbit reticulocytes were incubated in a nutritional medium containing 32Pi in the presence and absence of Bt2cAMP (1mM) and 3-isobutyl-1-methyl-xanthine (1mM). In the control cells, four phosphorylated derivatives of S6 were observed, with most of the radioactivity in the monophosphorylated form. Upon addition of cyclic nucleotide, a twofold increase in the phosphorylation of ribosomal protein S6 was observed. This was accompanied by an increase of radioactive phosphate in the diphosphorylated derivative. No alteration in protein synthesis was observed upon addition of cAMP and analogues of cAMP in conjunction with 3-isobutyl-1-methyl-xanthine or theophylline. The effects of sodium fluoride on phosphorylation of S6 and on protein synthesis were examined also. At 5 mM sodium fluoride, protein synthesis was inhibited by 85%. A 2.5-fold increase in the phosphorylation of ribosomal protein S6 was observed with an accumulation of 32Pi in the diphosphorylated, triphosphorylated and tetraphosphorylated derivatives. Inhibition of protein synthesis coincided with an increase in the more highly phosphorylated derivatives, whereas an increase of radioactive phosphate in the diphosphorylated derivative could not be correlated with an alteration in globin synthesis.

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Phosphorylation of proteins in the protein-synthesizing complex from rabbit reticulocyte lysates was examined under conditions of optimal protein synthesis and during inhibition of protein synthesis by hemin deprivation. A creatine phosphate -ATP -GTP -P, exchange system was used to maintain [ Y -~~P I A T P and [Y-~'P]GTP at high, constant specific activity. Phosphate incorporation into proteins in the 0.5 M KCl wash fraction and in salt-washed ribosomes was examined before and after inhibition of protein synthesis in hemin-deprived lysates and compared to phosphorylated proteins observed in hemin-supplemented lysates. Two of the phosphoproteins in the 0.5 M KCl wash fraction showed a twofold to fourfold increase in phosphate incorporation prior to inhibition of protein synthesis under conditions of hemin deprivation. One of these phosphoproteins was identified as the small subunit of initiation factor 2 (eIF-2), the initiation factor involved in the GTPdependent binding of Met-tRNAf to 40-S ribosomal subunits. A second protein, with a molecular weight of 55000, did not correspond to any of the highly purified initiation factors from reticulocytes, but was a major constituent of the salt-wash fraction. No changes in phosphate incorporation into the major phosphorylated 40-S ribosomal protein, S6, were observed; however, se\ era1 minor components, which were not completely removed by the high-salt wash, showed alterations in phosphorylation.Protein synthesis and phosphorylation were also examined in intact reticulocytu When iron and the iron-transporting protein, transferrin, were present, protein synthesis was lineal-for at least 2 h. When the cells were incubated in the nutritional media from which iron and transferrin had been omitted, inhibition occurred between 15 min and 30 min. Cells incubated in the absence of iron and transferrin showed increased phosphorylation of two proteins in the high-salt-wash fraction. These had niolecular weights of 38000 and 55000 and corresponded to the two proteins showing increased phosphorylation in the lysate system in the absence of hemin.Hemin has been identified as a translational regulator of globin synthesis in reticulocytes [1, 21 and reticulocyte lysates [3,4]. When lysates are incubated in the absence of hemin, formation of a hemincontrolled repressor occurs, which blocks peptide chain initiation [4 -71 resulting in a decrease in binding of Met-tRNAf to 40-S ribosomal subunits [8 -lo]. High concentrations of cIF-2. the initition factor involved in the GTP-de pendcnt binding o f met -t R N Af to 40-S ribosomal subunits [ l l -151, reverse inhibition produced by hemin deprivation [16,17] or by addition of the hemin-controlled repressor [17,18]. Several laboratories have identified a protein kinase activity associated with purified preparations of this repressor, which is highly specific for the cc subunit ( M , = 38000) Abbreviution. eIF-2 etc., eukaryotic initiation factor 2 etc of eIF-2 [19 -241. Phosphorylation of highly purified eIF-2 does not impair formation o...