Heme Deficiency and Phosphorylation of Ribosome‐Associated Proteins

Abstract

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...