Preparations of the hemin-controlled repressor (HCR) from rabbit reticulocytes contain 3':5'-cyclic-AMP-independent protein kinase activity for the smallest subunit of the peptide initiation factor eIF-2 and for proteins of reticulocyte 40S ribosomal subunits. Binding of the ternary complex formed between Met-tRNAf, GTP, and eIF-2 to 40S ribosomal subunits is shown to be inhibited by phosphorylation of either the ribosomal subunits or eIF-2. The protein kinase activity responsible for phosphorylation of eIF-2 has been separated from the activity for phosphorylation of 40S ribosomal subunits and shown to independently block the same (8) and shown recently (9-11) that HCR acts as a protein kinase. All three papers (9)(10)(11) demonstrated that preparations of HCR promote cAMP-independent phosphorylation of the smallest subunit of eIF-2. However, in addition, we reported (9) that a highly purified preparation of HCR also phosphorylated certain proteins associated with reticulocyte 40S ribosomal subunits that had been washed with 0.5 M KCI.One or two ribosomal proteins of the reticulocyte 40S ribosomal subunits have been shown to be phosphorylated in vivo or in vitro (12)(13)(14). However, no biological function could be attributed to the phosphorylated or dephosphorylated state of the ribosomes.In the present paper initiation factor and GTP-dependent binding of Met-tRNAf to reticulocyte 40S has been studied as a partial reaction of peptide chain initiation. The methylene analogue of GTP, GMP-P(CH2)P, can substitute for GTP in this Abbreviations: HCR, hemin-controlled repressor; eIF-2, the initiation factor that forms a ternary complex with Met-tRNAf and GTP; GMP-P(CH2)P, guanosine 5'-(&3-y-methylene)triphosphate; AMP-P(CH2)P, adenosine 5'-(,--y-methylene)triphosphate. * (15) and the purification of eIF-2 (9) have been described.Reticulocyte ribosomal subunits were prepared by a modification of the procedure described by Falvey and Staehelin (16). About 150 mg of reticulocyte ribosomes was adjusted to a concentration of 10 mg/ml in a solution containing 0.5 M KC1/3 mM MgCl2/20 mM Tris-HCl, pH 7.5/1 mM 2-mercaptoethanol and kept at 40 for 30 min. Then the solution was layered on a 550 ml 15-30% (wt/vol)

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Three functionally related components that block peptide initiation have been identified in lysates of rabbit reticulocytes. The components function consecutively in a cascade type sequence of reactions to cause phosphorylation of eukaryotic peptide initiation factor 2 (eIF-2). The eIF-2 kinase activated as part of this sequence has been tentatively identified as the same protein kinase that is activated by heme deficiency as part of the hemin-controlled repressor (HCR) system. The first component in the sequence is heat stable and can be reversibly activated by heat or pressure. It activates a second, heat-labile, component that in turn directly or indirectly activates the hemin-controlled eIF-2 kinase. This heat-labile component appears to function through proteolysis. This reaction sequence is not detectably affecte by heme or cyclic AMP and thus appears to provide an alternative mechanism, independent of heme, for activation of the cyclic AMP-independent eIF-2 kinase of the HCR system.

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