The control of protein synthesis by hemin in rabbit reticulocytes

Summary The control of protein synthesis by hemin in rabbit reticulocytes or lysates is mediated by the formation of a high molecular weight protein inhibitor of polypeptide chain initiation termed the hemin-controlled translational repressor (HCR). HCR becomes activated in the absence of hemin from a presynthesized precursor (prorepressor) in a manner that is still unclear but appears to involve a series of discrete conformational changes in a single protein. At a very early stage of activation, HCR (reversible) can be inactivated by hemin, at a somewhat later stage (intermediate HCR) it can still be inactivated in a GTP-dependent reaction by a soluble lysate protein termed the supernatant factor, and after more than several hours of warming, HCR (irreversible) can no longer be inactivated. Formation of HCR involves no detectable change in molecular size but may involve, directly or indirectly, disulfide bond formation or interchange, since activation occurs very rapidly in the presence of such sulfhydryl reagents as N-ethylmaleimide. Once activated, HCR (all three forms) acts by phosphorylating the 35,000 M_r () subunit of eIF-2, the initiation factor that mediates binding of Met-tRNA_f to 40 s ribosomal subunits. The protein kinase action of HCR is relatively specific for eIF-2, although HCR also autophosphorylates a 90–100,000 M_r component of itself. While most of the protein synthsized by rabbit reticulocytes is globin, the synthesis, at low levels, of other reticulocyte proteins is also reduced by HCR, consistent with its action on eIF-2, a factor that acts in initiation before mRNA is bound. At present, the mechanism by which phosphorylation of eIF-2 by HCR causes inhibition of polypeptide chain initiation is only partially understood. There is general agreement that the binding of Met-tRNA_f to 40 s ribosomal subunits is reduced, perhaps due to impaired interaction of eIF-2-P with other ribosomal protein components. There is also evidence that HCR causes the accumulation of 48 s intermediate initiation complexes, containing a 40 s ribosomal subunit, mRNA, and tRNA_f^met that is largely deacylated. This suggests that the joining of 48 s complexes with 60 s subunits to form 80 s initiation complexes is also blocked and results in the deacylation of subunit-bound Met-tRNA_f. Additional work will be required to delineate the precise molecular mechanisms by which HCR becomes activated in the absence of hemin and how the phosphorylation of eIF-2 interrupts the process of polypeptide chain initiation.Abbreviations HCR hemin-controlled translational repressor - eIF eukaryotic initiation factor