Membrane protein synthesis by rabbit reticulocytes

Intact rabbit reticulocytes synthesize several classes of membrane proteins ranging in molecular weight from 20,000 to 200,000. The synthesis is inhibited by cycloheximide, and treatment with ribonuclease does not significantly alter the gel pattern.     

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     

The initiation of haemoglobin synthesis in rabbit reticulocytes

1. The incorporation of labelled valine by rabbit reticulocytes into the N-terminal position of nascent haemoglobin was investigated by deaminating the nascent peptides with nitrous acid and isolating labelled alpha-hydroxyisovaleric acid and valine after acid hydrolysis. 2. The amount of radioactivity in alpha-hydroxyisovaleric acid relative to that in valine indicated the presence of 12.3% N-terminal valine having a free amino group. This high value suggests that most if not all nascent peptides contain valine in the N-terminal position. 3. Cell-free preparations containing reticulocyte ribosomes and pH5 enzymes incorporated alpha-hydroxy-[(14)C]isovaleryl-tRNA (where tRNA refers to transfer RNA), which was obtained by deamination of [(14)C]valyl-tRNA from yeast or liver with nitrous acid, into both soluble and nascent protein. 4. When the soluble protein was chromatographed on CM-cellulose, radioactivity was found to be associated with both the alpha-and beta-globin chains. 5. The kinetics of hydrolysis of [(14)C]valine, was also investigated. Most of the material was hydrolysed rapidly at pH10, but a minor component that was relatively stable appeared to be present to the extent of about 10% of the total valyl-tRNA. Valine was, however, the only hydrolysis product detected by paper chromatography. 6. It is concluded that chain initiation in haemoglobin synthesis involves valine as the N-terminal amino acid and that the amino group of nascent protein is probably not substituted.     

Characterization of protein synthesis factors from rabbit reticulocytes

As part of our efforts to characterize eukaryotic translation factors, we have sequenced a number of them chemically and inferred sequences from cDNA clones. To our surprise, there appears to be extensive identity of amino acid sequence in most factors characterized to date in that within mammalian species, usually greater than 99% identity is observed. Extreme examples are rabbit EF-1 alpha which is 100% identical to human EF-1 alpha and rabbit eIF-4AI and eIF-4AII which are 100% identical to mouse eIF-4AI and eIF-4AII for those amino acids sequenced (398/406 and 156/407, respectively). An extended analysis has been made of EF-1 alpha which in rabbit has three different post-translational modifications, dimethyllysine, trimethyllysine and glycerylphosphorylethanolamine. A comparison of the primary structure of EF-1 alpha to E. coli EF-Tu indicates an overall sequence identity of 33%. However, within the amino terminal 180 amino acids (the GTP-binding domain), there are found regions of much greater identity (50/85 = 59%).     

Membrane proteins synthesized by rabbit reticulocytes

Intact rabbit reticulocyte cells synthesize two predominant species of polypeptides which are components of the cell plasma membrane. Previous work (Lodish, H. F. 1973. Proc. Natl. Acad. Sci. U. S. A. 70:1526- 1530.) showed that these proteins were synthesized by polyribosomes not attached to membranes. We show here that both polypeptides are confined to the cytoplasmic surface of the cell membrane. These studies utilized iodination of whole cells and of membranes with lactoperoxidase, and digestion of whole cells and membranes with chymotrypsin, One of these proteins is synthesized as a precursor, and about 20-40 amino acids are removed after it is incorporated into the membrane, We discuss the probable sites of synthesis of these and other classes of membrane proteins.     

Evidence for an inhibitor of protein synthesis in rabbit reticulocytes activated by high pressure

Evidence is presented for an inhibitor of protein synthesis which is activated by subjecting rabbit reticulocyte postribosomal supernatant to high pressure (15,000 1b/in²). The inhibitor is antigenically and chromatographically distinct from the hemin-controlled repressor. The mechanism of inhibition by the pressure-treated postribosomal supernatant is not clear; however, the kinetics of inhibition seem to indicate that inhibition may be at the level of initiation.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.