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.     

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%).     

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     

Protein synthesis in rabbit reticulocytes. A study of the mechanism of Co-eIF-2 action

The characteristics of component activities in Co-eIF-2 (where eIF is eukaryotic initiation factor) protein complex have been studied. (i) At limiting concentrations, Co-eIF-2 promoted rapid GDP binding to eIF-2 and also GDP displacement from eIF-2 X GDP during ternary complex formation in the presence of GTP and Mg2+ (Co-eIF-2C activity) but did not significantly stimulate ternary complex formation by eIF-2. (ii) At higher concentrations, Co-eIF-2 significantly enhanced ternary complex formation by eIF-2 and also rendered the complex stable to aurintricarboxylic acid presumably as Co-eIF-2 became physically bound to the ternary complex (Co-eIF-2A activity). (iii) Ternary complex preformed in the presence of Co-eIF-2 and without Mg2+ dissociated upon subsequent addition of Mg2+ (Co-eIF-2B activity). This dissociation reaction was presumably due to loss of interaction of the Co-eIF-2A component in Co-eIF-2 with the ternary complex (reversal of Co-eIF-2A activity) as the complex became increasingly sensitive to aurintricarboxylic acid with increasing Mg2+ concentration. In another study, purified eIF-2 was freed of bound GDP by treatment with alkaline phosphatase and the characteristics of native and GDP-free eIF-2 were compared. (i) One mM Mg2+ inhibited (60%) ternary complex formation by native eIF-2 but not by GDP-free eIF-2. Addition of exogenous GDP rendered GDP-free eIF-2 sensitive to Mg2+ indicating that Mg2+ inhibition is due to eIF-2-bound GDP. (ii) In the presence of Mg2+, Co-eIF-2 stimulated similarly ternary and Met-tRNAf X 40 S X AUG complex formation by both native and GDP-free eIF-2. Such stimulatory activity in each case was strongly inhibited by prior phosphorylation of eIF-2 alpha subunit by heme-regulated translational inhibitor. (iii) Ternary complexes preformed using either native and GDP-free eIF-2 and excess Co-eIF-2A80 in the absence of Mg2+ did not form Met-tRNAf X 40 S X AUG complex. They required trace amounts of Co-eIF-2 for such activity.     

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.     

Protein synthesis in rabbit reticulocytes: characteristics of CO-eIF-2 protein complex.

A high molecular weight reticulocyte protein factor, named Co-eIF-2, contains Co-eIF-2A, Co-eIF-2B, and Co-eIF-2C activities and stimulates Met-tRNAf binding to eIF-2 both in the presence and absence of Mg2+. Some characteristics of this stimulation in the absence of Mg2+ are: (1) Stimulation is most pronounced at low eIF-2 levels. (2) Stimulation is partially resistant to heat and NEM treatment, and thus appears to be due to the combined action of both heat and NEM-insensitive Co-eIF-2A, and heat and NEM-sensitive Co-eIF-2C activities. (3) [3H]GDP bound in eIF-2 . [3H]GDP complex is rapidly displaced by unlabelled GTP during ternary complex formation Co-eIF-2 stimulates Met-tRNAf binding to eIF-2 even when added after the [3H]GDP from eIF-2 . [3H]GDP has been completely displaced. This indicates that Co-eIF-2-stimulation is not due to GDP displacement from eIF-2 . GDP. We propose that eIF-2 molecules become inactive in the presence of Mg2+ and at high dilution, and Co-eIF-2 restores the inactive eIF-2 molecules into an active form.     

Two fractions required for cell-free protein synthesis with components from rabbit reticulocytes

An extract was prepared from rabbit reticulocyte ribosomes after treatment with potassium chloride as described by Miller, Hamada, Yang, Cohen & Schweet (1967). This extract has been shown to convert monoribosomes into polyribosomes during protein synthesis in vitro (Cohen, 1968). The nature of this extract was studied in greater detail. Centrifugation of the extract through a sucrose density gradient separated the activity into a fast-sedimenting fraction. The two fractions were shown to have different functions in stimulating cell-free protein synthesis and their active components were shown to be protein or partly protein in nature. Each fraction was analysed by electrophoresis and in the analytical ultracentrifuge. It was concluded that the active component in the fast-sedimenting fraction had a sedimentation coefficient of 15.5s and that of the slow-sedimenting fraction 10.5s.     

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.