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     

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%).     

Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-¹⁴C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 μM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not δ-aminolevulinic acid, partly overcame the inhibition of ⁵⁹Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits δ-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 μM, 0.1 and 1 mM, inhibited ⁵⁹Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte ⁵⁹Fe uptake by about 25–30%. In succinylacetone-treated cells ⁵⁹Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and ⁵⁹Fe-transferrin-pretreated reticulocytes results in the transfer of ⁵⁹Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.     

Protein synthesis in rabbit reticulocytes: Evidence for the synthesis of initial dipeptides in the presence of pactamycin

The effects of pactamycin on peptide chain initiation were studied using a reconstituted system comprised of reticulocyte ribosomes, poly r(A-U-G) messenger and peptide chain initiation factors, and the transfer of methionine from precharged Met-tRNA_f^Met into di-, oligo- and polymethionine products was measured. In the presence of low concentrations of pactamycin (10^?6M), polymethionine synthesis in the above system was markedly reduced, and a concomitant increase in the synthesis of met-met and met-val dipeptides was observed. The latter dipeptide was presumably synthesized in response to endongenous hemoglobin messenger.Aurintricarboxylic acid and sparsomycin also inhibited polymethionine synthesis in response to poly r(A-U-G) messenger. Whereas aurintricarboxylic acid inhibited pactamycin induced dipeptide synthesis, sparsomycin had no significant effect on dipeptide synthesis in the presence of pactamycin.     

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.     

High pO2-activated inhibitor of protein synthesis in rabbit reticulocytes: its relationship to glutathione disulfide-induced inhibitor and to a approximately 23,000-Mr sulfhydryl protein

Fibronectin is a large dimeric glycoprotein found in plasma, on cell surfaces and as a component of the extracellular matrix which has been implicated in a variety of adhesive processes. The role of fibronectin in platelet function has not been clarified. The present investigation demonstrates that an excess of exogenously added fibronectin inhibits platelet aggregation induced by either thrombin or A23187 at a step subsequent to platelet activation and secretion. Similar concentrations of fibrinogen or von Willebrand factor, both of which also bind to the surface of activated platelets, are not inhibitory. These results are consistent with the concept that fibronectin is one of the important mediators of platelet aggregation.     

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.     

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.