Heat inhibition of reticulocyte protein synthesis. Evidence for a mechanism independent of the hemin-controlled repressor

Incubation of rabbit reticulocytes at 45 degrees C results in a prompt but reversible decrease in protein synthesis and a concomitant conversion of polyribosomes to smaller aggregates. These effects occur even in the presence of 100 micrometer hemin in the incubation medium. There is also inhibition of heme synthesis but this occurs at a later time than the effect on protein synthesis. The inhibtion of heme synthesis results from a decrease in activity of beta-aminolevulinic acid synthetase. This decrease of heme synthesis appears to be secondary to the inhibition of protein synthesis with resultant accumulation of intramitochondrial heme (which will decrease beta-aminolevulinic acid synthetase activity). An inhibitor of reticulocyte cell-free protein synthesis formed in the postribosomal supernatants of cells incubated at both 45 and 37 degrees C but not at 0 degrees C. No temporal or quantitative differences in the amount of this inhibitor from cells treated at either 37 or 45 degrees C was apparent. The inhibitor was not found in the fraction where the hemin-controlled repressor is isolated. It is concluded that heat inactivation of intact reticulocyte protein synthesis does not depend upon a decrease in heme synthesis, heme concentration or generation of the hemin-controlled repressor. Furthermore, it appears that the inhibitor formed in the post-ribosomal supernatant cannot be the sole cause of the heat inhibition of protein synthesis.     

A translational inhibitor activated in rabbit reticulocyte lysates under high pO2

An inhibitor of protein synthesis was activated under high oxygen partial pressure (pO2) in hemin-supplemented and glutathione disulfide-free lysates from rabbit reticulocytes. This inhibitor shared some common features with other translational inhibitors from rabbit reticulocytes; that is, hemin-controlled repressor, glutathione disulfide-activated inhibitor and high pressure-activated inhibitor. It caused biphasic kinetics of inhibition which could be potentiated by ATP. Its activation was prevented by cAMP or glucose 6-phosphate. The high pO2-inhibitor could be partially purified from post-ribosomal supernatant containing ribosomal salt wash by precipitation between 0-50% (NH4)2SO4-saturation, Sephadex G-100, and DEAE-cellulose chromatography.     

A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver.

A procedure is described for the preparation of free and bound polysomes from whole homogenates of rat liver tissue. Liver is homogenized in a conventional medium containing glutathione; then after a 12-min centrifugation at 131000g, the free polysomes in the supernatant are saved, while the membrane-bound polysomes in the pellet are suspended in a mixture of ribonuclease inhibitors (cell sap, 250 mM KCl, and glutathione), homogenized in the presence of detergent (Triton X-100), centirfuged for 5 min at 1470g, decanted, and treated with deoxycholate; the polysomes in the two supernatants are harvested by centrifugation through sucrose gradients containing 250 mM KCl and cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and virtually free of ribonuclease, membranous material, glycogen, deoxycholate, completed protein, and cross-contamination. The recovery of polysomes is approximately 95% and the distribution between the free and membrane-bound state is 25 and 75%, respectively. The molecular weight profiles after sodium dodecyl sulfate-acrylamide gel electrophoresis of the polypeptides completed and released by free and bound polysomes in vitro are different, indicating that there are quantitative differences in the synthesis of various size polypeptides between the two polysome classes. The differential centrifugation procedure is rapid and reproducible, requires much less ultracentrifugation than the isopycnic technique, and provides a nearly quantitative means of separating free and bound polysomes.     

Protein synthesis in homologous and heterologous cell-free systems from chick embryo connective tissues.

Two homologous systems for cell-free protein synthesis from chick embryo connective tissues are described. Both the skin polysomes and the wing-leg polysomes are active in collagen synthesis, but they have different requirements for optimum protein synthesis. Protein synthesis was not dependent on tissue-specific factors, since heterologous preparations of supernatant enzymes or initiation factors were able to stimulate maximum protein synthesis with each fraction of polysomes.     

Procollagen biosynthesis by embryonic-chick-bone polysomes. Estimation of the relative numbers of active proalpha1 and proalpha2 messenger ribonucleic acids.

Both total polysomes and polysomes of different size classes isolated from embryonic chick cranial bones were allowed to complete their nascent polypeptide chains in a cell-free system containing rabbit reticulocyte post-ribosomal supernatant fraction. In this system, no de novo initiation of polypeptide synthesis occurred. The product was analysed for relative content of proalpha1 and proalpha2, the precursors of the alpha chains of collagen, by dodecylsulphate-acrylamide gel electrophoresis as well as by paper electrophoresis following tryptic digestion. The results showed that the products of polysome protein synthesis contained proalpha1 and proalpha2 in the 2:1 ratio in which the corresponding alpha chains are present in native collagen, and that proalpha1 and proalpha2 synthesising polysomes are of the same size. These findings, in conjunction with results from a previous report (Vuust, J. and Piez, K.A. (1972) J. Biol. Chem. 247, 856-862) suggest that active messenger ribonucleic acids for the proalpha1 and proalpha2 chains, respectively, are present in the cells in a ratio of 2:1, and that the rates of initiation, elongation, termination and release from polysomes are all identical for the two chains.     

QUANTITATIVE ISOLATION AND PROPERTIES OF NEARLY HOMOGENEOUS POPULATIONS OF UNDEGRADED FREE AND BOUND POLYSOMES FROM RAT BRAIN1

Abstract— A procedure is described for the preparation of free and bound polysomes from whole homogenate of rat brain tissue. Brain is homogenized in a sucrose-polysome buffer medium high in KCl (250 mm). After a 12-min centrifugation at 135,000 g, the free polysomes in the supernatant are decanted and saved, while the membrane bound polysomes in the pellet are resuspended in homogenizing medium, homogenized in the presence of detergent (Triton X-100), centrifuged for 5min at 1470 g to remove nuclei, decanted, treated with deoxycholate and centrifuged for 10 min at 24,000 g to remove deoxycholate-insoluble material. Polysomes in the two supernatants are harvested by centrifugation through sucrose gradients prepared in high KCl polysome buffer, and with or without cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and largely free of the usual contaminants. Cross-contamination is minimal (>10%). The recovery of polysomes is at least 95%. The distribution of ribosomes and polysomes in rat brain is 58% free and 42% membrane-bound. The distribution of rat brain RNA is 65% ribosomal and 35% non-ribosomal. Conditions are described for the visualization and analysis of the entire complement of free and bound ribosomes. The size fractionation procedure is rapid and reproducible, requires much less ultracentrifugation than the density-gradient technique, and provides a nearly quantitative means of isolating undegraded free and bound polysomes of rat brain tissue.     

A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression

We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7-10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40-50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger-ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.     

Effect of temperature on protein and immunoglobulin synthesis and secretion in two mouse myeloma cell lines

Protein synthesis in differentiated MOPC-21 and MPC-11 mouse myeloma cells was studied to determine the basis for the differences in the temperature and actinomycin D sensitivity of translation between non-differentiated mouse L-cells and differentiated rabbit reticulocytes. The temperature dependence of total protein synthesis was similar to that of L-cells and reticulocytes, being biphasic in Arrhenius plots with apparent activation energies of approximately 25 and 42 kcal/mol, above and below 25 degress C. The dependence of the secretion process was different since it was not biphasic, having a single activation energy of about 22 kcal/mol. Myeloma polysomes were like L-cell polysomes in their response to lower temperature and reached a minimum level of 50% at 15 degress C. This response was also found for the specific polysomes synthesizing the IgG H- and L-chains. In the presence of actinomycin D, myeloma polysomes declined exponentially with a half-life of approximately 6 hours. These two L-cell-like responses were not found in reticulocytes. Translation of both the IgG mRNAs and the non-IgG mRNAs was reduced by lower temperatures and actinomycin D, even though the L-chain mRNA was slightly more resistant, suggesting that this mRNA is slightly more efficient. The results of these experiments suggest that the translational differences between L-cells and reticulocytes are not mRNA dependent, but are cell type differences.     

The influence of temperature upon polysomes of spermatids of rat testes

Incorporation of [³H]phenylalanine into protein by a reconstituted lysate subcellular system (ribosomes plus high-speed supernatant) from rat spermatids was measured at 34°C after 5 minutes preincubation of one component at 0°C while the other component was incubated at temperatures from 30°C to 40°C. Preincubation at temperatures above 34°C inhibits the ribosomal activity but not the high-speed supernatant activity. The incubation of lysate above 34°C results from a dissociation of polysomes to monosomes. These results indicate that ribosomes are the most sensitive component to the increased temperature on protein synthesis in lysate cell free system by spermatids and that the inhibition of protein synthesis in spermatids above 34°C is at least partly explained by the breakdown of polysomes in these cells.     

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from Friend leukemia cells.

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA^fMet to the ribosomal initiation complex and the NaF-sensitive reaction.     

Cell-free synthesis of tryptophanase from Escherichia coli. Use of ribonucleic acid isolated from induced cells and a comparison of the product from a system employing ribosomes with that from one employing ribosomes and exogenous ribonucleic acid

1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.-) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.     

Reversal of the inhibitory action of the hemin-controlled translational repressor by a post-ribosomal supernatant factor from rabbit reticulocyte lysate.

The inhibitory effect of the hemin-controlled translational repressor (HCR) on protein synthesis by rabbit reticulocyte lysates can be overcome by a factor in the post-ribosomal supernatant fraction. When chromatographed on Sepharose 6B, this supernatant factor migrates as a high molecular weight component that is distinct from the precursor of HCR (prorepressor). The supernatant factor does not appear to act by enzymatically degrading the repressor or by forming a stoichiometric complex with it, but may, rather, replace what has become limiting for protein synthesis due to repressor action.     

Synthesis of S100 Protein on Free and Membrane-Bound Polysomes of the Rabbit Brain

Abstract: Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [³⁵S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane- bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly- (A+)mRNA coding for SlOO protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.     

Purification and characterization of perchloric acid soluble protein from rat lung

We isolated a perchloric acid soluble protein from the post-mitochondria supernatant fraction of the rat lung and designated it as RLu-PSP1. The protein is soluble in 5% perchloric acid and was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The amino acid sequence of RLu-PSP was identical with that of rat liver PSP (RL-PSP). RLu-PSP inhibited protein synthesis in a rabbit reticulocyte lysate system. It was expressed mainly in cytoplasm of bronchioles and alveolar epithelial cells of the lung from 60-day-old rats. In 15-day-old rat embryos, the epithelial-lining of the terminal buds of the respiratory tree was immunopositive. The expression of RLu-PSP increased from the embryonic 15th day to the postnatal 40th day. This is the first report on the presence of a PSP in rat lung and on its involvement in the regulation of cellular growth and differentiation.     

Stimulation of yeast mitochondrial protein synthesis by postpolysomal supernatants from yeast,rat liver,and Escherichia coli

Isolated yeast mitochondria incubated with a protein-synthesizing mixture containing excess oxidizable substrate, amino acids, MgCl₂, an ATP-regenerating system, and optimal levels of [³H]leucine cease protein synthesis after 30 min. Postpolysomal supernatants from either yeast, rat liver, or Escherichia coli can restore protein synthetic activity to depleted yeast mitochondria; however the addition of bovine serum albumin to the incubation mixture did not restore activity. The restored incorporation activity was sensitive to chloramphenicol, insensitive to cycloheximide, and proportional to the protein concentration of the supernatants. Furthermore, addition of all three high-speed supernatants to isolated mitochondria at time zero stimulated the rate of protein synthesis to a greater extent than when these fractions were added to depleted mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the translation products obtained from mitochondria labeled in vitro in the presence of supernatant fractions were identical to the proteins labeled by mitochondria in vivo; however, the synthesis of the bands corresponding to subunit III of cytochrome oxidase, cytochrome b, and VAR-3 was stimulated to the greatest extent. The stimulatory activity in the supernatants was non-dialyzable, insensitive to treatment with ribonuclease A, but completely abolished by pretreatment with trypsin suggesting that the stimulatory factor(s) is of a protein nature. The postpolysomal supernatants did not incorporate amino acids into protein when incubated without mitochondria. These results suggest that the protein synthetic capacity of mitochondria is apparently limited by extramitochondrial proteins which are present in either yeast, rat liver, or E. coli.     

Ribosomal association of the yeast SAL4 (SUP45) gene product: implications for its role in translation fidelity and termination

The SAL4 gene of the yeast Saccharomyces cerevisiae encodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p-beta-galactosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post-ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the 'run-off' polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translation termination.     

Protein synthesis in postnuclear supernatants from mengovirus-infected Ehrlich ascites tumor cells.

The effect of mengovirus infection on the protein synthetic capacity of Ehrlich ascites tumor cells cultured in vitro was studied in vivo and in vitro employing postnuclear supernatants prepared at various times post-infection in the absence and in the presence of 1% Triton X-100. The amino acid incorporating activities of extracts obtained in the presence of the detergent were reduced by about 30% compared with the capacities of the corresponding postnuclear supernatants prepared in the absence of Triton X-100; but the course of the activity vs. time curve was not influenced by the detergent. Under the conditions employed, the postnuclear supernatants were unable to reinitiate protein synthesis once elongation of nascent polypeptide chains concomitant with ribosome runoff was completed. After mengovirus infection, a gradual disappearance of polysomes from postnuclear supernatants and a simultaneous accumulation of monosomes was observed. The protein-synthesizing activities of normal and infected cells were inversely proportional to the monosome concentrations of their corresponding extracts. Qualitatively, protein synthesis in intact cells and in postnuclear supernatants responded similarly to mengovirus infection. In both cases an initial reduction of host-specific amino acid incorporation was followed by a burst of viral protein synthesis. However, the two activity vs. time curves showed the following significant differences: 1) The activities of extracts from control cells and from mengovirus-infected cells nearly in the infectious cycle were low compared with the activities observed in vivo. 2) In the middle of the infectious cycle, the peak of viral protein synthesis occurred later and the activity was higher in vitro. 3) Finally, in the late period of the infectious cycle the postnuclear supernatants had considerable protein synthesizing activity, at a time when protein synthesis in vivo was nil.     

Age-dependent changes in brain protein synthesis in the rat

Brain protein synthesis was studied in vivo, in brain slices, and in cell-free systems in rats aged 1, 16, and 24 months. We observed a highly significant reduction in amino acid incorporation with advancing age. This reduction was observed in vivo, in slices, in postmitochondrial supernatant, microsomes, and membrane-bound polysomes. Free heavy polysomes showed no age-dependent decline but formed a smaller proportion of total ribosomes in older animals. These studies suggest that in the rat brain protein synthesis declines before senescence, possibly due to an impairment in the initiation process.     

Mechanism of inhibition of hepatic protein synthesis in rats by the carcinogen, methylazoxymethanol acetate.

Treatment of rats with the carcinogen, methylazoxymethanol acetate, results in a rapid, marked inhibition of hepatic protein synthesis and disaggregation of polysomes. Studies were undertaken to learn the mechanism by which this carcinogen induces these effects in rat liver. The data show that the inhibition of endogenous protein synthesis is not due to an effect on the high speed supernatant 'factors' but rather at the level of the polysome, and that both free and membrane-bound polysomes are affected. Poly(U)-directed polyphenylalanine synthesis by native ribosomal subunits is greater in preparations isolated from rats treated with carcinogen than it is in controls. Moreover, the native ribosomal subunit fraction from treated livers in response to added rabbit globin mRNA is able to synthesize a protein similar in molecular weight to globin. These studies show that methylazoxymethanol acetate does not induce significant alterations of ribosomal subunits or of initiation factors and suggest that the inhibition of protein synthesis and disaggregation of polysomes may be the results of an alteration of cytoplasmic mRNA, or its association with ribosomes.