In vitro translation of drosophila heat-shock and non-heat-shock mRNAs in heterologous and homologous cell-free systems

Upon heat shock, Drosophila Kc cells still contain normal cellular messenger RNAs in the cytoplasm. The distribution of these 25°C mRNAs between polysomes and the postpolysomal fraction of heat-shocked cells appears unaltered as compared with control cells. The translatability of these normal cellular messages isolated from heat-shocked and non-heat-shocked Kc cells is unaltered when analyzed by in vitro translation in the rabbit reticulocyte lysate. In contrast, homologous cell-free translation systems obtained from Kc cells effectively discriminate between the in vitro translation of normal cellular messages and heat-shock-specific mRNAs. In particular, a cell-free system from heat-shocked Drosophila Kc cells almost completely shuts down the translation of 25°C messenger RNA species, whereas the translatability of heat-shock-specific messenger RNA appears to be unaffected.     

The heat-shock response in Drosophila KC 161 cells. mRNA competition is the main explanation for reduction of normal protein synthesis

The effect of heat shock on protein synthesis in the Drosophila melanogaster KC 161 tissue culture cell line was examined with a view to investigating the mechanism underlying the acute reduction in normal cellular protein synthesis typical of heat-shocked Drosophila cells. However, at 36-37 degrees C, the optimum temperature for induction of the 70-kDa heat-shock protein, this cell line did not show such a response. The synthesis of a very limited number of proteins was abruptly turned off following heat shock in the presence or absence of actinomycin, but the rate of synthesis of the majority of normal cellular proteins declined slowly over a three-hour period. Incubation of heat-shocked cells in hypertonic media increased the relative proportion of protein synthesis directed towards heat-shock proteins (as opposed to normal cellular proteins). Incubation with low concentrations of cycloheximide had the converse effect and resulted in a preferential increase in the size of polysomes translating normal cellular mRNAs, greater than the increase in size of polysomes synthesising heat-shock proteins. Heat shock also resulted in some mRNAs being almost completely displaced from polysomes into the postribosomal supernatant. These observations suggest that competition between normal cellular mRNAs and increasing amounts of heat-shock mRNAs with a higher affinity for the translation machinery was the main explanation for the gradual reduction in the synthesis of normal cellular proteins, although a slight reduction in overall translation initiation rates cannot be excluded as a subsidiary cause. The results demonstrate that the acute reduction in normal cellular protein synthesis seen in other Drosophila cell lines is not an integral and necessary feature of the heat-shock response in this organism, which makes it unlikely that the mechanism of this acute shut-off is intimately connected with the mechanism of induction of heat-shock mRNAs.     

Cell-free protein synthesis in lysates of Drosophila melanogaster cells.

A procedure is described for preparing cell-free protein synthesizing lysates from Drosophila melanogaster tissue culture cells and embryos. Preparation of translationally active lysates from tissue culture cells is dependent on the presence of rat liver supernatant during cell lysis to inhibit ribonuclease activity. After micrococcal nuclease treatment of the lysate, protein synthesis is dependent on the addition of exogenous messenger RNA. The fidelity of translation is very high. The conditions for optimal translation have been determined. In addition, the effects on translation of a variety of supplements, including rat liver supernatant, have been analyzed. The products of translation by the Drosophila lysate have been compared with those of wheat germ extracts and of micrococcal nuclease treated rabbit reticulocyte lysates. Translation in vitro of bovine parathyroid hormone messenger RNA yielded two products tentatively identified as preproparathyroid hormone and proparathyroid hormone, as well as an unidentified third product. This result suggests that insect enzymes can accurately process mammalian precursor proteins.     

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates

When Drosophila tissue culture cells are shifted from 25 to 36°C (heat shocked) the pre-existing mRNAs (25°C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25°C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding α-tubulin, β-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25°C. Increased temperature should lead to increased output of protein per ribosome. However, the 25°C proteins are actually synthesized at less than 10% of 25°C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25°C mRNAs than they are on heat shock mRNAs in heat-shocked cells.     

Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich cells involves reduction of eukaryotic initiation factor 4F activity

Almost all living organisms studied respond to elevated temperature with a marked inhibition of overall protein synthesis but increased synthesis of a specific set of proteins, the so-called heat-shock proteins. We have prepared a cell-free protein synthesizing system (lysate) from heat-shocked Ehrlich ascites tumor cells that reflects the inhibition of protein synthesis in intact cells at elevated temperatures. We have isolated and partially purified a stimulator of the heat-shocked cell lysate from Ehrlich cells. Through four purification steps, the stimulator is chromatographically identical to eukaryotic initiation factor 4F (eIF-4F), an initiation factor which specifically binds mRNA cap structure. Therefore, we have tested the effects of highly purified reticulocyte eIF-4F on the heat-shocked cell lysate. Protein synthesis is strongly stimulated by addition of highly purified eIF-4F. Synthesis in the heat-shocked lysate is more inhibited at high (70 mM) KCl concentrations, than at lower concentrations, and stimulation by eIF-4F is correspondingly greater at higher KCl concentrations, so that the rate of protein synthesis is returned to control (non-heat-shocked lysate) levels at all KCl concentrations. Furthermore, at 70 mM KCl, in heat-shocked lysates, synthesis of the 68-kDa heat-shock protein is much less inhibited than synthesis of the bulk of non-heat-shock proteins, and eIF-4F stimulates synthesis of 68-kDa protein to a much lesser extent than non-heat-shock proteins. Thus, addition of purified eIF-4F reverses the effects of elevated temperatures on Ehrlich cells that are reflected in lysates. Therefore, we propose that the inhibition of translation in heat-shocked Ehrlich cells is the result of inactivation of eIF-4F function.     

Synthesis of heat shock proteins in Drosophila melanogaster embryos

The pattern of polypeptides synthesized in a cell-free protein synthesizing system containing polysomes isolated from heat-shocked (37 C) Drosophila embryos showed significant differences when compared with the pattern obtained when polysomes from normal embryos were used. The synthesis of normal embryonal proteins was reduced and the heat shock proteins were the major products of elongation. After short, 10 min, heat treatment mainly quantitative changes were observed suggesting that normal mRNAs were still present on polysomes, and their products could be completed in vitro in the heterologous cell-free system. The mRNAs coding for normal embryonal proteins were present in almost unchanged amounts in heat-shocked embryos as could be judged from the pattern of proteins synthesized in heterologous cell-free system supplemented with cytoplasmic RNA from normal and heat-shocked embryos. Thus the change in protein synthesis in heat-shocked embryos is not associated with degradation of normal embryonal mRNAs but with their inaccessibility for translation.     

Translational control in heat-shocked Drosophila embryos. Evidence for the inactivation of initiation factor(s) involved in the recognition of mRNA cap structure

Lysates from normally growing (25 degrees C) or heat shocked (37 degrees C, 45 min) Drosophila melanogaster embryos were obtained and the effect of analogues of the mRNA 5'-terminal cap, m7G(5')ppp(5')N structure and of potassium ions on their endogenous protein synthesis activity was studied. At optimal concentration of KCH3COO (75-80 mM), protein synthesis in normal lysates is strongly inhibited by cap analogues (m7GpppG, m7GDP, and m7GMP). At the same ionic conditions, heat shock lysates translate preferentially the heat shock messengers, and this translation is almost unaffected by the cap analogues. In contrast, residual synthesis of normal proteins in heat shock lysates was reduced by these compounds. By lowering the concentration of potassium ions it was possible to gradually reverse the inhibitory effect of the cap analogues in normal lysates and also to increase specifically the translation of normal mRNAs in heat shock lysates. Translation of normal mRNAs is also partial but specifically rescued by supplementing heat shock lysates with polypeptide chain initiation factors partially purified from rabbit reticulocytes. These data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, at least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the mRNA 5'-terminal cap structure.     

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.     

Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity.

Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption. Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate. This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control. Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells. When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2. However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control. When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed. Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.     

Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus.

After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.     

Translational control in early sea urchin embryogenesis: initiation factor eIF4F stimulates protein synthesis in lysates from unfertilized eggs of Strongylocentrotus purpuratus

We have used cell-free translation systems from unfertilized eggs and embryos of the sea urchin Strongylocentrotus purpuratus to analyze the mechanisms limiting protein synthesis in early embryogenesis. Unfertilized egg lysates supplemented with nuclease-treated reticulocyte lysate were stimulated 2-4-fold in incorporation of radioactive amino acid into protein. Thirty-minute zygote lysates supplemented in this way were not stimulated. These results suggested that a component limiting translation in the unfertilized egg lysate was provided by the nuclease-treated lysate and that this component was no longer limiting protein synthesis following fertilization. In view of these results, partially fractionated lysates and individual purified translational components from mammalian cells were tested for stimulation of the unfertilized egg lysate. A 1000000g supernatant devoid of ribosomal subunits also stimulated the unfertilized egg lysate. Thus, the stimulation was not due to the addition of active ribosomal subunits but to soluble elements in the reticulocyte lysate. Of the soluble components tested, only the cap-binding protein complex eIF4F caused a dramatic stimulation of the unfertilized egg lysate (2-3.5-fold). The 30-min zygote lysate was not stimulated by eIF4F or by any of the other components tested, supporting the hypothesis that a block in the translational machinery is removed at fertilization. A rabbit reticulocyte shift assay was used to analyze whether mRNA is limiting in early development. When unfertilized egg lysate was added to the shift assay, there was no shift in radioactivity from 43S to 80S complexes, indicating the unfertilized egg mRNA is not available for translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.     

Dephosphorylation of S6 and expression of the heat shock response in Drosophila melanogaster.

A basic ribosomal phosphoprotein of 30,000 molecular weight was rapidly dephosphorylated in cultured Drosophila melanogaster cells heat shocked at 37 degrees C. The protein was associated with the 40S ribosomal subunit and had an electrophoretic mobility similar to that of purified rat liver protein S6 on basic two-dimensional polyacrylamide gels as well as a similar partial proteolysis peptide map. In logarithmically growing cultures, this D. melanogaster S6 protein appeared to have a single phosphorylated species consisting of 30 to 40% of the total cellular S6. Thus, the nearly complete dephosphorylation of this protein observed in heat shock involves a large fraction of the cellular S6. The significance of this dephosphorylation in the expression of the heat shock response was investigated by examining the phosphorylation status of S6 in recovery from heat shock and in response to chemical inducers of the heat shock response. During recovery from a 30-min heat shock, the recovery of normal protein synthesis was almost complete in 2 to 4 hr, whereas there was no significant rephosphorylation of S6 for 8 h. Two chemical inducers of the heat shock response, canavanine and sodium arsenite, induced the synthesis of heat shock proteins in D. melanogaster cells. Sodium arsenite also caused an inhibition of normal protein synthesis similar to that observed in heat shock. Neither agent, however, caused significant dephosphorylation of S6. These results suggest that the dephosphorylation of S6, although invariably observed in heat-shocked cells, may in some cases be dissociated from both the induction of heat shock protein synthesis and the turnoff of normal protein synthesis which occur in a heat shock response.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Cellular localization of HSP23 during Drosophila development and following subsequent heat shock

The low-molecular-weight heat-shock protein HSP23 is synthesized in the absence of heat shock during Drosophila development. Here, I present a quantitative analysis of this phenomenon and describe the cellular localization of this protein during normal development and after a subsequent heat shock. HSP23 is first detected in the late third instar larvae and continues to accumulate reaching a maximum level in late pupae. In a 1-week-old adult, HSP23 can no longer be detected. Following lysis of whole pupae, HSP23 is found in the soluble lysate fraction in a form which sediments between 10 and 20 S. Exposure of larvae, pupae, and the adult fly to heat stress (37 degrees C) results in an increased amount of HSP23 which, however, is recovered in an insoluble particulate form following insect lysis. During recovery from heat shock, HSP23 is again found in the soluble 10- to 20-S lysate fraction. In pupae which are exposed to a severe heat stress (41 degrees C) HSP23 remains in the pellet fraction after the heat stress and no pupae are able to emerge as adult flies. However, when pupae are first exposed to a mild heat-shock treatment prior to the 41 degrees C stress, the thermotolerance process is induced and HSP23 is again rapidly found in the soluble lysate fraction during the recovery from heat shock. These observations suggest a possible correlation between the survival of pupae after heat shock and the recovery of HSP23 in the soluble lysate fraction as 10- to 20-S structures after the heat shock.     

Age-Dependent Metabolic Differences in Peripheral Hyphae of Rhizoctonia solani

Peripheral hyphae were separated from the remaining thallus of Rhizoctonia solani in exponential and stationary phases of growth. The QO(2) in whole cells of peripheral hyphae from young fungal colonies was on the average 2.6 times and the protein content 1.6 times greater than in peripheral hyphae from old fungal colonies. The overall rate of amino acid uptake was less in old than in young fungal colonies. In a polyuridylic acid-polyphenylalanine incorporating system, the two kinds of peripheral hyphae required ribosomes, supernatant fraction, polyuridylic acid, soluble ribonucleic acid, adenosine triphosphate, and pyruvate kinase. The rate of polyphenylalanine synthesis in old fungal colonies was slower than in the young fungal colonies. The ribosomes and supernatant fraction of the young and old fungal colonies were interchangeable and active. The factor responsible for deficient protein synthesis in old fungal colonies appears to be in the soluble fraction of the mycelium.     

Temperature sensitivity of protein synthesis initiation in the reticulocyte lysate system. Reduced formation of the 40 S ribosomal subunit - Met-tRNAf complex at an elevated temperature.

Analysis of protein synthesis in the rabbit reticulocyte lysate system revealed the existence of a temperature-sensitive step in chain initiation which became irreversibly inactivated in the incubation at 42 degrees C. This inactivation of initiation was accompanied by a marked reduction in formation of the 40 S ribosomal subunit - Met-tRNAf complex. Decreased protein synthesis and a decrease in formation of the 40 S complex were also evident in unfortified lysates which had been prewarmed at 42 degrees C prior to protein synthesis. Hemin did not protect such lysates. The addition of supernatant fraction of a fresh lysate did not promote recovery of the reduced protein synthesis by such prewarmed lysates. Moreover, the addition of supernatant fraction prewarmed at 42 degrees C in the presence of added hemin caused little inhibition of protein synthesis by fresh lysate. The results indicate that the supernatant fraction is not involved in the inactivation.     

Ubiquitin is a heat shock protein in chicken embryo fibroblasts.

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.     

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.