Inhibition Of Polypeptide Chain Initiation Research Articles

Ischemia-mediated neuronal injury.

Resuscitation of the brain after a period of global ischemia is limited by two classes of post-ischemic pathologies: hemodynamic disturbances which prevent the adequate re-oxygenation of the ischemic brain, and metabolic disturbances which may lead to delayed neuronal death in so-called selectively vulnerable brain regions. The hemodynamic disturbances can be classified into the no-reflow phenomenon and the post-ischemic hypoperfusion syndrome. The no-reflow phenomenon results from a combination of increased blood viscosity and perivascular edema; the severity increases with the duration of ischemia, and the treatment is by combining arterial hypertension with dehydration and anticoagulation. The post-ischemic hypoperfusion syndrome is independent of the duration of ischemia, it develops after a delay and is due to an impairment of the metabolic/hemodynamic coupling mechanisms; there is no specific treatment at the present. The most important metabolic disturbance leading to delayed neuronal death is prolonged inhibition of protein synthesis. The injury is manifested already after 5 min ischemia but it progresses little if ischemia is prolonged to 1 h. Inhibition occurs at the translation level due to selective inhibition of polypeptide chain initiation. After brief periods of ischemia, the disturbance can be reversed by various anesthetics and hypothermia but there is no treatment if ischemia is prolonged. Exitotoxity, free radical-mediated reactions, disturbances of polyamine metabolism, acidosis and selective disturbances of gene expression may also be involved but are probably of lesser importance.

Interactions between double-stranded RNA regulators and the protein kinase DAI.

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.

Interactions between double-stranded RNA regulators and the protein kinase DAI.

The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change.

A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.

Mechanism of inhibition of polypeptide chain initiation in calcium-depleted Ehrlich ascites tumor cells.

Protein synthesis in Ehrlich ascites tumor cells is inhibited when cellular calcium is depleted by the addition of EGTA to the growth medium. This inhibition is at the level of polypeptide chain initiation as evidenced by a disaggregation of polyribosomes accompanied by a significant elevation in 80-S monomers. To identify direct effects of calcium on the protein synthesis apparatus we have developed a calcium-dependent, cell-free protein-synthesizing system from the Ehrlich cells by using 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a recently developed chelator with a high (greater than 10(5)) selectivity for calcium (pKa = 6.97) over magnesium (pKa = 1.77). BAPTA inhibits protein synthesis by 70% at 1 mM and 90% at 2 mM. This effect was reversed by calcium but not by other cations tested. The levels of 43-S complexes (i.e., 40-S subunits containing bound methionyl-tRNAf.eIF-2.GTP) were significantly lower in the calcium-deprived incubations, indicating either inhibition of the rate of formation or decreased stability of 43-S complexes. Analysis of 43-S complexes on CsCl gradients showed that in BAPTA-treated lysates, 40-S subunits containing eIF-3, completely disappeared and the residual methionyl-tRNA-containing complexes were bound to 40-S subunits lacking eIF-3. Our results demonstrate a direct involvement of Ca2+ in protein synthesis and we have localized the effect of calcium deprivation to decreased binding of eIF-2 and eIF-3 to 40-S subunits.

A Mechanism by which Adenovirus Virus-Associated RNAI Controls Translation in a Transient Expression Assay

The mechanism by which adenovirus virus-associated RNAI stimulates translational efficiency in a transient-expression assay in 293 cells was investigated. We showed that DNA transfection leads to activation of a protein kinase that phosphorylates the alpha subunit of eucaryotic initiation factor 2 and, as a consequence, inhibition of polypeptide chain initiation. Cotransfection of a plasmid encoding adenovirus type 2 virus-associated RNAI recovered the translational capacity by preventing activation of the kinase.

A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay.

The mechanism by which adenovirus virus-associated RNAI stimulates translational efficiency in a transient-expression assay in 293 cells was investigated. We showed that DNA transfection leads to activation of a protein kinase that phosphorylates the alpha subunit of eucaryotic initiation factor 2 and, as a consequence, inhibition of polypeptide chain initiation. Cotransfection of a plasmid encoding adenovirus type 2 virus-associated RNAI recovered the translational capacity by preventing activation of the kinase.

Inhibition of polypeptide chain initiation in Daudi cells by interferons. Evidence that activity of initiation factor eIF-2 and availability of mRNA are unimpaired

The accompanying paper [McNurlan & Clemens (1986) Biochem. J. 237, 871-876] shows that the inhibition of proliferation of Daudi cells by human interferons is associated with impairment of the overall rate of protein synthesis. We have examined whether two of the mechanisms which are believed to control translation in interferon-treated virus-infected cells may be responsible for the inhibition of protein synthesis during the antiproliferative response in these uninfected cells. Although the rate of polypeptide chain initiation is lower in interferon-treated Daudi cells, as indicated by the disaggregation of polysomes, there is no significant inhibition of activity of initiation factor eIF-2 or of [40 S . Met-tRNAf] initiation complex formation in cell extracts. The phosphorylation state of the alpha subunit of eIF-2 remains unaltered. There is no major decrease in mRNA content as a proportion of total RNA up to 4 days of interferon treatment, as judged by poly(A) content, although the amount of total mRNA/10(6) cells eventually declines. The mRNA present in extracts from interferon-treated cells remains translatable when added to an mRNA-dependent reticulocyte lysate system. We conclude that neither the interferon-inducible eIF-2 protein kinase pathway nor the 2',5'-oligo(adenylate)-ribonuclease L pathway are responsible for the inhibition of polypeptide chain initiation. Rather, the data suggest impairment at the level of formation of [80 S ribosome X mRNA] initiation complexes.

Use of an antibody to characterize and determine the role of the major Met-tRNA f deacylase from rabbit reticulocyte ribosomes

Inhibition of polypeptide chain initiation in rabbit reticulocyte lysate by phosphorylation of eukaryotic initiation factor-2(α) results, secondarily, in the enzymatic deacylation of Met-tRNA f on the 48 S initiation complexes that accumulate. We have prepared an antibody to a highly purified preparation of the major Met-tRNA f deacylase activity on rabbit reticulocyte ribosomes, termed deacylase II. Antibody, but not similarly purified normal IgG, completely neutralizes the activity of Met-tRNA f deacylase II and has no effect on Met-tRNA f deacylase I, a separate, minor, reticulocyte activity with the same substrate specificity but very different physical and enzymatic properties, strongly suggesting that deacylase I and II are distinct proteins. We partially purified Met-tRNA f deacylase activities from rabbit liver, myocardium and bone marrow ribosomes and found them to be similar to each other and to reticulocyte deacylase I in their enzymatic properties and insensitivity to anti-deacylase II, suggesting that deacylase I may be a general form of this enzyme, present in many cells, while deacylase II may be induced specifically during erythroid differentiation. Addition of the antibody to reticulocyte lysate incubated in the absence of hemin or presence of hemin plus 0.1 μg/ml poly(I·C) did not reverse the inhibition of protein synthesis but did reduce the rate of turnover/utilization of Met-tRNA f and increase the level of Met-tRNA f bound to 48 S initiation complexes, demonstrating that the deacylase does not directly inhibit protein synthesis under these conditions but does mediate the deacylation, loss, and thus greater than expected turnover of Met-tRNA f in the 48 S complexes that accumulate.

Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits.

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).

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.

Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich ascites tumour cells.

The rate of polypeptide synthesis is inhibited by 80% in Ehrlich cells incubated at 43 degrees C compared to those at 37 degrees C. The regulatory site of translation resides at polypeptide chain initiation. Polypeptide synthesis does not recover at the higher temperature; however, the inhibition is reversed by returning the cells to 37 degrees C. Neither new RNA synthesis or protein synthesis is required for recovery at 37 degrees C, eliminating degradation of mRNA and irreversible denaturation of a protein essential for polypeptide chain initiation. The concentration of 40-S initiation complexes was found to be reduced markedly in heat-shocked cells compared to controls. This was confirmed in the cell-free protein-synthesizing systems prepared from heat-shocked and control cells. Reversible alteration in the activity of components affecting eIF2 function is, therefore, a likely mechanism of regulation in heat-shocked Ehrlich cells. In extracts from heat-shocked cells, Met-tRNA synthetase activity was unaltered compared to control extracts.

Resistance to inhibitors of mammalian cell protein synthesis induced by preincubation in hypertonic growth medium.

Incubation of L-929 cells in growth medium containing excess NaCl induces cellular protein synthesis to become resistant to the inhibitory action of growth medium made hypertonic by the addition of Na-acetate, KCl, choline chloride, D-alanine, or sucrose. Hypertonic preincubation also induces resistance to the inhibitory action of the drugs dimethyl sulfoxide, ethanol, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, but not to the action of NaF or the inhibitors of polypeptide chain elongation, emetine and cycloheximide. Induction is independent of the solute used to make the induction medium hypertonic and is not mediated by an elevated intracellular Na+ concentration. In a separate series of experiments, it was determined that the formation of functional methionyl-tRNAf.40 S ribosomal complexes is not impaired by exposure of uninduced cells to hypertonic growth medium. The combined results are evaluated with respect to the mechanism of action of hypertonic growth medium in the inhibition of polypeptide chain initiation and the adaptive mechanisms that animal cells have evolved to deal with alterations in their extracellular environments.

Rabbit reticulocyte double-stranded RNA-activated protein kinase and the hemin-controlled translational repressor phosphorylate the same Mr 1500 peptide of eukaryotic initiation factor 2α

The inhibition of polypeptide chain initiation that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin [ l--5] or in the presence of a low level (1 O-l 00 ng/ml) of dsRNA [ 1,6-81 appears to be mediated by the phosphorylation of the iW, 35 000 (a) subunit of eIF-2, the initiation factor that promotes binding of Met-tRNAf to 40 S ribosomal subunits [9-l 31, but involves the action of two distinct protein kinases [14]. The inhibitor formed in the absence of hemin (HCR) occurs in the post-ribosomal supernate, is rapidly activated by incubation with NEM [ 151, and is associated with the autophosphorylation of a M, 90 000-100 000 protein [1,16-191, whereas the dsRNA-activated protein kinase (ds1) is found in the ribosomal fraction,is activated by dsRNA, and is associated with the phosphorylation of a M, 67 000 protein(s) [ 1,7,8,20]. Peptide analyses of elF-2cu, phosphorylated either by HCR or dsI, have shown a similar pattern of phosphopeptides suggesting that these two protein kinases phosphorylate the same site or sites [20-231. When eIF-2ol, phosphorylated by HCR, is subjected to exhaustive trypsin digestion, almost all the phosphate can be localized to a single, M, 1500 peptide [24];we report here that this same single peptide is phosphorylated by dsl.

Processing of the precursor for the mitochondrial enzyme, carbamyl phosphate synthetase. Inhibition by rho-aminobenzamidine leads to very rapid degradation (clearing) of the precursor.

Pulse-chase experiments using liver explants incubated in modified Eagle's medium showed that newly synthesized precursor for carbamyl phosphate synthetase (pCPS) passes very rapidly through the cytosolic compartment en route to mitochondria (t 1/2 is approximately 2 min). Since even a small pool of precursor could not be detected in association with mitochondria, processing of pCPS must occur either coincident with or immediately following its transmembrane uptake by the organelle. Treatment of explants with the protease inhibitor, p-aminobenzamidine, however, inhibited normal processing of the precursor. But, rather than accumulate in the cell, newly synthesized pCPS was nonspecifically degraded with kinetics (t 1/2 of 2-3 min) which are consistent with the idea that the precursor was almost instantly degraded upon reaching the blocked processing enzyme in a mitochondrion. The protease inhibitor had little or no effect on synthesis of pCPS. This was determined by isolating polysomes aminobenzamidine; the two polysome preparations were about equally active in synthesizing pCPS in vitro in the presence of an inhibitor of polypeptide chain initiation, aurin tricarboxylic acid.

Amplification of translational control by membrane-mediated events: a pleiotropic effect on cellular and viral gene expression.

This review deals with the events which are triggered in tissue culture cells upon exposure to medium hyperosmolarity, to virus infection and to inducers of terminal differentiation. Increased medium osmolarity mimics, in several ways, events which follow infection of cells by cytopathogenic viruses. These are: inhibition of uptake of amino acids, glucose and uridine, the release or activation of a low molecular weight substance which mediates an immediate and specific inhibition of polypeptide chain initiation, and alteration in the phosphorylation state of ribosomal proteins. All these effects appear to be related to or be a consequence of membrane alterations. Similar alterations in transport and protein synthesis are initiated in Friend erythroleukemic cells upon induction of terminal differentiation.

Location of a collagen-binding domain in fibronectin.

Preferential labeling of COOH-terminal sequences in newly synthesized fibronectin was achieved by short term incorporation of radiolabeled amino acids in the presence of pactamycin, an inhibitor of polypeptide chain initiation. The labeled fibronectin was then cleaved with cathepsin D under conditions that yield a large (137,000-dalton) fragment that lacks collagen-binding properties, and a smaller (72,000-dalton) fragment that retains the ability of fibronectin to bind to collagen. Determination of the relative specific radioactivities of the two fragments leads us to conclude that the collagen-binding domain in fibronectin is located in the NH2-terminal third of the polypeptide chain and not in a COOH-terminal region as previously indicated by other structural studies.

The control of protein synthesis by hemin in rabbit reticulocytes.

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 Mr (α) subunit of eIF-2, the initiation factor that mediates binding of Met-tRNAf to 40 s ribosomal subunits. The protein kinase action of HCR is relatively specific for eIF-2α, although HCR also autophosphorylates a 90–100,000 Mr 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-2α by HCR causes inhibition of polypeptide chain initiation is only partially understood. There is general agreement that the binding of Met-tRNAf to 40 s ribosomal subunits is reduced, perhaps due to impaired interaction of eIF-2α-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 tRNAfmet 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-tRNAf. 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-2α interrupts the process of polypeptide chain initiation.

Regulation of protein synthesis.

Synthesis of globin in reticulocyte lysates depends on the presence of heme, the prosthetic group of hemoglobin. In the absence of heme, an inhibitor of polypeptide chain initiation is activated. The inhibitor is a cyclic AMP-independent protein kinase that catalyzes the phosphorylation by ATP of the small subunit of the initiation factor eIF-2. This blocks the interaction of eIF-2 with eIF-2-stimulating protein (ESP) that is essential for initiation. Our observations are consistent with the view that the inhibitor is activated by phosphorylation catalyzed by a cyclic AMP-dependent protein kinase. Heme inhibits this enzyme and, in this way, prevents activation of the inhibitor of chain initiation.

Control of protein synthesis by hemin An association between the formation of the hemin-controlled translational repressor and the phosphorylation of a 100 000 molecular weight protein

The control of protein synthesis by hemin in rabbit reticulocytes is mediated by the formation of a high molecular weight protein inhibitor of polypeptide chain initiation, termed the hemin-controlled translational repressor, from a presynthesized prorepressor. The prorepressor, purified approx. 600-fold, was used to study the mechanism of hemin-controlled translational repressor formation. When the prorepressor is converted to the hemin-controlled translational repressor, either by prolonged warming in the absence of hemin or by incubation with N-ethylmaleimide for 5 min, and then incubated briefly with [γ- 32P]-ATP and Mg 2+, a protein that migrates as a 100 000 molecular weight component on sodium dodecyl sulfate-polyacrylamide gels becomes phosphorylated. The extent of phosphorylation of this component is directly proportional to the amount of prorepressor converted to the hemin-controlled translational repressor. In addition, the 100 000 molecular weight protein is not labeled when phosphorylation is attempted with the prorepressor or prorepressor warmed in the presence of hemin, indicating that the protein kinase responsible is probably the hemin-controlled translational repressor. Since the 100 000 molecular protein copurifies with the prorepressor and since the phosphorylation reaction is very rapid (50% complete within 30 s at 34° C), relatively insensitive to dilution, and behaves like an intramolecular reaction, the data suggest that the hemin-controlled translational repressor, once activated, may autophosphorylate a 100 000 molecular weight subunit of itself. Approx. 5 mol phosphate are incorporated per mol of 100 000 molecular weight protein, when the prorepressor is completely converted to the hemin-controlled translational repressor by N-ethylmaleimide. Neither the rate of conversion of prorepressor to the hemin-controlled translational repressor nor the subsequent phosphorylation of the 100 000 molecular weight protein is enhanced by cyclic AMP or reduced by incubation with 3′:5′-cyclic nucleotide phosphodiesterase, indicating that cyclic AMP plays no role in hemin-controlled translational repressor formation.