Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion.

Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [³H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.     

Immunological studies of the fragments of bovine cartilage proteoglycan produced by chondroitinase-trypsin digestion.

The peptidyl transferase activity of polysomes from Escherichia coli, rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to 10-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.     

RIBOSOME-MEMBRANE INTERACTION : Nondestructive Disassembly of Rat Liver Rough Microsomes into Ribosomal and Membranous Components

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl₂, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [³H]puromycin into hot acid-insoluble material and from the release of [³H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (~15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.     

Ribosome-membrane association in fat body tissues from reproductively active females of Leucophaea maderae

Polysomal and microsomal profiles from fat body tissues of Leucophaea analysed by a combination of equilibrium sedimentation and sedimentation velocity centrifugations on sucrose density gradients revealed that microsomes from egg-maturing females are considerably more dense than those from allatectomized (reproductively inactive) females or from males. This greater density is conferred on the microsomes of reproductively active females by the binding of many more ribosomes (polysomes) to the membranes. Treatment of the microsomes with 500 mM K⁺ or 1 mM puromycin resulted in a removal of only a few ribosomes and polysomes from the membranes, as is documented with the electron microscope. Incubation of microsomes with a combination of 500 mM K⁺ and 1 mM puromycin resulted in a complete degranulation of the membranes. Such microsomes attained a density similar to those of the inactive tissues. From the available evidence it is concluded that the female specific protein (vitellogenin), which is induced by the juvenile hormone, is synthesized on ergastoplasmic membranes and released into the cisternae as is known for the synthesis of exportable proteins in several vertebrate systems.     

Nerve-dependent changes in content of ribosomes, polysomes, and nascent peptides in newt limb regenerates.

Regeneration of a newt limb requires a constant supply of adequate amounts of a neuronal contribution at the amputation site. Denervation during the early stages of regeneration precludes its growth and morphogenesis. It has been reported that denervation of a regenerating limb lowers the efficiency of incorporation of radioactive amino acids to 60% of contralateral control levels. To gain more insight into the mechanism responsible for this decrease, we examined the effects of denervation on the size distribution and quantity of regenerate polysomes. We characterized the [³⁵S]methionine-labeled nascent peptidyl-tRNA from polysomes by hydroxyapatite chromatography. Moreover, we show that the labeled nascent peptides on polysomes can serve as a measure to quantitate the relative amounts of ribosomes on polysomes and the relative size of the translational machinery. Thus, we report that [³⁵S]methionine-labeled nascent polypeptides on polysomes from denervated regenerates contain about 48% less radioactivity than those from controls. Despite decreased incorporation of [³⁵S]methionine into nascent peptides, the relative distribution of radioactivity across linear sucrose gradients is not significantly altered by denervation. Studies of polysomes labeled with [³H]uridine prior to denervation indicate that ribosome content is depressed by denervation. Our results suggest that the nerve-dependent decrease in protein synthesis is mediated by decreasing the number of ribosomes active in protein synthesis. In addition, similarities in the ratios of free monosomes to polysomes and the relative size distribution of polypeptides between denervated and innervated regenerates indicate that in denervated regenerates the number of translatable mRNA molecules decreases in a coordinate manner with the number of ribosomes active in protein synthesis.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons

Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.Polysomes were isolated from developing cotyledons 20–25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm³ (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.     

Vectorial discharge of nascent polypeptides attached to chloroplast thylakoid membranes.

Chloroplast thylakoids with attached ribosomes were isolated from Chlamydomonas reinhardti. They were allowed to incorporate labeled amino acids into polypeptides. Labeled membranes were recovered from the reaction mixture, and a portion was treated with puromycin. The amount of labeled polypeptides released to the medium, and to the membranes by puromycin was determined by comparing radioactivity in soluble protein before, and after untreated, and puromycin-treated membranes were solubilized with the detergent Nonidet P-40. About 20% of the radioactive protein associated with the membranes was in nascent chains which were terminated by puromycin. Essentially all of terminated nascent chains remained with the membranes, and thus, were vectorially released. The results support the hypothesis that polypeptides which are synthesized by thylakoid-bound ribosomes are being incorporated into the membranes as they are synthesized.     

Comparison of the activities for polypeptide synthesis of polysomes prepared with different detergents from normal and regenerating rat liver.

When prepared in the presence of deoxycholate, the activities for polypeptide synthesis of polysomes from normal and regenerating rat liver were similar. However, when the polysomes were prepared in the presence of either Triton X-100 or Lubrol WX, the polysomes from regenerating liver had about three to four times more activity than those from normal liver. On the other hand, the activities for polyphenylalanine synthesis of ribosomes from regenerating rat liver were similar irrespective of whether these ribosomes were prepared in the presence of deoxycholate or Triton X-100.     

Site of initial glycosylation of mannoproteins from Saccharomyces cerevisiae.

The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U-14C]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U-14C]mannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level.     

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [³⁵S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA_f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [³H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.     

Comparision of the structure and function of polysomal and helical ribosomes from Entamoeba invadens

Some structural and functional properties of ribosomes from polysomes and from helix aggregates of Entamoeba invadens have been compared by sucrose gradient analysis and assays of in vitro protein synthesis. Actively growing trophozoites, lacking helices, presented normal polysome profiles in sucrose gradients. The single large ribosomal helix aggregate (chromoatoid body) of cysts diappeared as the cells were disrupted. Gradient profiles of cyst extracts contained predominantly large and small ribosome subunit peaks and no evidence of remaining helix fragments of mRNA-bound polysomes. Sequential profiles of trophozoites incubated with NaF or cycloheximide (which both stimulate ribosome aggregation, but at different rates) showed that polysome breakdown occurred before aggregates appeared and, again, that helices broke down to subunits in vitro. Radioactive ribosomes synthesized during vegetative growth were collected into helices during encystation. Subunits of these ribosomes cosedimented with comparable particles isolated from trophozoites. Ribosomes from both trophozoites and cysts were active in cell-free protein synthesis, although activity in cyst extracts required the addition of trophozoite-soluble fraction. It was concluded that ribosomes from polysomes and helices in E. invadens were probably identical and that the ability to form helices was an intrinsic property of mature mRNA-free ribosomes of this organism.     

Regulation of mitochondrial protein synthesis at the polyribosomal level.

Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.     

Regulation of macromolecular synthesis during hyphal germ tube emergence from Mucor racemosus sporangiospores

Protein and RNA syntheses were examined during hyphal germ tube emergence from sporangiospores of a dimorphic phycomycete, Mucor racemosus. Both classes of macromolecules were synthesized immediately upon introduction of the dormant sporangiospores into nutrient medium. The specific rates of synthesis of both protein and RNA accelerated during initial germ tube emergence and reached a maximum when the emergence of new germ tubes ended. The specific rates of synthesis later decreased during further hyphal elongation. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by sucrose density gradient centrifugation, and the rate of amino acid addition to nascent polypeptide chains was calculated throughout the developmental sequence. The results showed that both the percentage of ribosomes active in protein synthesis and the velocity of ribosome movement along the mRNA were continuously adjusted throughout hyphal germ tube development. The free intracellular amino acid pools were measured throughout development. Alanine, glutamate, and aspartate were present at very high concentrations in the dormant spores but were rapidly depleted during hyphal germ tube emergence. The results of these studies are discussed in relation to hyphal germ tube development from yeast cells of Mucor and dormant spores of other fungal species.     

On the rate of ribosome translocation anisotropy and ribosome saturation in the polysome

This study examines the rate of ribosome translocation in the mammalian polysome engaged in protein synthesis by utilizing our knowledge of the hydrodynamic behavior of the rat liver polysomes, sedimenting in a linear sucrose density gradient. The average distance between adjacent ribosomes in the polysome was estimated assuming an extended linear configuration of the polysomes during sedimentation. Based on this estimate, the velocity of ribosome movement along the messenger RNA appears to be non-uniform and inversely related to the ribosome content of the polysome. Such non-uniformity prevails at stages of translation prior to ribosome “saturation” of the polysome. A correlation has been made between the results reported herein and previously published evidence on the rate of polypeptide chain synthesis. The steady-state condition for the polypeptide chain assembly is viewed as representing the state of ribosome “saturation”, characterized by a minimal ribosome velocity and a maximum density of ribosome distribution, both functions being uniform throughout the entire length of the polysome.     

A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

Glycosylation of nascent polypeptide chains in Aspergillus niger

Polysomes were isolated from Aspergillus niger and were characterized on sucrose gradients in several ways. First, they were found to be susceptible to degradation by treatment with RNase or EDTA. Second, they were labeled after treating mycelia with short pulses of [³H]uridine or [³H]leucine prior to polysome isolation. Third, they were capable of stimulating incorporation of [³H]leucine into trichloroacetic acid-precipitable material in a chick reticulocyte cell-free protein-synthesizing system. When isolated [³H]leucine pulse-labeled polysomes were treated with either EDTA-RNase or puromycin, 80–90% of the radioactivity was released, indicating that only the nascent polypeptide chains were labeled. After exposing mycelia for 1 min to [¹⁴C]mannose, the polysomes were exclusively labeled, indicating that initial glycosylation takes place on nascent polypeptide chains. Preincubation of mycelia with 2-deoxyglucose followed by pulse-labeling with [³H]leucine and [¹⁴C]mannose showed that 2-deoxy-d-glucose inhibits both protein synthesis and glycosylation. However, similar preincubation with tunicamycin caused an 80% drop in [¹⁴C]mannose label in the polysomes, but only a 10–20% drop of [³H]leucine label, suggesting that glycosylation of nascent chains in A. niger involves an oligosaccharide-lipid intermediate, since it has been shown that tunicamycin inhibits the synthesis of such an intermediate. When isolated polysomes were placed into an in vitro glycosylating mixture containing Mn²⁺, GDP-[¹⁴C]mannose, and smooth membranes from A. niger nascent chains were labeled. This reaction was shown to be dependent on addition of polysomes to the mixture and was not inhibited by 2-deoxy-d-glucose or tunicamycin. Both in vivo and in vitro glycosylated nascent chains were found to have about the same size range, and so it is suggested that in vitro no new oligosaccharide chains were synthesized, but preexisting chains were extended.     

Differences in the incorporation of [3H]-glucosamine into nascent polypeptide chains on free polysomes and two fractions of membrane-bound polysomes in mouse myeloma cells

The incorporation of [³H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after nitrogen cavitation of cells, remained bound to the endoplasmic reticulum (ER) associated with the nucleus (fraction 2). Polypeptide chains on membrane-bound polysomes in the microsomal fraction (fraction 1) and free polysomes contained much less radioactivity. Since nascent polypeptide chains contained within membrane-bound polysomes of fraction 2 are glycosylated at an earlier stage than those in fraction 1 it is likely that this represents a difference in type of proteins synthesized in the respective fractions of ER.     

Helical polysomes induced by aflatoxin B1 in vivo : A new hypothesis for helix formation by chemicals and carcinogens

We have studied the induction of helical polysomes by aflatoxin B₁ in liver and kidney cells from rat and mouse. We succeeded in giving to reticulocyte polysomes a shape resembling helices after in vitro treatment with O-methylthreonine which is used as an inhibitor of polypeptide chain termination. From this and knowing the site of action of aflatoxin B₁ on rat liver polysomes, we hypothesize that the induction of helical polysomes in tissues from adult animals treated by chemicals or carcinogens is due to the inhibition of release of ribosomes from the messenger RNA (mRNA). Theoretical studies of protein synthesis inhibition are in agreement with this new hypothesis.