Immunoglobulin synthesis by free polysomes of mouse myeloma cells

Free polyribosomes isolated from mouse myeloma cells in tissue culture synthesize immunoglobulin chains. The presence of these peptide chains in the cytoplasm of intact myeloma cells has been investigated. Some immunoglobulin chains were observed, but it could not be ruled out that these were originally inside cisternae of the endoplasmic reticulum, which were broken during hogenization. We have also investigated the transport of the hypothetical cytoplastic immunoglobulins into the cisternae of the endoplasmic reticulum after incubation with radioactive amino acids and subsequent chase in the absence of protein synthesis. A model to account for synthesis of immunoglobulins on free polysomes is presented. This model assigns specificity for translation on membrane-bound polysomes to the N-terminal region of secretory proteins.     

Cell-Free Translation of Free and Membrane-Bound Polysomes and Polyadenylated mRNA from Rabbit Brain Following Administration of d-Lysergic Acid Diethylamide In Vivo

Abstract: Free and membrane-bound polysomes and polyadenylated mRNA isolated from rabbit brain were translated in an mRNA-dependent rabbit reticulocyte lysate system. Electrophoretic analysis of the cell-free translation products demonstrated that although most of the nascent proteins were common to both free and membrane-bound brain polysomes, qualitative and quantitative differences were observed. Compared with the results obtained with purified polyadenylated mRNA, the addition of intact polysomes to the cell-free translation assay was more efficient and produced higher molecular weight products. Analysis of the translation products of free and membrane-bound polysomes revealed the appearance of 74K protein following either LSD administration or hyperthermia induced by elevated temperature treatment. The presence of this 74K protein was verified by analysis of the translation products by two-dimensional gel electrophoresis.     

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.     

The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes.

Free and membrane-bound polyribosomes were isolated from the forebrain of actively myelinating 24-day-old rats. The poly(A)+ RNA (polyadenylated RNA) extracted from both fractions was translated in vitro in reticulocyte lysates [Hall & Lim (1981) Biochem. J. 196. 327-336] in the presence or absence of a heterologous microsomal membrane fraction from dog pancreas. The rat myelin basic proteins synthesized in vitro were isolated by CM-cellulose chromatography and by immunoprecipitation with purified anti-(myelin basic protein) antibody. The large (mol.wt. 18 500) and small (mol.wt. 16 000) myelin basic proteins were translational products of poly(A)+ RNA from both free and membrane-bound polyribosomes. The identity of the myelin basic proteins was verified by analysis of peptides generated by the cathepsin D digestion of the immunoprecipitated proteins synthesized in vitro, in comparison with authentic rat myelin basic proteins. Although several other translational products of membrane-bound polyribosomal poly(A)+ RNA were modified when microsomal membranes were present during translation, molecular weights of the myelin basic proteins themselves were unchanged. The myelin basic proteins synthesized in vitro also did not differ significantly in size from the authentic myelin basic proteins, indicating that these membrane proteins are unlikely to be synthesized as substantially larger precursor molecules. The presence of the specific mRNA species on both free and membrane-bound polyribosomes is compatible with the extrinsic location of the myelin basic proteins on the cytoplasmic surface of the myelin membrane.     

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.     

Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

Protein synthesis by membrane-bound and free ribosomes of secretory and non-secretory tissues

1. Methods for the separation of membrane-bound and free ribosomes from rat brain (cortex) and skeletal muscle were described and the preparations characterized by chemical analysis and electron microscopy. The attachment of ribosomes to membranes is not an artifact of the separation procedure. 2. The rate of incorporation of l-[(14)C]leucine into protein in vitro by the membrane-bound and free ribosomes from these two predominantly non-protein-secreting tissues is compared with that by similar preparations from rat liver. With all three tissues the initial rate was higher for the membrane-bound preparations. 3. By using the technique of discharging nascent polypeptide chains by incubation with puromycin followed by treatment with sodium deoxycholate (Redman & Sabatini, 1966), a major difference was observed for the vectorial discharge of nascent protein synthesized both in vivo and in vitro on membrane-bound ribosomes from liver, on the one hand, and brain and muscle, on the other. Whereas a large part of nascent protein synthesized on membrane-bound liver ribosomes was discharged into the membranous vesicles (presumably destined for export from the cell), almost all nascent protein from membrane-bound ribosomes from brain and muscle was released directly into the supernatant. Incorporation of [(3)H]puromycin into peptidyl-[(3)H]puromycin confirmed these findings. There was thus no difference between membrane-bound and free ribosomes from brain on the one hand, and from free polyribosomes from liver on the other, as far as the vectorial release of newly synthesized protein was concerned. 4. Incubation with puromycin also showed that the nascent chains, pre-formed in vivo and in vitro, are not involved in the attachment of ribosomes to membranes of the endoplasmic reticulum. 5. The differences in vectorial discharge from membrane-bound ribosomes from liver as compared with brain and muscle are not due to the different types of messenger RNA in the different tissues. Polyphenylalanine synthesized on incubation with polyuridylic acid was handled in the same way as polypeptides synthesized with endogenous messenger. 6. It is concluded that there is a major difference in the attachment of ribosomes to the membranes of the endoplasmic reticulum of secretory and non-secretory tissues, which results in a tissue-specific difference in the vectorial discharge of nascent proteins.     

A simple and rapid procedure for high-yield isolation of essentially undegraded free and membrane-bound polysomes from rat liver

A procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4.1.2.13, and argininosuccinate synthetase, EC 6.3.4.5], one mitochondrial [ornithine carbamoyltransferase, EC 2.1.3.3] and one peroxisomal [catalase, EC 1.11.1.6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5 h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.     

Polymorphism in fowl serum albumin. VII. Distribution and activity of free and membrane-bound polysomes in developing fowl liver

The quantity and activities of membrane-bound and free polysomes in livers from chick embryos at successive stages of development were compared in cell-free protein-synthesizing systems. Membrane-bound polysomes increased 2-fold between 8 and 18 days of development, while total ribosome content remained constant. Free polysome activity also remained constant during this period, while that of membrane-bound (total--free) polysomes decreased, possibly because of an increase in ribonuclease activity in this fraction. Serum albumin biosynthesis occurred primarily on membrane-bound polysomes. With liver development, increased secretion of serum proteins may be correlated with synthesis of serum albumin on increasing numbers of membrane bound polyribosomes.     

Site of synthesis of the alpha and beta subunits of the Na,K-ATPase in brine shrimp nauplii

Developing nauplii (embryos) of the brine shrimp Artemia salina are an excellent model system for studying the biogenesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase). The nauplii exhibit a burst of Na,K-ATPase synthesis between 6 and 32 h of development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We have now determined the sites of synthesis of the alpha and beta subunits of the Na,K-ATPase in developing A. salina nauplii. Membrane-bound and free polysomes were isolated from nauplii, and RNA was extracted from the polysomes. The polysomal RNA was translated in vitro in a rabbit reticulocyte lysate, and the translation products were immunoprecipitated by anti-subunit antisera. The immunoprecipitated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Our data show that the alpha subunit precursor is synthesized on membrane-bound polysomes and the beta subunit precursor is synthesized on free polysomes. In addition, the alpha subunit precursor appears as two separate peptides on sodium dodecyl sulfate-polyacrylamide gels, which suggests that the two alpha subunit forms seen in mature brine shrimp Na,K-ATPase are products of two distinct messenger RNAs. The beta subunit precursor appears as a single discrete band, unlike the mature beta subunit, which appears as a diffuse band.     

Sites of synthesis of viral proteins in avian sarcoma virus-infected chicken cells.

We determined the sites of synthesis of avian sarcoma virus-specific proteins in infected chicken cells by immunoprecipitation of the products synthesized in vitro by free and membrane-bound polyribosomes; 85% of Pr76, the precursor of the viral internal structural proteins (group-specific antigens), was synthesized on free polyribosomes, and 15% was synthesized on membrane-bound polyribosomes. Pr92, the lycosylated precursor of the viral glycoproteins (gp85 and gp35), was synthesized exclusively on membrane-bound polyribomes, which is consistent with its role as a membrane protein. When we investigated the site of synthesis of pp60src, the product of the avian sarcoma virus src gene, we found that 90% was synthesized on free polyribosomes, whereas 10% was detected on membrane-bound polyribosomes. The implications of these results with respect to the subcellular location of pp60src are discussed.     

Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane-Bound Polysomes

Abstract: Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[³⁵S]methionine. The translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α¹, α², β¹, and α²) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two-dimensional gel. MB has a molecular weight and isoelectric point similar to α-tubulin. Only trace amounts of α- and β-tubulin and actin were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and α- and β-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α- and β-tubulin; MB yielded peptides not found in tubulin. We conclude that membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to α-tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.     

Free and membrane-bound polyribosomes in BHK cells infected with Sindbis virus.

The data presented in the paper demonstrate that in BHK cells infected with Sindbis virus virtually all the 42S mRNA not in nucleocapsid is associated with free polyribosomes, whereas the 26S mRNA is distributed between free and membrane-bound polyribosomes. We suggest that the 26S RNA polyribosomes are bound to the membranes through the nascent chains of the B1 protein and that a large percentage of 26S RNA polyribosomes free in the cytoplasm may be due to the small amount of rough endoplasmic reticulum in BHK cells. In addition, we found that intracellular nucleocapsid is in the nonmembrane fraction of the cytoplasm of infected cells.     

Intracellular site of prolactin synthesis in rat pituitary cells in culture

Free and membrane-bound polyribosomes were isolated from control and thyrotropin releasing hormone-treated GH₃ cells. The two polysome fractions were used to direct {³H}leucine incorporation into prolactin in both heterologous and homologous cell-free protein-synthesizing systems. Prolactin was measured by immunoprecipitation and SDS-disc gel electrophoresis of the reaction products. Only membrane-bound polysomes directed incorporation of {³H}leucine into labeled prolactin. In additon, intact cells were pulselabeled with {³H}leucine, free and membrane-bound polysomes were isolated, and newly synthesized prolactin associated with each polysome fraction was measured. In control cells, {³H}prolactin represented about 0.4 and 4.2% of total acid-insoluble radioactivity in free and membrane-bound polysomes, respectively; whereas, in thyrotropin releasing hormone-treated cells, these values were about 1 and 20%, respectively. Added {³H}prolactin did not associate nonspecifically with membrane-bound polysomes. We conclude that prolactin is synthesized predominantly on membrane-bound polysomes in GH₃ cells.     

Evidence for synthesis of alkaline phosphatase on membrane-bound polysomes in Escherichia coli.

Polysomes containing nascent chains of alkaline phosphatase have been isolated from a membrane-bound polysome preparation. Indirect immunoprecipitation using conformation-specific antibodies has been employed. This technique provides a good enrichment of these polyribosomes since routinely no more than than 10--15% of non-specific immunoprecipitation was observed. The yield of the procedure is generally 40% but can be increased if higher non-specific immunoprecipitation is tolerated. Antibodies, previously described, directed against uncoiled or folded monomers of alkaline phosphatase can be used as primary antibody to recognize the nascent chains contained in membrane-bound polysomes which suggests that these chains are partially folded.     

Storage Protein Synthesis in Maize: III. Developmental Changes in Membrane-bound Polyribosome Composition and in Vitro Protein Synthesis of Normal and Opaque-2 Maize

Zein synthesis accompanied an increase in large polyribosomes of maize (Zea mays) endosperm cells. The two classes of polyribosomes (free and membrane-bound) had dissimilar size class distributions. Membrane-bound polyribosomes were predominantly large size classes, which were not found in free polyribosomes. The ratio of large membrane-bound polysomes to total membrane-bound polysomes was highest when zein was being synthesized. Appearance of the large polysomes correlated with the onset of zein accumulation in vivo. These large size classes were nearly absent in the opaque-2 mutant at all stages of endosperm development. Similarly, rRNA content was reduced in the mutant from that in normal endosperm development. These differences were associated with reduced in vitro synthesis and in vivo accumulation of zein.     

Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland.

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.     

Import of rat liver mitochondrial malate dehydrogenase. Synthesis, transport, and processing in vitro of its precursor

Rat liver total RNA was translated in a reticulocyte lysate, and the precursor of rat liver mitochondrial malate dehydrogenase was identified by a monospecific antibody against the denatured mature enzyme. The precursor is about Mr = 1500 to 2000 larger than the monomeric form of the mature protein. The major spots of the two-dimensional peptide map of the two proteins were identical. The precursor was synthesized on free polysomes, but not membrane-bound polysomes. Upon fractionation by molecular sieve chromatography on Sephadex G-100, the size of the precursor was slightly larger than the dimeric form of the mature protein. Incubation of the precursor with isolated mitochondria from Chinese hamster ovary cells resulted in uptake and processing of the precursor to the mature size. The processed form was resistant to trypsin indicating that it was translocated into mitochondria. Processing was complete in 10 to 30 min at 30 degrees C. Rapid binding of the precursor to mitochondria was also observed at 0 or 30 degrees C. Processing but not binding was inhibited by an uncoupler.     

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.     

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate- cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S- translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.