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

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

Segregation of specific classes of messenger RNA into free and membrane-bound polysomes

Analysis of mRNA populations from rat liver rough microsomes and free polysomes by homologous and heterologous cDNA . mRNA hybridization shows that the two mRNA populations are distinct, demonstrating that specific mRNA classes are efficiently segregated for translation in association with endoplasmic reticulum membranes. We estimate that approximately 90% of the mRNA in membrane-bound polysomes contains a diverse set of messengers with a minimum of 500--2000 different species although approximately 5--8 messengers may constitute 25--30% of the mRNA mass. The complexity of the mRNA population of free polysomes appears to be comparable to that estimated for total liver poly(A) + mRNA by other investigators, and is likely to be substantially greater than that of the bulk of bound mRNA. In addition, mRNA in free polysomes lacks the high abundance class characteristic of mRNA-bound polysomes. The substantial complexity of the bound mRNA population suggests that the segregation of polysomes in rough microsomes is not limited to a small class specialized in manufacturing secretory proteins, but extends to polysomes engaged in the synthesis of proteins for intracellular distribution. The segregation of specific messengers into the free and membrane-bound classes was abolished when polysome disassembly was induced by administration of ethionine. Thus, messenger RNA molecules themselves lacked the capacity for segregation, although they contain information for segregation which is expressed during translation. These findings are consistent with the presence of signal sequences in nascent polypeptides which determine the attachment of ribosomes to endoplasmic reticulum membranes.     

Comparison of the Effect of Intravenous Administration of d-Lysergic Acid Diethylamide on Free and Membrane-Bound Polysomes in the Rabbit Brain

Abstract: The intravenous administration of LSD to young adult rabbits resulted in the disaggregation of both free and membrane-bound classes of brain polysomes. Based on the analysis of LSD dosage and the time course of the LSD-induced brain polysome shift, it was found that free polysomes were more sensitive to the drug than the membrane-bound polysome fraction. LSD-induced hyperthermia may be involved in the disaggregation of free and membrane-bound polysomes, since a correlation was found between the extent of LSD-induced hyperthermia and the degree of brain polysome shift. Prevention of LSD-induced hyperthermia by maintaining the animal at 4°C blocked the disaggregation of both polysome classes. Induction of hyperthermia by elevation of ambient temperature also resulted in a shift in free and membrane-bound polysomes. In all cases the disaggregation of polysomes to monosomes was not caused by RNase activation. During polysome disaggregation, polyadenylated mRNA associated with both free and membrane-bound polysomes was not degraded but was relocalized from polysomes to monosomes.     

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.     

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

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

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [¹²⁵I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [¹²⁵I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [¹²⁵I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[¹²⁵I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [¹²⁵I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[¹²⁵I]Fab to free polysomes was also obtained. The [¹²⁵I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[¹²⁵I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.     

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.     

Membrane-bound and free polysomes in transformed and untransformed fibroblast cells

A rapid and quantitative fractionation procedure has been used to measure the amounts of free and membrane-bound polysomes in growing and stationary Py3T3 and 3T3 (mouse) cells. A comparison of growing 3T3 and Py3T3 cells does not reveal any significant differences with regard to the ratio of the two polysome fractions. The amount of free and membrane-bound polysomes decreases in both 3T3 and Py3T3 cells as they approach the stationary state, an effect which is much more pronounced for free polysomes. At greatly reduced growth rates or in stationary cells, however, the amount of membrane-bound polysomes doubles in Py3T3 cells while it decreases even further in 3T3 cells. By contrast, the amount of free polysomes remains at a reduced level in both 3T3 and Py3T3 cells when cell multiplication is inhibited.Based on the hypothesis that membrane proteins are selectively synthesized on membrane-bound polysomes, an attempt is made to relate the results to accumulated data in the literature and discuss its possible significance with respect to the loss of growth control in Py3T3 cells.     

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.     

Direct association of messenger RNA-containing ribonucleoprotein particles with membranes of the endoplasmic reticulum in ethionine-treated rat liver.

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The fate of the mRNA molecules after polysome breakdown was investigated by measuring the amount of poly(A)-containing mRNA in membranous and non-membranous fractions obtained from the cytoplasm of ethionine-treated rat liver. The amount of poly(A)-containing mRNA in the membrane fraction of ethionine-treated liver was found to be the same as that of normal liver. When poly(A)-containing mRNAs from various fractions were translated in a wheat germ system and the products were isolated by immunoprecipitation, the albumin-specific mRNA was found exclusively in the membrane fraction of both normal and ethionine-treated livers. The membrane-bound mRNA in ethionine-treated liver, selectively labeled with [14C]orotate, was banded in CsCl gradient centrifugation at 1.42 g/ml which corresponds to the previously reported mRNA-containing ribonucleoprotein particles. From these results, we concluded that even after the polysome disaggregation by ethionine, most of the mRNA of membrane-bound polysomes remains attached to the endoplasmic reticulum membranes independently of ribosomes and the nascent polypeptide chains.     

Storage Protein Synthesis in Maize: II. Reduced Synthesis of a Major Zein Component by the Opaque-2 Mutant of Maize

Two zein proteins (Z1 and Z2) represent the majority of the protein synthesized during maize endosperm development. Undegraded membrane-bound polysomes isolated from normal maize synthesized these proteins when incubated in a cell-free protein-synthesizing system from wheat germ. The proteins synthesized in vitro were similar to authentic zein in ethanol solubility and electrophoretic mobility. Zein synthesis was associated with large size classes of membrane bound polysomes in normal maize.Membrane-bound polysomes isolated from developing kernels of opaque-2 mutant synthesized less total zein in vitro, and dramatically reduced incorporation into the Z1 component. The reduction in total zein corresponded to a 50% reduction in the level of membrane-bound polysomes in opaque-2, and the near absence of the large polysome size classes, which synthesized zein in normal maize. We concluded that the opaque-2 mutation results in a decreased "availability" of the zein mRNAs, reflected in a reduced level of membrane-bound polysomes.     

Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary

We have studied the shape and size distribution of membrane-bound polysomes in somatotropes and mammotropes, which are the sources, respectively, of growth hormone and of prolactin in the rat pituitary. The observations were made in conventional electron micrographs of these cells in situ, where occasional surface or en face views of the rough endoplasmic reticulum allow the polysomes to be seen as rows of ribosomes arranged in distinctive patterns on the membranes. It is possible by this means to characterize the shape and number of ribosomes for the total population of bound polysomes in the respective cell types. The great majority of membrane-bound polysomes in these two cell types (81% in somatotropes, 78% in mammotropes) have an approximately circular shape and contain an average of 6.8 (somatotropes) or 6.5 (mammotropes) ribosomes, which is an appropriate size for translation of the polypeptide hormones produced by these cells. About 17% of the membrane-bound polysomes in somatotropes and 20% in mammotropes have a spiral shape, resembling somewhat the letter "G," and contain about eight to nine ribosomes in both cell types. The preponderance of circular polysomes on the rough endoplasmic reticulum of somatotropes and mammotropes suggests the possibility that ribosomes (or the 40S ribosomal subunit) may recycle on the polysome after the translation of growth hormone or of prolactin.     

Dissociation of polysome aggregates by protease k

Apparent large size-classes of zein-synthesizing polysomes from developing kernels of Zea mays L. were converted to smaller polysomes after treatment with Protease K. The reduction in polysome size was not a result of ribonuclease activity, inasmuch as the enzyme did not affect the free polysomes or the size of the mRNA from the membrane-bound polysomes. High concentrations of MgCl(2) in polysome buffer inhibited ribonuclease activity and appeared to cause protein interaction between nascent zein polypeptides. Although Protease K inhibited the polysome's capacity for protein synthesis, it was a useful reagent for determining if polysomes were aggregated by protein.     

Polysome formation and stability in pea stem and root tissue

Polysome stability and the formation of various polysomal populations in pea stem and root tissue were examined. Both total ribosomal fraction and four polysome populations were isolated: FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). The content of above mentioned populations decreased in roots and stems during germination. In both roots and stems a gradual decrease of FP participation in the total polysomal population was also observed during germination. On the other hand, an obvious increase in participation of CMBP population in the total polysomes pool was observed in later stages of germination. Increase of CMBP participation in pea root and stem tissues in later stages of germination is probably due to intensive enzymatic protein synthesis taking place in them. These proteins may participate in elongating growth of cells. The results of investigation on polysomes stability showed that total polysomes isolated from pea roots appeared to be more resistant to digestion by exogenous ribonuclease (EC 3.1.27.5) than polysomes isolated from stems. As protein-mRNA interactions are widely known and ribosomes are also very adhesive structures, numerous non-ribosomal proteins are present in the polysome preparations. We suppose that changes in proteins bound to polysomes indicated by us previously, significantly influence both the stability and also translatability of polysomes isolated from different plant organs.     

Metabolism of Poly(A) in Plant Cells: Discrete Classes Associated with Free and Membrane-bound Polysomes

In the subapical region of dark-grown pea epicotyls about 40% of the total polysomes are associated with membranes. The presence of poly(A) in polysomal mRNA was detected by hybridization of unlabeled RNA with (3)H-poly(U). Both free mRNA and messenger ribonucleoprotein particles in polysomes hybridize with (3)H-poly(U) quantitatively. The binding of (3)H-poly(U) to polysomes is increased by treatment with the detergent sodium dodecyl sulfate. Since detergent influenced the (3)H-poly(U) binding more in membrane-bound polysomes than in free, there may be more protein(s) associated with the poly(A) portion of the mRNA in membrane-bound polysomes. Analysis of the poly(A) segments isolated from the mRNA of these two classes of polysomes indicates that there are discrete classes of poly(A) and they appear to be differentially associated with free and membrane-bound polysomes. Mean size distribution of poly(A) in free polysomes is larger than in membrane-bound polysomes.Following treatment (2 days) with the plant growth hormone indoleacetic acid, there is a gradual decrease in the mean length of total poly(A), which appears to correspond to a decrease in the size of the polysomes and their associated mRNA.     

Storage Protein Synthesis in Maize: Isolation of Zein-synthesizing Polyribosomes

Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane-bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mm Mg(2+) contained unique classes of large polysomes. These large polysomes were sensitive to ribonuclease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the principal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels.     

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.     

Labelling kinetics of RNA containing poly(a) in liver subcellular fractions

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

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.