Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts

Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus.     

Retention of mRNA on the endoplasmic reticulum membranes after in vivo disassembly of polysomes by an inhibitor of initiation

Membrane-bound ribosomes and messenger RNA remained associated with the microsomal membranes of human fibroblasts after cultures were treated with Verrucarin A, an inhibitor of initiation which led to extensive run-off of ribosomes from polysomal structures. When a membrane fraction from Verrucarin-treated cells containing such inactive ribosomes and mRNA was suspended in a medium of high salt concentration, extensive release of ribosomal subunits occurred without the need for puromycin. The mRNA nevertheless remained associated with the membranes. These results add support to the conclusion that, in human fibroblasts, mRNA is bound directly to ER membranes, independently of the ribosomes and nascent polypeptide chains.     

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.     

Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites

Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS-acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These observations suggest that ribophorins are in close proximity to the bound ribosomes, as may be expected from components of the ribosome-binding sites.     

Bound Ribosomes of Pea Chloroplast Thylakoid Membranes: Location and Release in Vitro by High Salt, Puromycin, and RNase

The mode of attachment of 70S ribosomes to thylakoid membranes from pea leaves was studied by determining the proportion of the bound RNA which was released by various incubation conditions. The results supported a model in which several classes of bound ribosomes could be distinguished: (a) very tightly bound, not released by any conditions yet tested (20% of the total); (b) monomeric ribosomes attached by electrostatic interaction with the membranes (30 to 40% of the total) and released by high salt; and (c) polysomes, with some of the ribosomes attached by a combination of electrostatic interactions and insertion of the nascent polypeptide chain into the membrane. These required a combination of puromycin and high salt for release. Other ("hanging") ribosomes of the polysomes were inferred to be attached through mRNA but not actually attached to the membranes directly; they could be released by RNase under low salt conditions, as well as by puromycin plus high salt.To obtain these results, chloroplasts had to be prepared in media containing 0.2 molar Tris at pH 8.5. Using Tricine buffers at pH 7.5 yielded thylakoid membranes whose ribosomes were removed almost completely by high salt alone; these showed no response to puromycin. However, pH 7.5 had to be used in all cases for ribosome dissociation in high salt media, as the ribosome structure appeared to be degraded by high salt at pH 8.5, and release then occurred without the need for puromycin.The kinetics of ribosome release by high salt showed a rapid initial phase with a half-life of 20 seconds. The extent of release by high salt was very dependent on the temperature of the incubation. Plotting the data according to the Arrhenius interpretation shows a significant break at about 15 C, with apparent activation energy of 20 kilocalories per mole below that temperature and 5 kilocalories per mole above that temperature. This result suggests that membrane fluidity might be an important factor permitting release of ribosomes under high salt conditions.Electron microscope pictures of the washed thylakoids showed polysomes closely associated with the outer membranes of grana stacks, and with the stroma lamellae. Following digitonin treatment of the membranes and centrifugation, fractions enriched in Photosystem I and presumed stroma lamellae were also enriched in bound RNA.     

Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin- KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.     

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.     

Association of messenger ribonucleic acid with mammalian microsomal membranes: characterization by analysis of cell-free translation products.

Total rough microsomes, isolated from the dog pancreas, were stripped of membranes-bound polysomes by treatment with either EDTA or puromycin and 0.5 M KCl. The stripped microsomal membranes were isolated relatively free from contamination, by using buoyant density centrifugation, and mRNA was isolated from both the membrane fraction and the released material. Depending on the method used to strip the rough microsomes, we found a variable but small percentage (3--15%) of the cellular poly(A)-containing mRNA attached to the microsomal membranes. Reextraction of isolated microsomal membranes with puromycin and 0.5 M KCl reduced the content of membrane-associated mRNA by approximately 50%, resulting in less than 2% of the total membrane-bound polysomal mRNA remaining associated with the microsomal membranes. The membrane-associated mRNA was characterized by translation in the wheat germ cell-free protein synthesizing system, and the products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The translation products of the membrane-associated mRNA were identical with those from the total pancreas mRNA and also with those obtained by using mRNA isolated from material released directly from the rough microsomes.     

Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.     

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of mitochondrial cytochrome P-450(SCC) and P-450(11 beta) and microsomal cytochrome P-450(C-21) in bovine adrenal cortex

Rabbit antibodies against cytochrome P-450 (SCC), P-450 (11 beta), and P-450 (C-21) from bovine adrenal cortex were prepared, and it was confirmed that these three cytochrome P-450 species are immunologically distinct from one another. Cytoplasmic sites of synthesis of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) in bovine adrenal cortex were determined by examining the presence of their nascent peptides on isolated free and bound ribosomes. Nascent peptides were released in vitro from ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) were isolated by immunoprecipitation. The nascent peptides of these three cytochrome P-450 species were found in both free and bound ribosomal fractions, suggesting that they share common sites of synthesis in the cytoplasm. However, the nascent peptides of mitochondrial P-450 (SCC) and P-450 (11 beta) were more concentrated in the free ribosomal fraction, whereas those of microsomal P-450 (C-21) were more abundant in the bound ribosomal fraction. The nascent peptides of the three cytochrome P-450 species were released from the membrane-bound ribosomes of rough microsomes into the cytoplasmic surface of microsomal vesicles by puromycin treatment.     

In vitro binding of 70S ribosomes to membrane structures of Escherichia coli

It was found that the maximal disattachment of the ribosomes from the membrane structures is observed upon their treatment with 10 mM tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 750 mM KCl, 5 mM magnesium acetate and 1 mM EDTA or puromycin. The most effective attachment of ribosomes to the membrane occurs in 10 mM tris-HCl buffer, pH 7.5, containing 5% sucrose and Mg2+. The increase of Mg2+ concentration in the medium from 0.5 mM up to 1 mM results in a 2-fold increase of the ribosomes bound to the membranes. The concentration of the ribosomal material involved in the reaction is very essential for ribosome binding to the membranes. The amount of ribosomes bound to the membranes increases proportionally to the increase of the ribosome concentration in the reaction mixture.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.     

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.     

RIBOSOMES BOUND TO CHLOROPLAST MEMBRANES IN CHLAMYDOMONAS REINHARDTII

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.     

Ribosomal-membrane interaction: in vitro binding of ribosomes to microsomal membranes

Rat liver rough microsomal membranes were stripped of bound ribosomes by treatment with puromycin and high concentrations of monovalent ions. Ribosomal subunits labeled in the RNA were detached from rough microsomes by the same procedure, recombined into monomers, and then incubated with stripped membranes in a medium of low ionic strength (25 mm-KCl, 50 mm-Tris-HCl, 5 mm-MgCl₂). These ribosomes readily attached to the stripped membranes, as determined by isopycnic flotation of the reconstituted microsomes. The binding reaction was complete after incubation for five minutes at 37 °C, but also proceeded at 0 °C, at a lower rate. Scatchard plots showed a binding constant of ~8 × 10⁷m^?1 and ~5 × 10^?8 mol binding sites per gram of membrane protein. Native rough microsomes showed a much lower binding capacity at 0 °C than stripped rough microsomes, but showed considerable uptake of ribosomes at 37 °C. Smooth microsomes, treated for stripping and incubated at 0 °C, accepted less than half as many ribosomes as stripped rough microsomes. Erythrocyte ghosts were incapable of binding ribosomes. Microsomal binding sites were heat sensitive, were destroyed by a brief incubation with a mixture of trypsin and chymotrypsin in the cold, and were unaffected by incubation with phospholipase C.Ribosome binding was decreased by increasing the concentration of monovalent ions and was strongly inhibited by 10^?4m-aurintricarboxylic acid. Experiments with purified ribosomal subunits revealed that at concentrations of monovalent ions close to physiological concentrations (100 to 150 mm-KCl), microsomal binding sites had a greater affinity for 60 S than for 40 S subunits.Stripped rough microsomes were also capable of accepting polysomes obtained from rough microsomes by detergent treatment. Although this binding presumably involves the correct membrane binding sites, polypeptides discharged from re-bound polymers were not transferred to the vesicular cavities, as in native microsomes. The released polypeptides remained firmly associated with the outer microsomal face, as shown by their accessibility to proteases.     

Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site.

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.     

Studies on the accessibility of nascent non-helical peptide chains on the ribosome

The accessibility of nascent polypeptides with special structural elements to the ribosome was investigated. Poly(C), poly(C, U) and poly(C, A) mRNAs were translated by E. coli ribosomes in vitro. The resulting peptides which were rich in prolines, remained on the ribosomal particles or were released after addition of puromycin. A protease from Aspergillus oryzae hydrolyzed the released peptides rapidly, whereas the degradation of the unreleased ones was only slightly affected. This result shows that the nascent peptides were protected against proteolytic attack by the ribosomal particles. Interestingly, the protease completely degraded the 30S particles whereas the 50S ones remained intact, even after prolonged incubation.     

A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression

We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7-10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40-50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger-ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.     

Study of the regulation of plastid development in Euglena gracilis. II. Functional localisation and synthesis of ribosomal chloroplast particles (author's transl)]

Chloroplast ribosomes in greening cells of Euglena gracilis are found either in the stroma or bound to thylakoid membranes. The membrane-bound chloroplast ribosomes are of two main types: those which can be released by 0.5 M KCl or by puromycin and 0.5 M KCl, and those which are released by detergent (deoxycholate or Triton X-100) and KCl. The ribosomes which are released by puromycin are presumably bound to chloroplast membrane by nascent peptide chains. Ribosomes released by puromycin are found only during the course of plastidial differentiation at the time of active thylacoid membrane synthesis. Following greening, those ribosomes remain bound to the membranes but can be removed by KCl alone. An analysis of RNA labelling showed that 30-S but not 53-S subunits of membrane-bound ribosomes are of uniform specific activity. This suggests that 30-S subunit exchange in a common pool while 53 S subunits remain membrane bound and do not exchange in a common pool. Membrane-bound chloroplast ribosomes which are released either by puromycin or by detergent are originally derived from loosely bound particles, released by 0.5 M KCl.     

PROTEIN SYNTHESIS BY CEREBRAL POLYSOMES PRETREATED WITH PUROMYCIN

Abstract— Polysomes prepared from rat cerebral microsomes, following preincubation with a high concentration of puromycin (2.5 mM) in the presence of rat liver soluble enzymes, were very similar to normal polysomes in yield, A 260_nm:A 280_nm ratio and in absorbance profile on sucrose density gradients. However, the capacity for amino acid incorporation was inhibited by more than 50 per cent by puromycin treatment. The extent of inhibition far exceeded what could be expected from the amount of residual puromycin bound to polysomes, suggesting that some essential step in polypeptide synthesis was damaged. An examination of the labelled polypeptides, using sucrose density gradient centrifugation, showed that most of the new chains synthesized by puromycin-polysomes were released into solution. However, small amounts of polypeptides of high specific radioactivity were distributed among the polysomal aggregates. In contrast to normal polysomes, the specific radioactivity of puromycin polysomes was the highest in aggregates of six or more ribosomes and declined sharply at the levels of trimers and dimers. It is suggested that cerebral polysomes pretreated with puromycin become defective in the termination mechanism with the consequence that even though they are capable of moving at least short distances on the messenger RNA and of releasing the polypeptide chains formed, a concomittant release of monomeric ribosomes is obstructed. This may result in the‘clogging’of the terminus of the mRNA, thus blocking further polypeptide synthesis.