The association in vitro of polyribosomes with ribonuclease-treated derivatives of hepatic rough endoplasmic reticulum. Characteristics of the membrane binding sites and factors influencing association

1. Pancreatic ribonuclease in dilute EDTA has been shown to condition rough-microsomal membranes from adult rat liver to accept exogenously added rat liver polyribosomes in vitro at 0-4 degrees C. Treated smooth membranes would not significantly interact with polyribosomes. 2. The conditioning process decreased the membrane RNA content and removed polyribosomes from vesicle surfaces as viewed electron-microscopically. 3. Binding to these conditioned membranes was shown to be uninfluenced by changes of temperature (0-37 degrees C) and pH (6.9-7.8) or the presence of cell sap, but was inhibited by increasing the concentration of potassium chloride. 4. Possession of a polyribosome-binding capacity by conditioned rough membranes was not dependent on adventitious materials that could be dislodged by high ionic strengths. 5. Trypsin treatment under mild conditions destroyed the binding capacity of ribonuclease-conditioned rough membranes. 6. A 2-10S residual RNA was recovered from ribonuclease-conditioned membranes, but its partial removal had no effect on the capacity of membranes to accept polyribosomes. However, some role for this residual RNA in attaching polyribosomes could not be discounted. 7. Evidence is considered that polyribosome-binding sites are intrinsic features of conditioned membranes isolated from rough-microsomal fractions, and that long-range ionic bonding is a primary factor in polyribosome interaction with these binding sites.     

Distribution of histone messenger RNA among free and membrane-associated polyribosomes of a mouse myeloma cell line

The distribution of histone messenger RNA among free polyribosomes and two classes of membrane-associated polyribosomes was studied in an immunoglobulin-secreting mouse myeloma cell line. The two classes of membrane polyribosomes were distinguished on the basis of their sensitivity to dissociation from membranes upon exposure to high concentrations of salt or during repeated centrifugation through sucrose. Histone messenger RNA is specifically excluded from that class of polyribosomes most tightly associated with cellular membranes.     

Biosynthesis of collagen and other proteins on tightly and loosely bound polyribosomes from chick embryos]

Free polyribosomes and polyribosomes bound to endoplasmic membranes were isolated from 10-day-old chick embryos by differential centrifugation. The tightly and loosely bound polyribosomal fractions were isolated from the membrane-bound polyribosomes using 0,5 M KCl. The synthesis of collagen and non-collagen proteins on the polyribosomes were studied in a homologous cell-free system. It was shown that the polyribosomes tightly bound to the membranes possess a lower protein-synthesizing activity as compared to free and loosely bound polyribosomes. The amount of bacterial collagenase-cleaved polypeptides in the protein product synthesized on the polyribosomes tightly and loosely bound to the memranes and on free polyribosomes is 31, 23 and 9%, respectively. The data obtained suggest that the loosely bound polyribosomes are actively involved in collagen synthesis and that this fraction is not a contamination of free polyribosomes in the preparations of totally bound polyribosomes. The role of tightly and loosely bound polyribosomes in the formation of the membrane polyribosomal complex is discussed.     

Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii.

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.     

Polyribosomal attachment to rat liver and hepatoma endoplasmic reticulum in vitro. A method for its study

A system for study and measurement of the attachment in vitro of exogenous polyribosomes to membranes has been presented. Its main features are use of low temperature, post-microsomal supernatant, pyrophosphate and citric acid to remove ribosomes from the surface of rough endoplasmic reticulum, and a method for quantitative separation of unattached from membrane-associated polyribosomes. The following were found. (1) Rough endoplasmic reticulum, from which ribosomes had been removed by treatment with pyrophosphate and citrate, bound over 50% of added polyribosomes, whereas the untreated (or control) rough and smooth endoplasmic reticulum and the smooth endoplasmic reticulum treated with pyrophosphate-citrate did not bind polyribosomes. (2) The polyribosome-binding capacity of rough endoplasmic reticulum stripped of its ribosomes decayed upon storage of the membranes at 0-4 degrees C. The half-life of this decay was about 6 days whereas that of the polyribosome-binding capacity of hepatoma stripped rough endoplasmic reticulum was about 1.5 days. (3) Preparations of stripped rough endoplasmic reticulum after reassociation with polyribosomes in vitro were quite similar to preparations of native rough endoplasmic reticulum as viewed with the electron microscope. Evidence is presented to support the contention that association of polyribosomes with membranes was the result of polyribosomal reattachment to the membranes rather than trapping of the polyribosomes between vesicles of the membranes.     

Localization of Polyribosomes Containing Alkaline Phosphatase Nascent Polypeptides on Membranes of Escherichia coli

A procedure has been developed for extracting membranes from bacterial cells under conditions that keep a large fraction of bacterial polyribosomes intact. Freeze-thawing spheroplasts in the presence of deoxyribonuclease, followed by differential centrifugation, permits a separation of free and membrane-associated polyribosomes. The latter fraction contains as much as 40% of cell ribosomal ribonucleic acid (RNA) and 55% of cell messenger RNA (mRNA). Nascent polypeptides were divided almost equally between the two fractions, but 70 to 80% of alkaline phosphatase nascent chains, detected both chemically and immunologically, were derived from polyribosomes associated with the bacterial membrane. Analysis of the fractions for mRNA specific for the lac and trp operons by RNA-deoxyribonucleic acid hydridization showed somewhat larger amounts on membrane than on free polyribosomes, but enrichment for nascent alkaline phosphatase (a secreted protein) on membranes was consistently greater, suggesting that polyribosomes making secreted proteins are more tightly bound to membranes. Electron micrographs of the membrane preparations show relatively intact membranes with clusters of polyribosomes on their inner surfaces.     

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.     

IUPAC Commission on the Nomenclature of Organic Chemistry (CNOC) and IUPAC-IUB Commission on Biochemical Nomenclature (CBN). Nomenclature of cyclitols. Recommendations, 1973.

The amino acid-incorporating activities of free polyribosomes, rough membranes and rough membranes reconstituted in vitro, derived from rat liver, were compared. The amino acid-incorporating activity of the two membrane fractions were very similar in their response towards changes in pH, Mg2+ concentration and temperature, but differed from the response of the amino acid-incorporating activity of free polyribosomes. Free polyribosomes irreversibly lost part of their amino acid-incorporating capacity after they had become bound to rough membrane, from which the original ribosomes had been removed. Ribonuclease activity present in the membrane fraction may be responsible for this loss.     

Functional interaction of free polyribosomes with the membrane of the endoplasmic reticulum in a cell-free protein-synthesizing system from plasmacytoma X5563

In cell-free protein synthesis by the murine plasmacytoma X5563, which had become a nonproducing mutant, mixed systems with free polyribosomes and mirosomes incorporated ¹⁴C-amino-acid into protein 3–8 times greater than the sum of the incorporations in the individual system irrespective of S-100 concentrations. This enhancement was inhibited by lecithinase A and was markedly reduced at high KCl concentrations. Smooth endoplasmic membranes had more stimulatory activity than rough endoplasmic membranes. The results indicate that the membrane of the endoplasmic reticulum and free polyribosomes interact in the cell-free protein-synthesizing system, resulting in the enhancement of protein synthesis.     

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro.

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.     

Is the cytoskeleton-plasma membrane complex involved in lens protein biosynthesis?

Calf lens fiber cells contain a population of polyribosomes that direct, at leastin vitro, the synthesis of a specific plasma membrane protein MP26. This protein may serve as a marker in terminal differentiation, since it is absent in the lens epithelium but appears in lens fiber plasma membranes. The MP26 manufacturing polyribosomes are found to be associated with a structural complex in which also the cytoskeleton and plasma membranes participate. They can be released from the complex by treatment with DNAse I. This result presumably reflects the involvement of actin in the linkage of the MP26 synthesizing polyribosomes to the cytoskeleton-membrane complex.     

Cytoplasmic microtubules are essential for the formation of membrane-bound polyribosomes

Colchicine, at low intracellular concentrations, causes a rapid depolymerization of membrane-associated polyribosomes. Poly(A+) mRNA is rapidly lost from these polysomes, and 80 S monomers are left attached to the membranes of the endoplasmic reticulum. Binding studies and measurements of intracellular colchicine concentrations indicate that the drug is acting via depolymerization of cytoplasmic microtubules. The vinca alkaloids, vincristine and vinblastine, have the same effect on polyribosomes, whereas lumicolchicine is ineffective. Furthermore, cordycepin and actinomycin D are without effect on polyribosomes indicating that colchicine is not simply inhibiting the production or transport of new mRNA. It appears that disruption of the cytoplasmic microtubule network prevents membrane-associated ribosomes from reinitiating protein synthesis resulting in the rapid loss of mRNA.     

Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton.

Gel filtration with 1% agarose (Bio-Gel A-150m) separates polyribosomes bound to microsomal membranes from 'free' polyribosomes when these fractions are prepared by standard centrifugal techniques. However, when polyribosomes contained in an unfractionated postmitochondrial supernatant are run on an identical column, over 90% of the total polyribosomes are present as aggregates, designated 'membrane-cytomatrix', which are eluted in the column void volume. Polyribosomes are not released from these aggregates on removal of microsomal phospholipids by treatment of postmitochondrial supernatant with 1% Triton X-100, a neutral detergent. The aggregates are disrupted by the usual ultracentrifugation techniques used in subcellular fractionation. After treatment of membrane-cytomatrix with Triton X-100 to remove phospholipids and membrane proteins, 58% of the polyribosomes still remain associated with protein-containing complexes in the form of a cytomatrix and are not 'free'. Preparations of both membrane-cytomatrix and cytomatrix are capable of sustained protein synthesis. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the cytoskeletal proteins actin and myosin are present in the cytomatrix. Incubation of cytomatrix preparations with the actin-depolymerizing agent deoxyribonuclease I caused release of the polyribosomes. Polyribosome release by deoxyribonuclease I was prevented by prior incubation with phalloidin, which is known to stabilize F-actin. Thus polyribosomes are associated with cytoskeletal elements in rat liver, and this association is dependent on polymeric forms of actin.     

Free and membrane-bound polyribosomes in normal and Rauscher-virus-infected mouse spleen cells

1. Free and membrane-bound polyribosomes and ribosomal monomers were isolated from normal and Rauscher-virus-infected mouse spleens by means of discontinuous sucrose density gradients. 2. The addition of ribonuclease inhibitor from rat liver was essential to protect these polyribosomes from degradation. To separate the smooth and rough membranes from ribosomal monomers an additional centrifugation step through a continuous sucrose density gradient was necessary. 3. After infection a marked increase in rRNA from both membrane-bound and free polyribosomes was observed. Treatment of the membrane-bound polyribosomes with sodium deoxycholate yielded only 80S particles even when ribonuclease inhibitor was added. 4. A striking feature of the infected spleen was the occurrence of large polyribosomes. Up to 40 monomers per polyribosome could be counted on electron micrographs.     

In vitro synthesis of A-particle structual protein by membrane-bound polyribosomes.

On the basis of association with endoplasmic reticulum membranes, poyribosomes isolated from mouse myeloma MOPC-104E were separated into two classes, membrane bound and free. The membrane-bound and free polyribosomes were then compared for their capacity to incorporate [35S]methionine into A-particle proteins in vitro. As revealed by a radioimmunological assay method, labeling of A-particle protein occurred with the membrane-bound polyribosomes but not with the free polyribosomes. Peptide mapping of the immunoprecipitated, in vitro [35S]methionine-labeled product confirmed that A-particle protein had been synthesized in vitro.     

Study of membrane-bound ribosomes during pea chloroplasts formation]

Membrane-bound ribosomes of chloroplasts, isolated from pea seedlings during grana formation, can be partially liberated by 0.5 M KCl and 0.001 M puromycin. In case of mature chloroplasts, after the completion of grana formation process these agents are inefficient, and liberation of ribosomes and polyribosomes may be achieved only after solubilization of thylakoid membranes by 1% Triton X-100. Electron microscopic study of the heavy membrane fraction of young chloroplasts reveals electron-transparent membranes, containing rings and discs of thylakoids with a diameter of about 2 mum. These rings are liberated together with ribosomes under the action of 0.5 M KCl; Triton X-100 liberates equally-sized annular polyribosomes. The rings detected in chloroplast membranes at early stages of development are regarded as structures, precursor grana thylakoids, and the annular polyribosomes included into them as immediate participants of thylakoid morphogenesis.     

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.     

Membrane-bound ribosomes of myeloma cells. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane- bound ribosomal particles

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.     

The effect of cycloheximide on incorporation of newly formed mRNA into membrane-bound polyribosomes in rat liver cells]

Incorporation kinetics of new synthesized mRNA into free and endoplasmic membrane-bound polyribosomes in the absence of normal translation (when protein synthesis in inhibited by 98% with cycloheximide) is studied. mRNA is found to incorporate into both free and bound polyribosomes. Relative content of new synthesized membrane-bound polyribosomes in the presence of cycloheximide within 2.5-4.5 hours is by 30-40% lower as compared with the control. This fact can be explained either by the absence of a growing peptide of a sufficient length, which is necessary for the formation of a part of membrane-bound polyribosomes, or by a restricted number of attachment sites on membranes as a result of delayed translation of mRNA in pre-existed polyribosomes. It is suggested that 1) the growing peptide in liver cells is responsible for the recognition of a membrane only under the formation of only one type of membrane-bound polyribosomes, or 2) the formation of all bound polyribosomes has a single mechanism and the growing peptide does not participates in the membrane recognition.