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.     

Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.     

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.     

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.     

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.     

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.     

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.     

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.     

Biogenesis of endoplasmic reticulum membrane in rat liver cells. III. Biosynthesis of NADPH-cytochrome c reductase and cytochrome b5 by loosely-bound ribosomes.

Membrane-bound ribosomes were separated into two distinct classes (loosely-bound and tightly-bound ribosomes) by treatment with 0.6 M KCl, 1 mM puromycin, 0.05% DOC, or 10 mM EDTA. It was also confirmed that any one of these reagents except for EDTA dissociated the same class of ribosomes from the membrane. A population of lighter microsomal vesicles was formed from rough microsomes upon the dissociation of loosely-bound ribosomes by treatment with these chemicals. Rough microsomes were subfractionated into lighter and heavier fractions, L-rMs and H-rMs, by centrifugation using a discontinuous gradient of sucrose consisting of 1.3 M, 1.5 M, and 2.1 M solutions. It was found that L-rMs was rich in loosely-bound ribosomes, whereas H-rMs contained a high proportion of tightly-bound ribosomes. It is likely that loosely-bound and tightly-bound ribosomes are heterogeneously distributed among rough microsomal vesicles. Loosely-bound ribosomes and tightly-bound ribosomes synthesize different kinds of proteins. Two microsomal membrane proteins, NADPH-cytochrome c reductase and cytochrome b5, were exclusively synthesized by loosely-bound ribosomes, whereas serum albumin, which is a major component of the secretory proteins of hepatocytes, was synthesized only by tightly-bound ribosomes. Since the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 are released from bound ribosomes to the cytoplasmic surface of endoplasmic reticulum, while those of secretory proteins are discharged into the lumen across the membrane, the strength of the association between ribosomes and microsomal membrane seems to be correlated with the direction of release of nascent peptides.     

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.     

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of NADPH-adrenodoxin reductase and adrenodoxin of bovine adrenal cortex mitochondria

Cytoplasmic free and membrane-bound ribosomes were isolated from bovine adrenal cortex, and characterized. Contributions of free and bound ribosomes to the synthesis of NADPH-adrenodoxin reductase (AdR) and adrenodoxin (Ad) were determined by examining the presence of their nascent peptides on isolated ribosomes. Nascent peptides were released from the ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of AdR and Ad were separately isolated by immunoprecipitation using antibodies. AdR nascent peptides were associated with free and loosely-bound ribosomes, whereas Ad nascent peptides were associated with free, loosely-bound and tightly-bound ribosomes. Smaller nascent peptides of AdR were carried by free ribosomes, whereas larger nascent peptides were preferentially carried by loosely-bound ribosomes. In the case of Ad, smaller nascent peptides were more abundant in free ribosomes than in bound ribosomes. The nascent peptides of Ad were released from bound ribosomes of rough microsomes to the aqueous milieu by puromycin treatment, suggesting the release of completed Ad peptides into the cytoplasm in cells.     

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.     

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.     

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.     

Vectorial discharge of nascent polypeptides attached to chloroplast thylakoid membranes.

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

fMet-tRNA F Met binding and peptidyl transferase function in free and bound ribosomes from normal and puromycin aminonucleoside-treated rats.

Treatment of rats with the aminonucleoside of puromycin, which increases the incorporation of labelled phenylalanyl-tRNA into polypeptide chains in liver ribosome preparations studied in vitro, did not change the factor-dependent binding of fMet-tRNA f Met to ribosomes nor the peptidyl transferase function of the ribosomes. Peptidyl transferase function, as measured by fMet-tRNA f Met-puromycin formation, was comparable in the free and bound ribosome preparations. Similarly, the factor-dependent binding of fMet-tRNA f Met to ribosomes was the same in free ribosome preparations obtained from rat liver as it was in bound ribosome preparations that had been freed of membranes by puromycin incubation and high salt wash.     

Identification of a Novel Stage of Ribosome/Nascent Chain Association with the Endoplasmic Reticulum Membrane

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.     

A protein-conducting channel in the endoplasmic reticulum

The existence of a protein-conducting channel in the endoplasmic reticulum membrane was demonstrated by electrophysiological techniques. Pancreatic rough microsome (RM) vesicles were fused to one side (cis) of a planar lipid bilayer separating two aqueous compartments of 50 mM salt. This exposed the cytoplasmic surface of the RMs, with its attached ribosomes, to the cis chamber. Addition of 100 microM puromycin to the cis side caused a large increase in membrane conductance, presumably the result of puromycin-induced clearance of nascent protein chains from the lumen of protein-conducting channels. When puromycin was added at low concentrations (0.33 microM), single channels of 220 pS were observed. These closed when the salt concentration was raised to levels at which ribosomes detach from the membrane (150-400 mM), indicating that the attached ribosome keeps the channel in an open conformation. A mechanism for a complete cycle of opening and closing of the protein-conducting channel is suggested.     

Controlled proteolysis of nascent polypeptides in rat liver cell fractions. II. Location of the polypeptides in rough microsomes

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.     

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.