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.     

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.     

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.     

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.     

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.     

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

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

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.     

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl₂. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [¹⁴C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.     

ATTACHMENT OF RIBOSOMES TO MEMBRANES DURING POLYSOME FORMATION IN MOUSE SARCOMA 180 CELLS

Addition of nutrients to starved mouse S-180 cells leads to rapid conversion of ribosomal monomers to polysomes. During this process, a portion of the ribosomes originally found in the 17,000 g (10 min centrifugation) supernatant of cell lysates becomes firmly attached to structures sedimenting at 500 g (5 min centrifugation). Electron microscopy of sections of the intact cells showed the change from randomly distributed ribosomal particles to clusters. Association with membranes also became evident. The material sedimenting at 500 g comprised nuclei enclosed in an extensive endoplasmic reticulum (ER) network. This fraction prepared from recovering cells showed numerous ribosome clusters associated with the ER network. The appearance of many of these clusters indicated that the ribosomal particles were not directly bound to the membranes. RNase treatment released about 40% of the attached ribosomes as monomers, and ethylenediaminetetraacetic acid released 60% as subunits. It is suggested that during polysome formation a portion of the ribosomes becomes attached to the membranes through the intermediary of messenger RNA.     

Characterization of cytoplasmic and chloroplast ribosomal proteins of Euglena gracilis.

Active cytoplasmic ribosone subunits 41 and 62S were prepared by treatment with 0.1 mM puromycin in the presence of 265 mM KCl. Active chloroplast subunits 32 and 49S were obtained after dialysis of chloroplast ribosomal preparations against 1 mM Mg(2+)-containing buffer. Proteins from these different ribosomal particles were mapped by two-dimensional gel electrophoresis in the presence of urea. The 41S small cytoplasmic ribosomal subunit contains 33-36 proteins, the 62S large cytoplasmic ribosomal subunit contains 37-43, the 32S small chloroplast ribosomal subunit contains 22-24, and the 49ts large chloroplast ribosomal subunit contains 30-34 proteins. Since some proteins are lost during dissociation of monosomes into subunits, the 89S cytoplasmic monosome would have 73-83 proteins and the 68S chloroplast monosome, 56-60. The amino acid composition of ribosomal proteins shows differences between chloroplast and cytoplasmic ribosomes.     

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.     

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.     

Inhibition of the Peptide Bond Synthesizing Cycle by Chloramphenicol

To study the mechanism by which chloramphenicol inhibits bacterial protein synthesis, we examined the kinetics of the puromycin-induced release of peptides from transfer ribonucleic acid (tRNA) in the presence and in the absence of chloramphenicol. Washed Escherichia coli ribosomes with nascent peptides which had been radioactively labeled in vivo were used for this study. When such ribosomes were incubated in the presence of 10 mug of puromycin per ml, approximately one-fourth of the radioactive peptide material was rapidly released from tRNA. This rapid, puromycin-dependent reaction is assumed to be equivalent to the peptidyl transferase reaction. Chloramphenicol inhibited the extent of the puromycin-induced release of peptides by only 50%, demonstrating that some of the peptide chains which are present on active ribosomes react with puromycin, even in the presence of chloramphenicol. The addition of the supernatant fraction and guanosine triphosphate (GTP) increased the extent of the puromycin-induced release; this additional release was completely inhibited by chloramphenicol. Peptidyl chains on washed ribosomes prepared from chloramphenicol-inhibited cells were not released by puromycin in the presence of chloramphenicol and reacted slowly with puromycin in the absence of chloramphenicol. The release of peptidyl groups from these ribosomes became largely insensitive to chloramphenicol after preincubation of the ribosomes with GTP and the supernatant fraction. We conclude that chloramphenicol does not inhibit the peptidyl transferase reaction as measured by the puromycin-induced release of peptides from tRNA, but rather inhibits some step in the peptide synthesis cycle prior to this reaction.     

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.     

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.     

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.     

EFFECTS OF CHLORAMPHENICOL ON CHLOROPLAST AND MITOCHONDRIAL ULTRASTRUCTURE IN OCHROMONAS DANICA

The effect of chloramphenicol (CAP) on cell division and organelle ultrastructure was studied during light-induced chloroplast development in the Chrysophyte alga, Ochromonas danica. Since the growth rate of the CAP-treated cells is the same as that of the control cells for the first 12 hr in the light, CAP is presumed to be acting during that interval solely by inhibiting protein synthesis on chloroplast and mitochondrial ribosomes. CAP markedly inhibits chloroplast growth and differentiation. During the first 12 hr in the light, chlorophyll synthesis is inhibited by 93%, the formation of new thylakoid membranes is reduced by 91%, and the synthesis of chloroplast ribosomes is inhibited by 81%. Other chloroplast-associated abnormalities which occur during the first 12 hr and become more pronounced with extended CAP treatment are the presence of prolamellar bodies and of abnormal stacks of thylakoids, the proliferation of the perinuclear reticulum, and the accumulation of dense granular material between the chloroplast envelope and the chloroplast endoplasmic reticulum. CAP also causes a progressive loss of the mitochondrial cristae, which is paralleled by a decline in the growth rate of the cells, but it has no effect on the synthesis of mitochondrial ribosomes. We postulate that one or more chloroplast ribosomal proteins are synthesized on chloroplast ribosomes, whereas mitochondrial ribosomal proteins are synthesized on cytoplasmic ribosomes.     

The release and reassociation of 5.8 S rRNA with yeast ribosomes.

Yeast 5.8 S rRNA is released from purified 26 S rRNA when it is dissolved in water or low salt buffer (50 mM KCl, 10mM Tris-HCl, pH 7.5); it is not released from 60 S ribosomal subunits under similar conditions. The 5.8 S RNA component together with 5 S rRNA can be released from subunits or whole ribosomes by brief heat treatment or in 50% formamide; the Tm for the heat dissociation of 5.8 S RNA is 47 degrees C. This Tm is only slightly lower when 5 S rRNA is released first with EDTA treatment prior to heat treatment. No ribosomal proteins are released by the brief heat treatment. A significant portion of the 5.8 S RNA reassociates with the 60 S subunit when suspended in a higher salt buffer (e.g.0.4 m KCl, 25 mM Tris-HCl, pH 7.5, 6 mM magnesium acetate, 5 mM beta-mercaptoethanol). The Tm of this reassociated complex is also 47 degrees C. The results indicate that in yeast ribosomes the 5.8 S-26 S rRNA interaction is stabilized by ribosomal proteins but that the association is sufficiently loose to permit a reversible dissociation of the 5.8 S rRNA molecule.     

The puromycin reaction mediated by yeast ribosomes in high salt

The extend of the reaction between puromycin and yeast peptidyl-tRNA prelabeled in vitro was determined by measuring the distribution of trichloroacetic acid precipitable material in isokinetic sucrose gradients in the presence of 0.5 M KCl.Thus it was found that increasing amounts of puromycin remove increasing amounts of peptidyl-tRNA from the 80S position in the gradient. The extend of the reaction, however, was independent of pretreatment of the ribosomes with inhibitors of the translocation indicating that peptidyl-tRNA at the donor and at the acceptor site of the ribosomes are equally accessible to puromycin at 0.5 M KCl.The exposure of both ribosomal binding sites to puromycin in high salt is accompanied by an enhanced reactivity of puromycin towards peptidyl-tRNA. The ED₅₀ determined by measuring the inhibition by puromycin of the poly-U dependent phenylalanine incorporation drops from 5×10^-5 M below 250 mM KCl to 5×10^-6 M at 300 mM and higher concentrations of KCl.