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.     

A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

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.     

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.     

Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [(14)C]leucine and delta-amino[(14)C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.     

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.     

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.     

Biosynthesis of aldolase B by free ribosomes in rat liver

Free ribosomes and membrane-bound ribosomes were prepared from rat livers, and the contributions of these two types of ribosomes to the synthesis of aldolase B were studied by the immunoprecipitation of [3H]puromycin-labeled nascent peptides with a rabbit antibody to this enzyme. Although rat liver aldolase was recovered in both cytosolic and microsomal fractions by the fractionation of liver homogenate, the microsomal aldolase was immunologically identical with its cytosolic counterpart as confirmed by Ouchterlony immunodiffusion test. We examined the nascent peptide fractions prepared from free and bound ribosomes, and found that the nascent peptides of aldolase were mainly localized in free ribosomes. About 0.5% of the total nascent peptides of free ribosomes and 0.08% of those of bound ribosomes was aldolase. The site of synthesis of serum albumin was also examined as a reference standard by the immunoprecipitation of labeled nascent peptides, and the nascent peptides of this secretory protein were mainly associated with bound ribosomes, as reported by other workers. These observations confirm that aldolase B is mainly synthesized by free ribosomes in rat liver cells.     

The binding of ribosomal subunits to endoplasmic reticulum membranes

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60-80% of added 60S subunits and 10-15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane-60S subunit-40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.     

Effect of a modified separation procedure on the size and protein-synthetic activity of membrane-bound liver polyribosomes

1. Various subcellular fractions containing ribosomes were isolated from rat liver. 2. In the presence of [(14)C]leucine and Sephadex-treated cell sap the radioactivity incorporated into the synthesized protein resulting from the incubation of microsomal preparations or deoxycholate-treated polyribosomes was dependent on the amount of rRNA incubated. In contrast, when Sephadex-treated post-mitochondrial supernatant was incubated, the radioactivity incorporated into the synthesized protein was independent of the amount of rRNA incubated. 3. Microsomal preparations and membrane-bound ribosomes, prepared by the standard procedure, incorporated less [(14)C]leucine into protein, per mg of rRNA incubated, than free or deoxycholate-treated polyribosomes; accordingly, polyribosomes associated with the former fractions were found mainly as monomers. 4. If microsomal fractions or membrane-bound ribosomes were prepared by a simple modification of the standard procedure, i.e. by centrifugation on to a ;cushion' of 2m-sucrose, their protein-synthesizing activity was of the same order as that of the original post-mitochondrial supernatant, and membrane-free and deoxycholate-treated polyribosomes; in this case polyribosome profiles showed that very little degradation had occurred and compared well with those obtained for post-mitochondrial supernatant and isolated polyribosomes. 5. A method is described (Appendix) that provides a rapid and reliable assessment of the concentration of rRNA in subcellular fractions.     

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.     

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.     

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.     

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.     

Measurement of the protein-synthetic activity in vivo of various tissues in rats by using [3H]Puromycin.

The validity of a new technique was examined for estimating the protein-synthetic activity of various tissues in vivo. The basic assumption underlying the method is that the number of peptide chains growing on each active ribosome would increase as the protein-synthetic activity of each tissue increases. The principle of the procedure, which was devised originally by Wool & Kurihara [(1967) Proc. Natl. Acad. Sci. U.S.A. 58, 2401-2407] to determine in vitro the number of functional ribosomes in skeletal muscle, is as follows. Puromycin is known to bind easily to the C-terminal end of the growing peptide on ribosomes and thus stop further chain elongation. Hence, if the number of puromycin molecules attached to the nascent peptide is determined by using radioactive puromycin as a tracer, one can estimate the number of growing peptides, i.e. the activity of tissue protein synthesis. By using this technique, it is shown that both starvation and the feeding of a protein-free diet caused marked decreases in the relative rate of formation of peptidyl-puromycin, i.e. activity of protein synthesis in liver, skeletal muscle, heart, spleen, testis, lung, kidney and intestine.     

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.     

In vitro exchange of ribosomal subunits between free and membrane-bound ribosomes

Studies on the distribution of isotopieally labeled ribosomal subunits between free and membrane-bound ribosomes from rat liver showed that, upon release of nascent polypeptides in vitro, the small subunits of membrane-bound ribosomes could exchange with small subunits derived from free polysomes. However, under the same conditions, the large subunits of membrane-bound ribosomes did not exchange efficiently with large subunits derived either from free or bound polysomes; instead, the addition of large subunits caused a transfer of microsomal small subunits into a newly formed pool of free monomers.The small subunit exchange required a macromolecular fraction of the cell sap, was stimulated by ATP or GTP, and occurred at low concentrations of magnesium ions.Sodium dodecyl sulfate, polyacrylamide gel electrophoresis revealed close similarities between the protein complement of subunits from free and membrane-bound ribosomes, with the exception of one protein band which was more intense in free large subunits.     

Vectorial Discharge and Localization of Nascent Polypeptides in Rough Endoplasmic Reticulum of Developing Neuronal Perikarya of Rat Brain Cortex

Rough endoplasmic reticulum (RER) prepared from bulk-isolated neuronal perikarya of rat brain cortex of different postnatal ages was found to be active in vectorial discharge of nascent proteins through the membrane; this activity increased with the increasing age of animals and reached maximal values in adults. RER isolated from whole cortical tissue (containing all cell types) exhibited vectorial release only up to 18 days of age; the preparation from adult animals was essentially devoid of secretory activity. Controlled proteolysis of various preparations suggested that in neuronal RER of 8-day-old rats the proportion of nascent proteins operationally retained in the intravesicular space was about twice that retained by cortical preparations. For the purpose of comparison, these parameters were studied also in liver RER.     

Vectorial transport of proteins by membrane-bound ribosomes of nongrowing and growing Jerusalem artichoke tuber cells

Both nongrowing (water-incubated) and growing (hormonally stimulated) Jerusalem artichoke tuber cells contain membrane-bound (mb) ribosomes. Using a rapid flotation procedure, a membrane fraction was prepared from both types of cells. This fraction was enriched in mb ribosomes, contained NADH cytochrome c reductase activity, had RNA:phospholipid and RNA:protein ratios similar to those reported for rough microsomes from animal tissues, and supported synthesis of preinitiated proteins in vitro. Using puromycin and detergent release, vectorial transport of labelled polypeptides was measured in the in vitro system. Of proteins made by mb ribosomes from nongrowing cells, on 12% remained associated with microsome membranes following chain termination. The comparable figure for proteins from mb ribosomes of growing tissue was 42%. The membrane-associated proteins were preferentially protected from protease digestion. Some possible reasons are suggested for the correlation between cell growth and the association of newly synthesized proteins with microsomes. The role of proteins synthesized by mb ribosomes but not vectorially transported, in both growing and nongrowing cells, is unknown.     

Comparison of the protein content of free and membrane-bound rat liver polysomes and of the derived subunits

Ribosomal proteins from pure free and membrane-bound rat liver polysomes were analyzed with a highly resolutive two-dimensional gel electrophoresis technique, using sodium dodecyl sulfate in the second dimension. Three acidic proteins found in free polysomes were always absent from the membrane-bound polysomes. Their molecular weights were estimated to be 20 000, 19 500 and 18 500. When free ribosomes were dissociated into subunits, the three protein spots were still found in the 60S subunit pattern, but they were weaker than in polysomes. A possible involvement of these three proteins in the attachment of ribosomal structures to the membranes is proposed.