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.     

Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes

A cell fractionation procedure is described which allowed, by use of MOPC 21 (P3K) mouse plasmocytoma cells in culture, the separation of the cytoplasmic free and membrane-bound ribosomes in fractions devoid of mutual cross-contamination, and in which the polyribosomal structure was entirely preserved. This was achieved by sedimentation on a discontinuous sucrose density gradient in which the two ribosome populations migrate in opposite directions. A variety of controls (electron microscopy, labeling of membrane lipids, further repurification of the isolated fractions) provided no evidence of cross- contamination of these populations. However, when an excess of free 60S or 40S subunits, labeled with a different isotope, was added to the cytoplasmic extract before fractionation, the possibility of a small amount of trapping and/or adsorption of free ribosomal particles by the membrane fraction was detected, especially in the case of the 60S subunits; this could be entirely prevented by the use of sucrose gradients containing 0.15 M KC1. EDTA treatment of the membrane fraction detached almost all the 40S subunits, and about 70% of the 60S subunits. 0.5 M KC1 detached only 10% of the ribosomal particles, which consist of the native 60S subunits and the monoribosomes, i.e. the bound particles inactive in protein synthesis. Analysis in CsC1 buoyant density gradients of the free and membrane-bound polyribosomes and of their derived 60S and 40S ribosomal subunits showed that the free and membrane-bound ribosomal particles have similar densities.     

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.     

Cerebral ribosomal protein phosphorylation in experimental hyperphenylalaninaemia.

Investigations were carried out on the effects of phenylalanine loading on ribosomal protein phosphorylation in cerebral cortices of infant rats. Administration of L-phenylalanine intraperitoneally, in doses of 1 or 2 mg/g body wt., resulted within 30 min in a significant decrease in incorporation of radioactivity from intracisternally administered [32P]Pi into constitutive ribosomal proteins of the cerebral 40S subunit. This phenomenon was not accompanied by significant variations in 32P uptake into the cerebral cytosol. Incorporation of radioactivity into ribosomal proteins of the cerebral 60S subunit exhibited only minor variations under these circumstances. Alterations in the phosphorylation state of cerebral 40S ribosomal proteins induced by phenylalanine loading involved principally the S6 protein, which exists in multiple states of phosphorylation. The proportions of the more highly phosphorylated congeners of this protein were markedly decreased, as detected by two-dimensional electrophoretograms and autoradiographs of the cerebral 40S ribosomal proteins. Phenylalanine loading also altered the relative extent of phosphorylation of the S6 protein in cerebral polyribosomes and monoribosomes. In control animals, the specific radioactivity of 40S proteins in cerebral polyribosomes was five to ten times that of 40S proteins in the monoribosome population. At 1 h after phenylalanine administration, the specific radioactivities of 40S proteins in the two ribosome populations tended to approach equality. These alterations in ribosomal protein phosphorylation were accompanied by a decrease in the proportion of polyribosomes in purified ribosome preparations isolated from cerebral cortices of phenylalanine-treated infant rats. In animals given the higher dose of phenylalanine (2 mg/g body wt.), subsequent administration of a mixture of seven neutral amino acids, which resulted in partial recovery of polyribosomes, also tended to reverse the changes in ribosomal protein phosphorylation. Variations in the activities of ribonuclease enzymes in the cerebral cytosol were also observed under these conditions. Administration of phenylalanine increased the activities of cerebral ribonucleases, whereas subsequent treatment with the amino acid mixture partly reversed this effect. The results suggest that alterations in cerebral ribosomal protein phosphorylation, ribosome aggregation and ribosome function are interrelated in experimental hyperphenylalaninaemia.     

Assembly of Membrane-bound Polyribosomes

The 60S large ribosomal subunit binds directly to membranes of the endoplasmic reticulum. Experiments with myeloma cells in tissue culture suggest that membrane-bound polyribosomes are assembled by attachment of small ribosomal subunits and mRNA to membrane-bound 60S subunits.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.     

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.     

Comparison of native and KCl treated 40S ribosomal subunits from the silkmoth Antherea pernyi and mammals

Native 40S ribosomal subunits and 18S ribosomal RNA from ovarian follicles of the silkmoth A. pernyi showed a lower sedimentation coefficient in comparison to ascites cells, in contrast to the KCl treated 40S ribosomal subunits where no difference was observed in both tissues. Moreover the silkmoth native 40S ribosomal subunits--in contrast to the KCl treated ones--could not reassociate with radioactive ascites cell 60S ribosomal subunits. These results, combined with the great similarities in the two dimensional electrophoretic patterns of 40S ribosomal proteins from silkmoth follicles and other mammalian cells lead to the possibility of the existence of a specific RNase associated with the 40S ribosomal subunit.     

Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide.

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.     

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.     

Phosphorylation of ribosomal proteins in rat cerebral cortex in vitro.

Incubation of cerebral cortical tissue from immature rats in the presence of [32P]orthophosphate resulted in similar rates of incorporation of radioactivity into the proteins of free and membrane-bound ribosomes. Incorporation of label into ribosomal proteins of both species continued actively for at least 3 hours. Since recovery of membrane-bound ribosomes from rat cerebral cortex is quite low, further analyses of the radioactive phosphoproteins were restricted to the free ribosome population. A significant fraction of the radioactivity which was precipitated with trichloroacetic acid was not removed by heating in trichloroacetic acid at 90 degrees or extracted with organic solvents and therefore was presumed to be covalently bound to protein. The radioactive phosphoryl groups present in the ribosomal proteins were mainly in ester linkages since they were readily removed by exposure to 1 N NaOH, relatively unaltered by 1N HCl, and unaffected by hydroxylamine. This conclusion was supported by the isolation of labeled o-phosphoserine and o-phosphothreonine residues from hydrolysates of ribosomal proteins. A significant fraction of the labeled phosphoproteins in the purified ribosomes appeared to be bound tightly to the ribosome structure since only 40% of the radioactivity could be removed by extraction of these ribosomes with 1 M KCl. Phosphorylation of proteins of cerebral monoribosomes was more rapid than the same process in polyribosomes from the same source. Eight radioactive phosphoprotein bands could be detected by electrophoresis of proteins obtained from unfractionated cerebral ribosomes on unidimensional polyacrylamide gels containing sodium dodecyl sulfate. The protein nature of these materials was confirmed by pronase digestion. Proteins of subribosomal particles isolated from the total free ribosomal population were labeled differentially. When dissociation was carried out in the presence of EDTA, the small subunit contained four radioactive phosphoprotein bands, whereas the large subunit contained five. Three of the radioactive phosphoprotein components of the small subunit were removed when dissociation of cerebral ribosomes which were previously washed with high salt media was carried out in the presence of puromycin and high salt. However, only the largest labeled phosphoprotein band of the large subunit was removed by this procedure. This component exhibited the same electrophoretic mobility as one of the radioactive phosphoprotein bands which was removed from the small subunit by high salt treatment..     

Synthesis of ribosomal proteins in developing Dictyostelium discoideum cells is controlled by the methylation of proteins S24 and S31.

Ribosomal protein mRNAs left over from growth are selectively excluded from polyribosomes in the first half of Dictyostelium discoideum development. This is due to the fact that they are sequestered by a class of free 40S ribosomal subunits, characterized by possessing a methylated S24 protein. At the time of formation of tight cell aggregates, the methylated S24 is substituted by an unmethylated S24, while protein S31 of the same or other 40S subunits becomes methylated. This leads to a rapid degradation of the ribosomal protein mRNAs.     

Metabolism of 18S-rRNA in rat liver cells in different functional states of the protein-synthesizing apparatus

The ratio of absolute radioactivities of 28S and 18S ribosomal RNA in membrane-bound and free polysomes and in free ribosomes of rat liver were studied under conditions of translation inhibition by cycloheximide, insulin and cAMP. Insulin and cAMP, in contrast with cycloheximide, did not induce selective degradation of 18S-rRNA. The data obtained are discussed in terms of the feasible role of S6 protein phosphorylation in degradation of the 40S ribosomal subunit.     

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.     

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.     

Heterogeneity of native ribosomal 60-S subunits in Ehrlich ascites tumor cells cultured in vitro.

Native large ribosomal subunits in cultured Ehrlich ascites tumor cells analyzed by high-resolution CsCl isopycnic centrifugation consist of at least two classes of particles with densities of 1.57 g/cm3 (LI) and 1.59 g/cm3 (LII), respectively. A wash with 0.5 M KCl converts LI into particles with the density of LII particles. Incubation of derived large subunits (density 1.59 g/cm3) with 0.5 M KCl wash of reticulocyte ribosomes leads to the formation of particles with the density of LI particles. A protein with a molecular weight of 57000 present in the high-KCl wash of 60-S native subunits was virtually absent in the KCl wash of 40-S subunits or polyribosomes suggesting that specific protein factors may be present on some native 60-S subunits. Possible functions of these protein factors are discussed.     

Effect of ribonuclease T1 on ribosomal subunits of rat liver.

The accessibility of 28S RNA within the ribosomal subunits to ribonuclease T1 was studied, in comparing results obtained after enzyme treatment of compact, K+ deficient 60S subunits and of EDTA-treated 60S subunits. RNA, extracted from the subunits, using a mixture of sodium dodecyl sulfate and phenol was analyzed on sucrose gradients. The RNA from active subunits was only degraded in high enzyme concentrations. In the K+ deficient subunits, RNA is more accessible since it breaks down into 6 well-defined fragments, sedimenting between 4S and 18.5S. Within the EDTA-subunits, there is no more protection of the RNA. In fact, it is degraded by weak enzyme concentrations, as is the free 28S RNA, giving heterogeneous fragments. Comparison of the melting curves of subunits and free 28S RNA showed that it is only in EDTA subunits that proteins do not stabilize the secondary structure of RNA. In the case of 40S subunits, the action of ribonuclease T1 combines with the action of the endogenous nuclease which makes the degradation process more difficult to analyze.     

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-¹⁴C]orotic acid and inorganic [³²P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.