Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes.

PG (prostaglandin) E1 inhibits the uptake of iridine, thymidine, 2-deoxy-D-glucose and L-isoleucine into human diploid WI38 fibroblasts. The inhibition occurs within seconds of the addition of the prostaglandin to the culture. PGE2, PGF1alpha and PGF2alpha behave similarly. Arachidonic acid and 8,11,14-eicosatrienoic acid also decrease uptake in the presence or absence of indomethacin. Other unsaturated fatty acids such as oleic acid, linoleic acid and linolenic acid are essentially inactive. Ricinoleic acid (the 9-hydroxyoleic acid), however, inhibits uptake to about the same degree, at concentrations similar to those of the prostaglandins. Results indicate that this rapid blockage by the prostaglandins and certain fatty acids is not cyclic AMP-mediated. For example, although PGF1alpha and PGF2alpha are much poorer stimulators of cyclic AMP formation than are PGE1 and PGE2, they are nevertheless effective inhibitors of substrate uptake. Adrenaline, a very effective stimulator of cyclic AMP formation in the cells, is not inhibitory. Also, the addition of 8-methylthioadenosine 3':5'-cyclic monophosphate (methylthio cyclic AMP) to the culture, methylthio cyclic AMP decreases the uptake of nucleotides into cultures undergoing active cell division, approximately to values found in quiescent cultures. PGE1 also has this effect on cells undergoing active growth. This gradual decrease is substrate uptake caused by PGE1 appears to be a separate event from its initial rapid inhibition of uptake.     

Identification of transcripts translated on free or membrane-bound polyribosomes by differential display

Differential display has been widely and successfully used to identify differentially expressed genes based on physiological treatments or genetic variation. We used differential display to identify genes based on their site of translation. Identification was made based on the fractionation of RNA from soybean leaves into total RNA, free polyribosomal RNA, and membrane-bound (MB) polyribosomal RNA. Sequences were identified representing RNAs uniquely translated on free or MB polyribosomes. The compartmentalization was confirmed on RNA blots. Differential display of free and MB polyribosomal RNA from genetic mutants or physiological studies has 2 potential advantages. First, the sensitivity of the method is increased. Second, localization of mRNAs to the free or MB compartments may identify genes that are controlled at the level of translation or that switch compartments in response to a treatment.     

The binding of monoribosomes, oligoribosomes and polyribosomes to reticular membranes from rat liver.

Proteoglycans from pig laryngeal cartilage prepared by dissociative extraction in guanidine hydrochloride were studied in dilute solution by light-scattering and ultracentrifugation. In buffered 150mM-NaCl, pH7.4, the proteoglycan particle weights were about 5x10(6) daltons, but at 100mM-, 200mM- and 300mM-NaCl particle weights of 2.5x10(6)--3.0x10(6) daltons were observed. These results, together with corroborative evidence from sedimentation-velocity experiments, were interpreted in terms of proteoglycans self-associating at physiological ionic strength. The data were examined by using a proteoglycan monomer-dimer model. Proteoglycan preparations that had thiol groups partially carboxymethylated gave particle weights of 3.2x10(6)--3.5x10(6) daltons in 150mM-NaCl, which suggested that carboxymethylation inhibited multimerization and hence that the protein core is implicated in the binding site. Further studies showed that the multimers were stable to 60 degrees C, unlike the hyaluronate-proteoglycan complex.     

Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma.

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.     

Induction of rat liver metallothionein mRNA and its distribution between free and membrane-bound polyribosomes.

Total, membrane-bound and free polyribosomes were purified from livers of Zn2+-treated and control rats. Polyadenylated RNA was separated from the polyribosomal RNA extracts by oligo(dT)--cellulose chromatography and translated in a wheat-germ cell-free translation system. Newly synthesized 35S-labelled metallothionein was isolated from the other [35S]methionine-labelled translation products by activated-thiol--Sepharose 4B chromatography. The purity of the 35S-labelled metallothionein product was substantiated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Zinc administration resulted in an elevation of metallothionein mRNA activity to 11% of the total polyribosomal mRNA activity. The vast majority of biologically active metallothionein mRNA was localized in the free polyribosomal pool, at least 94% and 97% in control and zinc-treated rats respectively. The increase in the percentage of polyribosomal mRNA coding for metallothionein after zinc administration was 3-fold, whether measured directly in total polyribosomal mRNA or as a combination derived from membrane-bound and free polyribosomal mRNA. These data indicate that the induction of metallothionein mRNA by zinc involves only free polyribosomes and suggest that the function of metallothionein is limited to intracellular processes.     

Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver.

We have examined the distribution of ferritin mRNA to free and endoplasmic reticulum (ER)-bound liver polyribosomes during inflammation and iron treatment of rats. Postnuclear tissue supernatants were fractionated on a discontinuous sucrose gradient developed to separate free and bound polyribosomes. Total RNA recovered averaged 3.2 mg/g tissue, 40% of which was with ER and 30% with the free polyribosomes, about 25% being with the postribosomal/RNP fraction. Slot-blot hybridization of equal portions of RNA revealed that 12 h after injection of turpentine to induce inflammation, ferritin mRNA was concentrated on the ER-bound polyribosomes, while it was concentrated on the free polyribosomes 2 h after injection of ferric ammonium citrate. Differences were highly significant, based on multiple determinations and densitometry. Profiles of ferritin mRNA distribution on linear sucrose gradients corroborated the differential findings. Concentrations of total ferritin mRNA per gram liver doubled with iron treatment but were not significantly different 12 h after turpentine treatment. At the same time point after turpentine, ferritin protein synthesis was increased twofold, as measured by the 1 h incorporation of [14C]leucine. We conclude that a significant portion of ferritin mRNA always associates with the ER-bound polyribosomes, and that inflammation and iron differentially alter the polysomal distribution of ferritin mRNA, suggesting that two different kinds of mRNA may be involved.     

Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human fibroblasts and chick embryos

Using immunochemical methods, the fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied. It was demonstrated that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts makes up to 4.9%, while that from chicken embryos--1.1% of the total amount of the de novo synthesized proteins (as compared to 1.0 and 0.3% in free polyribosomes, respectively). Fibronectin monomers (Mr = 330 kD) were detected only in the newly synthesized (in the presence of spermidine) material of the cell-free system containing heavy fractions of membrane-bound polyribosomes.     

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.     

Characterization of translation products of the polyadenylated RNA of free and membrane-bound polyribosomes of rat forebrain.

Poly(A)+ RNA (polyadenylated RNA) isolated from membrane-bound and free polyribosomes was translated in reticulocyte lysates, and the products were analysed by two-dimensional gel electrophoresis. Several translation products were specific to membrane-bound polyribosomal mRNA, including polypeptides of 47kDa, 35kDa and 21 kDa, whereas others (e.g. of 37 kDa, 17 kDa and 14 kDa) were specific to free polyribosomal mRNA. Although many products were common to both mRNA species, cross-contamination could be ruled out on the basis of the presence of these and other specific products. The common products included a 68 kDa microtubule-associated protein, tubulin, actin, the brain form of creatine kinase, neuron-specific enolase and protein 14-3-3 and calmodulin, all of which were identified on the basis of two-dimensional gel and peptide analyses. The 35 kDa protein product of membrane-specific mRNA was co-translationally processed in vitro by microsomal membranes, resulting in its cleavage to 33 kDa (and partial glycosylation). The 33 kDa processed protein (but not the 35 kDa precursor) was integrated into both dog pancreas and rat brain microsomal membranes. The occurrence of the enzymes and calmodulin as products of membrane-bound polyribosomal mRNA is discussed in the light of their presence on rat brain synaptic plasma membranes [Lim, Hall, Leung, Mahadevan & Whatley (1983) J. Neurochem. 41, 1177-1182] and their existence in a specific component of axonal flow. It is suggested that some of these translation products of the rough endoplasmic reticulum may represent proteins destined for the plasma membrane. However, the identity and location of the 35 kDa membrane-specific product (or its processed form) still remain unestablished.     

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.     

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

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

Expression and developmental regulation of two unique mRNAs specific to brain membrane-bound polyribosomes.

Translation in vitro of membrane-bound polyribosomal mRNAs from rat brain has shown several to be developmentally regulated [Hall & Lim (1981) Biochem. J. 196, 327-336]. Here we describe the isolation and characterization of cDNAs corresponding to two such brain mRNAs. One cDNA (M444) hybrid-selected a 0.95 kb mRNA directing the synthesis in vitro of a 21 kDa pI-6.3 polypeptide, which was processed in vitro by microsomal membranes. A second cDNA (M1622) hybridized to a 2.2 kb mRNA directing the synthesis of a 55 kDa pI-5.8 polypeptide. Both mRNAs were specific to membrane-bound polyribosomes. Restriction maps of the corresponding genomic DNA sequences are consistent with both being single copy. The two mRNAs were present in astrocytic and neuronal cultures, but not in liver or spleen or in neuroblastoma or glioma cells. The two mRNAs were differently regulated during brain development. In the developing forebrain there was a gradual and sustained increase in M444 mRNA during the first 3 weeks post partum, whereas M1622 mRNA appeared earlier and showed no further increase after day 10. In the cerebellum the developmental increase in M444 mRNA was biphasic. After a small initial increase there was a decrease in this mRNA at day 10, coincident with high amounts of M1622 mRNA. This was followed by a second, larger, increase in M444 mRNA, when amounts of M1622 mRNA were constant. The contrasting changes in these two mRNAs in the developing cerebellum are of particular interest, since they occur during an intensive period of cell proliferation, migration and altering neural connectivity. As these mRNAs are specific to differentiated neural tissue, they represent useful molecular markers for studying brain differentiation.