Interaction between membrane functions and protein synthesis in reticulocytes: specific cleavage of 28-S ribosomal RNA by a membrane constituent

A factor isolated from rabbit reticulocyte white ghosts by Triton X-100 treatment blocks protein synthesis at the elongation-termination stage. Factor-treated ribosomes were found to have an identical buoyant density to that of control ribosomes. When incubated with either reticulocyte ribosomes or ribosomal RNA, the factor products specific cuts in the 28-S ribosomal RNA compenent without damaging the 18-S RNA. Incubations of pancreatic or T1 RNase, with ribosomal RNA, at similar protein-synthesis inhibitory concentrations effected a complete breakdown to oligo and mononucleotides. When challenged with isolated 28-S or 18-S reticulocyte ribosomal RNA, the highly purified factor only attacked the 28-S RNA species. There was no accumulation of nucleotides or oligonucleotides and we concluded that the membrane factor causes inhibition of protein synthesis by having a specific endonucleolytic cleavage activity.     

Protein synthesizing cell apparatus in malignant growth and pharmacotherapy by chlofiden

Data on the influence of a new antitumor preparation chlofiden on the general contents of rat liver ribosomes and sarcoma 45 and their division on free and membrane of membrane bound and decrease of free ribosomes during tumor growth supposed synthesis of specific proteins bound are given in the paper. It was shown that in the liver of tumor bearing rats total and membrane bound ribosomes decreased and the level of free ribosomes increased. High contents of free ribosomes in sarcoma 45 may testify increase of intracellular protein synthesis including processes of cell growth and division as well as the tendency for increase. Chlofiden normalized total contents, increased free and decreased liver membrane bound ribosomes contents, during tumor growth supposed synthesis of specific proteins. Increase of free ribosomes and decrease of their specific radioactivity in sarcoma 45 testified membrane damage by chlofiden and inhibition of intracellular protein synthesis which are essential in cell division.     

Decreased ratio of alpha- to beta-globin mRNA in reticulocyte membrane RNA

The amount of RNA obtained from rabbit reticulocyte membrane-bound ribosomes by direct phenol extraction of washed membranes was inversely related to the hematocrit of the animals. Translation of the RNA in the reticulocyte translation system showed that the Mr = 30,000 protein reported to be a marker of membrane polysomes was also made by an endogenous mRNA in this translation system. Analyses of the translation products made in the wheat germ system on Triton X-100 acid urea gels show that membrane RNAs display a characteristic alpha- to beta-globin ratio of 0.77 which differentiates them from RNAs prepared from cytoplasmic polysomes and from the postpolysomal supernatant. These results show that free and membrane-bound ribosomes can be distinguished by the main protein that they produce.     

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.     

Effect of food deprivation and hypophysectomy on in vitro protein synthesis by membrain-bound and free hepatic ribosomes.

The protein synthetic activities of membrane-bound and free hepatic ribosomes isolated from intact rats fed ad libitum, and normal rats subjected to food restriction to match that of hypophysectomised (Hx) rats were compared to the in vitro protein synthetic capacity of hepatic ribosomes isolated from Hx rats. Hypophysectomy resulted in decreased protein synthetic ability of bound ribosomes, whether protein synthesis was directed by endogenous messenger RNA (mRNA) (p less than 0.05) or by polyuridylic acid (polyU) (p less than 0.01). In contrast, the protein synthetic activity of free hepatic ribosomes from Hx rats was reduced when protein synthesis was directed by endogenous mRNA (p less than 0.05) but, when polyU was substituted as the messenger, the protein synthetic activity of these free ribosomes was equal to that of control rats. On the other hand the effects of food restriction on hepatic ribosomal function could be clearly differentiated from the effects observed following hypophysectomy. Thus, the reduced protein synthetic activity of hepatic bound ribosomes isolated from food restricted normal rats was not demonstrable, when polyU was used to direct protein synthesis. Further, food restriction had no effect on the protein synthetic activity of free hepatic ribosomes, and this was true when protein synthesis was directed by either endogenous or artificial messenger. It is concluded that hypophysectomy reduces the protein synthetic ability of both bound and free hepatic ribosomes, and this change of ribosomal function of Hx rats cannot be attributed to their decreased food intake.     

Isolation and properties of polyribosomes and fragments of the endoplasmic reticulum from rat liver

1. A centrifugation method for the fractionation of the postmitochondrial fraction from rat-liver homogenates is described. The technique, in which no detergent is used, may be used as a tool to discriminate between two classes of ribosomes. One class is firmly bound to membranes and the other consists either of free polysomes or of ribosomes attached by weaker forces to the membranes of the endoplasmic reticulum. 2. Electron-micrograph studies revealed that the polysomes were not contaminated with bound ribosomes or with membranous fragments. 3. The separated fractions were characterized by their RNA, protein, ribonuclease and phospholipid content. 4. The influence of starvation on the RNA and protein contents of the different fractions was investigated. 5. Labelling of the various centrifugal fractions in vivo revealed no difference in uptake of radioactive amino acid between the two classes of ribosomes. 6. Incorporation of radioactive leucine in vitro and the polyuridylic acid-directed phenylalanine incorporation were similar for both classes of ribosomes.     

Amino acid regulation of synthesis of ribonucleic acid and protein in the liver of rats

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.     

Degradation of messenger RNA, ribosomal RNA and 2-5A-dependent inhibition of protein biosynthesis

In rabbit reticulocyte lysates the addition of exogenous 2-5A leads after 10-20 minutes to the inhibition of protein synthesis. This inhibition can be blocked by rat antiserum to 2-5A. In intact ribosomes the ribosomal RNA is cleaved after 2-5A addition, but this cleavage is not in correlation with the protein synthesis shutoff. Ribosomal 5S RNA and 5,8S RNA are not cleaved even after several hours of incubation with 2-5A. The degradation of polysome associated mRNA correlates with the protein synthesis inhibition as revealed by Northern blot hybridization of globin mRNA with 32P-labelled p beta G plasmid. The addition of 2-5A antiserum to the rabbit reticulocyte lysate also inhibits the degradation of polysome bound globin mRNA.     

Extraction from free ribosomes of a factor mediating ribosome detachment from rough microsomes.

A salt extract of rabbit reticulocyte free monosomes or polysomes contains a factor with an activity that detaches membrane bound monosomes but not polysomes from dog pancreas rough microsomes. It is proposed that this activity, referred to as detachment factor, functions in the dissociation of membrane bound ribosomes from the microsomal membrane after each round of translation. In addition to free ribosomes, the factor is also present in a ribosome-free, high speed supernatant, the cell fractionation equivalent of the cytosol. The factor can be extracted from free ribosomes of a variety of tissues and species, and is able to function on ribosome membrane junctions homologous as well as heterologous with respect to its source.     

Peptidyl-puromycin synthesis by free and membrane-bound ribosomes.

The peptidyl transferase reaction, as measured by the formation of peptidyl-puromycin, was compared for free ribosomes and ribosomes bound to two types of membrane, the endoplasmic reticulum and the outer nuclear membrane. In most respects the reaction catalyzed by the three types of ribosome was similar, demonstrating that interaction of the 60 S ribosomal subunit with the membrane has little effect on the functioning of peptidyl transferase, a 60 S protein. However, both the rate and extent of synthesis of peptidyl puromycin were lower for ribosomes bound to the nuclear membrane than for free or microsome-bound ribosomes. This difference appears to be a direct consequence of the ribosome-membrane interaction, since ribosomes stripped from the nuclear membrane could not be distinguished from the other classes of ribosome.     

Incomplete polypeptides are formed in vitro by premature chain termination

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell-free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5' AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high-speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.     

THE IN VIVO PROTEIN SYNTHETIC ACTIVITIES OF FREE VERSUS MEMBRANE-BOUND RIBONUCLEOPROTEIN IN A PLASMA-CELL TUMOR OF THE MOUSE

Cytoplasmic extracts of the transplantable RPC-20 plasma-cell tumor were fractionated by sucrose density gradient centrifugation. Four major fractions were distinguished: (a) microsomes and mitochondria; (b) membrane-free polyribosomes; (c) free monomeric ribosomes; and (d) soluble fraction. The fractions were analyzed for RNA and lipid phosphorus, and their particulate components were characterized by electron microscopy. Particular attention was paid to the problem of membrane contamination of the free polyribosome fraction. It was shown that this contamination was small in relation with the total content of ribosomes in the fraction, and that it consisted primarily of smooth-surfaced membranes which were not physically associated with the polyribosomes themselves. In vivo incorporation studies were carried out by injecting tumor-bearing animals intravenously with leucine-C¹⁴, removing the tumors at various times thereafter, and determining the distribution of protein radioactivity among the gradient-separated cytoplasmic fractions. The free polyribosome and the microsome-mitochondria fractions constituted active centers for protein synthesis. It was shown that nascent protein of the free polyribosome fractions was not associated significantly with the contaminating membranes. The kinetics of labeling during incorporation times up to 11 min suggested that protein synthesized on the free polyribosomes was rapidly transferred in vivo to the soluble fraction of the cell, while protein synthesized by the microsomes and mitochondria remained localized within these elements. It was estimated that the free polyribosome fraction and the microsome-mitochondria fraction accounted for approximately equal proportions of the total cytoplasmic protein synthesis in vivo.     

Effect of Fluoride on Ribosomes from Corn Roots. Changes with Growth Retardation

Growth inhibition caused by fluoride was further studied by analyzing free and bound ribosomes, and the ribosomal components in corn roots. Ribosomes were isolated by differential centrifugation. Ribosomal components were analyzed by column chromatography and electrophoresis. Fluoride reduces the amounts of both free and bound ribosomes. Fluoride also modifies the RNA to protein ratio of bound ribosomes more than that of free ribosomes. Fluoride does not affect base ratios of the ribosomal RNA, but reduces its content and changes the structure of the ribosomal protein moiety.     

Characterizaton and Biosynthesis of the Plasma Membrane Proteolipid Protein in Neural Tissue

In this study we have characterized, in brain, the expression of a plasma membrane proteolipid protein (PM-PLP) complex that can form cation-selective channels in lipid bilayers. We isolated PLP fractions from synaptic plasma membrane and glial microsomes and found a high degree of similarity in both size and amino acid composition to the complex we had previously isolated from kidney. Antibodies specific to the kidney PM-PLP were prepared, and, on the basis of immunoblot and immunoprecipitation studies, the PM-PLP complex isolated from neural membranes was shown to be immunologically related to the kidney PM-PLP. These proteolipid proteins exhibited a molecular weight of approximately 14K and contained a high percentage of hydrophobic amino acids with an apparent absence of cysteine. The biogenesis of PM-PLP in brain was studied by in vitro translation of free and bound polysomes and total RNA in a rabbit reticulocyte lysate followed by immunoprecipitation of the translation products. From these studies it is concluded that the PM-PLP complex is synthesized on the rough endoplasmic reticulum. On the basis of the identical electrophoretic mobility of material isolated from plasma membranes and material immunoprecipitated after translation of bound polysomes and isolated RNA, it appears that the PM-PLP does not undergo detectable posttranslational processing between its site of synthesis and its incorporation into the plasma membrane.     

Interaction between membrane functions and protein synthesis in reticulocytes. An elongation-stage inhibitor of protein synthesis extracted from the reticulocyte membrane.

A component of the reticulocyte cell membrane was found to inhibit protein synthesis severely in a reticulocyte lysate system. An investigation into the mode of action of the membrane inhibitor revealed the following facts. (1) The binding of the tertiary initiation complex (methionyl-tRNAfMet-Initiation Factor 2-GTP) to the 40S ribosomal subunit was unaffected by the membrane inhibitor. (2) The membrane component did not interfere with the binding of the 40S initiation complex to the AUG initiation codon and subsequent attachment of the 60S ribosomal subunit. (3) Elongation of the peptide chain, as assayed by peptidyl-puromycin formation, was markedly affected by the membrane inhibitor. Surprisingly, the membrane component caused a considerable increase in peptidyl-puromycin formation. (4) Reticulocyte ribosomes that had been reisolated by high-speed centrifugation, after preincubation with the membrane component, were found to be highly defective when assayed in a cell-free protein-synthesizing system. These results indicated that an extract of the reticulocyte cell membrane inhibited protein synthesis by interacting with the ribosome and thus interfered with the correct functions of the elongation stage of protein synthesis. The implications of this conclusion are discussed in the light of data showing that a highly purified preparation of the membrane inhibitor also displayed an endonucleolytic activity highly specific for 28S RNA.     

Studies on the protein-synthesizing activity of the ribosomes of rat liver. The activity of free polysomes

1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.     

Translocation and proteolytic processing of nascent secretory polypeptide chains: two functions associated with the ribosomal domain of the endoplasmic reticulum

Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP-receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent-disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar-Costesec A. & Beaufay H. (1981) J. Theor. Biol. 89, 217-230].     

In Vitro Synthesis of the Myelin Basic Proteins: Subcellular Site of Synthesis

Abstract: Brains of 3-week-old C57BL/6J mice were homogenized and fractionated into several subcellular components, each of which was examined for ability to synthesize the myelin basic proteins (MBPs) in vitro. Myelin basic proteins were purified from incubation mixtures by conventional means. That the products of synthesis were the myelin basic proteins was established by solubility at pH 3, co-chromatography with authentic proteins on carboxymethylcellulose and co-migration with standards in two different polyacrylamide gel electrophoretic systems. The fractions examined for their ability to synthesize MBPs were the whole homogenate, postnuclear supernatant, postmitochondrial supernatant, crude mitochondrial pellet, free ribosomes and bound ribosomes. Although there was no requirement for exogenous energy sources for protein synthesis in the whole homogenate, as the homogenate was fractionated an increasing requirement emerged. Most of the label in the MBP preparations from whole homogenate and postnuclear supernatant incubations migrated with the large (L) and small (S) MBPs on gel electrophoresis; however, as the homogenate was subfractionated and incubated, a greater percentage of the label migrated more slowly than L and S on acetic acid-urea gels. To show synthesis of the MBPs the L and S bands were cut out of these gels and rerun on sodium dodecylsulfate gels. Alternatively, MBP preparations were subjected directly to two-dimensional gel electrophoresis and the bands corresponding to L and S were excised and counted. With this method only the whole homogenate, postnuclear supernatant, postmitochondrial supernatant and free ribosomes were observed to synthesize the MBPs in vitro. The "bound" ribosomes were not observed to synthesize significant amounts of the MBPs, incubated either intact or released from the membrane. It was concluded that the free ribosomes are the principal site of synthesis of the myelin basic proteins in the brain.     

Study of the type of RNA, degraded by polynucleotide phosphorylase in polyribosomal fraction of rat liver]

The type of RNA is studied, which is degraded by polynucleotide phosphorylase (PNPase) in the fraction of free ribosomes and ribosomes released from endoplasmic reticulum membranes with Triton X-100. Beta-32P labelled ADP, UDP, GDP and CDP are found among the degradation products of endogenous RNA of free and bound ribosomes in vitro in the presence of 32P-ortophosphate. An analysis of molar ratio of beta-32P-NDP isolated revealed that PNPase degrades RNA of GC type in both ribosome fractions. The amount of PNPase-degraded RNA in bound ribosimes is 4-fold as high as that in free ribosomes under the same conditions. Analysis of stable 32P-RNA and rapidly labelled 32-P-dRNA, isolated from bound ribosomes after the incubation with and without inorganic phosphate, revealed that PNPase attacks the 28S fragment of RNA, which consists of about 370 nucleotides, and dRNA having a sedimentation coefficient less than 12S. The rate of dRNA degradation is considerably higher than that of rRNA. 5'-RNAase, hydrolysing synthetic homopolyribonucleotides to oligonucleotides with free 3'-OH terminal group, apparently participates, together with PNPase, in dRNA and rRNA degradation.     

Molecular action of tricholin, a ribosome-inactivating protein isolated from Trichoderma viride

An extracellular protein was isolated from a species of soil-borne fungi (Trichoderma viride) and its amino acid composition has been determined. The protein is acidic with a molecular mass of 14,200 daltons and is given the trivial name tricholin. Tricholin is a potent inhibitor of cell-free protein synthesis. When rabbit reticulocyte lysate was incubated with tricholin at a concentration of 6.3 x 10(-7) M, it completely abolished the capacity of the lysate to support protein synthesis. The inhibition appears to be due to its reaction to ribosomes, since it generates a specific cleavage product, an alpha-sarcin RNA fragment, from reticulocyte ribosomal RNA. This reaction to ribosomes mimics that of alpha-sarcin. The antibody of alpha-sarcin strongly cross-reacts with tricholin, while the antibody of tricholin shows a weak reaction with alpha-sarcin.