Membrane-bound Polysomes Research Articles

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.

C-myc mRNA in cytoskeletal-bound polysomes in fibroblasts

3T3 fibroblasts were treated sequentially with 25 mM-KCl/0.05% Nonidet P40, 130 mM-KCl/0.05% Nonidet P40 and finally with 1% Nonidet P40/1% deoxycholate in order to release free, cytoskeletal-bound and membrane-bound polysomes respectively. The membrane-bound fraction was enriched in the mRNA for the membrane protein beta 2-microglobulin, whereas the cytoskeletal-bound polysomes were enriched in c-myc mRNA. Actin mRNA was present in both free and cytoskeletal-bound polysomes. The results suggest that cytoskeletal-bound polysomes are involved in the translation of specific mRNA species.

The characterization of free, cytoskeletal and membrane-bound polysomes in Krebs II ascites and 3T3 cells

Polysomes from Krebs II ascites and 3T3 cells were separated into three populations by using a sequential extraction method. Free polysomes were released by using a combination of low salt (25 mM KCl) and NP-40 detergent in the lysis buffer. The cytoskeletal bound polysomes were subsequently released by raising the salt concentration to 130 mM and finally, polysomes bound to the membranes of the endoplasmic reticulum were extracted by the combined treatment with Triton X-100 and deoxycholate. The results presented here illustrate that the three polysome-containing fractions differ in many parameters such as polysome profiles, cytoskeletal components and phospholipid content. When polyA-containing mRNA was isolated from the three polysome fractions and translated in an in vitro system, some differences were observed in the patterns of proteins being synthesized.

The distribution of apolipoprotein B in endoplasmic reticulum and Golgi subfractions of rabbit liver

The liver is the site of synthesis and secretion of very-lowdensity lipoproteins (VLDL), the vehicle for the transport of endogenous triacylglycerol (TAG) and the precursor of lowdensity lipoproteins (LDL). Elevated plasma levels of VLDL and LDL are recognized risk factors in the development of atherosclerosis. The production of VLDL by the liver is considered to be the most appropriate therapeutic target for the reduction of circulating lipoprotein levels. An understanding of the molecular and topographical events in the assembly of VLDL is thus of fundamental importance. Apolipoprotein B (apo B) is the major non-exchangeable protein of VLDL, and is essential for their secretion. Studies in HEP-G2 cells have indicated that apo B is synthesized by membrane-bound polysomes [I]. Newly synthesized apo B remains associated with the endoplasmic reticulum (ER) membrane for a period of time before being transferred to the cisternal space [2]. Consistent with this, we have previously shown that apo B does not associate with TAG-richVLDL precursors in the ER cisternae, and that the main site of packaging of VLDL lipid and protein is the Golgi cisternae [3]. To examine the intracellular events in apo B synthesis and translocation in more detail, we have determined the intracellular location and amount of apo B within the secretory pathway. The intracellular location of apo B was determined by SDS/PAGE and immunoblotting of subcellular fractions. ER and Golgi fractions were prepared from rabbit liver, and separated into membrane and cisternal content fractions by sodium carbonate treatment 14, 51. Fractions were delipidated and separated on 3-20% (w/v) polyacrylamidegradient gels [6], and then electroblotted on to nitrocellulose membrane [7]. Apo B- 100, the only apparent species of apo B secreted by rabbit liver, was detected by immunoblotting using sheep anti-rabbit apo B (primary antibody), and biotinylated anti-sheep IgG (secondary antibody). The secondary antibody was detected colorimetrically by the highly sensitive Vector ABC-alkaline phosphatase system. Apo €3- 100 was detectable in both ER and Golgi fractions (Fig. 1 ). When ER was separated into membranes and cisternal contents, apo B-100 was found in both fractions at apparently similar levels. When Golgi was separated into membranes and cisternal contents, although apo B- 100 could be detectcd in membranes, most of the protein appeared to be located in the contents. Apo €3- 100 in subcellular fractions was quantified by radioimmunoassay. LDL were isolated from rabbit plasma (d 1 .O 19-d 1.063). and radiolabclled with lz51 [ 8, 01. Apo B-I00 was the only protein detectable in LDL by SDS/ PAGE. and was found to be 'z51-labelled. Using a fixed concentration of labelled LDL with varying concentrations of

Differential association of membrane-bound and non-membrane-bound polysomes with the cytoskeleton

We report here a differential release of specific mRNAs from the cytoskeleton by cytochalasin D treatment. Non-membrane-bound polysomal mRNAs, such as histone mRNA and c-fos mRNA, are readily released from the cytoskeleton of HeLa cells during cytochalasin D treatment. Over 90% of H3 and H4 histone mRNA is associated with the cytoskeleton in control cells and only 25% in cells treated with cytochalasin D (40 μg/ ml). In contrast, the membrane-bound polysomal mRNAs for HLA-B7 and chorionic gonadotropin-α are inefficiently released from the cytoskeletal framework by cytochalasin D alone; approximately 98% of the HLA-B7 mRNA in control cells is associated with the cytoskeleton, whereas approximately 65% of the HLA-B7 mRNA is retained on the cytoskeleton in cells treated with cytochalasin D (40 μg/ml). Disruption of polysome structure with puromycin during cytochalasin D treatment results in the efficient release of HLA-B7 mRNA from the cytoskeleton. Under these conditions, only 25% of the HLA-B7 mRNA remains associated with the cytoskeletal framework. Thus, membrane-bound polysomes appear to be attached to the cytoskeleton through a cytochalasin D-sensitive site as well as through association with the nascent polypeptide and/or ribosome. These results demonstrate a complex association of polysomes with the cytoskeleton and elements of the endoplasmic reticulum.

The secretion of amylase, phospholipase and protease from Aeromonas salmonicida, and the correlation with membrane-associated ribosomes

Abstract. The following extracellular enzymes have been readily detected in the culture filtrates from Aeromonas salmonicida: amylase, phospholipase, lysophospholipase and ribonuclease. Amylase and phospholipase have been partially characterized. Evidence suggests that glycogen may be the natural substrate for amylase, and that the role of the enzyme in natural infection is to digest glycogen present in fish muscle. The secretion of amylase activity is suppressed by the addition of glucose to the growth medium. The amounts of amylase, phospholipase and protease that can be detected in culture filtrates decreases with increase in the growth temperature from 25 to 32°C. This marked decrease in secretion of hydrolytic enzymes occurs although the initial growth rates at 25 and 32°C are similar. Free and membrane associated ribosomes have been isolated from cultures grown at 25 and 32°C. At 32°C there is a smaller proportion of membrane-associated ribosomes and this is consistent with the hypothesis that extracellular enzymes from Aeromonas salmonicida are secreted on membrane-bound polysomes.

Multiple types of mRNA‐cytoskeleton interactions

Nearly all actively translated mRNAs are associated with the cytoskeleton in HeLa cells and the nature of this association is poorly understood. To gain insight into this association, we have examined and compared the cytoskeleton-mRNA interactions of a signal peptide-histone fusion mRNA (membrane-bound polysomal mRNA) to those of endogenous histone mRNA (nonmembrane-bound polysomal mRNA). We report here the detection of a cytoskeleton attachment site within the signal peptide-histone fusion mRNP/mRNA nucleotide sequence that is not present in wild-type histone mRNA or in HLA-B7 and chorionic gonadotropin-alpha membrane-bound polysomal mRNAs. These results support the possibility that there are multiple mechanisms for the attachment of specific classes of mRNAs to the cytoskeleton.

Translational discrimination of mRNAs coding for human insulin-like growth factor II.

Sucrose gradient and Northern analyses were used to study the translational status of endogenous multiple mRNAs encoding the prepropeptide for human insulin-like growth factor II. The results showed that a minor 4.8-kilobase mRNA was exclusively engaged in the synthesis of the prepropeptide on membrane-bound polysomes, whereas a major 6.0-kilobase mRNA was present in a cytoplasmic particle which was stable in EDTA. We conclude that the translational discrimination between the mRNAs is dictated by their different 5'-untranslated regions.

Role of messenger RNA subcellular localization in the posttranscriptional regulation of human histone gene expression

Histone mRNAs are naturally localized on non-membrane-bound polysomes and selectively destabilized during inhibition of DNA replication. Targeting histone mRNA to membrane-bound polysomes, by incorporating sequences coding for a signal peptide into the message, results in the stabilization of the histone fusion mRNA when DNA synthesis is interrupted (Zambetti et al.: Proceedings of the National Academy of Sciences of the United States of America 84:2683-2687, 1987). A single nucleotide substitution that abolishes the synthesis of the signal peptide results in the localization of the histone fusion mRNA on non-membrane-bound polysomes to the same extent as endogenous histone mRNA and fully restores the coupling of histone fusion mRNA stability to DNA replication. Signal peptide-histone fusion mRNAs containing two point mutations that result in the incorporation of two positively charged amino acids into the hydrophobic domain of the signal peptide are partially retained on non-membrane-bound polysomes and are partially destabilized during inhibition of DNA synthesis. These data indicate that the degree to which the signal peptide-histone fusion mRNAs are associated with non-membrane-bound polysomes is correlated with the extent to which the mRNAs are degraded during inhibition of DNA synthesis. These results suggest that the subcellular location of histone mRNA plays an important role in the posttranscriptional regulation of histone gene expression.

Insulin and step-up conditions cause a redistribution of polysomes among free, cytoskeletal-bound and membrane-bound fractions in krebs II ascites cells

From 30 min to 1h of step-up conditions there was a redistribution of polysomes between free, cytoskeletal-bound and membrane-bound fractions such that more polysomes were recovered bound to the cytoskeleton and less in the free fraction. After 1h incubation with insulin there was a higher proportion of polysomes in the cytoskeletal fraction with a decrease occurring in the membrane-bound fraction. At 2h little change was observed in the presence of insulin while a large increase occurred in the cytoskeletal-bound fraction and a decrease in membrane-bound polysomes was seen in cells incubated in the absence of insulin. The results indicate that the proportions of polysomes in the three different fractions can be modulated by physiological stimuli, such as media replenishment and insulin.

The signal peptide cleavage site of a B1 hordein determined by radiosequencing of the in vitro synthesized and processed polypeptide

The major storage proteins which accumulate in the endosperm of the developing barley grain are the hordein polypeptides. They are synthesized on membrane-bound polysomes and co-translationally transported into the lumen of the ER and are ultimately deposited in the vacuole. The processing step, which accompanies transport across the ER membrane, was studied in vitro with the B1 hordein gene, λhor2-4 in the expression plasmid pDS6. Microsomes were prepared from barley endosperm which transported and processed the B1 hordein polypeptide to a mature form with a high degree of efficiency. Because the B1 hordein was previously found to be NH2-terminally blocked, the site of processing was unknown. The in vitro synthesized precursor and processed B1 hordein polypeptides were subjected to N-chlorosuccinimide peptide mapping and NH2-terminal radiosequencing. The signal peptide cleavage site, predicted from the deduced amino acid sequence, was confirmed.

Biosynthesis of the Tonoplast H+-ATPase from Oats

To determine whether the tonoplast-type H(+)-ATPase was differentially synthesized in various parts of the oat seedling, sections of 4-day-old oat (Avena sativa L. var Lang) seedlings were labeled in vivo with [(35)S]methionine and ATPase subunits were precipitated with polyclonal antisera. ATPase subunits were detected in all portions of the seedling with the exception of the seed. Lesser amounts of the 60 and 72 kilodalton polypeptides of the ATPase were found in apical regions (0-5 millimeter) than in maturing regions (10-15, or 20-25 millimeter from the tip) of the roots or shoots. To initiate a study of the biosynthesis of the ATPase, the intracellular site of synthesis for two peripheral ATPase subunits was investigated. Poly(A) RNA from either free or membrane-bound polysomes was isolated and translated in vitro. Message encoding the 72 kilodalton (catalytic) subunit was found predominantly in mRNA isolated from membrane-bound polysomes. In contrast, the message for the 60 kilodalton (putative regulatory) subunit was found predominantly on free polysomes. Polypeptides synthesized in vivo or obtained from RNA translated in vitro exhibited no apparent size differences (limit of resolution, approximately 1 kilodalton), suggesting the absence of cleaved precursors for the 72 or 60 kilodalton subunits. These data suggest a complex mechanism for the synthesis and assembly of the tonoplast ATPase.

A developmentally regulated gene product from Dictyostelium discoideum shows high homology to human α-L-fucosidase

A cDNA library of poly(A +)-RNA has been prepared from membrane-bound polysomes of Dictyostelium discoideum and screened for clones hybridizing to mRNA species that encode developmentally regulated proteins. The clone investigated in this paper recognizes a 1.8 kb transcript that accumulates strongly between the growth phase and aggregation stage. Stimulation of cells with pulses of cAMP enhances the accumulation. The amino acid sequence derived from a complete cDNA and from a genomic clone displays extensive sequence identity to human liver α-L-fucosidase. The D. discoideum DNA sequence encodes a 50.5 kDa polypeptide with a hydrophobic signal peptide at the N-terminus. Antibodies against a synthetic peptide corresponding to amino acids 262–275 of the deduced protein sequence recognize a developmentally regulated 50 kDa protein in D. discoideum that is recovered in the particulate fraction.

Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds

A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 +/- 1 degrees C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme.

Intracellular Protein Sorting and the Formation of Protein Reserves in Storage Tissue Cells of Plant Seeds

Similar to the cells of other eukaryotes plant cells are compartmentalized by several membranebounded organelles.These compartments as well as their bounding membranes contain different sets of proteins which are related to functional and structural differences and can be used in compartment diagnosis.The great majority of the proteins in the different compartments is synthesized at cytoplasmic polysomes and has to be transferred from this site of formation into the compartments of destination.This intracellular protein sorting represents one of the most important steps on the way of genetic character formation.Targeting signals which reside in special regions of the amino acid sequences of the respective polypeptides are inherited with the corresponding gene and represent one component of the signal systems underlying the intracellular protein sorting.These sequence signals interact with receptors in the surface membrane of the target compartment.Receptors are inherited with the cellular compartments which are distributed from the mother to the daughter cells during mitosis and meiosis. Storage proteins are synthesized at membrane-bound cytoplasmic polysomes and transferred from their sites of formation into protein bodies where they are deposited.Predominantly, the storage protein precursor polypeptides growing vectorially into the ER-lumen after cotranslational cleavage of the Nterminal signal peptide are transferred through the Golgi apparatus, which forms transfer vesicles, into the vacuolar compartment that finally generates the protein bodies.A different mechanism underlies the prolamin deposition in the endosperm of maize kernels, and it remains still unresolved whether a different pathway also exists for the deposition of other storage proteins in plant seeds.The present knowledge on protein sorting during protein deposition in storage tissue cells of plant seeds is reviewed and controversal results as well as open questions are discussed with respect to future research.

Synthesis of phospholipid transfer proteins from maize seedlings

The synthesis of phospholipid transfer proteins has been studied in vitro after isolation of poly(A)+RNAs from maize seedlings and by in vivo labelling of coleoptiles. After immunoprecipitation of translation products in wheat germ or in reticulocyte lysate systems, the analysis by electrophoresis revealed two bands of molecular mass 9 kDa and 12 kDa. The in vitro synthesized 12 kDa protein is a precursor of the 9 kDa purified protein from maize seedlings as suggested by competition experiments with the pure protein. After immunoprecipitation of in vivo labelled proteins, two bands were detected. One of them, having a molecular mass of 7 kDa, could be related to the in vitro synthesized 9 kDa protein, the other corresponding to the purified protein. Furthermore, biosynthesis of both precursors occurs on membrane-bound polysomes. Presumably a post translational process occurs, yielding to the mature forms.

Genes expressed during imaginal disc morphogenesis: IMP-E1, a gene associated with epithelial cell rearrangement

A unique set of genes that encodes hormone-inducible transcripts found on membrane-bound polysomes is expressed during ecdysone-dependent morphogenesis of Drosophila imaginal discs cultured in vitro. The pattern of expression of one of these genes, IMP-E1, was analyzed in tissues from late third instar larvae and white prepupae by hybridization of asymmetric RNA probes to tissue sections. The IMP-E1 transcript was detected in all anterior and posterior imaginal discs except the ommatidial region of the imaginal eye disc. Within the imaginal leg disc, the IMP-E1 transcript is expressed more abundantly in the proximal than in the distal portions of the epithelium. The distribution of transcripts is consistent with the hypothesis that the IMP-E1 gene product is involved in the cell rearrangements associated with morphogenesis of the disc epithelium. The IMP-E1 transcript is also expressed at pupariation in glial cell layers that ensheath the brain. This glial cell transcript is the same size (7.5 kb) and polarity as the imaginal disc transcript, and is also transcribed in response to 20-hydroxyecdysone. Similarities between the morphogenetic changes in imaginal disc and glial cell layers during metamorphosis are discussed.

The influence of tetracyclines on the aggregation of free and membrane-bound polysomes and on the RNA content of the liver of mice

Tetracycline (100 micrograms/g) and, to a lesser extent, doxycycline (50 and 100 micrograms/g) inhibited the aggregation of free but not of membrane-bound polysomes in the livers of female NMRI mice. In addition tetracycline decreased the RNA content of the fraction of membrane-bound polysomes probably due to detachment from their membrane sites.

Genes expressed during imaginal discs morphogenesis: IMP-L2, a gene expressed during imaginal disc and imaginal histoblast morphogenesis

We describe our analysis of IMP-L2, one of a set of six ecdysone-inducible genes in imaginal discs of Drosophila whose transcripts are associated with membrane-bound polysomes. The spatial and temporal patterns of expression of the IMP-L2 transcript were analyzed. This transcript is first expressed in imaginal discs in areas that are precursors of head and thoracic epithelium, particularly the peripodial epithelia. It is later expressed in the imaginal histoblasts, precursors of the adult abdomen. The appearance of IMP-L2 transcript in each of these tissues precedes the spreading and fusion of the separate imaginal anlagen to form the continuous adult epidermis.

The half-life of immunoglobulin mRNA increases during B-cell differentiation: a possible role for targeting to membrane-bound polysomes.

The increase in abundance of immunoglobulin heavy-chain (IgH) mRNA that accompanies development of a B cell into a plasma cell is mainly due to post-transcriptional events. By constructing mu genes, whose expression is under inducible control, we determined the half-life of pulsed mu transcripts to be approximately 20 hr in plasmacytoma hosts and approximately 3 hr in B-cell lymphomas. Interestingly, a mu gene with a mutated signal sequence that is found on free (rather than membrane-bound) polysomes decays in the plasmacytoma host with a shortened half-life of 3 hr. Thus, a change in the turnover rate of IgH mRNA plays an important role in the cell-type specificity of immunoglobulin gene expression; this change may be a consequence of the fact that IgH mRNA is located in the membrane-bound polysome fraction.