Polysomes membrane-bound

Translating ribosomes in eukaryotes are located in different places in the cell depending on the fate of their proteins. Free polysomes are in the cytoplasm and synthesize cytoplasmic proteins and those that are bound for most intracellular organelles, for example, the nucleus. Members of the second class of polysomes, membrane-bound polysomes, are attached to the endoplasmic reticulum (forming the rough ER), and synthesize exported proteins. In cells that are actively secreting enzymes or hormones (for example, those in the pancreas), most of the protein synthesis occurs on the rough ER. 

Processing of insulin. Insulin is synthesized by membrane-bound polysomes in the /3 cells of the pancreas. The primary translation product is preproinsulin, which contains a 24-residue signal peptide preceding the 81-residue proinsulin molecule. The signal peptide is removed by signal peptidase, cutting between Ala (—1) and Phe (+1), as the nascent chain is transported into the lumen of the endoplasmic reticulum. Proinsulin folds and two disulfide bonds crosslink the ends of the molecule as shown. Before secretion, a trypsinlike enzyme cleaves after a pair of basic residues 31, 32 and 59, 60 then a carboxypeptidase B-like enzyme removes these basic residues to generate the mature form of insulin. 

From a critical study of the metabolism of poly (A) in auxin-treated pea epicotyl, Verma and Maclachlan (73) showed that discreet classes of poly (A) (presumably part of mRNA s) are differentially associated with free and membrane-bound polysomes. The induction of specific mRNA s, the decline in the rate of synthesis of mRNA s, the polysome content per cell, and the formation of cellulase were all related to the membrane-bound polysomes. Although the rate of in vivo enzyme synthesis is... 

Verma, D. P. S./ MacLachlan, G. A. Metabolism of poly(A) in plant cells. Discrete classes associated with free and membrane-bound polysomes. Plant Physiol. 1976, 58, 405-410. 

Vectorial translation [31,32]. Polypeptides are made on membrane-bound polysomes. Many of these proteins are synthesized with a 16-30 amino acid extension at the NH2-terminus. This signal sequence is hydrophobic in nature. Protein synthesis and translocation, into or across the membrane, are obligatorily linked. Therefore, the transmembrane movement is co-translational and it is coupled to the elongation of the polypeptide chain. Consequently, the completed polypeptide chain is never present in the compartment where it is synthesized. The polypeptides that do not yet cross the membrane are shorter than the mature protein. Addition of inhibitors of protein synthesis immediately arrest movement of the polypeptide across the membrane. 

In higher eukaryotes messenger RNAs for chloroplast and mitochondrial proteins have been localized exclusively to free and not membrane-bound polysomes [4,68]. In yeast, the situation is not as clear a portion of the polysomes for some of the proteins is found to be bound to mitochondria [69,70], This may only reflect the speed at which the import process occurs in the different organisms. Most of the cytoplasmically-synthesized precursors have amino terminal extensions varying in size and nature. The size variation goes from no extension (cytochrome c and ATP-ADP translocator) to approximately 12000 Da (proteolipid of the proton... 

Poly(dT)-cellulose was used to isolate a poly(A) containing RNA fraction from membrane-bound polysomes of mouse myeloma tumour... 

Ovalbumin expressed in E. coli is synthesized on membrane-bound ribosomes and is localized to the periplasm, but in a strain producing a shortened ovalbumin lacking the first 126 amino acids, the protein is synthesized on free polysomes and is not exported (Baty et al., 1981). 

The low affinity of the SRP receptor for the ribosome, and the substoi-chiometric ratio of SRP and SRP receptor to membrane-bound polysomes, suggest that interaction of the SRP and SRP receptor with the ribosome is transient (Gilmore and Blobel, 1983). Thus, the observed binding of the ribosome to the membrane must occur by some other means. Two integral membrane glycoproteins, ribophorins I and II, have been identified (Kreibich et al., 1978, 1983). They appear to be required for translocation (Amar-Costesec et al., 1984), and may be responsible for binding of the ribosome to the membrane. 

There is a detailed review of the effect of ions and detergents on the release of subcellular mRNAs during lysis,20 indicating that the inclusion of sodium deoxycholate is crucial for efficient release of membrane-bound polysomes. However, based on Northern analysis of the debris following lysis in 140 mM KC1,1.5 mM MgCl2... 

In the case of several D-JH integrations, transcripts have been detected that initiate a few nucleotides upstream of the rearranged D segment [31,32], Some of these DJh-Cm transcripts have been shown to be translated into polypeptides that are presumed to be synthesized on membrane-bound polysomes as the nucleotide sequences suggest the presence of a hydrophobic leader at the amino-terminal end of the polypeptide [31] and, at least in one cell line, the protein has been shown to be glycosylated [32],... 

Some precautions should be taken during the isolation of membrane-bound polysomes. For example, some D-RNPs, including nuclear 308 particles, are not stable and dissociate in the presence of the anionic detergent deoxycholate. The nonionic... 

Ribonucleoprotein complexes containing mRNA have been isolated from free polysomes of reticulocytes (Huez et al., 1967 Weisberger and Armentrout, 1966), of rat liver (Henshaw, 1968), from the membrane-bound polysomes of rat liver (Leytin et al., 1970), from thyroid gland (Cartouzou et al., 1968) and from L-cells (Perry and Kelley, 1968). 

A very important question concerns the nature of the proteins of polysomal mRNP. This question has not yet been answered because of the difficulties in the isolation of polysomal mRNPs. Since reticulocyte mRNPs can now be obtained in a pure state, the question may be answered in the near future. In another approach, Schweiger and Hannig (1970) tried to find proteins typical of nuclear D-RNP by direct analysis of polysomes. They separated the total polysomal protein into several fractions using free flow electrophoresis and then compared the fractions with nuclear D-RNPs obtained by means of polyacrylamide gel electrophoresis. In free polysomes and in membrane-bound polysomes, proteins similar to proteins of the nuclear D-RNP were found. If the membrane-bound polysomes are separated from the membranes by treatment with pure deoxycholate, these proteins are dissociated from polysomes and thus resemble the proteins of nuclear D-RNP in their sensitivity to deoxycholate. 

Approximately 25% of the total polysomes in poliovirus-infected and uninfected HeLa cells are membrane-bound (10). The membrane-bound polysomes are about five times more active in translation per unit mass than free polysomes, as determined by incorporation of amino acids in cell-free extracts (11). The exact role of the membrane in this process has not yet been defined. 

T2 Tanaka, T., Takagi, M. and Ogata, K. Metabolism of RNA of free and membrane-bound polysomes from rat liver. Biochim. Biophys. Acta, 224, 507-517 (1970)... 

Attachment of membrane-bound polysomes to the protein bodies might occur during extraction. 

Spielman, L. L., and Bancroft, F. C., 1977, Pregrowth hormone Evidence for conversion to growth hormone during synthesis on membrane-bound polysomes. Endocrinology 101 651. 

One mechanism for the transfer of particular translation products across membrane barriers which has received extensive experimental support is the vectorial release of secretory proteins into the cisternal space of the endoplasmic reticulum. In this system, secretory proteins are synthesized selectively on polysomes attached to the rough endoplasmic reticulum and are subsequently released from the membrane-bound ribosomes through the adjacent membrane into the cisternum of the reticulum for later transport from the cell. ... 

Slow cooling of a culture of cells presumably results in some runoff of ribosomes from membrane-bound polysomes. More extensive runoff results from starving the cells for 1 h. The addition of cycloheximide to a log-phase culture of cells, either prior to harvesting or prior to starvation, prevents the translocation step of polypeptide chain elongation and thereby prevents runoff of ribosomes from polysomes. As a result, mitochondria isolated from a log-phase culture of cells to which 200 Mg/ml cycloheximide was added 10 min prior to harvesting, had a density of 1.264 g/ml (Fig. 17E), a density greater than that observed for mitochondria isolated from log-phase cells without prior incubation of the culture with cycloheximide (Fig. 17A). Moreover, the addition of cycloheximide to a log-phase culture of cells prior to starving the culture for 1 hr, completely prevented the decrease in mitochondrial density noted above (Fig. 17F). 

We discussed earlier that one mechanism for the transfer of particular translation products across the membrane barriers is the process of vectorial translation as carried out by polysomes attached to the rough endoplasmic reticulum of secretory cells. An in vitro assay for this process has been devised by Redman and Sabatini. In order to carry out this assay, it is necessary to release labeled, nascent polypeptide chains from membrane-bound ribosomes by reaction with puromycin and assess their distribution between the medium and the membrane compartment. We followed this procedure to assay for vectorial release of nascent chains from bound 80 S polysomes attached to the outer mitochondrial membrane. 

These questions must be examined with regard to local circumstances of biosynthesis. In cells, proteins are synthesized either by free polysomes or by rough endoplasmic reticulum (RER) membrane-bound polysomes. For nascent polypeptide chains, these two different situations have to be considered since, in the second case, compartmentalization leads to a vectorial transport and discharge through the membrane. Bound and free polysomes seem to derive from a common precursor pool of ribosomes. Several lines of evidence indicate that membrane-bound ribosomal subunits readily exchange in vivo and no major differences in the rRNA sequences and organization of free and bound ribosomes have been found (Fern and Garlik, 1976 see Shore and Tata, 1977). 

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