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D D Sabatini

Publications and source records attributed to D D Sabatini.

At least 91 records · Page 5Linked to original sources
PubMed1978

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins.

Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the vectorial transfer of nascent polypeptides into the microsomal lumen.

Animals↗
PubMed1977

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum.

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High-salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase-treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.

Animals↗
PubMed1977

Release of poly A(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes.

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

Animals↗
PubMed1976

Retention of mRNA on the endoplasmic reticulum membranes after in vivo disassembly of polysomes by an inhibitor of initiation.

Membrane-bound ribosomes and messenger RNA remained associated with the microsomal membranes of human fibroblasts after cultures were treated with Verrucarin A, an inhibitor of initiation which led to extensive run-off of ribosomes from polysomal structures. When a membrane fraction from Verrucarin-treated cells containing such inactive ribosomes and mRNA was suspended in a medium of high salt concentration, extensive release of ribosomal subunits occurred without the need for puromycin. The mRNA nevertheless remained associated with the membranes. These results add support to the conclusion that, in human fibroblasts, mRNA is bound directly to ER membranes, independently of the ribosomes and nascent polypeptide chains.

Anti-Bacterial Agents↗
PubMed1975

Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts.

Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus.

Adenosine Monophosphate↗
PubMed1974

On the spatial arrangememt of proteins in microsomal membranes from rat liver.

Rat liver rough microsomes were labeled enzymatically with (125)I using lactoperoxidase and glucose oxidase. In intact microsomes only proteins exposed on the outside face of the microsomal membrane were iodinated. Low concentrations of detergent (0.049% deoxycholate) were used to allow entrance of the iodination system into the vesicles without disassembling the membranes. This led to iodination of the soluble content proteins and to an increased labeling of the membrane proteins. The distribution of radioactivity in microsomal proteins was analyzed after separation by sodium dodecyl sulfate acrylamide gel electrophoresis. Most membrane proteins were labeled when intact microsomes were iodinated. No major membrane proteins were exclusively labeled in the presence of low detergent concentrations or after complete membrane disassembly. Therefore it is unlikely that there are major membrane proteins, other than glycoproteins, present only on the inner membrane face or completely embedded within the microsomal membrane. Microsomal proteins were also labeled by incubating rough microsomes with [(3)H]-NaBH(4) after reaction with pyridoxal phosphate. Microsomal membranes were permeable to these small molecular weight reagents as shown by the fact that proteins in the vesicular cavity as well as membrane proteins were labeled with this system.

Animals↗
PubMed1974

Selective release of content from microsomal vesicles without membrane disassembly. II. Electrophoretic and immunological characterization of microsomal subfractions.

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [(3)H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [(3)H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [(3)H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.

Animals↗
PubMed1973

Ribosome-membrane interaction. Nondestructive disassembly of rat liver rough microsomes into ribosomal and membranous components.

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl(2), 50 mM Tris.HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [(3)H]puromycin into hot acid-insoluble material and from the release of [(3)H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25-100 mM) as at high (500-1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction ( approximately 15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.

Amino Acids↗
PubMed1973

Selective release of content from microsomal vesicles without membrane disassembly. I. Permeability changes induced by low detergent concentrations.

Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025-0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.

Animals↗