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D D Sabatini

Publications and source records attributed to D D Sabatini.

At least 73 records · Page 4Linked to original sources
PubMed1982

Synthesis and incorporation of myelin polypeptides into CNS myelin.

The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath.

Animals↗
PubMed1982

Identification of ribophorins in rough microsomal membranes from different organs of several species.

Microsomes prepared from several animal sources were analyzed for the presence of proteins corresponding to the ribophorins (I and II) which have been previously characterized in rat liver rough microsomes and appear to be involved in the binding of polysomes to endoplasmic reticulum membranes. In rough microsomal membranes from rat lacrimal gland, rabbit liver, dog and chicken pancreas, and mouse myeloma, ribophorin-like polypeptides with similar electrophoretic mobilities were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In all cases the polypeptides remained associated with sedimentable polysomes after solubilization of the microsomal membranes with nonionic detergents. Ribophorin-like polypeptides were absent from smooth microsomes. Antibodies raised against each rat liver ribophorin, purified by preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoprecipitated only the corresponding polypeptide, indicating no crossreactivity between ribophorins I and II. These antibodies also immunoprecipitated the homologous ribophorins found in microsomal preparations from other organs and species.

Animals↗
PubMed1981

In vitro synthesis and posttranslational uptake of cytochrome c into isolated mitochondria: role of a specific addressing signal in the apocytochrome.

Administration of the thyroid hormone 3,3,5'-triiodo-L-thyronine (T3) to rats leads to a marked increase in hepatic levels of mRNA for cytochrome c. Messenger RNA prepared from the free polysomes of T3-treated rats directed the in vitro synthesis of a polypeptide which only differed in amino acid sequence from mature cytochrome c in that it contained an NH2-terminal methionine. The in vitro product was incorporated specifically into purified rat liver mitochondria and became inaccessible to added trypsin when the mitochondria were added after translation was completed. Horse heart apocytochrome c, but not the holocytochrome, could compete with the in vitro synthesized polypeptide for its uptake into mitochondria. This suggests that the primary structural features of apocytochrome c, which serve as an addressing signal for mitochondria, are masked after the acquisition of heme and that this process occurs in the mitochondria. The addressing signal seems to be contained in a specific segment of the cytochrome polypeptide because only one fragment generated by CNBr cleavage of horse apocytochrome c, extending from residue 66 to the carboxy end of the molecule, could compete with the in vitro product for its transfer into mitochondria.

Animals↗
PubMed1981

Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes.

Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA-dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the interior of the polypeptide. a mechanism that explains the final transmembrane disposition of band 3 in the plasma membrane as resulting from the mode of its incorporation into the ER is presented.

Animals↗
PubMed1980

Biosynthesis of erythrocyte membrane protein band 3 in DMSO-induced Friend erythroleukemia cells.

The major integral membrane protein of red blood cells, the mouse equivalent of human band 3, was purified and used to raise a specfic antiserum. The murine protein resembles its human counterpart in several of its properties, including susceptibility to digestion by chymotrypsin added to intact cells and an ability to bind to concanavalin A. The synthesis of 35S-labeled band 3 was detected in Friend erythroleukemia cells treated with DMSO by immuneprecipitation followed by SDS gel electrophoresis and fluorography. Induction with DMSO led to a greater than tenfold increase in the synthesis of band 3 and maximal synthesis was reached 3 to 4 days after the beginning of induction.

Animals↗
PubMed1980

Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes.

Treatment of rats with phenobarbital leads to a substantial increase in levels of translatable liver cytochrome P-450 mRNA. This mRNA is primarily associated with ribosomes bound to endoplasmic reticulum membranes which in an in vitro system synthesized approximately 10 times more cytochrome P-450 than did free polysomes from the same animals. Cytochrome P-450 synthesized by rough microsomes in vitro appears to be directly inserted into the membranes because it was not released by a treatment with low detergent concentrations that released albumin and other microsomal content proteins. The amino-terminal amino acid sequence of cytochrome P-450 synthesized in an mRNA-dependent system resembles in hydrophobicity the signal segment of presecretory proteins and therefore may serve to insert the polypeptide into the membrane during synthesis. In contrast to the situation with secretory proteins and several other membrane proteins, however, the putative insertion signal of cytochrome P-450 is not removed by a membrane-associated peptidase and remains in the mature polypeptide.

Animals↗
PubMed1979

In vitro synthesis of the Ca2+ transport ATPase by ribosomes bound to sarcoplasmic reticulum membranes.

The calcium transport ATPase (M(r) 100,000) from sarcoplasmic reticulum membranes was synthesized in a cell-free translation system containing rough microsomes or detergent-treated bound polysomes from 14- to 16-day old chicken embryo muscles. Immunoprecipitates obtained from total translation mixtures treated with anti-ATPase antiserum contained 1.5% of the total radioactivity incorporated in vitro. A polypeptide with the electrophoretic mobility, isoelectric point, and [(35)S]methionine-labeled tryptic peptide pattern of the mature ATPase was a major component of these immunoprecipitates. By contrast, free polysomes from the same source, which were capable of high levels of in vitro protein synthesis, did not yield immunoprecipitable ATPase. ATPase synthesized in rough microsomes was not released by treatment with 10 mM EDTA in a high-salt medium (0.5 M KCl) which removes ribosomes and peripheral membrane proteins. Furthermore, labeled ATPase remained associated with the microsomes after these were treated with low concentrations of deoxycholate (0.1 mg/mg of protein in 0.3 ml) which release the luminal content of the vesicles. Only with higher deoxycholate concentrations (0.5 mg/mg of protein in 0.3 ml), which cause membrane dissolution, was the labeled ATPase found on the detergent extracts. These observations indicate that newly synthesized ATPase discharged from bound ribosomes is transferred directly to the sarcoplasmic reticulum membranes where it is incorporated as an integral membrane protein.

Animals↗
PubMed1978

Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding.

Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detergent Kyro EOB; iii) in intact rough microsomes ribophorins can be cross-linked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton-X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and "rough-inverted" vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosomes when these aggregate without detaching. Measurements of the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents suggest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them.

Animals↗
PubMed1978

Asymmetric budding of viruses in epithelial monlayers: a model system for study of epithelial polarity.

Infection of two different lines of polarized epithelial cells grown as monolayers with several types of enveloped viruses results, for each virus type, in a characteristic asymmetric budding of virions. Influenza virus (WSN strain), simian virus 5, and Sendai virus bud exclusively from the free (apical) surface of the cells, while vesicular stomatitis virus acquires its envelope only from the basolateral plasma membrane. Because different viruses select specific domains of plasma membrane in the same cell type, virus-infected epithelial monolayers can provide an excellent model system for studies of the mechanisms that generate regional differences in the distribution of plasma membrane components of epithelial cells.

Cell Line↗
PubMed1978

Localization of eukaryotic initiation factor 3 on native small ribosomal subunits.

The localization of eukaryotic initiation factor 3(eIF-3) on native small ribosomal subunits has been established by electron microscopy through a comparison of native small ribosomal subunits with derived subunits and with native subunits stripped of eIF-3. Small subunits derived from reticulocyte ribosomes by the puromycin/KCl method are seen in electron micrographs as elongated particles, divided by a heavily stained partition into approximately one-third and two-third domains. Most particles (60-70%) observed in electron micrographs of native small subunit preparations resemble derived small subunits, but have an additional mass attached to one side, thus producing profiles with a three-lobed appearance. The mass measures approximately 160 x 100 x 60 A, and its particle weight is estimated to be about one-third to one-half that of a 40S subunit. The site of attachment of the additional mass is located on a prominence extending from the central part of the small subunit and is separated by a cleft from the smaller third of the subunit. The remaining particles in preparations of native subunits resemble the profiles seen in electron micrographs of derived subunits. After removal of eIF-3 by treatment with high concentrations of salt, profiles observed in electron micrographs of washed, native subunits were indistinguishable from those of derived subunits. Since removal of eIF-3 coincided with removal of a mass of the correct molecular weight, subunits with the three-lobed appearance are identified as native small subunits carrying eIF-3.

Animals↗
PubMed1978

Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes.

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin-KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.

Animals↗
PubMed1978

Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites.

Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS-acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These observations suggest that ribophorins are in close proximity to the bound ribosomes, as may be expected from components of the ribosome-binding sites.

Animals↗
PubMed1978

Polarized monolayers formed by epithelial cells on a permeable and translucent support.

An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (10(6) cells/cm2) on collagen-coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division. An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 +/- 1.8 omega-cm2 after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na+ and Cl- ions. The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na+ and Cl- caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrates and of medium field strength. Measurements of Na+ permeability of electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/deltapsi curves does not support the involvement of carriers. The discrimination between Na+ and Cl- was severely but reversibly decreased at low pH, suggesting that Na+-specific channels which exclude Cl- contain acidic groups dissociated at neutral pH. Bound Ca++ ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA. Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.

Animals↗
PubMed1978

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes.

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.

Animals↗