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D I Meyer

Publications and source records attributed to D I Meyer.

At least 55 records · Page 3Linked to original sources
PubMed1986

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

Binding Sites↗
PubMed1985

A structural and functional analysis of the docking protein. Characterization of active domains by proteolysis and specific antibodies.

Docking protein is a 73-kDa integral membrane protein of the rough endoplasmic reticulum. It is essential for translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Monoclonal and polyclonal antibodies have been generated which, in conjunction with limited proteolysis, have been used to characterize several subspecies of docking protein. These proteolytic fragments have been analyzed with respect to the various functions ascribed to docking protein which can be assayed in vitro. Proteolytic digestion of membrane-associated or of affinity-purified intact docking protein showed that: elastase cleavage generates a 59-kDa soluble fragment and one of 14 kDa which contains the membrane anchoring domain; trypsin as well as endogenous proteolysis generates a 46-kDa fragment, leaving a 27-kDa domain containing the membrane anchor. This 27-kDa fragment can be reduced to a 13- and a 14-kDa piece by elastase digestion. The characteristics of these various subspecies were examined. The 59-kDa soluble fragment, which can reconstitute full translocation activity to docking protein-depleted microsomes (Meyer, D. I., and Dobberstein, B. (1980) J. Cell Biol. 87, 503-508) was capable of releasing a signal recognition particle-mediated translation arrest. The 46-kDa fragment was neither able to reassociate with nor to reconstitute the activity of docking protein-depleted microsomes. Moreover this fragment was unable to release a signal recognition particle-mediated arrest. This suggests that the 13-kDa fragment (the difference between 46 and 59 kDa) is both essential for association with the membrane, and for the release of translation arrests.

Animals↗
PubMed1985

Immunochemical analysis of rough and smooth microsomes from rat liver. Segregation of docking protein in rough membranes.

Docking protein (or signal recognition particle receptor) is an integral membrane protein essential for translocation of nascent polypeptides across the membrane of the endoplasmic reticulum. It serves as the receptor for the signal recognition particle, and represents the site of interaction between the translation and the translocation systems. Results presented here demonstrate that this protein is localized exclusively in rough microsomal membranes. Rough and smooth microsomes were prepared and their content of various marker proteins was determined by immunochemical techniques. Whereas a number of proteins, including cytochromes b5 and P-450 and their reductases were evenly distributed between rough and smooth microsomes, docking protein was found at 20-fold higher levels in the rough fraction. On the basis of these results it is concluded that docking protein is a functionally characterized integral protein specifically restricted to rough microsomal membranes.

Animals↗
PubMed1985

Membrane insertion of alpha- and beta-subunits of Na+,K+-ATPase.

Insertion of the alpha- and beta-subunits of amphibian epithelial Na+,K+-ATPase into pancreatic microsomes in cell-free systems was shown to be the same as into membranes of intact cells. The glycoproteic beta-subunit was observed to be cotranslationally inserted into endoplasmic reticulum membranes and to adopt a different pattern of N-linked core and terminal sugars in two different amphibian species. The beta-subunit lacks a cleavable signal sequence but quantitative membrane integration required membrane addition at the start of synthesis. Proteolysis of beta-subunit assembled in vitro indicated a cleavable cytoplasmic domain of about 2000 daltons. The catalytic 98-kilodalton alpha-subunit was also membrane-associated during its synthesis in an alkali-resistant fashion and independent of newly synthesized beta-subunit. In contrast to the beta-subunit, membrane integration of the alpha-subunit was possible as late as a time point in its synthesis which corresponded to about 1/3-1/2 of completion of the nascent chain. A small 34 kDa trypsin-resistant fragment of the alpha-subunit was produced at an early stage of synthesis both in the intact cell and in the cell-free system. These results suggest that membrane insertion of both alpha- and beta-subunit occurs during their synthesis but with a different time course.

Animals↗
PubMed1985

Signal recognition particle-dependent insertion of coronavirus E1, an intracellular membrane glycoprotein.

The membrane insertion of the E1 protein of a coronavirus, mouse hepatitis virus A59, was studied in a wheat germ cell-free translation system. E1 is a transmembrane protein spanning the lipid bilayer several times. It is synthesized without a cleavable signal sequence, localized intracellularly, and not transported to the cell surface. It thus represents a model intracellular protein. We found that the synthesis of E1 is specifically and stably blocked by the addition of signal recognition particle to the wheat germ system. Subsequent addition of salt-extracted pancreatic microsomes resulted in the full release of this arrest as well as the completion and the correct membrane integration of E1. Such signal recognition particle-induced arrests failed to produce shorter peptides of a defined length. Addition of signal recognition particle to a synchronized translation at any time during the synthesis of about the first two thirds of E1 (150 amino acids) blocked further translation, suggesting that the most C-terminal of the three internal hydrophobic domains of E1 could function as its signal sequence.

Animals↗
PubMed1985

Signal recognition particle (SRP) does not mediate a translational arrest of nascent secretory proteins in mammalian cell-free systems.

The ability of the signal recognition particle (SRP) to induce translational arrests in wheat germ, reticulocyte and HeLa cell-free translation systems was examined. In accordance with published data, SRP caused a complete arrest of secretory protein (IgG light chain) translation in wheat germ. In contrast, SRP had no effect on translation in either reticulocyte or HeLa cell lysates, even at 5-fold higher SRP levels than needed for complete arrest in wheat germ. The existence of a "docking-protein-like" releasing activity was ruled out, in the case of reticulocyte lysate, by experiments in which reticulocyte subfractions were added to blocked translations in wheat germ. In the absence of additional evidence to the contrary, it seems as if the translational arrest is peculiar to the wheat germ cell-free system.

Animals↗
PubMed1985

Restriction of docking protein to the rough endoplasmic reticulum: immunocytochemical localization in rat liver.

Docking protein (or SRP receptor) is an integral membrane protein essential for translocation of nascent polypeptides across the membrane of the endoplasmic reticulum (ER). Anti-docking protein antibodies were used to localize this protein in situ in thin frozen sections using protein A-gold detection methods. The majority of gold particles was restricted to ribosome-studded membranes, whereas particles were rarely seen in areas rich in smooth ER. Quantitative evaluation of labeling suggests that there is one molecule of docking protein for roughly 10 to 20 bound ribosomes. On the basis of these results we conclude that docking protein is the first functionally-characterized integral marker protein specific for the rough membranes of ER.

Animals↗
PubMed1984

Pushing the signal hypothesis: what are the limits?

Recent advances in understanding the in vitro translocation of nascent polypeptides across the endoplasmic reticulum (ER) membrane have established a molecular basis for the initial reactions predicted by the signal hypothesis. The first two events involve a transient arrest of nascent chain elongation, followed by a docking maneuver with the ER membrane which releases this block. It is not clear, however, that such signal sequence-mediated transfer occurs in the case of all proteins, or for that matter in all cell-free translation systems.

Animals↗
PubMed1982

Characterization of molecules involved in protein translocation using a specific antibody.

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane-bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.

Animals↗
PubMed1980

A membrane component essential for vectorial translocation of nascent proteins across the endoplasmic reticulum: requirements for its extraction and reassociation with the membrane.

Previous reports have shown that rough microsomes treated with high salt (Warren and Dobberstein, 1978, Nature, 273:569-571) or proteases (Walter et al., 1979, Proc. Natl. Acad. Sci, U. S. A., 76:1,795) are unable to vectorially translocate nascent proteins. Readdition of the high salt or protease extracts restored activity to such inactive rough microsomes. A detailed study was carried out to determine how this factor interacts with the rough microsomal membrane. Proteolytic cleavage was found to be necessary but not sufficient to remove this factor from the membrane. A subsequent treatment with high salt had to be carried out. Endogenous (pancreatic) protease could effect the required cleavage, but low levels of trypsin, clostripain, or elastase were far more efficient. Several proteases were not effective. The minimum level of salt (after proteolysis) required to solubilize the active factor was approximately 200 mM KCl. Salt extracts prepared by treatment with one of the effective proteases were capable of restoring activity to inactive microsomes produced by treatment with one of the others.

Animals↗
PubMed1980

Identification and characterization of a membrane component essential for the translocation of nascent proteins across the membrane of the endoplasmic reticulum.

When rough microsomes are subjected to limited proteolysis and high salt, a soluble fraction can be separated from the membrane. Neither fraction alone is capable of vectorially translocating nascent peptides. When the soluble extract is recombined with the residual membrane fraction, translocating activity is restored. Standard biochemical techniques were used to identify and characterize the active component derived by treating rough microsomes with elastase and high salt. The active factor is a peptide fragment with an apparent molecular weight of 60,000. It represents the cytoplasmic domain of a larger membrane protein. The fragment is basic and has at least one accessible sulfhydryl group. These characteristics facilitated its purification and identification as a membrane component required for translocation of nascent peptides across microsomal membranes.

Animals↗
PubMed1979

Isolation of a protein from the plasma membrane of adrenal medulla which binds to secretory vesicles.

Solubilized proteins of the plasma membrane of bovine adrenal medulla were fractionated on the basis of their affinity for secretory vesicles. The isolation procedure included preparation of a highly purified fraction of plasma membranes, its solubilization in detergent, and application to a column prepared from glutaraldehyde-fixed chromaffin granules. Using this technique, one major polypeptide (80% of the material bound) was isolated. This protein has been shown to originate from the plasma membrane and has no affinity for fixed bovine adrenal medullary mitochondria or lysosomes. It is eluted most effectively by low pH (3.0) and can be rebound and re-eluted from fixed secretory granules. In sodium dodecyl sulfate and beta-mercaptoethanol it has an apparent molecular weight of 51,000. In addition, two minor components, comprising about 20% of the material bound were detected having apparent molecular weights in sodium dodecyl sulfate of 14,000 and 62,000. It is suggested that such a molecule could function as a plasma membrane-located receptor for chromaffin granules during the secretory process.

Adrenal Medulla↗
PubMed1976

The chromaffin granule surface. Localization of carbohydrate on the cytoplasmic surface of an intracellular organelle.

Intact chromaffin granules from bovine adrenal medulla are shown to have complex carbohydrates on their external (cytoplasmic) surface. This is demonstrated by the facts (1) that granules can be agglutinated by wheat germ agglutinin, and (2) that significant amounts of sialic acid can be removed from the granule surface with neuraminidase. Glycoproteins located in the granule membrane, and not glycolipids, are the molecules that mediate wheat germ agglutinin agglutination. The possible involvement of granule surface carbohydrate in the process of exocytosis is discussed.

Adrenal Medulla↗