Rough Endoplasmic Reticulum Membranes Research Articles

A quantitative electron microscope autoradiographic study on 125I-human choriogonadotropin internalization in bovine luteal slices

Very few silver grains were seen on the cell surface and none intracellularly after incubation for 2 h at 4 °C. However, numerous grains were seen in various subcellular organelles when the tissues were incubated for 2 h at 22 ° or 38 °C. The grain distribution was qualitatively similar, but quantitatively, there were fewer grains at 22 ° than at 38 °C. Co-incubation of 125I-hCG with excess unlabelled hCG resulted in the virtual disappearance of silver grains from all the subcellular organelles. Excess unlabelled human luteinizing hormone (but not follicle-stimulating hormone or prolactin) inhibited the appearance of silver grains in luteal tissue. There were no silver grains in bovine liver slices incubated with 125I-hCG. The plasma membrane-associated grains progressively decreased, while intracellular organelle-associated grains increased with time at 38 °C. There were no grains in nuclei at 5 min, but they appeared at 10 min and increased until 120 min. After correction for radiation spread by three-step mask analysis, smooth endoplasmic reticulum and mitochondria did not contain any grains. The grain density was the highest in Golgi, followed by lysosomes, rough endoplasmic reticulum, nuclei, and plasma membranes after incubation for 2 h at 38 °C. Thus, the electron microscope autoradiography approach confirmed our biochemical data in the preceding paper (Chegini et al., Exp cell res 151 (1984) 466 [5]) on time, temperature dependency and specificity of 125I-hCG internalization, association of internalized hormone with a variety of intracellular organelles, and the highest uptake in Golgi.

Subcellular fractions in rubella immunoassays.

Rubella virus-infected cells were fractionated by differential and sucrose gradient centrifugations. Rubella virus antigens distributed into all fractions but particulate material in the 100,000 x g pellet was shown to be enriched about two-fold for rubella virus antigen. Similarly, sucrose gradient fractions for rough endoplasmic reticulum and smooth cellular membranes were enriched for rubella virus antigens. The 100,000 x g pellet and the isolated cellular membranes proved to be useful when different fractions were used in solid-phase immunoassays for rubella virus-specific IgG or IgM. These fractions were equal in quality of the semipurified rubella virus preparations in the IgG assays but inferior to those in the IgM assays. However, simultaneous use of 35/25% sucrose fractions from infected and non-infected cells reveals non-specific binding of IgM to the antigens and renders the IgM tests more specific for rubella virus.

Retinal pigment epithelial fine structure in the nighthawk (Chordeiles minor).

The retinal pigment epithelial (RPE) layer of a nocturnally active bird, the nighthawk (Chordeiles minor), has been investigated by electron microscopy. This layer is composed of a single layer of low cuboidal cells joined laterally by tight junctions. Basally (sclerally), the epithelial cells display deep infoldings while apically (vitreally) numerous processes enclose photoreceptor outer segments. Internally, the nucleus is large, vesicular and basally located. Smooth endoplasmic reticulum and mitochondria are abundant. Rough endoplasmic reticulum (RER) is scarce but numerous polysomes are present. Myeloid bodies are widespread and often appear to be continuous with the RER membranes. Phagosomes of outer segment material are present but not numerous in the light-adapted condition. Large spindle-shaped melanosomes as well as numerous rounded tapetal spheres are present in the apical region and apical processes of the epithelial cells. The composition of the reflective tapetal spheres is unknown. Bruch's membrane (complexus basalis) in most locations displays a pentalaminate appearance while in some regions it is trilaminate. The endothelium of the choriocapillaris facing Bruch's membrane is extremely thin but only sparsely fenestrated.

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium.

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis--probably through the compartment of coated pits and coated and uncoated vesicles.

Mechanism of glycosylation in the Golgi apparatus.

A major role of the Golgi apparatus in liver is the terminal glycosylation of secreted serum proteins and of plasma membrane glycoproteins. Galactosyltransferase is a membrane-bound Golgi enzyme that transfers galactose directly from uridine diphosphogalactose (UDP-Gal) to terminal N-acetylglucosamine groups of N-asparagine-linked glycoproteins during secretion. Sialytransferase then transfers sialic acid from cytidine monophosphosialic acid (CMP-NAN) to the newly added terminal galactose of the glycoprotein. In the cell, the transfer reaction must occur on the lumen side of the Golgi membrane. UDP-Gal is synthesized mainly in the cytoplasm and CMP-NAN is synthesized in the nucleus in liver. An important question for understanding the mechanism is, how do these nucleotide sugars gain access to the transferases? A second question involves uridine diphosphate (UDP), a highly inhibitory product of galactosyltransferase. How is UDP removed from the lumen of the Golgi fast enough to prevent product inhibition of the galactosyltransferase? We have shown that isolated Golgi, although vesiculated, retains its original orientation. The vesicles are oriented with greater than 90% of both galactosyltransferase and sialyl-transferase on the luminal side of the vesicles. Using intact vesicles, we can show that UDP-Gal is taken up via a saturable carrier system present in the Golgi membrane. During galactosylation in vitro, UDP formed in the lumen of Golgi vesicles is rapidly converted to UMP by a nucleoside diphosphatase in the lumen. Uridine monophosphate, which is much less inhibitory to the galactosyltransferase than UDP, is then transported out of the lumen by a second carrier and is broken down further to uridine by 5'-nucleotidase on the cytoplasmic side of the Golgi vesicles. The transport of nucleotides appears unique to the Golgi membranes, since neither rough endoplasmic reticulum nor plasma membrane vesicles from rat liver accumulate these nucleotides.

Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates.

In eukaryotic cells, secretory proteins and glycoproteins migrate from the rough endoplasmic reticulum, their site of synthesis, through Golgi vesicles before being released from the cell. Cellular and viral integral plasma membrane glycoproteins are co-translationally inserted into the rough endoplasmic reticulum membrane and follow a similar pathway to the cell surface. Previous studies using endoglycosidase H (Endo H) suggested that in rat hepatoma cells the vesicular stomatitis virus (VSV) G protein, albumin and transferrin migrate from the rough endoplasmic reticulum to the Golgi apparatus at different rates. Here we show directly that in human hepatoma HepG2 cells, five secreted proteins mature from the rough endoplasmic reticulum to Golgi vesicles at characteristic rates which differ at least threefold. The results are incompatible with bulk-phase movement of the luminal contents of the endoplasmic reticulum, and suggest that there is a membrane-bound receptor that selectively mediates the transport of secretory proteins from the rough endoplasmic reticulum to the Golgi.

Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti.

Ineffective alfalfa nodules formed by Rhizobium meliloti nif::Tn5 mutants were examined by light and electron microscopy. R. meliloti nifH::Tn5 mutants formed nodules that were similar in structure to wild-type nodules except that nifH- bacteroids accumulated a compact, electron-dense body. In contrast to nodules induced by wild type and nifH mutants, nifDK- R. meliloti mutants induced nodules which contained numerous starch grains and prematurely senescent bacteroids. In addition, meristematic activity in nifDK- nodules ceased significantly earlier than in nifH- nodules. All mutant nodules exhibited elevated levels of rough endoplasmic reticulum and Golgi membranes compared to wild-type nodule cells. These elevated levels may reflect either a response to nitrogen starvation in the ineffective nodules or an abnormal synthesis and export of nodule-specific proteins during later developmental stages.

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes.

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence.

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.

Medroxyprogesterone Induced Changes in Rat Liver: A Light and Electron Microscopic Study

Medroxyprogesterone acetate (MPA) induced morphological alterations in the normal female rat liver and after carbon tetrachloride exposure were assessed by light and electron microscopy and morphometry. Administration of MPA increased the smooth endoplasmic reticulum (SER) in normal rat. Carbon tetrachloride, injected intraperitoneally for four weeks, caused fatty accumulation, enlargement of the cell nucleus and decrease of the rough endoplasmic reticulum (RER) and increase of the SER. After one week of recovery, without treatment, the fatty infiltration and the SER membranes decreased and the RER increased. MPA, following injury, induced a decrease in the fatty accumulation and mitochondrial volume density and the hepatocyte nuclear and cytoplasm size normalized. The SER membrane volume density increased and the surface density decreased, the RER membrane volume density increased while the surface density did not alter significantly. The volume densities of the mitochondria and the peroxisomes decreased in the MPA treated CCl4 exposed groups. The results demonstrate that MPA induces a proliferation of the SER in normal rat liver and after hepatic injury the compound has a beneficial effect on the regeneration.

7] Methods for the study of protein translocation across the RER membrane using the reticulocyte lysate translation system and canine pancreatic microsomal membranes

This chapter presents the methods developed to elucidate this mechanism for the transport of pancreatic exocrine proteins across the rough endoplasmic reticulum (RER) membrane. The methods are optimized for the simultaneous study of 17 discrete pancreatic secretory proteins, which include individual members of several families of enzymes and proenzymes. The results show that secretory proteins synthesized by dog pancreas mRNA in the presence of nuclease treated microsomal membranes were similar to those synthesized in vivo. The findings indicate that microsomal membranes isolated from dog pancreas and added to the reticulocyte lysate system are unstable in the presence of 50 μg/ml each of trypsin and chymotrypsin. Using polyacrylamide gel electrophoresis in SDS, the chapter demonstrates that the addition of micrococcal nuclease-treated rough microsomes from dog pancreas results in complete proteolytic processing of pancreatic presecretory proteins to polypeptide chains with apparent molecular weights identical with authentic secretory enzymes and zymogens.

Immunoelectron microscopy of the outer membrane of rat hepatocyte nuclear envelopes in relation to the rough endoplasmic reticulum.

Prefixed nuclei were isolated from rat liver after perfusion with dilute glutaraldehyde. These nuclei sometimes were associated with the rough endoplasmic reticulum (ER), and occasionally continuity of the outer nuclear membrane with rough ER membrane was found. When these prefixed nuclei were incubated with ferritin antibody conjugates against cytochrome P-450, the cytoplasmic face of the outer nuclear membrane was labeled with ferritin similar to the labeling of the rough ER membrane. In the nuclear pore region, ferritin particles were present on the outer membrane up to the outer annuli of the pore complexes. When unfixed nuclei were incubated at 0-4 degrees C or at 20 degrees C, some outer nuclear membranes were detached from the nuclear surface. In this case the nuclear pore complexes remained associated with the inner nuclear membrane, and small inside-out vesicles were formed at these pore regions. We also found some rough vesicles heavily labeled with ferritin particles within the nuclear matrix. They probably were derived from the rough ER or from the outer nuclear envelope which was internalized artificially during incubation.

The regulation of acetylcholine receptor expression in mammalian muscle.

The synthesis of functional AChRs can be described as a pathway leading from the translation of subunit mRNAs to the plasma membrane forms of extrajunctional and junctional receptors (Fig. 9). We have not included in this scheme pretranslational steps for the synthesis and processing of RNA coding for receptor subunits because very little is known about such processes. Several aspects of Figure 9 are worthy of note: It is now well established that polypeptide synthesis is initiated on free cytoplasmic polysomes and that once sufficient nascent subunits bearing signal peptides at the amino terminus is formed, polysomes assemble with the membranes of the rough endoplasmic reticulum via a mechanism that employs the signal recognition particle (Anderson et al. 1982). Nascent subunits undergo cotranslational insertion through the rough endoplasmic reticulum membrane, signal peptide removal, and core glycosylation (Anderson and Blobel 1981; Merlie et al. 1981; Anderson et al. 1982; Sebbane et al. 1983). Anderson and Blobel (this volume) have demonstrated that subunits synthesized in vitro and inserted into membrane vesicles do not undergo heterologous subunit-subunit associations. We have shown that alpha- and beta-subunits newly synthesized in vivo are not associated with each other. Our data indicate that the alpha-subunit is initially present in vivo in a conformation that is radically different from its native conformation in the mature receptor complex. We assume that beta-, gamma-, and delta-subunits also are synthesized as conformationally "immature" forms, but verification of this point must await the availability of new monoclonal antibody specificities. Our data indicate that only a fraction of the newly synthesized alpha-subunit undergoes conformational maturation to the 5S species which binds both alpha-bungarotoxin and anti-main immunogenic region monoclonal antibodies. alpha-Subunits synthesized during a 5-minute pulse labeling require 30 minutes for completion of this process. alpha-Subunits that do not undergo conformational maturation are degraded rapidly (t1/2 = 0.5 hr) ( Merlie et al. 1982). Assembly of alpha- and beta-subunits synthesized during a 5-minute pulse labeling lags for approximately 30 minutes and is not complete until 90 minutes. Finally, assembled receptors are transported to the surface and appear in the plasma membrane. These processes occur during expression of AChRs in differentiated myoblasts. We do not know how undifferentiated myogenic cells, in vivo or in tissue culture, differ with regard to any of these steps.(ABSTRACT TRUNCATED AT 400 WORDS)

Freeze-fracture images of distribution of filipin-cholesterol complexes in the oviduct epithelium of mice.

In order to clarify the distribution of cholesterol in the plasma-and cytomembranes of the oviduct epithelium of mice, freeze-fracture images of the filipin-treated tissues were observed. The filipin-sterol complexes were distributed in high density on the fractured plasma membranes of both secretory and ciliated cells except for special areas. The limiting membranes of secretory granules of the secretory cell were very rich in complexes. In both types of cells, membranes of nucleus and rough endoplasmic reticulum were virtually free of the complexes, though very small aggregates of a few complexes were infrequently scattered. Most Golgi stacks were labeled slightly with filipin, but small vesicles and a stack in the trans-side showed a fairly large number of the complexes to form small aggregates. No complexes were recognized on the outer and inner mitochondrial membranes of both type cells. Although most of the ciliary membrane contained numerous complexes, only the ciliary necklace portion lacked the complexes. The area of zonula occludens was generally free of the complexes. The membrane enclosed by the strands of zonula occludens was devoid of the complexes, though filipin extensively labeled the membrane surrounding the zonula occludens. Round patches studded beneath the zonula occludens, which might correspond to desmosomes, did not bear the complexes.

62] Quantitative assay for signal peptidase

Signal peptidase is the enzyme responsible for removing the signal peptide portion of nascent presecretory and presumably also prelysosomal and premembrane proteins during their cotranslational translocation across the rough-endoplasmic reticulum (RER) membrane. This chapter describes the quantitative assay for signal peptidase that involves the translation of mRNA encoding a secretory protein in a cell-free in vitro translation system containing microsomal membranes. In this assay, signal peptidase activity is coupled to both the translation and translocation of the presecretory protein. [35S]Prolactin produced by posttranslational cleavage of [35S]preprolactin, in an assay containing 1.0% Triton X-100 and 2.5 mM phospholipid, is detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) followed by autoradiography and is quantitated by counting the portion of the gel containing the prolactin band or by densitometry of the autoradiograph. Utilizing a qualitative assay for signal peptidase, signal peptidase can be inactivated by delipidation, and activity can be restored to the delipidated enzyme by readdition of phospholipid.

The Golgi complex and the sorting of membrane and secreted proteins

All proteins and glycoproteins synthesized by eukaryotic cells are either secreted or become localized to the cytoplasm or to specific membranes or organelles. Proteins that are encoded by nuclear DNA (the vast majority of cellular proteins) are synthesized on cytoplasmic ribosomes: depending on the protein being synthesized, the ribosomes are either bound to the rough endoplasmic reticulum membrane, or are free in the cytosol, unbound to membranes. All proteins must be sorted, or targetted, to their proper destinations during or after synthesis.

56] In vitro processing of plant preproteins

This chapter proposes to develop a cell-free system for the translation, processing and packaging of plant preproteins that demonstrates the synthesis, processing, and deposition of zein polypeptides and α-amylase. The method uses a nuclease-treated rough endoplasmic reticulum (RER)-enriched preparation derived from developing maize endosperm in combination with a wheat germ translation system. The procedure involves material required for membrane preparation, isolation of membranes, preparation of treated membranes, and in vitro protein synthesis and processing. Nonpigmented kernels, such as Illinois High Protein, are preferred to colored kernels. In vivo , zein is synthesized in the maize endosperm on polyribosomes of the RER cisternae that subsequently enlarge to form globular protein bodies. The procedures for the isolation and preparation of treated maize RER membranes can probably be readily adapted to other plant species. Before setting up a synthesis and processing reaction, the membranes are treated with micrococcal nuclease to destroy endogenous zein messages.

Biosynthesis of Basement Membrane by Parietal Yolk Sac Cells

The biosynthesis of basement membrane (Reichert's membrane) by parietal yolk sac cells of the mouse embryo was studied using immunohistochemistry and autoradiography with the electron microscope. Fab' fragments of characterized antisera to either a mixture of type IV collagen and laminin, or to type IV collagen alone, were conjugated to horseradish peroxidase for the immunohistochemical study; radiolabeled proline, leucine, and glucosamine were utilized either in vivo or in isolated yolk sac membranes in vitro for autoradiography. Whereas neither of the two antisera employed localized in either the Golgi apparatus or in the cytosol, both localized in the rough endoplasmic reticulum (RER) and in the extracellular basement membrane. Furthermore, no evidence of passage of isotopically labeled precursors of basement membrane from the RER to the Golgi apparatus was observed. Consequently, it is concluded that the basement membrane precursors are synthesized in the RER of parietal yolk sac cells and neither traverse the Golgi apparatus nor diffuse across the cytosol of the cell in the process of secretion. By elimination, secretion must be via direct communications between RER and plasma membrane or via small vesicles observed in the cytosol between RER and plasma membrane. These data are not in accord with the concept of the universality of the Golgi apparatus in the biosynthesis of glycoprotein. Parietal yolk sac cells do not use the Golgi apparatus in the biosynthesis of basement membrane, but do use the Golgi apparatus for the biosynthesis of other proteins.

Three mutations in Zea mays affecting zein accumulation: a comparison of zein polypeptides, in vitro synthesis and processing, mRNA levels, and genomic organization.

We studied three mutations, opaque-2 (o2), opaque-7 (o7), and floury-2(fI2), each of which causes a depression in zein synthesis. We examined the processing efficiencies of the rough endoplasmic reticulum membranes in vitro, the levels of RNA transcription using cloned zein probes, and the genomic organization of the zein sequences as possible sites for the genetic defects. The results obtained indicate that the steps in prezein translation and processing occurring on the protein body membranes are not accountable for the lowered zein content in any of the mutations. The o2 mutation that typically shows a paucity of 22.5-kdalton zein polypeptides was found to have a concomitant reduction in a particular subgroup of mRNAs coding for this size class. Southern analyses suggest that the o2 mutation is not the result of a large deletion of tandem-linked zein genes.

A quantitative ultrastructural study of normal rat erythroblasts and reticulocytes.

Early, intermediate and late erythroblasts and reticulocytes were studied by electron microscopic morphometry. The volumes of mitochondria, Golgi zone and autophagosomes, as well as the surface areas of membranes of rough endoplasmic reticulum (RER) and of mitochondrial cristae and the numbers of ribosomes per unit volume cytoplasm were calculated. the results revealed 3 phases in erythroid maturation: (1) early and intermediate erythroblasts, (2) late erythroblasts, and (4) reticulocytes. The only significant difference between early and intermediate proliferating erythroblasts was a decrease in the surface area of the RER in the latter. After the last mitotic division in late erythroblasts significant reductions occurred in the RER, the Golgi apparatus, in the mitochondria and the number of ribosomes. The numbers of mitochondria and ribosomes were further reduced at the reticulocyte stage (clustered ribosomes more rapidly than single ones). Morphometric analysis showed no evidence of degradation of erythroid mitochondria whilst they are free in the cytoplasm, but there was some evidence of degradation after their uptake into autophagosomes in late erythroblasts and in reticulocytes.