Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

Localization and partial characterization of acyltransferases present during rapid membrane formation in the Drosophila melanogaster embryo

Rapid membrane assembly occurs in the early, syncytial Drosophila embryo when 3500 cells are separated within a period of 90 min by the formation of plasma membranes. Acyltransferases catalyzing two of the early steps in phospholipid synthesis were studied during the course of this membrane formation. The enzymes appeared to be similar to mammalian acyltransferases in pH and substrate optima. The activity was inhibited by sulfhydryl-binding reagents and stimulated by low concentrations of magnesium, calcium, and managnese. The enzymes incorporated α-l-glycerophosphate and palmityl coenzyme A into both phospholipids and neutral lipids. The acyltransferases were also localized cytochemically under conditions shown to preserve a substantial proportion of the enzyme activity. Early in embryo development, the activity was localized in the rough endoplasmic reticulum and nuclear envelope. Reaction products were particularly frequent around mitotic nuclei. During the first phase of plasma membrane formation, the activity was localized at the furrow regions of the plasma membrane and in the apical nuclear envelope. During the second (fast) phase, the reaction products appeared mainly in the basal nuclear envelope and rough endoplasmic reticulum internal to the nuclear layer. The data were consistent with the hypothesis that phospholipids for membrane assembly can be formed in situ in several subcellular compartments.     

Subcellular distribution and regulation of hepatic bilirubin UDP-glucuronyltransferase

We have investigated the subcellular location and regulation of hepatic bilirubin UDP-glucuronyltransferase, which has been presumed to be located largely in the smooth endoplasmic reticulum. Purity of subcellular membrane fractions isolated from rat liver was assessed by electron microscopy and marker enzymes. Bilirubin UDP-glucuronyltransferase activity was measured by radiochemical assay using a physiologic concentration of [14C]bilirubin, and formation rates of bilirubin diglucuronide and monoglucuronides (C-8 and C-12 isomers) were determined. Activity of the enzyme was widely distributed in subcellular membranes, the majority being found in smooth and rough endoplasmic reticulum, with small amounts in nuclear envelope and Golgi membranes. No measurable activity was found in plasma membranes or in cytosol. Synthesis of bilirubin diglucuronide as a percentage of total conjugates and the ratio of C-8/C-12 bilirubin monoglucuronide isomers formed were comparable in all membranes, suggesting that the same enzyme is present in all locations. However, the regulation of bilirubin UDP-glucuronyltransferase activity differed among intracellular membranes; enzyme activity measured in the presence of the allosteric effector uridine 5'-diphospho-N-acetylglucosamine exhibited latency in smooth endoplasmic reticulum and Golgi membranes, but not in rough endoplasmic reticulum and nuclear envelope. Since rough membranes comprise 60% of hepatocyte endoplasmic reticulum and bilirubin UDP-glucuronyltransferase activity in vitro is maximal in this membrane fraction under presumed physiologic conditions, it is likely that the rough endoplasmic reticulum represents the major site of bilirubin glucuronidation in hepatocytes.     

Light- and electron-microscopic studies on acetylcholinesterase activity in Auerbach's plexus of the developing rat colon

The distal portions of rat colon from 14-, 16-, 18-, and 21-day fetuses, newborns, and adults were histochemically examined for acetylcholinesterase (AChE) activity by light and electron microscopy. The specificity of AChE activity in Auerbach's plexus was confirmed by specific and/or nonspecific cholinesterase inhibition tests. Enzyme activity was first detectable after 18 days of gestation and became stronger with age. The reaction product was demonstrated by electron microscopy in and between the plasma membranes of the nerve fibers and their terminals. Ganglion cells also showed positive activity in the plasma membrane, nuclear envelope, and rough endoplasmic reticulum. The distribution pattern of the reaction product in fetal and newborn rat colons was basically the same as in adult rat colon. Therefore, the localization of AChE activity is considered to be a good marker for identifying premature ganglion cells in Auerbach's plexus, and the degree of AChE staining is a good indication of the degree of maturation of the plexus.     

Histochemical localization of adenosine triphosphatase activity in thymus: A light microscopical and ultrastructural study

Summary Ultrastructural localization of ATPase at high pH in the presence of Ca²⁺ showed that activity in thymocyte precursors was stronger than in mature thymocytes. The activity was localized in the nuclear envelope, rough endoplasmic reticulum, Golgi apparatus and mitochondria. The difference in activity was attributed to a marked decrease in ATPase-containing organelles, mainly the endoplasmic reticulum in the mature thymocytes. This appears to be related to the proliferative activity of the cells rather than to the immunological maturity of the thymocytes. A very strong activity, also localized in the same organelles, was present in the macrophages and interdigitating cells which might have a secretory function and possibly contribute to thymocyte maturation. The Ca²⁺—ATPase activity in the nuclear envelope—endoplasmic reticulum system suggests that these may be the sites for storage and regulation of cytoplasmic calcium.     

The fine structural localization of peroxidase activity in digestive organs of rats and mice

Summary An endogenous peroxidase activity is demonstrated in acinar cells of the salivary gland and epithelial cells of the colonic crypt of normal rats and mice using electron microscopic histochemistry. The main site of the enzymatic activity is cisternae of the rough endoplasmic reticulum including those of the nuclear envelope, while the intensity of the activity is greatly variable among cell types. Some vesicular and cisternal elements of the Golgi apparatus and secretory granules exhibit the reaction, but it is not consistent in all cells with the peroxidase-positive endoplasmic reticulum. It is very interesting that the peroxidase activity is positive in the rough endoplasmic reticulum-Golgi complex-secretory granule system (EGG system) of the cells located at the beginning and the end of the digestive tract. This suggests a peroxidase-dependent anti-infectious mechanism.Some large and small membrane-limited non-secretory granules and mitochondria also reacted.     

Cytochemical evidence of the origin of the dense tubular system in the mouse platelet

Summary Glucose-6-phosphatase (G6Pase) was used as a marker enzyme for the endoplasmic reticulum in mouse megakaryocytes and platelets. G6Pase activity was localized in the dense tubular system of the platelets. Enzyme activity was also observed in the nuclear envelope, and in the rough endoplasmic reticulum of the megakaryocytes. However, the Golgi apparatus of the megakaryocyte was never involved. The present study has added new cytochemical evidence for the hypothesis that the dense tubular system of the platelet originates from the endoplasmic reticulum of the megakaryocyte.     

False localization of acid phosphatase activity in the nuclear envelope and endoplasmic reticulum of peritoneal macrophages

Summary Acid phosphatase cytochemistry using lead salt methods was performed on rat peritoneal macrophages obtained by the intraperitoneal injection of dextran five days previously. Lead precipitate was present in the nuclear envelope, the rough endoplasmic reticulum, Golgi apparatus and lysosomes in about 50% of these cells. The formation of reaction product appeared to be substrate-specific and was sensitive to sodium fluoride in all these sites. However, only in the nuclear envelope, the rough endoplasmic reticulum and Golgi apparatus could lead salt precipitation be prevented by (a) omission of the washing procedure following the incubation step, (b) postincubation in a medium containing sodium fluoride, or (c) washing in buffer containing lead salt. It is concluded that precipitation of lead salt does not prove the presence of acid phosphatase activity in these organelles. The formation of precipitate in these sites is probably due to a local matrix effect, facilitated by the persistence of acid phosphatase activity in the lysosomes and a suboptimal trapping efficiency of phosphate ions during the washing procedure which follows in the incubation step.     

Dilated cisternae of nuclear envelope and rough endoplasmic reticulum in the proventriculus of Drosophila auraria larvae

In the cells of the middle layer of the proventriculus of Drosophila auraria larvae, the nuclear envelope and the rough endoplasmic reticulum are always found in the form of dilated cisternae. The length of these cisternae can reach 10 mu. There are indications that materials from the outer membrane of the nuclear envelope are directly transported to the Golgi complex of the examined cells.     

Light- and electron-microscopic studies on acetylcholinesterase activity in Auerbach's plexus of the developing rat colon

Summary The distal portions of rat colon from 14-, 16-, 18-, and 21-day fetuses, newborns, and adults were histochemically examined for acetylcholinesterase (AChE) activity by light and electron microscopy. The specificity of AChE activity in Auerbach's plexus was confirmed by specific and/or nonspecific cholinesterase inhibition tests. Enzyme activity was first detectable after 18 days of gestation and became stronger with age. The reaction product was demonstrated by electron microscopy in and between the plasma membranes of the nerve fibers and their terminals. Ganglion cells also showed positive activity in the plasma membrane, nuclear envelope, and rough endoplasmic reticulum. The distribution pattern of the reaction product in fetal and newborn rat colons was basically the same as in adult rat colon. Therefore, the localization of AChE activity is considered to be a good marker for identifying premature ganglion cells in Auerbach's plexus, and the degree of AChE staining is a good indication of the degree of maturation of the plexus.     

Differentiation and Ultrastructure of the Protophloem Sieve Elements of Minor Veins in the Maize Leaves: Changes in the Protoplast

Protophloem sieve element differentiation in the minor veins of the maize ( Zea mays L. ) leaves was first evidenced as an increase of the wall thickness, which began in the comers of the cell and then extended to other parts of the wall, and the appearance of long rough endoplasmic reticulum cistemae distributed throughout the cytoplasm, and then the presence of characteristic crystalloid inclusions within the plastids. As differentiation progressed, long cisternae of rough endoplasmic reticulum appeared to transform into shorter forms and eventually aggregated into small stacks, losing their ribosomes during the process. The nuclei degenerated, although frequently persisted until very late in differentiation the stages of maturation, as darkly stained amorphous aggregates surrounded by double nuclear envelope or only inner membrane of nuclear envelope. Subsequently, the nuclear envelope collapsed and became discontinuous. At the beginning of nuclear degeneration the perinuclear spaces were partly dilated and sometimes the outer nuclear envelope in the dilated portions then ruptured, and was accompanied by the disappearance of the cytoplasmic portion near it. During the peried of nuclear degeneration, in addition to the endoplasmic reticulum, plastids and mitochondria underwent structural modification, while components such as ribosomes, cytoplasmic ground substances, vacuoles and dictyosomes disintegrated and disappeared. At maturity, the surviving protoplasmic components, including plasmalemma, mitochondria, small stacked smooth endoplasmic reticulum and P-type plastids with crystalloids, became parietal in position. As differentiation of adjacent metaphloem sieve elements proceeded, the protoplasmic components of the mature protophloem sieve elements progresively degenerated and finally obliterated.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.     

Different concentrations of thyroid peroxidase and thyroglobulin in the nuclear envelope and the endoplasmic reticulum throughout the cytoplasm.

Thyroid peroxidase (TPO) and thyroglobulin (TG) represent two major glycoproteins of thyroid follicular cells performing biological functions such as iodination, transcytosis of thyroglobulin, and formation of thyroid hormones. They are involved in thyroid autoimmunity and thyroid inborn metabolic disorders. Studying these processes at a molecular level includes the determination of their precise intracellular distribution. An evaluation of the relative concentrations of TG and TPO in different subcellular compartments was carried out in stimulated human follicular cells using thin-frozen sections and the immunogold technique. It is documented that TG is transported from the endoplasmic reticulum and the Golgi apparatus to the follicular lumen by transport vesicles; most of it being present in the expanded endoplasmic reticulum throughout the cytoplasm. On the other hand, gold particles indicating TPO are adjacent to the membranes of the exocytotic pathway. They do not label the basolateral membrane but show the strongest density in the nuclear envelope and the apical membrane. The labeling density of TPO is about four times higher in the nuclear envelope than in the endoplasmic reticulum throughout the cytoplasm. In contrast, TG is concentrated three times higher in the rough endoplasmic reticulum throughout the cytoplasm than in the nuclear cisternae. Our results give the first quantitative evidence that TPO and TG are concentrated in different subcompartments of the endoplasmic reticulum. Because previous studies demonstrated the nuclear envelope as the site where the synthesis of endogenous peroxidase (Br?kelmann, J., D. W. Fawcett, Biol. Reprod. 1, 59-71 (1969)) begins, we suggest that synthesis of these functionally related proteins happens in specialized parts of the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholinesterase activity in the myotome of the early chick embryo

Summary The myotome of early chick embryos was investigated histochemically by means of the acetylcholinesterase (AChE) reaction.Light-microscopically, at the cervical level, the myotome was first recognized and AChE activity demonstrated at stage 13 (2 day-old embryo). Subsequently, the myotome elongated ventro-laterally along the inner surface of the dermomyotome and reached the ventro-lateral end of the dermomyotome at stage 17 to 18 (3 day-old embryo). AChE activity in the myotome showed subsequent increase in intensity during the course of development. The myotome consisted mainly of AChE-positive cells displaying enzymatic activity along the nuclear membrane and within the cytoplasm. In contrast, almost all cells of the dermomyotome and the interstitial cells were AChE-negative.Electron-microscopically, the myotome cells of the 2 day-old embryo and the cells in the dorso-medial portion of the myotome of the 3 day-old embryo were morphologically undifferentiated; AChE activity was detected in the nuclear envelope and in single short profiles of the endoplasmic reticulum (ER). On the other hand, in the 3 day-old embryo the cells in the ventro-lateral portion of the myotome showed AChE activity in the nuclear envelope, numerous profiles of the ER and some Golgi complexes. These AChE-positive cells were regarded as developing myogenic cells based on their morphological characteristics.The present findings indicate (i) that the appearance of AChE activity in the cytoplasm is the first sign of the differentiation of myogenic cells, and (ii) that in these myogenic cells the increase in AChE activity is based on the development of the ER.     

Acetylcholinesterase activity and type C synapses in the hypoglossal, facial and spinal-cord motor nuclei of rats. An electron-microscope study

Using the electron-microscope technique of Lewis and Shute, we studied the localization of the acetylcholinesterase (AChE) activity in the hypoglossal, facial and spinal-cord motor nuclei of rats. The technique used selectively detects synapses with subsynaptic cisterns (type C synapses) as well as heavy deposits of reaction products in the rough endoplasmic reticulum, in fragments of the nuclear envelope, in some Golgi zones and on parts of the pericaryal plasma membrane, the axolemma and the dendritic membrane. In C synapses, AChE activity was located in the synaptic cleft and on the membrane of presynaptic boutons. Some C synapses exhibited distinct synaptic specialization in the form of multiple 'active zones'. These zones were characterized by dense presynaptic projections, short dilations of the synaptic cleft, and postsynaptic densities localized between the postsynaptic membrane and the outer membrane of the subsynaptic cistern. Within the postsynaptic densities, rows of rod- or channel-like structures were observed. The subsynaptic cisterns were continuous with the positive rough endoplasmic reticulum. The results are discussed in terms of the possible role of C synapses in the regulation of AChE synthesis in postsynaptic cholinergic neurons and/or in the regulation of AChE release into the extracellular space as well as in the establishment of new synaptic contacts.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p involves the nuclear envelope

The reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p was examined. Using a Pex3-green fluorescent protein (Pex3-GFP) fusion protein, expressed under the control of an inducible promoter, it was observed that, initially on induction of Pex3-GFP synthesis, GFP fluorescence was localized to the endoplasmic reticulum and the nuclear envelope. Subsequently, a single organelle developed per cell that increased in size and multiplied by division. At these stages, GFP fluorescence was confined to peroxisomes. Fractionation experiments on homogenates of pex3 cells, in which the endoplasmic reticulum and nuclear envelope were marked with GFP, identified a small amount of GFP in peroxisomes present in the initial stage of peroxisome reassembly. Our data suggest a crucial role for the endoplasmic reticulum/nuclear envelope in peroxisome reintroduction on complementation of pex3 cells by the PEX3 gene.     

Acetylcholinesterase and pseudocholinesterases in developing chick adrenal

The distribution of acetylcholinesterase (AChE) and pseudocholinesterases (PsChE) in chick adrenal gland during the first phases of organogenesis was studied. Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates for the two enzymes, respectively, whereas BW284c51 (1,5 bis (4-allyldimethylammonium-phenyl)pentan-3-one-dibromide) and ISO-OMPA (tetraisopropylpyrophosphoramide) were used as respective inhibitors of AchE and PsChE. AchE was present on the plasma membrane, in the perinuclear cisterna and in some cisternae of the rough endoplasmic reticulum of both interrenal and chromaffin cells; moreover enzymatic activity was found in the same sites of ganglion cells and mesenchymatic undifferentiated cells, i.e. on the inside and in the proximity of the glandular anlage. PsChE activity was localized in the perinuclear space and in the rough endoplasmic reticulum of all types of cells in the anlage. It is suggested that these enzymatic activities may be implicated in morphogenetic mechanisms.     

Peroxidase-positive endothelial cells in sinusoids of the mouse liver

The cytochemical localization of endogenous peroxidase activity in sinus lining cells of mouse liver has been investigated. Kupffer cells, as identified by their exclusive ability to phagocytize large (0.8 micron) latex particles, exhibited strong peroxidase activity in nuclear envelope and endoplasmic reticulum. In addition, weak to moderate peroxidase activity was found in 57% of all endothelial cells. The enzyme in endothelial cells was also localized in nuclear envelope and endoplasmic reticulum, with a negative reaction in the Golgi apparatus. These observations indicate that peroxidase staining, as a marker for identification of Kupffer cells in mouse liver, is only of limited value and should be used in conjunction with other methods (e.g., latex phagocytosis).     

THE APPEARANCE OF ACETYLCHOLINESTERASE IN THE DORSAL ROOT NEUROBLAST OF THE RABBIT EMBRYO : A Study by Electron Microscope Cytochemistry and Microgasometric Analysis with the Magnetic Diver

In the nine day old embryo, acetylcholinesterase (AChE) is found in the reticulum, i.e. the nuclear envelope, endoplasmic reticulum, and Golgi complex, of a few cells in the neural crest. When the neurite first enters the neural tube, reticulum-bound enzyme is present also in the varicosity of the growth cone of the bipolar neuroblast. At later stages, AChE in the neuroblast has a dual distribution; in addition to the reticulum, activity also appears at the axolemmal surface. The axolemmal activity is found initially on the distal portions of axons in the posterior fasciculus and then progressively appears along the nerve roots in a distal to proximal direction. Very little reticulum-bound enzyme is present within the axon proper. After the 13th day the levels of AChE activity in the posterior fasciculus greatly exceed those in the dorsal root or in the ganglion. Enzymatic activity in the dorsal root equals or exceeds that in the posterior fasciculus by day 16, and both areas are considerably more active than the ganglion.