Compartmentalization of intracellular membrane glycocomponents is revealed by fracture-label

We used thin-section fracture-label to determine the distribution of wheat-germ agglutinin binding sites in intracellular membranes of secretory and nonsecretory rat tissues as well as in human leukocytes. In all cases, analysis of the distribution of wheat germ agglutinin led to the definition of two endomembrane compartments: one, characterized by absence of the label, includes the membranes of mitochondria and peroxisomes as well as those of the endoplasmic reticulum and nuclear envelope; the other, strongly labeled, comprises the membrane of lysosomes, phagocytic vacuoles, and secretory granules, as well as the plasma membrane. The Golgi apparatus was weakly labeled in all studied tissues.     

Three-dimensional architecture of the tubular endocytic apparatus and paramembranous networks of the endoplasmic reticulum in the rat visceral yolk-sac endoderm

The three-dimensional architecture of the tubular endocytic apparatus and the endoplasmic reticulum in the rat yolk-sac endoderm was investigated after loading with horseradish peroxidase-conjugated concanavalin A by intrauterine administration. After 30 min, small vesicles (50–150 nm in diameter), small tubules (80–100 nm in diameter) and large vacuoles (0.2–1.0 m in diameter) in the apical cytoplasm were labeled with the tracer, but lysosomes (1.0–3.5 m in diameter) in the supranuclear cytoplasm were not labeled until 60 min after loading. Stereo-viewing of the labeled small tubules in thick sections revealed that they were not isolated structures but formed three-dimensional anastomosing networks, which were also confirmed by scanning electron microscopy after maceration with diluted osmium tetroxide. Their earlier labeling with the endocytic tracer, localization in the apical cytoplasm and three-dimensional network formation indicated that the labeled small tubules represented tubular endosomes (tubular endocytic apparatus). These well-developed membranous networks provided by the tubular endosomes are suggested to facilitate the receptor-mediated endocytosis and transcytosis of the maternal immunoglobulin in the rat yolk-sac endoderm. Scanning electron microscopy further revealed lace-like networks of the smooth endoplasmic reticulum near the lateral plasma membrane. Their possible involvement in transport of small molecules or electrolytes is discussed.     

Evidence for glycoprotein components of the hepatocellular bile acid transporter

The hepatocellular transporter, responsible for the uptake of bile acids and some foreign substances, can be shown to contain carbohydrate moieties. The hepatocellular uptake of cholate and phallotoxin is immediately inhibited by addition of wheat-germ agglutinin. Concanavalin A and lentil lectin reduce the uptake in a time-dependent manner. Apparently sialic acids or N-acetylglucosamine residues are involved in the translocation process. Polypeptides (Mr 50,000, 54,000) of the above transport system, identified by affinity labeling with [3H]isothiocyanatobenzamido cholate and [3H2]diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid, are heterogenously glycosylated. Binding of 80-90% of the 54, 50 kDa polypeptides to all immobilized lectins tested suggests that both high-mannose and complex type oligosaccharides with fucose and terminal sialic acid residues occur as carbohydrate chains. A 67 kDa labeled polypeptide is not glycosylated. Pilot experiments for purification of the above glycosylated membrane proteins on concanavalin A, lentil lectin and wheat-germ lectin columns are described. However, lectin affinity chromatography is not suitable as a one-step purification procedure for the labeled polypeptides.     

Morphological changes of the terminations of afferent pathways in a neurodegenerative lesion induced by kainic acid

30 days after kainic acid injection into the rat ventrobasal thalamus, lemniscal afferents were labeled using wheat-germ agglutinin conjugated to HRP. They appeared considerably swollen in the area where neuronal post-synaptic targets had been eliminated. Electron microscopic analysis of the lesioned tissue revealed the presence of large profiles containing numerous organelles, particularly smooth endoplasmic reticulum, and giving rise to thin excrescences filled with neurofilaments. Since these morphological features are typical of regenerating "growth cones", we conclude that afferent terminals deprived of their post-synaptic targets undergo morphological changes preparing them for new synapses.     

Ultrastructural immunocytochemistry and lectin histochemistry of the subcommissural organ in the snake Natrix maura with particular emphasis on its vascular and leptomeningeal projections

The ependymal cells of the subcommissural organ (SCO) of the snake Natrix maura display long basal processes which terminate either on blood vessels or on the leptomeninges. The cell body and the basal processes contain a secretory material detectable immunocytochemically at the light-microscopic level using an antibody raised against bovine Reissner's fiber. The present investigation deals with the ultrastructural location in these cells of the (i) immunoreactive material; (ii) concanavalin A (Con A)- and wheat-germ agglutinin (WGA)-binding sites. In the subnuclear region the immunoreactive material was located within dilated cisternae of the rough endoplasmic reticulum and had affinity for Con A but not for WGA. In the supranuclear region the secretory material was exclusively located within numerous granules. Since all these granules showed affinity for WGA, they can be regarded as "post-Golgi" elements. Thus, at variance with the situation in the mammalian SCO, in the ophidian SCO most of the secretion is stored in secretory granules rather than in dilated cisternae of the rough endoplasmic reticulum. In the perivascular and leptomeningeal endings the immunoreactive material was located within granules which, because of their affinity for WGA, should also be regarded as true secretory granules derived from the Golgi apparatus. It is concluded that these granules are transported along the basal processes and accumulated in the perivascular and leptomeningeal endfeet. This observation favours the view of a local release of the content of these granules, since there is no evidence for a reverse transport of these granules all the way back from the distal termination to the apical pole, to be finally released into the ventricle.     

Glycosylated polypeptides of soybean root endomembranes

Summary Endoplasmic reticulum, Golgi apparatus, plasma membrane and mitochondria vesicles were isolated from the roots of four-day-old dark-grown soybean [Glycine max (L.) Merr. cv. Wells II] seedlings and characterized by marker enzyme analyses. Glycoproteins of enriched membrane fractions were identified by concanavalin A (con A)-peroxidase staining of polypeptides separated by two-dimensional IEF-SDS-PAGE and transferred to nitrocellulose.Con A bound to many polypeptides in each endomembrane-enriched fraction with several glycopolypeptides common to all fractions. The mitochondria-enriched fraction possessed few glycopolypeptides and those appeared to be highly glycosylated contaminants of endomembrane origin. Comparison of the endomembrane con A-binding patterns revealed changes in relative stain intensity, molecular weight and isoelectric point of several membrane glycopolypeptides suggestive of processing reactions of the endomembrane complex.Abbreviations con A concanavalin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum     

A rapid isolation procedure of plasma membranes from human neutrophils using self-generating Percoll gradients. Importance of pH in avoiding contamination by intracellular membranes

In this study we report an overall procedure for the isolation of both human polymorphonuclear neutrophils and their plasma membrane, by means of self-generating Percoll gradients. After efficient purification (40% yield), neutrophils were lysed by nitrogen cavitation and cellular structures quickly isolated in a one-step procedure. Plasma membrane recovery was monitored by [3H]concanavalin A and 5'-nucleotidase (EC 3.1.3.5) activity. We showed the latter activity is indeed present in human neutrophils. The procedure resulted in a good yield of plasma membrane, since 45% and 55% of total 5'-nucleotidase and [3H]concanavalin A activity, respectively, were recovered within two gradient fractions. Depending on the final pH of the Percoll gradient medium, endoplasmic reticulum markers contaminated either the plasma membrane or the granule fractions. At pH 9.05, NADH-ferricyanide reductase activity clearly separated from plasma membrane markers and displayed the same profile as CDPcholine:diacylglycerolcholine phosphotransferase (EC 2.7.8.2), a typical enzyme of endoplasmic reticulum. These results emphasize the need for strict monitoring of the pH of the gradient medium in subcellular fractionation of neutrophils.     

Concanavalin A binds to the endoplasmic reticulum and the starch grain surface of root statocytes

Using Concanavalin A (Con A) labeled with fluorescein isothiocyanate, we studied the intracellular localization of receptor molecules in the calyptra of 24-h dark-grown cress roots. Fixation in glutaraldehyde gave positive binding of the distal complex of the endoplasmic reticulum and the nucelus in the statocytes. In contrast, fixation in formaldehyde did not preserve the membrane-associated receptors, but revealed Con A affinity of the starch grain surface within the amyloplasts. Treatment of glutaraldehydefixed sections with non-ionic detergents led to partial solubilization of membrane components: the starch grain surface turned positive, though the positive binding of Con A to the endoplasmic reticulum and the nucleus remained unaffected. We therefore conclude that the Con A receptor in the membrane is a glycoprotein tightly inserted in other components of the compartment.Abbreviations Con A Concanavalin A - ER endoplasmic reticulum - FITC fluorescein isothiocyanate - NP 40 nonidet P40     

Ultrastructural immunocytochemistry and lectin histochemistry of the subcommissural organ in the snake Natrix maura with particular emphasis on its vascular and leptomeningeal projections

Summary The ependymal cells of the subcommissural organ (SCO) of the snake Natrix maura display long basal processes which terminate either on blood vessels or on the leptomeninges. The cell body and the basal processes contain a secretory material detectable immunocytochemically at the light-microscopic level using an antibody raised against bovine Reissner's fiber. The present investigation deals with the ultrastructural location in these cells of the (i) immunoreactive material; (ii) concanavalin A (Con A)-and wheat-germ agglutinin (WGA)-binding sites. In the subnuclear region the immunoreactive material was located within dilated cisternae of the rough endoplasmic reticulum and had affinity for Con A but not for WGA. In the supranuclear region the secretory material was exclusively located within numerous granules. Since all these granules showed affinity for WGA, they can be regarded as post-Golgi elements. Thus, at variance with the situation in the mammalian SCO, in the ophidian SCO most of the secretion is stored in secretory granules rather than in dilated cisternae of the rough endoplasmic reticulum. In the perivascular and leptomeningeal endings the immunoreactive material was located within granules which, because of their affinity for WGA, should also be regarded as true secretory granules derived from the Golgi apparatus. It is concluded that these granules are transported along the basal processes and accumulated in the perivascular and leptomeningeal endfeet. This observation favours the view of a local release of the content of these granules, since there is no evidence for a reverse transport of these granules all the way back from the distal termination to the apical pole, to be finally released into the ventricle.     

Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and golgi apparatus with [3H]leucine

Summary Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [³H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.     

Chitosomes lack concanavalin-A-binding sites

Whether intact or dissociated with digitonin, chitosomes isolated from the fungusMucor rouxii lack the ability to bind concanavalin A. The absence of external or internal concanavalin A-binding sites distinguishes the chitosome membrane no only from plasma membrane but also from membranes of other organelles (endoplasmic reticulum, mitochondrion, vacuole). This differential binding ability was used to partially separate chitosomal chitin synthetase from major membranes in a crude cell-free extract ofM. rouxii.     

Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule- associated tubular organelles.     

An enzyme-linked immunoassay for the detection of antibodies to endoplasmic reticulum

A simple, sensitive solid-phase assay for the detection of antibodies to endoplasmic reticulum is described. The assay is dependent upon the amount of antigen bound to the solid support and upon the amount of antibody bound to the support via the relevant antigen. The assay can be used to measure both polyclonal and monoclonal antibody to endoplasmic reticulum. It has been used to isolate several monoclonal antibodies which can recognize and precipitate specific proteins of the endoplasmic reticulum. In addition, it has been used to probe the membrane orientation of endoplasmic reticulum antigens.     

Isolation and characterization of plasma membranes from krebs II ascite cells using Percoll gradient

1. Plasma membranes were isolated from Krebs II ascite cells grown in the mouse. Cells were disrupted by nitrogen cavitation in an isotonic alkaline buffer containing magnesium and ATP. Isolation was performed in an alkaline-buffered self-generating gradient of Percoll with an angular rotor. At each step of the preparation, the pH appeared as the critical aspect of our procedure. 2. External membrane markers were concanavalin A and 5'-nucleotidase (EC 3.1.3.5). They reached a relative specific activity of 10, whereas this value was only of 0.7 for the endoplasmic reticulum marker, NADH dehydrogenase (EC 1.6.99.3). 3. Plasma membrane from 4 ml packed cells were isolated within 1 h after homogenization with good yield: 50% and 67% of total [3H]concanavalin A and 5'-nucleotidase, respectively, were recovered in the two plasma membrane fractions. 4. Electron microscopy examination showed the presence of vesicles of different sizes devoid of other structural contaminants. 5. Using the specific binding of concanavalin A to the external cell membrane, it was calculated that about 50% of the total cell phospholipid and 10% protein are located in the plasma membrane. Their sphingomyelin content is much higher than in the whole cell, in contrast to phosphatidylinositol, known as a more specific endoplasmic reticulum phospholipid.     

Receptor-mediated gonadotropin action in the ovary. Inhibitory actions of concanavalin A and wheat-germ agglutinin on gonadotropin-stimulated cyclic AMP and progesterone responses in ovarian cells.

Pretreatment of ovarian cells with concanavalin A and wheat-germ agglutinin blocked the gonadotropin-induced cyclic AMP and progesterone responses and this effect was time- and concentration-dependent. Basal production of either cyclic AMP or progesterone, however, was not affected by treatment of cells with lectin. The effect of concanavalin A on gonadotropin-mediated cyclic AMP and progesterone responses was blocked by alpha-methyl D-mannoside and alpha-methyl d-glucoside. Similarly the inhibitory effect of wheat-germ agglutinin was reversed by N-acetyl-D-glucosamine. Pretreatment of ovarian cells with concanavalin A or wheat-germ agglutinin had no effect on protein synthesis in the ovary as monitored by [3H]proline incorporation studies. Concanavalin A and wheat-germ agglutinin did not affect steroid production in response to dibutyryl cyclic AMP and 8-bromo cyclic AMP, indicating that the inhibitory action of lectin was occurring at a step before cyclic AMP formation. Lectins specific for L-fucose, D-galactose and N-acetyl-D-galactosamine, gorse seed agglutinin, peanut agglutinin and Dolichos biflorus agglutinin respectively, did not interfere with gonadotropin-induced cyclic AMP and progesterone responses. The present studies suggest that gonadotropin receptors may be glycoprotein in nature or closely associated with glycoprotein structures with the carbohydrate chain containing N-acetyl-D-glucosamine, mannose and possibly N-acetylneuraminic acid.     

Localization of the ethylene receptor ETR1 to the endoplasmic reticulum of Arabidopsis

The ethylene receptor ETR1 of Arabidopsis contains transmembrane domains responsible for ethylene binding and membrane localization. Sequence analysis does not provide information as to which membrane system of the plant cell ETR1 is localized. Examination by aqueous two-phase partitioning, sucrose density-gradient centrifugation, and immunoelectron microscopy indicates that ETR1 is predominantly localized to the endoplasmic reticulum. Localization of ETR1 showed no change following a cycloheximide chase. Ethylene binding by ETR1 did not affect localization to the endoplasmic reticulum, based upon analysis of plants treated with the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid and by examination of a mutant receptor that does not bind ethylene. Determinants within the amino-terminal half of ETR1 are sufficient for targeting to and retention at the endoplasmic reticulum. These data support a central role of the plant endoplasmic reticulum in hormone perception and signal transduction.     

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [³H]glucosamine and [³⁵S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.     

Modification of yeast plasma membrane density by concanavalin A attachment. Application to study of chitin synthetase distribution

Yeast protoplasts were coated with different amounts of concanavalin A. Upon subsequent lysis and centrifugation in isopycnic density gradients, it was found that the buoyant density of plasma membranes was progressively increased from 1.125 to 1.21, according to the amount of bound concanavalin A. Enzymes that are attached to the plasma membrane showed the same density modifications and could thus be distinguished from constituents of intracellular membranes and organelles. With this methodology, it was confirmed that about two-thirds of yeast chitin synthetase is associated with the plasma membrane. The remainder of the enzyme was found in a peak at a lower density. Vanadate-sensitive ATPase showed a similar pattern, whereas GDP-mannose dolichyl-phosphate mannosyltransferase, an enzyme attached to the endoplasmic reticulum, remained in the same position in the gradients, irrespective of the amount of concanavalin A associated with the plasma membrane. Potential applications of this technique to the determination of plasma membrane markers and to the separation of subcellular organelles are discussed.     

Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells

We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.     

Immunocytochemical localization of concanavalin A in developing jack-bean cotyledons

The lectin, concanavalin A (Con A), was localized in the cotyledon of developing jack beans (Canavalia ensiformis (L.) DC) by electron-microscope immunocytochemistry. In mature seeds, Con A was present in protein-storage vacuoles (protein bodies) of storage-parenchyma cells. Although protein bodies could be seen in other cell types, only protein bodies in storage-parenchyma cells contained Con A. During seed development, Con A was also localized on the endoplasmic reticulum and Golgi apparatus, presumably en route toward deposition within the protein bodies. The intensity of labeling of the endoplasmic reticulum was much greater during the developmental stage of protein-body filling (66% final seed weight) than in mature seeds.Abbreviations Con A concanavalin A - ER endoplasmic reticulum - IgG immunoglobulin G