Showing papers on "Cellular compartment published in 1984"

Identification of the acidic compartment of Plasmodium falciparum-infected human erythrocytes as the target of the antimalarial drug chloroquine.

Abstract: Chloroquine (CQ), the most widely used antimalarial drug, is an acidotropic agent (De Duve, 1983) which accumulates to high levels in malaria-infected erythrocytes. A possible site of accumulation of the drug, the parasite's food vacuole, has been implicated in the mode of action of CQ. We have defined the various compartments of Plasmodium falciparum-parasitized human erythrocytes in terms of their pH and capacity to accumulate bases. The host cell and the parasite cytosols were differentially labeled in situ with pH-sensitive fluorescein, and the parasite food vacuole was revealed by targeting fluoresceinated dextran via endocytosis. The pH of the various compartments obtained from fluorescence excitation spectra were 6.9 for the cytosol of normal and infected erythrocytes and 5.2 for the parasite food vacuole. Determination of CQ and methylamine accumulation in infected erythrocytes, in conjunction with morphometric determination of the relative sizes of the various cellular compartments, provided an independent assessment of the vacuolar pH, yielding a value of 5.0-5.2. Perturbation of the proton gradient, either by lowering extracellular pH or by alkalinization of the food vacuole with NH4Cl or monensin, resulted in a concomitant and reversible decrease in accumulation of the probe. We conclude that drug accumulation in malaria-infected erythrocytes can be fully accounted for by the steady-state proton gradients across the barriers delineating the various cellular compartments and the acidotropic properties of the drug.

Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization.

Abstract: Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer chains and the 260,000-mol-wt monomers dimerized by interchain disulfide bond formation. The other processing steps have been localized to the Golgi apparatus and later compartments (e.g., Weibel-Palade bodies). High mannose carbohydrate was converted to the complex type, leading to the appearance of a larger precursor subunit of 275,000 mol wt. The 275,000-mol-wt species was not formed if carbohydrate processing was inhibited by the ionophore monensin. From the large pool of dimers of precursor subunits, the high molecular weight multimers were built. These dimer molecules appeared to have free sulfhydryls which might have been involved in the interdimer disulfide bond formation. Simultaneously with multimerization, the precursor subunits were cleaved to the 220,000-mol-wt form. The cleavage of the pro-sequence was not likely to be an absolute requirement for von Willebrand protein multimerization or secretion, as the 275,000-mol-wt precursor subunit was present in secreted high molecular weight multimers of the protein.

Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation.

Abstract: The O-linked oligosaccharides of mucin-type glycoproteins contain N-acetyl-D-galactosamine (GalNAc) that is not found in N-linked glycoproteins. Because Helix pomatia lectin interacts with terminal GalNAc, we used this lectin, bound to particles of colloidal gold, to localize such sugar residues in subcellular compartments of intestinal goblet cells. When thin sections of low temperature Lowicryl K4M embedded duodenum or colon were incubated with Helix pomatia lectin-gold complexes, no labeling could be detected over the cisternal space of the nuclear envelope and the rough endoplasmic reticulum. A uniform labeling was observed over the first and several subsequent cis Golgi cisternae and over the last (duodenal goblet cells) or the two last (colonic goblet cells) trans Golgi cisternae as well as forming and mature mucin droplets. However, essentially no labeling was detected over several cisternae in the central (medial) region of the Golgi apparatus. The results strongly suggest that core O-glycosylation takes place in cis Golgi cisternae but not in the rough endoplasmic reticulum. The heterogenous labeling for GalNAc residues in the Golgi apparatus is taken as evidence that termination of certain O-oligosaccharide chains by GalNAc occurs in trans Golgi cisternae.

Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.

Abstract: Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.

Isolation of plasma membrane, Golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver

Abstract: Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2-3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K+-stimulated p-nitrophenyl phosphatase and (Na+K+) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.

Synthesis of prenyl lipids in cells of spinach leaf. Compartmentation of enzymes for formation of isopentenyl diphosphate.

Abstract: Purified spinach chloroplasts incorporate [1-14C]isopentenyl diphosphate into prenyl lipids in high yields. The immediate biosynthetic precursors of isopentenyl diphosphate (hydroxymethylglutaryl-CoA, mevalonate, mevalonate-5–phosphate, mevalonate-5-diphosphate), on the other hand, are not accepted as substrates and the corresponding enzymes hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase are not present in the organelles. These enzymes can only be detected in a membrane-bound form at the endoplasmic reticulum (hydroxymethylglutaryl-CoA reductase) and as soluble activities in the cytoplasm. The concept is developed that isopentenyl diphosphate is formed in the cytoplasm as a ‘central intermediate’ and is distributed then to other cellular compartments (endoplasmic reticulum, plastids, mitochondria) for further biosynthetic utilization.

Endocytosis in Entamoeba histolytica. Evidence for a unique non-acidified compartment.

Abstract: Our studies on endocytosis in Entamoeba histolytica trophozoites suggest that there are two vacuolar compartments in this organism. The first compartment consists of large vacuoles (greater than 2 microns diameter). As measured by the fluid phase markers, fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP), this compartment is a rapid equilibrium with the external milieu and is constantly exchanging (1-2 h) its contents with the external medium. The contents of these vacuoles are not acidified. This together with the absence of degradation of fluid phase markers clearly differentiates these vacuoles from lysosomes of eucaryotes. By labeling externally disposed peptides on the surface membrane of trophozoites with 125I, we could show that the surface membrane was rapidly internalized over a 2-h period and then reached a plateau. All major 125I surface proteins, with the exception of a set of peptides in the 40,000 molecular weight range, were interiorized and approximately 60% of the total radiolabel were found to be in the internal membrane fraction at any given time. The kinetics of this process were similar to those for the uptake of fluid phase markers and are best explained by cycling of the surface membrane into the vacuolar compartment(s) and then back to the cell surface. The second vacuolar compartment consisted of small vesicles (less than 2 microns diameter) with acidified contents as indicated by acridine orange uptake. The endocytic nature of these vesicles was shown by their slow (days) labeling with FITC-dextran, and spectral analysis of internalized FITC-dextran confirmed that this second compartment is acidified (pH 5.2).

Mobilization of Oil and Wax Reserves

Abstract: Publisher Summary This chapter presents the mobilization of oil and wax reserves. The conversion of lipid to carbohydrates (gluconeogenesis) is a main pathway in all oil seeds examined, ranging in its predominance from a quantitative conversion in the endosperm of castor beans to about 70% in cotyledons. Sucrose is a main (though not exclusive) product of gluconeogenesis and typically is transported to the embryo axis to support seedling growth. Other products synthesized from lipid catabolites in cotyledons are retained and used for maintenance and differentiation into photosynthetic organs. Gluconeogenesis involves four main cellular compartments: lipid bodies, glyoxysomes, mitochondria, and cytosol. Glyoxysomes, possessing β -oxidation and glyoxylate cycle enzymes, have been studied extensively and shown to metabolize fatty acids and acetyl-CoA similarly in all species (including jojoba) and storage tissues (scutellum, endosperm, cotyledons) examined. Glyoxysomal enzymes are synthesized de novo and are added post-translationally to glyoxysomes during the period of lipid degradation. These glyoxysomes persist through desiccation and seed germination and may be the recipients of enzymes synthesized on cytosolic polysomes following imbibition. In cotton, maize, rape, and mustard, lipid body lipases with pH optima between pH 5 and 7 develop activities, which can account for lipolysis, and true glyoxysomal lipases may or may not be present. In soybean, a glyoxysomal lipase appears to hydrolyze the lipid and a true lipase does not seem to be associated with the lipid bodies. The control of lipid mobilization in seeds has not been studied thoroughly. Source-sink relationships between sugars and gluconeogenesis enzymes seem to be important as high sugar concentrations tend to slow the mobilization processes.

Isolation and compositional analysis of secretion granules and their membrane subfraction from the rat parotid gland.

Abstract: A secretory granule fraction has been isolated from rat parotid by discontinuous gradient centrifugation using hyperosmotic sucrose-Ficoll solutions of low ionic strength. The secretion granule fraction comprises 25% of the total tissue α-amylase activity and is judged to be of high purity, both morphologically and by its low level of contamination by enzyme activities associated with other organelles. Secretion granules were lysed by capitalizing on their lability in KCl-containing media, and the low density granule membranes were separated from residual organelle and soluble contaminants by flotation in a sucrose gradient. Residual, poorly extractable secretory contaminants of the granule membrane subfraction were selectively removed by a saponin- (10 μg/ml) Na2SO4 (0.3m) wash, apparently with negligible disruption of granule membrane structure. Based on detailed consideration of the extent of contamination by residual mitochondria and incompletely removed secretory polypeptides, it is possible to estimate that ∼95% of the protein associated with the purified secretion granule membrane is bona fide granule membrane protein. Further analyses indicate that γ-glutamyltransferase constitutes a marker enzymatic activity shared by granule membranes and the apical domain of the plasma membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretograms of radio-iodinated granule membrane polypeptides are characterized by 20–25 radioactive bands of which 5–6 are suggested to be glycoproteins by virtue of their binding of concanavalin A. The limited polypeptide composition of the secretion granule membrane (in comparison to membranes of other cellular compartments) and the high phospholipid-protein ratio (4.4 mg/mg) may reflect the functional specialization of this storage container for secretory proteins.

Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes

Abstract: Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of Ca2+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2+ concentration, as indicated by the activation of phosphorylase, Ca2+ is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2+ homeostasis by pyridine nucleotide oxidation associated with tert-butyl hydroperoxide metabolism, prevents the ATP-dependent cellular Ca2+ accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3-hydroxybutyrate in the medium, prevents this hydroperoxide-induced release of intracellular Ca2+. Under conditions of impaired mitochondrial Ca2+ sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2+ from the mitochondria to the endoplasmic reticulum. Our results provide additional evidence for the critical involvement of the plasma membrane Ca2+-extruding system in the physiological regulation of the cytosolic free Ca2+ concentration in hepatocytes, and suggest that the mitochondria play a more important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca2+ pump is inhibited.

The intracellular pathway of vitellogenin secretion in the frog hepatocyte as revealed by protein A-gold immunocytochemistry.

Abstract: The protein A-gold immunocytochemical technique was applied to the localization of vitellogenin in the hepatocyte of the bullfrog, Rana catesbeiana, eight days after treatment with estradiol-17 beta. Specific labeling was present in cellular compartments involved in protein secretion and was shown to progress in sequence through RER, Golgi apparatus, immature secretory granules, and mature secretory granules. Labeling intensities were quantitated and the values ranged from 34.6 to 172 gold particles/micron 2. In contrast, low background labeling was observed over mitochondria, nuclei, lipid droplets, and bile canaliculi. These observations support the hypothesis that vitellogenin synthesis and secretion in the frog hepatocyte lies exclusively along the RER-Golgi-granule secretory pathway. In addition to the cellular compartments involved in protein secretion, labeling was found over the majority of the lysosomes. The intensity of lysosomal labeling was intermediate between that of RER and Golgi apparatus....

Resolution of cellular compartments involved in membrane potential changes accompanying IgE-mediated degranulation of rat basophilic leukemia cells.

Abstract: The overall membrane potential of rat basophilic leukemia cells (RBL-2H3) calculated from the transmembrane distribution of the lipophilic, tritium-labelled cation tetraphenyl-phosphonium [( 3H]TPP+) was resolved into its mitochondrial and plasma membrane potential components. Using the mitochondrial uncoupler carbonylcyanide-p-trifluormethoxyphenyl hydrazone (FCCP) which collapses the mitochondrial potential, it was shown that about one third of the overall potential resulted from the mitochondrial contribution. Degranulation of the RBL cells induced by two different IgE-cross-linking agents (specific antigen and anti-IgE antibodies), was accompanied by, and well correlated with, a decrease in the overall potential. However, evaluation of the source of these observed potential changes revealed that the FCCP-insensitive fraction of the overall potential, delta psi P, (representing the plasma membrane potential), was not affected. In contrast, the FCCP-sensitive component due to the mitochondrial potential decreased when receptor cross-linking increased. Thus, the observed decrease in the overall potential is most probably a secondary event in the sequence leading from stimulus to secretion. Indeed, exposure of the RBL cells either to a high external concentration of K+ ions or to a high amount of external TPP+, both causing depolarization, failed to trigger degranulation. It is suggested that the apparent decrease in the measured overall potential is a reflection of the mitochondrial membrane depolarization. The latter is most probably caused by mitochondrial Ca2+ uptake initiated by the increase in the intracellular concentration of Ca2+ which follows cells activation.

General and selective inhibition of pancreatic enzyme discharge using a proteinase inhibitor (FOY-305).

Abstract: The guanidino acid esters (FOY, FOY-305) represent a new class of potent proteinase inhibitors and are thought to have a beneficial effect on the course of acute pancreatitis. Because of their structure and low molecular size they might enter cells and interfere with cellular processes. To test this possibility in the case of the exocrine pancreas a series of in vivo and in vitro studies was carried out to analyse intracellular transport and discharge of pancreatic enzymes in the presence of FOY-305. The infusion of FOY-305 to conscious rats led to a transient inhibition of protein and enzyme discharge from the cannulated pancreas accompanied by lower serum enzyme levels and increased enzyme content in the pancreas. An identical inhibition of discharge of newly synthesized proteins was observed in vitro in the presence of 1 µM FOY-305. The analysis of the release of individual enzymes using separation on two-dimensional gels showed a pronounced inhibition of mainly the release of acidic proteins. FOY-305 not only interfered with discharge of serine proteinases (trypsinogen, chymotrypsinogen, proelastase) but also with procarboxypeptidases and lipase. It was concluded that FOY-305 enters the acinar cell and due to an unspecific binding to acidic proteins interferes with the intracellular transport of individual enzyme proteins during their passage through the membrane-bound cellular compartments. This charge-dependent effect is independent of the inhibitory effect on enzymatic activity of serine proteinases.

Light-induced Biosynthesis of Chlorophyll-Binding Proteins in Euglena

Abstract: Light-induced chloroplast development in Euglena requires a massive de novo synthesis of membrane specific components. This differentiation process involves a highly coordinate participation of the different cellular compartments, nucleocytoplasm, chloroplast and mitochondria. Little is known about the regulation mechanism which controls the expression of genes in the two informational systems, the nucleus and the chloroplast. On a structural and functional levels, the most important proteins are the chlorophyll-binding proteins. There are evidences for the nuclear origin of the LHCP apoproteins and chloroplastic origin of the proteins involved in the reaction center PS I and PS II. It is of special interest to determine the sequence of events occurring in the early stage of chloroplast development. To understand the biogenetic regulation it will be essential to examine the membrane polypeptide composition and the translationable mRNA species of the cells after the onset of illumination. In this paper, we report on the use of specific antibodies raised against the chlorophyll-binding proteins in order to establish a precise kinetic of their biosynthesis and insertion into the thylakoids.

The pH of mitochondria of fibroblasts from a hyperornithinaemia, hyperammonaemia, homocitrullinuria-syndrome patient

Abstract: The intracellular pH of control fibroblasts and of fibroblasts of a HHH-syndrome patient have been determined. Values of 6.94±0.15 and 7.05±0.14 for control and patient fibroblasts, respectively, were found. By means of analyses of malate in the cytosolic and particulate fractions of the fibroblasts the differences in pH between these two cellular compartments were estimated to be 1.34±0.12 and 1.38±0.18 for control and patient, respectively. Neither difference was statistically significant. The decrease in the rate of ornithine uptake by mitochondria of the patient's fibroblast is therefore not due to an increased intramitochondrial pH.

Localization of filipin-sterol complexes in cell membranes of eosinophils

Abstract: The polyene antibiotic filipin was used as a probe for the detection of cholesterol in the cell membranes of eosinophils isolated from the peritoneal exudate of rats. A homogenous distribution of filipin-sterol complexes was observed, both in thin sections and freeze-fracture replicas throughout the whole plasma membrane but not in the membrane of pynocytic vesicles, Golgi complex, endoplasmic reticulum, mitochondria and the nucleus. Few complexes were seen in freeze-fracture replicas showing the membrane of the specific granules. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.

Autoantibody activity against evolutionary conserved juxtanuclearly arranged antigen in mice with tumors induced by a progressor Moloney sarcoma virus transformant.

Abstract: Adult STU mice with tumors induced by a progressor Moloney sarcoma virus transformant developed serum antibodies against juxtanuclearly arranged structures (JNS) 14 days after tumor cell transplantation the antibody activity against JNS could be observed During repeated tumor transfers anti-JNS development always accompanied tumor proliferation In indirect immunofluorescence microscopy JNS were recognized by these antibodies not only in cultured cells of murine origin (normal as well as transformed cells), but also in cells of other mammalian species (rat, bat, hamster, dog, goat, cattle, horse) and even in cells of members of two further vertebrate classes: avian embryo fibroblasts and a fish-derived cell line In the various cell lines tested, the morphology of the JNS differed, but they were always arranged close to the nucleus The distinct perinuclear and juxtanuclear position is indicative for the Golgi region; the staining pattern after osmium impregnation and immunoelectron microscopy supported the localization of the recognized structures in this cellular compartment Drugs like monensin or colcemid, which are known to influence Golgi morphology and function, altered the staining pattern of the antibodies drastically The widespread occurrence of this antigenic determinant(s) in cultured cells from different species suggests that this Golgi component is highly conserved in evolution According to immunoprecipitation studies four proteins with apparent molecular weights of 250,000, 96,000, 53,000, and 38,000 daltons were recognized by these antisera It remains to be determined, if these molecules are involved in the described serological reaction

Studies of membrane glycoproteins in the rough endoplasmic reticulum.

Abstract: Studies on membrane glycoproteins which have the RER as their final destination are comparatively rare; thus it has not been possible to examine the initial stages of protein compartmentalization. The roles of the RER and the Golgi apparatus in membrane protein sorting need to be elucidated. It has been suggested, for example, that RER proteins would be returned to the endoplasmic reticulum while the other species would collect in the trans aspect of the Golgi apparatus before being shuttled to their ultimate sites (Rothman, 1981). Viral model systems for exploring RER membrane proteins have not yet been produced and such information as exists concerns ribophorin, a glycoprotein which is quite difficult to work with because of its low abundance and slow rate of turnover. Analogous to the plasma membrane studies, a RER-maturing virus could serve as a useful probe in the elucidation of the behaviour of membrane proteins. Because many lytic viruses completely shut off host RNA and protein synthesis, the addition of radioactive amino acids or sugars leads to the labelling of only virus-specified molecules, which are present in high abundance in the infected cells. The intracellular dynamics of molecules of interest can then be readily followed. This paper discusses various glycoproteins which are located in the RER and their carbohydrate processing. We also describe a new viral model, rotavirus, for studying the behaviour of membrane proteins.

Physiological regulations and compartments in cells

Abstract: Quantitative short-term regulation of a chemical component (glucose) in a single cell (hepatocyte of rat) is represented by an "equilibration diagram," which relates concentration of the regulated component or substrate (glucose) to loss and to gain of the same substrate (glucose). Loss and gain are separately active through specific enzymatic processes. Evidence indicates that some of these processes require the presence of subcellular compartments for their participation.