Showing papers on "Cellular compartment published in 1997"

Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments.

Abstract: Copper is an essential trace element in prokaryotes and eukaryotes and is strictly regulated by biological mechanisms. Menkes and Wilson diseases are human disorders that arise from disruption of the normal process of copper export from the cytosol to the extracellular environment. Recently a gene for Wilson disease (WD)(also named the ATP7B gene) was cloned. This gene encodes a copper transporter of the P-type ATPase. We prepared monoclonal and polyclonal anti-(WD protein) antibodies and characterized the full-length WD protein as well as a shorter form that is produced by alternative splicing in the human brain. We found that the WD protein is localized mainly in the Golgi apparatus, whereas the shorter form is present in the cytosol. These results suggest that the alternative WD proteins act as key regulators of copper metabolism, perhaps by performing distinct roles in the intracellular transport and export of copper.

Characterization of transport of newly assembled, T cell-stimulatory MHC class II-peptide complexes from MHC class II compartments to the cell surface.

Abstract: In human B cells MHC class II molecules acquire antigenic peptides in lysosome-related compartments called the MHC class II compartments (MIIC). How assembled complexes, capable of activating T cells, then reach the cell surface has not been fully resolved. We have used selective ablation of early and recycling endosomes to determine whether newly peptide-loaded class II molecules require functional recycling endosomes to exit to the cell surface. Cellular compartments accessed by transferrin-horseradish peroxidase conjugates were functionally inactivated by cross-linking with diaminobenzidine and hydrogen peroxide. Cells with ablated endosomal compartments were unable to recycle transferrin to the cell surface and could not deliver exogenous Ag for processing and presentation to T cells. In contrast, cells that had taken up Ag and assembled intracellular class II-peptide complexes before endosome ablation were still able to deliver class II-peptide complexes to the cell surface and stimulate T cell proliferation. This delivery was abolished in the presence of brefeldin A. These data show that the parts of the endocytic apparatus necessary for Ag delivery to the MIIC are not required for functional class II-peptide complexes to reach the cell surface. Moreover, the brefeldin A sensitivity of this final step in the class II molecule biosynthetic pathway suggests a vesicular intermediate for transport between the MIIC and the plasma membrane.

Differential phosphorylation of the beta2 integrin CD11b/CD18 in the plasma and specific granule membranes of neutrophils.

Abstract: Neutrophil aggregation is mediated by the beta2 integrin CD11b/CD18, which has limited expression on the surface membrane of resting cells but is recruited from intracellular organelles after cell activation. We have previously found that CD11b/CD18 newly translocated to the plasma membrane does not contribute to adhesion but must be modified to be functional. Because neutrophil aggregation induced by phorbol myristate acetate (PMA) is accompanied by de novo phosphorylation of the CD18 cytoplasmic tail, we sought to determine whether CD11b/CD18 phosphorylation is separately regulated in the different cellular compartments. Accordingly, [32P]-labeled CD11b/CD18 was immunoprecipitated from purified neutrophil-specific granule or plasma membrane lysates. In plasma membrane fractions, as in whole cell lysates, CD18 became phosphorylated in cells exposed to PMA but not in untreated cells or cells treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The alpha chain, CD11b, was phosphorylated under all conditions. In contrast, only marginal phosphorylation of specific granule-associated CD18 or CD11b was observed. Calyculin A, an inhibitor of serine/threonine phosphatases (pp1 > pp2a), induced strong phosphorylation of CD18 in the plasma membrane but not in the specific granules. Addition of intact specific granule membranes to the plasma membranes from PMA-treated neutrophils markedly decreased phosphorylation in both CD11b and CD18 subunits. These data suggest that the phosphorylation of CD11b/CD18, which accompanies neutrophil activation, is limited to plasma membrane-associated molecules. Phosphorylation, either constitutive or induced, is absent in the specific granule membranes. The difference may be due to a specific granule-associated phosphatase, probably distinct from ppl. Therefore adhesion-competent plasma membrane CD11b/CD18 and adhesion-incompetent specific granule CD11b/CD18 differ in their state of phosphorylation.

Differential fate of glycoproteins carrying a monoglucosylated form of truncated N-glycan in a new CHO line, MadIA214214, selected for a thermosensitive secretory defect.

Abstract: A temperature sensitive secretory line, MadIA214, was selected from mutagenized Chinese hamster ovary cells that express two heterologous export marker proteins: a secretory form of the human placental alkaline phosphatase (SeAP), and the Kd heavy chain of mouse MHC class I. SeAP secretion in MadIA214 was extremely reduced at elevated temperature (40 degrees C), while the export of functional H-2Kd molecules to the plasma membrane was only slightly affected. This mutant constitutively transferred onto newly synthesized proteins a truncated oligosaccharide core, Man5GlcNAc2, which was monoglucosylated in the protein-bound form. Nevertheless, the final oligosaccharide-structures associated to mature SeAP and H-2Kd were similar in mutant and wild-type glycoproteins. The inaccessibility in MadIA214 endoplasmic reticulum (ER) of one or more components required for oligosaccharide chain elongation is supported by the reconstitution of a correct core structure, obtained after disruption of cellular compartments, but not after cell permeabilisation or blocking ER-to-Golgi transport. The increased association of the ER-chaperone BiP with immature SeAP correlated with the thermodependent decrease in SeAP secretion. The retention of incompletely folded polypeptides in MadIA214 parallels both a marked ER-dilation and an important glycoprotein degradation documented by the formation of soluble oligomannosides with one GlcNAc residue. Our data provide the first in vivo evidence that the initial step in N-glycosylation differentially governs glycoprotein maturation, transport and degradation.

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes.

Abstract: Ca2+ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca2+-dependent enzymes. However, excessive mitochondrial Ca2+ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca2+ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca2+ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca2+ levels ([Ca2+]c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca2+ content (Ca2+ m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca2+]c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca2+ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca2+ m that was not further increased during prolonged incubations, despite a sustained [Ca2+]c elevation. By contrast, thapsigargin induced a net Ca2+ accumulation in mitochondria that continued for up to 30 min and reached Ca2+ m levels 10–20 fold over basal. Accumulation of mitochondrial Ca2+ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca2+ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca2+ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca2+ through artificial means, enabling mitochondria to function as a Ca2+ sink under these conditions. (Mol Cell Biochem 174: 173–179,1997)

Two-hybrid Cloning of a Gene Encoding TNF Receptor-Associated Protein 2, a Protein That Interacts With the Intracellular Domain of the Type 1 TNF Receptor: Identity With Subunit 2 of the 26S Protease

Abstract: A protein that binds the intracellular domain of the type 1 TNFR (TNFR-1IC) has been identified by two-hybrid cloning. The 97-kDa TNFR-associated protein, TRAP2, shows sequence identity with internal amino acid sequences from subunit 2 of the 26S protease. TRAP2 antiserum recognizes subunit 2 of the 26S protease, which is consistent with the identity of these proteins. TRAP2 antiserum interacted with the 97-kDa protein in HeLa cell lysates and cytosol, the latter observation showing that TRAP2 resides in the same cellular compartment as TNFR-1IC. A fusion of glutathione-S-transferase and TNFR-1IC (GST-TNFR-1IC) precipitated TRAP2 from a HeLa cell lysate; conversely, GST-TRAP2 precipitated TNFR-1 from such a lysate. These observations show that the proteins interact in the cellular milieu. After in vitro transcription/translation and 35S labeling, TRAP2 was precipitated from a cellfree system by GST-TNFR-1IC, showing that TNFR-1IC and TRAP2 interact directly. TRAP2 was also precipitated from the cellfree translation system by a GST fusion containing the N-terminal half of TNFR-1IC, but not by a GST fusion containing the C-terminal half of TNFR-1IC that contains a "death domain" that plays an obligatory role in signaling cytotoxicity. The ability of deletion mutants of TNFR-1IC to interact with TRAP2 was tested using the two-hybrid system. This also showed that the amino acid sequences that mediate binding reside outside of the death domain in TNFR-1IC. The demonstration that a subunit of the 26S protease binds TNFR-1 may identify a novel TNF-signaling pathway.

Characterization of a putative 82 kDa nuclear ligand for the N-acetylglucosamine-binding protein CBP70

Abstract: Since nuclear lectins were first characterized several years ago, six lectins have been isolated. Furthermore, the existence of nuclear glycoproteins containing N-linked complex-oligosaccharide chains or O-linked GlcNAc residues was evidenced. These latter are abundant in the nucleus and are well-studied so far. The presence of both glycoprotein and lectin in the cell nucleus led us to postulate that these two proteins could interact and play a role in some nuclear activities such as the modulation of transcription and/or nuclear cytoplasmic exchanges or by the disruption of protein-protein interactions. In such context, the recent data concerning the GlcNAc-binding activity of CBP70 argued this postulate. However, to study the possible role of a glycoprotein-lectin complex, it was critical to isolate the two partners. Because CBP70 was also a cytoplasmic protein, the lectin was isolated in both cytoplasmic and nuclear compartments in order to investigate the putative ligand in the two cellular compartments. The results obtained with cross-linking experiments on isolated and membranedepleted nuclei incubated with the CBP70 bearing an iodinatable, cleavable, photoreactive cross-linking agent (sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3'-dithiopropionate) and immunoprecipitation experiments with polyclonal antibodies raised against CBP70, revealed that both nuclear and cytoplasmic CBP70 have the same 82 kDa nuclear ligand which is absent in the cytoplasmic fraction. In addition, this ligand is glycosylated, containing GlcNAc residues, and, therefore, the complex between CBP70 and the 82 kDa polypeptide could be due to a glycoprotein-lectin interaction. These results raised the possibility that nuclear glycoprotein-lectin interaction could be involved in nuclear activities.

Lysozyme expression in the rat parotid gland: light and electron microscopic immunogold studies.

Abstract: Lysozyme (muramidase) is capable of direct bacteriolytic action by hydrolyzing glycosidic bonds in bacterial cell walls. Although it is broadly distributed in vertebrate tissues and secretions, the cellular and subcellular localizations of the enzyme are still not well known. The present study examines the distribution of lysozyme expression in the various cell types of LR gold-embedded rat parotid gland, applying a postembedding immunogold-silver staining technique for light microscopy. Simultaneously, a postembedding immunogold method for electron microscopy was used to determine the cellular compartments engaged in the biosynthesis and exocytosis of lysozyme. Silver-amplified immunogold staining for lysozyme demonstrated identical localization in both paraffin and semithin LR-gold sections: in the supranuclear parts of acinar and intercalated duct cells. Staining intensity varied even between adjacent cells. In the electron microscope, immunogold labeling was detected over the cell compartments associated with protein synthesis and exocytosis in acinar and intercalated duct cells. Lysozyme antigenic sites were visible over endoplasmic reticulum and throughout the Golgi apparatus, being intense over the trans-Golgi network, but even stronger in the condensing vacuoles and most prominent over secretory granules in both cell types. The findings provide the first immunocytochemical evidence of the synthesis and secretion of lysozyme in parotid acinar and intercalated duct cells.

Secretory Lysosomes and the Production of Exosomes

Abstract: Eukaryotic cells secrete proteins either by the so-called constitutive secretion involving vesicular transport and exocytosis or by the regulated secretion of storage granules upon proper stimulation.1,2 Only recently has one become aware that alternative mechanisms operate that may account for the secretion of specific membrane and cytotoxic proteins. Indeed, accumulating evidence indicates that cellular compartments displaying intralumenal membrane vesicles, collectively named multivesicular bodies (MVBs), fuse with the plasma membrane in an exocytic manner. During exocytosis, the 60 to 80 nm membrane vesicles present in the lumen of MVBs are released into the extracellular environment. The secreted membrane vesicles are called exosomes.3–6

Vesicular pH Is Sensitive to Changes in Cell Volume

Abstract: As shown previously, osmotic swelling of proximal renal tubules, vascular smooth muscle cells and of hepatocytes leads to an increase of the pH in acidic cellular compartments. This vesicular alkalinization is reflected by an increase of acridine orange fluorescence intensity and a corresponding alteration of the FITC-dextran fluorescence ratio. The present study has been performed to test whether this phenomenon can be observed in other cell types. Osmotic cell swelling (–30 mOsm) increased acridine orange fluorescence intensity in cardiac myocytes as demonstrated using fluorescence imaging. Microspectrophotometry was used to demonstrate a similar increase in acridine orange fluorescence intensity following treatment with hypotonic solution in MDCK cells, macrophages, brown fat cells, dendritic cells, pancreatic β-cells, alveolar cells and normal and ras oncogene expressing fibroblasts. Swelling of MDCK and L2 cells, induced by a block of K+ channels with Ba2+ or the elevation of extracellular K+, also led to vesicular alkalinization. The uptake of various amino acids in MDCK cells, resulting in cell swelling, similarly increased the acridine orange fluorescence intensity. Further, the acridine orange results were confirmed and quantified with FITC-dextran in brown fat cells, where treatment with hypotonic solution increased the vesicular pH by as much as 0.53 ± 0.04 (n = 4). It is concluded that osmotic swelling of a wide variety of cells increases the pH of acidic cellular compartments, which in turn is expected to modify the respective activity of lysosomal proteases and trafficking of vesicles and receptors.

Osmotic cell swelling alkalinizes acidic cellular compartments of pancreatic islet and RINm5F cells.

Abstract: To the Editor: Recent studies in liver cells have demonstrated that the pH of acidic intracellular compartments in various cell types is highly sensitive to cell volume, i.e., cell swelling leads to an alkalinization of the acidic cellular compartments (1-3). Hepatic proteolysis resides largely within acidic lysosomes and is accomplished by pH-sensitive lysosomal proteinases (4). Thus, the alkalinization of acidic intracellular compartments could couple cell volume to proteolysis, which has been shown to be inhibited by cell swelling (1). The present experiments were performed to test whether cell volume similarly influences the pH of acidic intracellular compartments in endocrine cells, such as cells from pancreatic islets. To this end, acridine orange and fluorescein isothiocyanate (F1TC)-dextran have been utilized as fluorescent indicators of pH in intracellular acidic compartments.

Upregulation of a Myristylated 74-kDa Protein by Interferon Treatment of Daudi Cells

Abstract: Labeling of unstimulated human Daudi B lymphoblastoid cells with exogenously added [3H]myristate resulted in acylation of a broad spectrum of different proteins, most of which are currently unknown. Among this array of labeled proteins, a unique 74-kDa acylated protein was induced in interferon (IFN)-treated cells. In the present study, we defined the myristylation kinetics of this protein and examined the subcellular distribution before and after activation with IFN-alpha/beta. This acylated protein was detected only at a very low level in the membrane fraction of untreated cells, and its level increased 3-4-fold by treatment with IFN. This induction occurred over a short period of time and was IFN-alpha/beta dose-dependent. No significant induction was observed with IFN-gamma. Incorporation of [3H]myristate was completely abolished by cycloheximide. The fatty acid associated with this protein was probably linked to a nascent chain through an amide linkage, as it was not released by alkaline hydroxylamine treatment and was identified as myristic acid by HPLC after its release from the polypeptide chain by acid methanolysis. In contrast to other IFN-induced proteins, whose synthesis started at 10 h and was maintained for 20 h, this protein was present in the plasma membrane for a short period of time, between 4 and 6 h after IFN-alpha/beta treatment, and was no longer present in this cellular compartment. This event appears to be transient and suggests that a degradation or a negative regulation of transcription starts from 6-7 h after continuous IFN treatment. As many other myristylated proteins are implicated in cellular regulation, it is possible that this 74-kDa protein may have a regulatory role in cell proliferation and the inhibition of viral replication.