Showing papers on "Cellular compartment published in 1999"

Control of the terminal step of intracellular membrane fusion by protein phosphatase 1.

Abstract: Intracellular membrane fusion is crucial for the biogenesis and maintenance of cellular compartments, for vesicular traffic between them, and for exo- and endocytosis. Parts of the molecular machinery underlying this process have been identified, but most of these components operate in mutual recognition of the membranes. Here it is shown that protein phosphatase 1 (PP1) is essential for bilayer mixing, the last step of membrane fusion. PP1 was also identified in a complex that contained calmodulin, the second known factor implicated in the regulation of bilayer mixing. The PP1-calmodulin complex was required at multiple sites of intracellular trafficking; hence, PP1 may be a general factor controlling membrane bilayer mixing.

Protein Sorting by Directed Maturation of Golgi Compartments

Abstract: How does the Golgi stack mediate transport of cargo from the endoplasmic reticulum (ER) to the cell surface? A possibility is that cargo-containing vesicles derived from the ER form early Golgi compartments that then mature by retrieval of processing enzymes from later Golgi compartments. Maturation continues at terminal Golgi compartments by retrieval of transport components from the endocytic pathway to promote sorting of cargo to multiple cellular destinations. Hence, retrograde movement may integrate exocytic and endocytic pathways in eukaryotic cells and coordinate membrane flow and cargo transport through the Golgi stack.

Reactive oxygen species activate a Ca2+-dependent cell death pathway in the unicellular organism Trypanosoma brucei brucei.

Abstract: Here we examine a cell death process induced by reactive oxygen species (ROS) in the haemoflagellate Trypanosoma brucei brucei. Ca2+ distribution in cellular compartments was measured with stable transformants expressing aequorin targeted to the cytosol, nucleus or mitochondrion. Within 1.5 h of ROS production, mitochondrial Ca2+ transport was impaired and the Ca2+ barrier between the nuclear envelope and cytosol was disrupted. Consequently the mitochondrion did not accumulate Ca2+ efficiently in response to an extracellular stimulus, and excess Ca2+ accumulated in the nucleus. The terminal transferase deoxytidyl uridine end labelling assay revealed that, 5 h after treatment with ROS, extensive fragmentation of nuclear DNA occurred in over 90% of the cells. Permeability changes in the plasma membrane did not occur until an additional 2 h had elapsed. The intracellular Ca2+ buffer, EGTA acetoxymethyl ester, prevented DNA fragmentation and prolonged the onset of changes in cell permeability. Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase 3, caspase 1, calpain, serine protease, cysteine protease or proteasome activity. Moreover, trypanosomes expressing mouse Bcl-2 were not protected from ROS even though protection from mitochondrial dysfunction and ROS have been reported for mammalian cells. Overall, these results demonstrate that Ca2+ pathways can induce pathology in trypanosomes, although the specific proteins involved might be distinct from those in metazoans.

Intracellular Trafficking and Localization of the Pseudorabies Virus Us9 Type II Envelope Protein to Host and Viral Membranes

Abstract: The Us9 protein is a phosphorylated membrane protein present in the lipid envelope of pseudorabies virus (PRV) particles in a unique tail-anchored type II membrane topology. In this report, we demonstrate that the steady-state residence of the Us9 protein is in a cellular compartment in or near the trans-Golgi network (TGN). Through internalization assays with an enhanced green fluorescent protein epitope-tagged Us9 protein, we demonstrate that the maintenance of Us9 to the TGN region is a dynamic process involving retrieval of molecules from the cell surface. Deletion analysis of the cytoplasmic tail reveals that an acidic cluster containing putative phosphorylation sites is necessary for the recycling of Us9 from the plasma membrane. The absence of this cluster results in the relocalization of Us9 to the plasma membrane due to a defect in endocytosis. The acidic motif, however, does not contain signals needed to direct the incorporation of Us9 into viral envelopes. In this study, we also investigate the role of a dileucine endocytosis signal in the Us9 cytoplasmic tail in the recycling and retention of Us9 to the TGN region. Site-directed mutagenesis of the dileucine motif results in an increase in Us9 plasma membrane staining and a partial internalization defect.

Polyunsaturated fatty acid biosynthesis: a microsomal-peroxisomal process.

Abstract: The synthesis of 22-carbon fatty acids, with their first double bond at position 4, requires the participation of enzymes in both peroxisomes and the endoplasmic reticulum as well as the controlled movement of fatty acids between these two cellular compartments. It has been observed that there is generally an inverse relationship between rates of peroxisomal β-oxidation vs those for the microsomal esterification of fatty acids into 1-acyl- sn -glycero-3-phosphocholine. With a variety of different substrates it was found that when a fatty acid is produced in peroxisomes, with its first double bond at position 4, its preferred metabolic fate is to move to microsomes for esterification rather than to serve as a substrate for continued degradation. The required movement, and the associated reactions, in peroxisomes and microsomes is not restricted to the synthesis of 4,7,10,13,16-docosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid. When microsomes and peroxisomes were incubated with NAD, NADPH and malonyl-CoA it was found that 6,9,12-octadecatrienoic acid was metabolized to linoleate. Collectively our findings suggest that there may be considerably more recycling of fatty acids between peroxisomes and the endoplasmic reticulum than was previously recognized.

High-level expression of IκB-β in the surface epithelium of the colon: in vitro evidence for an immunomodulatory role

Abstract: The intestinal epithelium is spatially segregated into two compartments, one containing undifferentiated cells in a proliferative state and one with non-proliferative differentiated cells. Al- though this epithelium can produce many immune- modulating substances, emerging evidence suggests that the differentiated cell compartment is less immune responsive. Indeed, it is the differentiated cellular compartment that represents the interface between the highly antigenic luminal environment and the mucosal immune system. The NF-kB/rel family of transcriptional activators play a critical role in regulating the inflammatory response by activating a wide variety of immune-modulating genes. These transcription factors are maintained in an inactive state in the cytoplasmic compartment by interaction with inhibitory proteins of the IkB family. In this study we show by immunohistochem- istry that IkB-b is expressed at high levels specifi- cally in the differentiated surface epithelium of the colonic mucosa. Using a naturally occurring com- pound found in the colon of vertebrates, butyrate, we provide evidence in an intestinal cell line that alteration of IkB-b expression can modulate the transcriptional activation of the interleukin-8 (IL-8) gene by preventing the nuclear translocation of NF-kB proteins. Therefore, the expression of IkB-b in the differentiated surface epithelium of the colon may help these cells act as an immunological barrier to prevent activation of the mucosal im- mune system. J. Leukoc. Biol. 66: 1049-1056; 1999.

The cytoplasmic chaperone hsp104 is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum

Abstract: Severe heat stress causes protein denaturation in various cellular compartments. If Saccharomyces cerevisiae cells grown at 24°C are preconditioned at 37°C, proteins denatured by subsequent exposur...

Effects of chronic salt stress on the ultrastructure of Dunaliella bioculata (Chlorophyta, Volvocales): mechanisms of response and recovery

Abstract: The ultrastructural changes taking place in Dunaliella bioculata after chronic exposure to a sodium chloride-induced stress were examined. Hyperosmotic shock was induced by raising the sodium chloride concentration of the culture medium from 0·3 to 1·3 M, which affected a number of cellular organelles during the initial stages of the stress period, i.e. 24, 48 and 72 h. Changes in whole-cell volume were recorded, as well as alterations in the size of the following components: starch grains and sheath, lipid and plastoglobuli, chloroplast, pyrenoid, nucleus, mitochondria, cytoplasm, Golgi apparatus and endoplasmic reticulum. Cells were examined using transmission electron microscopy and changes to their fine structure quantified via image analysis of the electron micrographs. The image analysis program was designed to measure various geometric parameters for all the cell components within individual algal cells. Quantitative image analysis of cells subjected to a chronic salt stress revealed marked increases in the cross-sectional areas of the Golgi apparatus and the endoplasmic reticulum. The enhanced production of the Golgi apparatus within the algal cells was thought to be the direct result of a salt-stress-induced endoplasmic reticulum production within the cells. The increase in the endoplasmic reticulum was manifested as extensive networks of cortical endoplasmic reticulum. It is suggested that the endoplasmic reticulum serves both physiological and structural roles during chronic salt stress by providing the driving force behind increased synthetic/Golgi apparatus activities of the cells, and by providing a type of ‘cellular scaffolding’ to limit the degree of cell contraction in the face of long-term salt stress.

[Introduction of the lipofuscin-fluorophor A2E into the lysosomal compartment of human retinal pigment epithelial cells by coupling to LDL particles. An in vitro model of retinal pigment epithelium cell aging].

Abstract: PURPOSE Lipofuscin accumulates with age and in association with various retinal diseases. To investigate cellular effects of lipofuscin components in an in vitro RPE cell model, specific loading of the lysosomal compartment is required. Herein a major lipofuscin fluorophor was complexed to LDL and the subsequent subcellular localization of the retinoid was examined. METHODS The lipofuscin component N-retinylidene-N-retinylethanolamine (A2-E) was synthesized and coupled to LDL. Human RPE cell cultures were loaded with the A2-E/LDL complex over 4 weeks. Thereafter, RPE cells were harvested by trypsinization and disrupted by nitrogen cavitation. After ultra-centrifugation, the postnuclear supernatant was fractionated on a self-generating gradient and fractions were analyzed by measuring marker enzyme activities of various cellular compartments. RESULTS A2-Eaccumulated almost exclusively in the lysosomal compartment, as indicated by the identical peaks of the marker enzyme ss-hexosaminidase and the relative fluorescence of A2-E. Only a small amount of A2-E appeared to associate with the cell membrane, as shown by a minor peak of A2-E corresponding to the distribution of phosphodiesterase activity. The lysosomal marker enzyme was not present in the cytosolic fraction. CONCLUSIONS The feeding of A2-E/LDL complexes to cultured RPE-cells proved to be highly effective in specific loading of the lysosomal compartment, providing a suitable in vitro cell culture model for RPE aging and the investigation of A2-E-effects on lysosomal functions in RPE cells. Such a model may contribute to the understanding of the pathogenesis of degenerative diseases of the outer retina associated with excessive lipofuscin accumulation, including age-related macular degeneration, Stargardt's disease and Best's disease.

Complex formation among rat pancreatic secretory proteins under mild alkaline pH conditions.

Abstract: Previous in vitro studies have demonstrated that enzyme proteins liberated from isolated zymogen granules of the rat pancreas aggregate already at neutral or slightly basic pH and form small particles which in the acidic pH range progressively condense into dense cores of about the size of zymogen granules. To characterize the protein composition of the original particles in more detail non-denaturing agarose gel electrophoresis was employed. Five major protein complexes were identified which upon separation of individual complexes in 1-D or 2-D gel electrophoresis were shown to be composed of a distinct set of known enzymes and several unknown proteins. Complexes 1-4 quickly dissociated when enzyme activation was induced by enterokinase, but complex 5 was resistant even to this treatment. All 5 complexes revealed a distinct fine structure when eluted from the gels and studied in negative staining electron microscopy. These findings suggest that pancreatic zymogens form complexes already in the lumen of the rough endoplasmic reticulum and are transported as such to the Golgi complex where they aggregate into granule cores due to the internal acidic pH. Complex formation may thus facilitate zymogen sorting within the rough endoplasmic reticulum and may prevent premature enzyme activation within cellular compartments.

Overview of Protein Trafficking in the Secretory and Endocytic Pathways

Abstract: Proteins migrate from the site of their synthesis to intracellular, surface, or extracellular sites depending on their function. This well-illustrated overview provides an introduction to the pathways of protein trafficking. It includes descriptions of the relevant cellular compartments, modifications associated with the movement of proteins through these compartments, signals used for targeting and translocation, mechanisms for transit between compartments, and methods for sorting proteins.

Lectin-induced retardation of subcellular organelles during preparative density gradient electrophoresis: selective purification of plasma membranes

Abstract: Plasma membranes (PM) are difficult to separate by conventional means from other cellular compartments. Using a density gradient electrophoresis (DGE) apparatus (7 cm, x 2.2 cm), mammalian subcellular organelles were separated from a total postnuclear supernatant. The sialic acid-binding lectin wheat germ agglutinin (WGA) permitted 1.5-fold electrophoretic retardation of plasma membranes lagging far behind endoplasmic reticulum, endosomes, Golgi and lysosomes (in order of increasing electrophoretic mobility). Mobilities of the latter organelles were not affected by wheat germ agglutinin. The retarded plasma membrane was monitored by surface iodination, the presence of Ca(++)- and Na+/K(+)-ATPases and by the presence of clathrin-coated pits using Western immunoblotting. In the presence of WGA two clathrin-containing compartments were detected; in the absence of WGA three clathrin populations were seen in the electropherogram: clathrin-coated vesicles, clathrin-coated pits (on the PM) and clathrin-coated structures on the trans-Golgi network (TGN). Both in the presence and absence of WGA, plasma membrane domains of different electrophoretic mobilities were detected.

Wrapping the package.

Abstract: The membranes of living cells are in a state of dizzying flux. Lipid vesicles, often containing receptors and wrapped generally in protein coats, shuttle between cellular compartments at such a rate that, for example, the substance of the plasma membrane is typically turned over in less than an hour. Of the known classes of vesicles, the most extensively studied, by reason of their abundance and ease of preparation, are the clathrin-coated vesicles. These are the vehicles through which cell-surface receptors are internalized and delivered to the endosomal compartment, and they also transport lysosomal enzymes from the trans-Golgi network to the endosomes (1).