scholarly journals Specific Heterodimer Formation Is a Prerequisite for Uroplakins to Exit from the Endoplasmic Reticulum

2002 ◽  
Vol 13 (12) ◽  
pp. 4221-4230 ◽  
Author(s):  
Liyu Tu ◽  
Tung-Tien Sun ◽  
Gert Kreibich
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Posttranslational Modifications ◽  
Cell Layer ◽  
Building Blocks ◽  
Apical Plasma Membrane ◽  
Membrane Bound ◽  
Umbrella Cells ◽  
Vesicular Compartment ◽  
Lower Urinary

Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER.

Studies of the endoplasmic reticulum and plasma membrane-bound ribosomes in erythropoietic cells

1978 ◽  
Vol 31 (1) ◽  
pp. 165-178
Author(s):  
J.A. Grasso ◽  
A.L. Sullivan ◽  
S.C. Chan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Exact Role ◽  
Whole Cells ◽  
Cytoplasmic Face ◽  
Close Proximity ◽  
Haem Biosynthesis ◽  
Hypotonic Buffer ◽  
Membrane Bound ◽  
Hypotonic Lysis

Erythropoietic cells of 5 species, including man, contain endoplasmic reticulum present as individual cisternae or tubules scattered throughout the cytoplasm of all stages except mature RBCs. The endoplasmic reticulum is mainly agranular but occurs frequently as a variant of granular ER which is characterized by an asymmetrical and irregular distribution of ribosomes along one cytoplasmic face. In most cells, the endoplasmic reticulum occurs in close proximity to mitochondria or the plasma membrane, suggesting that the organelle may be involved in functions related to these structures, e.g. haem biosynthesis. Endoplasmic reticulum is more abundant in early than in late erythroid cells. Its exact role in RBC development is unclear. Since endoplasmic reticulum could account for ‘plasma membrane-bound ribosomes’ reported in lysed reticulocytes, studies were performed which ruled out this possibility and which suggested that such ribosomes were an artifact of the lysing conditions. Hypotonic lysis in less than 20 vol. of magnesium-containing buffers yielded ghosts variably contaminated by ribosomes and other structures. Lysis of reticulocytes in 20–30 vol. of magnesium-free buffer or homogenization of whole cells or crude membrane fractions in hypotonic buffer removed virtually all contaminating ribosomes from the purified membrane fraction.

scholarly journals Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1975 ◽  
Vol 152 (2) ◽  
pp. 291-302 ◽  
Author(s):  
Richard Harwood ◽  
Michael E. Grant ◽  
David S. Jackson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Subcellular Fractions ◽  
Anterior Lens Capsule ◽  
Cartilage Cells ◽  
Membrane Bound ◽  
And Cartilage

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [14C]lysine were assayed for hydroxy[14C]lysine and hydroxy[14C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[14C]lysine and glucosylgalactosylhydroxy[14C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [14C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. 14C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5′-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

scholarly journals Influenza A Virus M2 Protein Apical Targeting Is Required for Efficient Virus Replication

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Nicholas Wohlgemuth ◽  
Andrew P. Lane ◽  
Andrew Pekosz
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Apical Plasma Membrane ◽  
M2 Protein ◽  
Particle Release

ABSTRACTThe influenza A virus (IAV) M2 protein is a multifunctional protein with critical roles in virion entry, assembly, and budding. M2 is targeted to the apical plasma membrane of polarized epithelial cells, and the interaction of the viral proteins M2, M1, HA, and NA near glycolipid rafts in the apical plasma membrane is hypothesized to coordinate the assembly of infectious virus particles. To determine the role of M2 protein apical targeting in IAV replication, a panel of M2 proteins with basolateral plasma membrane (M2-Baso) or endoplasmic reticulum (M2-ER) targeting sequences was generated. MDCK II cells stably expressing M2-Baso, but not M2-ER, complemented the replication of M2-stop viruses. However, in primary human nasal epithelial cell (hNEC) cultures, viruses encoding M2-Baso and M2-ER replicated to negligible titers compared to those of wild-type virus. M2-Baso replication was negatively correlated with cell polarization. These results demonstrate that M2 apical targeting is essential for IAV replication: targeting M2 to the ER results in a strong, cell type-independent inhibition of virus replication, and targeting M2 to the basolateral membrane has greater effects in hNECs than in MDCK cells.IMPORTANCEInfluenza A virus assembly and particle release occur at the apical membrane of polarized epithelial cells. The integral membrane proteins encoded by the virus, HA, NA, and M2, are all targeted to the apical membrane and believed to recruit the other structural proteins to sites of virus assembly. By targeting M2 to the basolateral or endoplasmic reticulum membranes, influenza A virus replication was significantly reduced. Basolateral targeting of M2 reduced the infectious virus titers with minimal effects on virus particle release, while targeting to the endoplasmic reticulum resulted in reduced infectious and total virus particle release. Therefore, altering the expression and the intracellular targeting of M2 has major effects on virus replication.

scholarly journals Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors.

1986 ◽  
Vol 34 (1) ◽  
pp. 5-16 ◽  
Author(s):  
D S Papermaster ◽  
B G Schneider ◽  
D DeFoe ◽  
J C Besharse
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Photoreceptor Cells ◽  
Disk Surface ◽  
Light Sensitivity ◽  
Apical Plasma Membrane ◽  
Rod Photoreceptor ◽  
Electron Microscopic ◽  
Retinal Rod ◽  
Membrane Bound

Retinal rod photoreceptor cells absorb light at one end and establish synaptic contacts on the other. Light sensitivity is conferred by a set of membrane and cytosol proteins that are gathered at one end of the cell to form a specialized organelle, the rod outer segment (ROS). The ROS is composed of rhodopsin-laden, flattened disk-shaped membranes enveloped by the cell's plasma membrane. Rhodopsin is synthesized on elements of the rough endoplasmic reticulum and Golgi apparatus near the nucleus in the inner segment. From this synthetic site, the membrane-bound apoprotein, opsin, is released from the Golgi in the membranes of small vesicles. These vesicles are transported through the cytoplasm of the inner segment until they reach its apical plasma membrane. At that site, opsin-laden vesicles appear to fuse near the base of the connecting cilium that joins the inner and outer segments. This fusion inserts opsin into the plasma membrane of the photoreceptor. Opsin becomes incorporated into the disk membrane by a process of membrane expansion and fusion to form the flattened disks of the outer segment. Within the disks, opsin is highly mobile, and rapidly rotates and traverses the disk surface. Despite its mobility in the outer segment, quantitative electron microscopic, immunocytochemical, and autoradiographic studies of opsin distribution demonstrate that little opsin is detectable in the inner segment plasma membrane, although its bilayer is in continuity with the plasma membrane of the outer segment. The photoreceptor successfully establishes the polarized distribution of its membrane proteins by restricting the redistribution of opsin after vectorially transporting it to one end of the cell on post-Golgi vesicles.

Evidence for a Vacuolar-Type Proton ATPase in Entamoeba histolytica

1990 ◽  
Vol 45 (3-4) ◽  
pp. 229-232 ◽  
Author(s):  
Ursula Löhden-Bendinger ◽  
Tilly Bakker-Grunwald
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Entamoeba Histolytica ◽  
Specific Inhibitor ◽  
Eukaryotic Cells ◽  
Proton Atpase ◽  
Membrane Bound ◽  
Higher Eukaryotes ◽  
Type V ◽  
Pinocytic Vesicles

Abstract Entamoeba histolytica Entamoeba histolytica is a primitive eukaryote that lacks mitochondria, Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes. Similar to the latter, the amebal vesicles are acidified. We report here that bafilomycin AI, a specific inhibitor of vacuolar-type (V-) ATPases, suppressed this acidification at submicromolar concentrations; concom itantly, it inhibited pinocytosis. These results strongly suggest the presence of a V-ATPase in pinocytic vesicles of E. histolytica, and thereby support the notion that the V-ATPases in the organelles of higher eukaryotes are derived from an archaic plasma membrane-bound form.

Ultrastructure of sex pheromone gland cells in Lobesia botrana Den &Schiff. (Lepidoptera: Olethreutidae)

1977 ◽  
Vol 55 (4) ◽  
pp. 672-680 ◽  
Author(s):  
B. Lalanne-Cassou ◽  
Jean Percy ◽  
J. A. MacDonald
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Sex Pheromone ◽  
Lipid Droplets ◽  
Lobesia Botrana ◽  
Apical Plasma Membrane ◽  
Pheromone Gland ◽  
Gland Cells ◽  
Sex Pheromone Gland ◽  
Tubular Endoplasmic Reticulum

In newly emerged and 48-h-old female adults of Lobesia botrana the sex pheromone gland cells are columnar. Organelles characteristic of lipid-producing cells are very prominent. Smooth tubular endoplasmic reticulum is extensively developed but few lipid droplets are observed. Numerous microbodies are present and are located, along with the Golgi complexes, primarily in the vicinity of the nucleus. The apical plasma membrane is folded. The cuticle overlying the gland cells differs from unmodified cuticle in the number and location of epicuticular filaments. They originate near the bases of the furrows between the apical folds.

scholarly journals Temporal and spatial distribution of V-ATPase and its mRNA in the midgut of moulting Manduca sexta.

1996 ◽  
Vol 199 (5) ◽  
pp. 1019-1027
Author(s):  
D Jäger ◽  
F J Novak ◽  
W R Harvey ◽  
H Wieczorek ◽  
U Klein
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Manduca Sexta ◽  
Rough Endoplasmic Reticulum ◽  
Larval Instar ◽  
Goblet Cells ◽  
Apical Plasma Membrane ◽  
Tissue Preparation ◽  
Temporal And Spatial Distribution ◽  
Columnar Cells

The spatial and temporal distribution of the plasma membrane V-ATPase and its encoding mRNA in the midgut of Manduca sexta were investigated during the moult from the fourth to the fifth larval instar. Digoxigenin-labelled RNA probes were used for in situ hybridization of V-ATPase mRNA of both peripheral and integrated subunits; monoclonal antibodies to subunits of the peripheral sector of the purified plasma membrane V-ATPase were used for immunocytochemistry. Extensive mRNA labelling was found in both mature columnar and goblet cells of intermoult and moulting larvae. Hybridization screening in several tissues suggested that only cells with increased V-ATPase biosynthesis were labelled by our hybridization method. Mature goblet cells contain a large amount of V-ATPase in the apical plasma membrane and were therefore expected to contain V-ATPase mRNA. The intense mRNA signal found in mature columnar cells was unexpected. However, after refining the techniques of tissue preparation, immunolabelling in apical blebs of columnar cells was demonstrated. Since this immunoreactivity did not appear to be membrane-associated, it suggested a cytosolic localization of peripheral V1 subunits. The mRNA encoding subunit A of the peripheral V1 sector was distributed unevenly in columnar cells with a strong apical preference, whereas the mRNA for the proteolipid of the integral V0 sector was evenly distributed in the cytosol. This spatial pattern reflected the distribution of free ribosomes and rough endoplasmic reticulum in the cell, supporting the view that V1 subunits are synthesized at free ribosomes, whereas the V0 subunits are synthesized at the rough endoplasmic reticulum. All undifferentiated cells exhibited intense mRNA signals for V-ATPase subunits of both holoenzyme sectors from the start of proliferation and thus precursors of columnar and goblet cells could not be distinguished.

Apical plasma membrane-bound enzymes of rabbit uterine epithelium

1987 ◽  
Vol 87 (6) ◽  
pp. 517-529 ◽  
Author(s):  
I. Classen-Linke ◽  
H. -W. Denker ◽  
E. Winterhager
Keyword(s):  
Plasma Membrane ◽  
Uterine Epithelium ◽  
Apical Plasma Membrane ◽  
Membrane Bound ◽  
Membrane Bound Enzymes

scholarly journals A NET4-RabG3 couple mediate the link between actin and the tonoplast and is essential for normal actin cytoskeletal remodelling in stomatal closure to flg22

2021 ◽  
Author(s):  
Timothy J HAWKINS ◽  
Michaela Kopischke ◽  
David Mentlak ◽  
Patrick Duckney ◽  
Johan Kroon ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Guard Cell ◽  
Actin Filaments ◽  
Plant Immunity ◽  
Stomatal Closure ◽  
Actin Network ◽  
Actin Reorganization ◽  
Downstream Effectors ◽  
Membrane Bound

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. They tether the cell's plasma membrane (PM) to the actin network. Moreover, in a similar manner, they are also involved in the tethering of membrane bound organelles to the actin cytoskeleton; the endoplasmic reticulum (ER) and the ER to the PM. This raises the question as to whether NET proteins are involved in actin cytoskeletal remodelling. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function as downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure depends on a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune signalling events and proper actin cytoskeletal remodelling.

Ultrastructure of dormant basidiospores of Agaricus campestris

1988 ◽  
Vol 66 (3) ◽  
pp. 583-587 ◽  
Author(s):  
Donald G. Ruch ◽  
Mary C. North
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Short Distance ◽  
Mitochondrial Membrane ◽  
Lipid Droplets ◽  
Wall Material ◽  
Outer Mitochondrial Membrane ◽  
The Third ◽  
Membrane Bound ◽  
Agaricus Campestris

The basidiospore wall of Agaricus campestris Fr. consists of three distinct layers. The outer two layers are continuous around the spore, while the third layer originates only a short distance from the hilar appendage and quickly thickens to form the bulk of the wall material of the hilar appendage. The protoplast is surrounded by a typical plasma membrane which lacks distinct invaginations. Centrally located nonmembrane-bound lipid droplets comprise the bulk of the protoplasm. Spores are binucleate, but the two nuclei do not exhibit any distinct relationship to each other. Sausage-shaped mitochondria with only a few but well-delineated plate-like cristae are present. Scant endoplasmic reticulum occurs just beneath the plasma membrane. Ribosomes occur regularly attached to the endoplasmic reticulum and outer mitochondrial membrane, as well as being densely packed throughout the cytoplasm. The structure and possible functions of single membrane bound vacuoles and microbody-like organelles are discussed in relation to other basidiospores.

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