scholarly journals Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells.

1996 ◽  
Vol 133 (3) ◽  
pp. 543-558 ◽  
Author(s):  
A Müsch ◽  
H Xu ◽  
D Shields ◽  
E Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mdck Cells ◽  
Current Model ◽  
Gh3 Cells ◽  
Plasma Membrane Proteins ◽  
Vesicular Release ◽  
Vesicular Stomatitis

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.

scholarly journals The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein.

1988 ◽  
Vol 107 (5) ◽  
pp. 1707-1715 ◽  
Author(s):  
J E Bergmann ◽  
P J Fusco
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
M Protein ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

Using monoclonal antibodies and indirect immunofluorescence microscopy, we investigated the distribution of the M protein in situ in vesicular stomatitis virus-(VSV) infected MDCK cells. M protein was observed free in the cytoplasm and associated with the plasma membrane. Using the ts045 mutant of VSV to uncouple the synthesis and transport of the VSV G protein we demonstrated that this distribution was not related to the presence of G protein on the cell surface. Sections of epon-embedded infected cells labeled with antibody to the M protein and processed for indirect horseradish peroxidase immunocytochemistry revealed that the M protein was associated specifically with the basolateral plasma membrane. The G and M proteins of VSV have therefore evolved features which bring them independently to the basolateral membrane of polarized epithelial cells and allow virus to bud specifically from that membrane.

scholarly journals Pseudotypes of Vesicular Stomatitis Virus with CD4 Formed by Clustering of Membrane Microdomains during Budding

2005 ◽  
Vol 79 (11) ◽  
pp. 7077-7086 ◽  
Author(s):  
Erica L. Brown ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
Vesicular Stomatitis ◽  
Membrane Components

ABSTRACT Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

scholarly journals Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.

1986 ◽  
Vol 102 (6) ◽  
pp. 2147-2157 ◽  
Author(s):  
L Puddington ◽  
C E Machamer ◽  
J K Rose
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Heavy Chain ◽  
Cytoplasmic Domain ◽  
Integral Membrane Proteins ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Hybrid Proteins ◽  
Vesicular Stomatitis ◽  
Immunoglobulin Mu

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.

Different properties of two isoforms of annexin XIII in MDCK cells

2000 ◽  
Vol 113 (14) ◽  
pp. 2607-2618 ◽  
Author(s):  
S. Lecat ◽  
P. Verkade ◽  
C. Thiele ◽  
K. Fiedler ◽  
K. Simons ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Functional Data ◽  
Mdck Cells ◽  
Amino Terminal ◽  
Influenza Virus Hemagglutinin ◽  
Vesicular Stomatitis ◽  
Amino Terminal Domain

Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.

scholarly journals Transcytosis of the G protein of vesicular stomatitis virus after implantation into the apical plasma membrane of Madin-Darby canine kidney cells. I. Involvement of endosomes and lysosomes.

1984 ◽  
Vol 99 (3) ◽  
pp. 796-782 ◽  
Author(s):  
M Pesonen ◽  
W Ansorge ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Vesicular Stomatitis ◽  
Darby Canine Kidney ◽  
Canine Kidney

The G protein of vesicular stomatitis virus, implanted into the apical plasma membrane of Madin-Darby canine kidney cells, is rapidly transcytosed to the basolateral membrane. In this and the accompanying paper (Pesonen, M., R. Bravo, and K. Simons, 1984, J. Cell Biol. 99:803-809.) we have studied the intracellular route by which the G protein traverses during transcytosis. Using Percoll density gradient centrifugation and free flow electrophoresis we could demonstrate that the G protein is endocytosed into a nonlysosomal compartment with a density of approximately 1.05 g/cm3, which has many of the characteristics of endosomes. Transcytosis to the basolateral membrane appeared to occur from this compartment. No direct evidence for the involvement of lysosomes in the transcytotic route could be obtained. No G protein was detected in the lysosomes when transcytosis of G protein was occurring. Moreover, at 21 degrees C when passage of G protein to the lysosomes was shown to be arrested, transcytosis of G protein could still be demonstrated.

Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy

1994 ◽  
Vol 52 ◽  
pp. 220-221
Author(s):  
Greg Martin ◽  
Rohit Cariappa ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Polarized Epithelial Cells

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.

Cell-free reconstitution of the transport of viral glycoproteins from the TGN to the basolateral plasma membrane of MDCK cells

1996 ◽  
Vol 109 (7) ◽  
pp. 1667-1676 ◽  
Author(s):  
A. Mayer ◽  
I.E. Ivanov ◽  
D. Gravotta ◽  
M. Adesnik ◽  
D.D. Sabatini
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Great Promise ◽  
Plasma Membrane Proteins ◽  
In Vitro System ◽  
Viral Glycoproteins ◽  
Basolateral Plasma Membrane

An in vitro system to study the transport of plasma membrane proteins from the TGN to the basolateral plasma membrane of polarized MDCK cells has been developed in which purified cell fractions are combined and transport between them is studied under controlled conditions. In this system, a donor Golgi fraction derived from VSV or influenza virus-infected MDCK cells, in which 35S-labeled viral glycoproteins were allowed to accumulate in the TGN during a low temperature block, is incubated with purified immobilized basolateral plasma membranes that have their cytoplasmic face exposed and are obtained by shearing-lysis of MDCK monolayers grown on cytodex beads. Approximately 15–30% of the labeled glycoprotein molecules are transferred from the Golgi fraction to the acceptor plasma membranes and are recovered with the sedimentable (1 g) beads. Transport is temperature, energy and cytosol dependent, and is abolished by alkylation of SH groups and inhibited by the presence of GTP-gamma-S, which implicates GTP-binding proteins and the requirement for GTP hydrolysis in one or more stages of the transport process. Endo H-resistant glycoprotein molecules that had traversed the medial region of the Golgi apparatus are preferentially transported and their luminal domains become accessible to proteases, indicating that membrane fusion with the plasma membrane takes place in the in vitro system. Mild proteolysis of the donor or acceptor membranes abolishes transport, suggesting that protein molecules exposed on the surface of these membranes are involved in the formation and consumption of transport intermediates, possibly as addressing and docking proteins, respectively. Surprisingly, both VSV-G and influenza HA were transported with equal efficiencies to the basolateral acceptor membranes. However, low concentrations of a microtubular protein fraction preferentially inhibited the transport of HA, although this effect was not abolished by microtubule depolymerizing agents. This system shows great promise for elucidating the mechanisms that effect the proper sorting of plasma membrane proteins in the TGN and their subsequent targeting to the appropriate acceptor membrane.

scholarly journals Block of vesicular stomatitis virus endocytic and exocytic pathways by 1-cinnamoyl-3,11-dihydroxymeliacarpin, a tetranortriterpenoid of natural origin

2004 ◽  
Vol 85 (2) ◽  
pp. 483-493 ◽  
Author(s):  
Andrea A. Barquero ◽  
Laura E. Alché ◽  
Celia E. Coto
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Vero Cells ◽  
Melia Azedarach ◽  
Antiviral Action ◽  
Leaf Extracts ◽  
Acridine Orange Staining ◽  
Multiplication Cycle ◽  
Vesicular Stomatitis

Previously, it has been shown that 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), a natural compound isolated from leaf extracts of Melia azedarach L., inhibits the vesicular stomatitis virus (VSV) multiplication cycle when added before or after infection. Here, we have established that the lack of VSV protein synthesis in CDM pre-treated Vero cells is ascribed to the inhibition of an initial step during virus multiplication, although indirect immunofluorescence (IFI) studies confirmed that the binding and uptake of [35S]methionine-labelled VSV was not affected by CDM pre-treatment. Instead, our findings revealed that this compound impedes the uncoating of VSV nucleocapsids in pre-treated Vero cells, since the antiviral action of CDM was partially reversed by inducing VSV direct fusion at the plasma membrane, and VSV M protein fluorescence was confined to the endosomes, even 2 h post-internalization. Furthermore, CDM induced cytoplasmic alkalinization, as shown by acridine orange staining, consistent with the inhibition of virus uncoating. Although VSV proteins are synthesized when CDM is added after infection, IFI studies revealed that G protein was absent from the surface of infected cells and co-localized with a Golgi marker. Therefore, CDM inhibits the transport of G protein to the plasma membrane. Taken together, these findings indicate that CDM exerts its antiviral action on the endocytic and exocytic pathways of VSV by pre- or post-treatment, respectively.

scholarly journals Transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles.

1980 ◽  
Vol 86 (1) ◽  
pp. 162-171 ◽  
Author(s):  
J E Rothman ◽  
H Bursztyn-Pettegrew ◽  
R E Fine
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Coated Vesicles ◽  
Transmembrane Glycoprotein ◽  
Vesicular Stomatitis ◽  
Two Stages

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Pulse-chase experiments suggest that G is transported to the cell surface in two successive waves of clathrin-coated vesicles. The oligosaccharides of G protein carried in the early wave are of the "high-mannose" (G1) form, whereas the oligosaccharides in the second, later wave are of the mature "complex" (G2) form. the early wave is therefore proposed to correspond to transport of G in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, and the succeeding wave to transport from the Golgi apparatus to the plasma membrane. The G1- and G2-containing coated vesicles appear to be structurally distinct, as judged by their differential precipitation by anticoated vesicle serum.

scholarly journals EFR3s are palmitoylated plasma membrane proteins that control responsiveness to G-protein-coupled receptors

2014 ◽  
Vol 128 (1) ◽  
pp. 118-128 ◽  
Author(s):  
N. Bojjireddy ◽  
M. L. Guzman-Hernandez ◽  
N. R. Reinhard ◽  
M. Jovic ◽  
T. Balla
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Plasma Membrane Proteins ◽  
G Protein Coupled
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