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 Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach

1983 ◽  
Vol 210 (1) ◽  
pp. 37-47 ◽  
Author(s):  
B T Pan ◽  
R Blostein ◽  
R M Johnstone
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Plasma Membranes ◽  
Plasma Membrane Proteins ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Cell Plasma ◽  
A Subunit

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.

275 EVIDENCE FOR α5 AND αV INTEGRINS ON SPERM PLASMA MEMBRANES AND THEIR ROLE IN BOVINE FERTILIZATION

2010 ◽  
Vol 22 (1) ◽  
pp. 294
Author(s):  
R. F. Gonçalves ◽  
R. P. Bertolla ◽  
V. H. Barnabe
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Weighted Least Squares ◽  
Gradient Centrifugation ◽  
Plasma Membrane Proteins ◽  
Western Blots ◽  
Integrin Αv ◽  
Sds Page

Sperm-egg interaction is a complex molecular process leading to gamete fusion mediated by a series of molecular interactions. Some integrin subunits, which are adhesion molecules, are expressed on human and mouse sperm, but major questions about the roles of integrins in sperm-oocyte fusion remain unsolved. This study was conducted to determine the presence of α5 and αV integrins on cattle (Bos indicus and Bos taurus) sperm, and whether fertilization might be affected by treating sperm with antibodies to these integrin subunits. To determine if integrin subunits were present on sperm, sperm plasma membrane proteins were subjected to 1-dimensional SDS-PAGE and Western blot analysis. Frozen-thawed sperm, donated by ABS Pecplan, were centrifuged at 700 × g for 10 min, washed twice with warm PBS (Nutricell®, Campinas, Sao Paulo, Brazil), and resuspended in Jones buffer (0.4% deoxyclolic acid, 8.9 M sucrose, 0.1 M Tris, pH 8.5) for 60 min at 4°C to solubilize sperm plasma membranes. Plasma membrane proteins were then separated by SDS-PAGE and transferred to nitrocellulose. The resulting blots were probed with αV integrin antibody (Calbiochem®, San Diego, CA, USA) or α5 integrin antibody (Calbiochem) and developed using ECL. Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h in fertilization medium (FM; 1), FM with anti-integrin αV IgG (2), and FM with anti-integrin a5 IgG (3). In vitro-matured cattle oocytes were incubated (39°C, 5% CO2 in air) with 1 × 105 washed, pretreated spermatozoa per 25 oocytes for 18 h. The oocytes were fixed in acid alcohol, stained with 1% acetate-orcein, and observed to determine the presence of pronuclei. Each experiment was repeated 4 times and data from each experiment were pooled. Approximately 80 to 90 oocytes per treatment for fertilization were evaluated in each replicate. Weighted least squares means were used to analyze fertilization data (SAS software, SAS Institute, Cary, NC, USA). The significance level for all tests was P < 0.05. Both antibodies for α5 (35 kDa) and αV (34 kDa) integrins showed immunoreactivity on Western blots of sperm membrane proteins. Addition of anti-integrin αV, and anti-integrin α5 decreased fertilization (P < 0.05) compared with the control: (1) 94.1 ± 1.0%; (2) 18.2 ± 1.0%; (3) 12.2 ± 1.0%. These findings show that αV and α5 integrins are expressed by cattle spermatozoa and may be involved in sperm-oocyte fusion and fertilization. This study was supported by FAPESP grants (2007/00363-5 and 2006/06008-0, Brazil). We acknowledge Nutricell and ABS Pecplan for their generous contribution.

scholarly journals Biosynthesis of plasma-membrane proteins during myogenesis of skeletal muscle in vitro

1978 ◽  
Vol 174 (3) ◽  
pp. 873-881 ◽  
Author(s):  
G A Cates ◽  
P C Holland
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Myoblast Fusion ◽  
Plasma Membrane Proteins ◽  
Membrane Markers ◽  
Surface Labelling

1. Surface labelling of plasma-membrane proteins with 125I, catalysed by lactoperoxidase, and radioactive l-fucose incorporation into glycoprotein were used as plasma-membrane markers for skeletal-muscle cells in culture. 2. Plasma membranes were prepared at various stages of myogenesis in vitro and rates of synthesis and accumulation of proteins in the membranes were compared. 3. Increased synthesis and accumulation of a protein of apparent mol.wt. 70000 occurred in the plasma-membrane fraction concomitant with the onset of myoblast fusion. 4. In cultures in which fusion of myoblasts was inhibited by 5′-bromo-2-deoxyuridine, synthesis and accumulation of the protein of apparent mol.wt. 70000 was selectively inhibited. 5. It is suggested the protein of apparent mol.wt. 70000 may be involved in the process of myoblast fusion.

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 Proteomic Analysis of Plasma Membrane Proteins in an In Vitro Blood-Brain Barrier Model

10.5772/30543 ◽  
2012 ◽  
Author(s):  
Sophie Duban-Deweer ◽  
Johan Hachani ◽  
Barbara Deracinois ◽  
Romo Cecchelli ◽  
Christophe Flahaut ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Blood Brain Barrier ◽  
Proteomic Analysis ◽  
Brain Barrier ◽  
Plasma Membrane Proteins ◽  
Blood Brain ◽  
Blood Brain Barrier Model ◽  
Barrier Model

scholarly journals Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

1992 ◽  
Vol 127 (3) ◽  
Author(s):  
TheoJ.M. Schoenmakers ◽  
PeterH.M. Klaren ◽  
Gert Flik ◽  
RobertA.C. Lock ◽  
PeterK.T. Pang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Calcium Uptake ◽  
Plasma Membrane Proteins ◽  
Fish Intestine ◽  
Basolateral Plasma Membrane

Myogenesis of normal and dystrophic chick embryonic skeletal muscle cells in vitro: biosynthesis of plasma membrane proteins

1980 ◽  
Vol 58 (10) ◽  
pp. 1156-1164 ◽  
Author(s):  
Paul C. Holland ◽  
George A. Cates ◽  
Byron S. Wenger ◽  
Barbara L. Raney
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Protein Biosynthesis ◽  
Sodium Dodecyl ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Plasma Membrane Proteins ◽  
Dystrophic Muscle

Plasma membranes were prepared from primary cell cultures of normal and genetically dystrophic chick embryonic pectoral muscle. These membranes were analyzed both by one-dimensional sodium dodecyl sulphate – polyacrylamide slab gel electrophoresis and by two-dimensional electrophoresis using isoelectric focusing in the first dimension. No marked and reproducible abnormalities could be detected in the synthesis, or accumulation, of plasma membrane proteins of dystrophic muscle cells maintained in culture for periods of up to 6 days. Analysis of the relative rates of protein turnover, analysis of fucose incorporation into plasma membrane proteins, and comparison of iodinated cell surface proteins also failed to reveal distinct abnormalities in plasma membranes derived from cultured dystrophic muscle cells. Although the results obtained do not rule out an early defect in plasma membrane protein biosynthesis during the development of dystrophic skeletal muscle in vivo, they do demonstrate that the synthesis and assembly of at least the major plasma membrane proteins occur normally during the initial phases of terminal differentiation of isolated dystrophic skeletal muscle cells in tissue culture.

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