scholarly journals Molecular characterization of gp40, a mucin-type glycoprotein from the apical plasma membrane of Madin–Darby canine kidney cells (type I)

1997 ◽  
Vol 326 (1) ◽  
pp. 99-108 ◽  
Author(s):  
Gert ZIMMER ◽  
Friedrich LOTTSPEICH ◽  
Andrea MAISNER ◽  
Hans-Dieter KLENK ◽  
Georg HERRLER
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Apical Plasma Membrane ◽  
Type I ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
A Cell ◽  
Polarized Transport ◽  
Darby Canine Kidney ◽  
Canine Kidney

gp40 has been recently identified as a major apical cell-surface sialoglycoprotein of type-I Madin–Darby canine kidney cells, a cell line widely used for the study of polarized transport. The determination of two internal amino acid sequences of the purified glycoprotein by Edman degradation enabled us to isolate the cDNA encoding the 18.6 kDa protein backbone of gp40. Sequence analysis revealed that gp40 is a type-I membrane protein which has several characteristics in common with glycophorin A and other mucin-type glycoproteins. At least 14 serine/threonine residues were found to be used for O-glycosylation. No potential sites for N-glycosylation were detected. gp40 turned out to represent the canine homologue of a cell-surface antigen expressed by various epithelial and non-epithelial cells in rat and mouse. Potential O-glycosylation sites, transmembrane and cytoplasmic domains were found to be highly conserved in the three species. gp40 was detected in canine lung, intestine, kidney, brain and heart but not in liver and spleen. The subline II of Madin–Darby canine kidney cells was found not to express gp40. Stable expression of gp40 in transfected type-II cells revealed that gp40 is predominantly delivered to the apical plasma membrane. N-Glycans and a glycosylphosphatidylinositol anchor, both proposed apical targeting signals, are absent from gp40, indicating that other determinants are responsible for its polarized transport.

scholarly journals An efficient method for introducing defined lipids into the plasma membrane of mammalian cells.

1983 ◽  
Vol 97 (5) ◽  
pp. 1365-1374 ◽  
Author(s):  
G van Meer ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
Efficient Method ◽  
Mammalian Cells ◽  
Lipid Vesicles ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Ha Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).

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.

scholarly journals Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. II. Immunological quantitation.

1983 ◽  
Vol 97 (3) ◽  
pp. 638-643 ◽  
Author(s):  
M Pesonen ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Binding Assay ◽  
Protein A ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The envelope of vesicular stomatitis virus was fused with the apical plasma membrane of Madin-Darby canine kidney cells by low pH treatment. The fate of the implanted G protein was then followed using a protein A-binding assay, which was designed to quantitate the amount of G protein in the apical and the basolateral membranes. The implanted G protein was rapidly internalized at 31 degrees C, whereas at 10 degrees C no uptake was observed. Already after 15 min at 31 degrees C, a fraction of the G protein could be detected at the basolateral membrane. After 60 min 25-48% of the G protein was basolateral as measured by the protein A-binding assay. At the same time, 25-33% of the implanted G protein was detected at the apical membrane. Internalization of G protein was not affected by 20 mM ammonium chloride or by 10 microM monensin. However, the endocytosed G protein accumulated in intracellular vacuoles and redistribution back to the plasma membrane was inhibited. We conclude that the implanted G protein was rapidly internalized from the apical surface of Madin-Darby canine kidney cells and a major fraction was routed to the basolateral domain.

Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells

1990 ◽  
Vol 96 (1) ◽  
pp. 143-149
Author(s):  
J.M. Wilson ◽  
N. Fasel ◽  
J.P. Kraehenbuhl
Keyword(s):  
Plasma Membrane ◽  
Common Mechanism ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Transfected Cells ◽  
Glycosyl Phosphatidylinositol ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.

scholarly journals Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence.

1983 ◽  
Vol 97 (3) ◽  
pp. 627-637 ◽  
Author(s):  
K Matlin ◽  
D F Bainton ◽  
M Pesonen ◽  
D Louvard ◽  
N Genty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Basolateral Membrane ◽  
Viral Envelope ◽  
Morphological Evidence ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The G protein of vesicular stomatitis virus was implanted in the apical plasma membrane of Madin-Darby canine kidney cells by low pH-dependent fusion of the viral envelope with the cellular membrane. The amount of fusion as determined by removal of unfused virions, either by tryptic digestion or by EDTA treatment at 0 degree C, was 22-24% of the cell-bound virus radioactivity. Upon incubation of cells after implantation, the amount of G protein as detected by immunofluorescence diminished on the apical membrane and appeared within 30 min on the basolateral membrane. At the same time some G protein fluorescence was also seen in intracellular vacuoles. The observations by immunofluorescence were confirmed and extended by electron microscopy. Using immunoperoxidase localization, G protein was seen to move into irregularly shaped vacuoles (endosomes) and multivesicular bodies and to appear on the basolateral plasma membrane. These results suggest that the apical and basolateral domains of Madin-Darby canine kidney cells are connected by an intracellular route.

scholarly journals rab4 Regulates Transport to the Apical Plasma Membrane in Madin-Darby Canine Kidney Cells

2002 ◽  
Vol 277 (12) ◽  
pp. 10474-10481 ◽  
Author(s):  
Karin Mohrmann ◽  
Richtje Leijendekker ◽  
Lisya Gerez ◽  
Peter van der Sluijs
Keyword(s):  
Plasma Membrane ◽  
Apical Plasma Membrane ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Inhibition by brefeldin A of protein secretion from the apical cell surface of Madin-Darby canine kidney cells.

1991 ◽  
Vol 266 (27) ◽  
pp. 17729-17732 ◽  
Author(s):  
S.H. Low ◽  
S.H. Wong ◽  
B.L. Tang ◽  
P. Tan ◽  
V.N. Subramaniam ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Secretion ◽  
Apical Cell ◽  
Brefeldin A ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.

1987 ◽  
Vol 105 (4) ◽  
pp. 1623-1635 ◽  
Author(s):  
G van Meer ◽  
E H Stelzer ◽  
R W Wijnaendts-van-Resandt ◽  
K Simons
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Mdck Cells ◽  
Laser Fluorescence ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Basolateral Side ◽  
Darby Canine Kidney ◽  
Canine Kidney

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.

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