GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells

1993 ◽  
Vol 104 (4) ◽  
pp. 1281-1290 ◽  
Author(s):  
M. Garcia ◽  
C. Mirre ◽  
A. Quaroni ◽  
H. Reggio ◽  
A. Le Bivic
Keyword(s):  
Intracellular Transport ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Membrane Microdomains ◽  
Ph Variation ◽  
Glycosyl Phosphatidylinositol ◽  
Apical Sorting ◽  
Triton X

In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4 degrees C but fully soluble in n-octyl-glucoside. Resistance to Triton X-100 extraction was not observed in the endoplasmic reticulum but appeared during transport through the Golgi complex. It was not dependent upon N-glycosylation processing, or pH variation from 6.5 to 8.5, and was not affected by sterol-binding agents. Other apical or basolateral transmembrane proteins were well solubilized in TX-100, with the exception of sucrase-isomaltase, which was partly insoluble. We isolated a membrane fraction from Caco-2 cells that contained GPI-anchored proteins and sucrase-isomaltase but no antigen 525, a basolateral marker, or dipeptidylpeptidase IV, an apical one. These data suggest that GPI-anchored proteins cluster to form membrane microdomains together with an apical transmembrane protein, providing a possible apical sorting mechanism for intestinal cells in vitro that might be related to apical sorting in MDCK cells, and that other mechanisms might exist to sort proteins to the apical membrane.

scholarly journals In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1784-1792 ◽  
Author(s):  
Gianluca Civenni ◽  
Samuel T. Test ◽  
Urs Brodbeck ◽  
Peter Bütikofer
Keyword(s):  
Membrane Vesicle ◽  
Human Erythrocytes ◽  
Membrane Extraction ◽  
Membrane Microdomains ◽  
Membrane Insertion ◽  
Vesicle Formation ◽  
Calcium Loading ◽  
Lipid Moiety ◽  
Triton X

Abstract In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

scholarly journals N-Glycans Mediate the Apical Sorting of a Gpi-Anchored, Raft-Associated Protein in Madin-Darby Canine Kidney Cells

1999 ◽  
Vol 146 (2) ◽  
pp. 313-320 ◽  
Author(s):  
Jürgen H. Benting ◽  
Anton G. Rietveld ◽  
Kai Simons
Keyword(s):  
Mdck Cells ◽  
Apical Cell ◽  
Chimeric Protein ◽  
Secretory Proteins ◽  
Madin Darby Canine Kidney ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Apical Sorting ◽  
Darby Canine Kidney ◽  
Canine Kidney

Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.

scholarly journals Clathrin and clathrin adaptor AP-1 control apical trafficking of megalin in the biosynthetic and recycling routes

2019 ◽  
Vol 30 (14) ◽  
pp. 1716-1728 ◽  
Author(s):  
Diego Gravotta ◽  
Andres Perez Bay ◽  
Caspar T. H. Jonker ◽  
Patrick J. Zager ◽  
Ignacio Benedicto ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Transmembrane Protein ◽  
Low Density Lipoprotein ◽  
Basolateral Membrane ◽  
Density Lipoprotein ◽  
Apical Surface ◽  
Density Lipoprotein Receptor ◽  
Clathrin Adaptor ◽  
Apical Sorting ◽  
Apical Localization

Megalin (gp330, LRP-2) is a protein structurally related to the low-density lipoprotein receptor family that displays a large luminal domain with multiligand binding properties. Megalin localizes to the apical surface of multiple epithelia, where it participates in endocytosis of a variety of ligands performing roles important for development or homeostasis. We recently described the apical recycling pathway of megalin in Madin–Darby canine kidney (MDCK) cells and found that it is a long-lived, fast recycling receptor with a recycling turnover of 15 min and a half-life of 4.8 h. Previous work implicated clathrin and clathrin adaptors in the polarized trafficking of fast recycling basolateral receptors. Hence, here we study the role of clathrin and clathrin adaptors in megalin’s apical localization and trafficking. Targeted silencing of clathrin or the γ1 subunit of clathrin adaptor AP-1 by RNA interference in MDCK cells disrupted apical localization of megalin, causing its redistribution to the basolateral membrane. In contrast, silencing of the γ2 subunit of AP-1 had no effect on megalin polarity. Trafficking assays we developed using FM4-HA-miniMegalin-GFP, a reversible conditional endoplasmic reticulum–retained chimera, revealed that clathrin and AP-1 silencing disrupted apical sorting of megalin in both biosynthetic and recycling routes. Our experiments demonstrate that clathrin and AP-1 control the sorting of an apical transmembrane protein.

scholarly journals Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts.

1989 ◽  
Vol 108 (3) ◽  
pp. 821-832 ◽  
Author(s):  
J E Skibbens ◽  
M G Roth ◽  
K S Matlin
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Disulfide Bond ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Transport Pathway ◽  
Influenza Virus Hemagglutinin ◽  
Triton X

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.

scholarly journals Analysis of the Transmembrane Domain of Influenza Virus Neuraminidase, a Type II Transmembrane Glycoprotein, for Apical Sorting and Raft Association

2000 ◽  
Vol 74 (14) ◽  
pp. 6538-6545 ◽  
Author(s):  
Subrata Barman ◽  
Debi P. Nayak
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Apical Plasma Membrane ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Influenza Virus Neuraminidase ◽  
Apical Sorting

ABSTRACT Influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. Previously, it was shown that the transmembrane domain (TMD) of NA provides a determinant(s) for apical sorting and raft association (A. Kundu, R. T. Avalos, C. M. Sanderson, and D. P. Nayak, J. Virol. 70:6508–6515, 1996). In this report, we have analyzed the sequences in the NA TMD involved in apical transport and raft association by making chimeric TMDs from NA and human transferring receptor (TR) TMDs and by mutating the NA TMD sequences. Our results show that the COOH-terminal half of the NA TMD (amino acids [aa] 19 to 35) was significantly involved in raft association, as determined by Triton X-100 (TX-100) resistance. However, in addition, the highly conserved residues at the extreme NH2 terminus of the NA TMD were also critical for TX-100 resistance. On the other hand, 19 residues (aa 9 to 27) at the NH2 terminus of the NA TMD were sufficient for apical sorting. Amino acid residues 14 to 18 and 27 to 31 had the least effect on apical transport, whereas mutations in the amino acid residues 11 to 13, 23 to 26, and 32 to 35 resulted in altered polarity for the mutant proteins. These results indicated that multiple regions in the NA TMD were involved in apical transport. Furthermore, these results support the idea that the signals for apical sorting and raft association, although residing in the NA TMD, are not identical and vary independently and that the NA TMD also possesses an apical determinant(s) which can interact with apical sorting machineries outside the lipid raft.

GPI-anchor deficiency in myeloid cells causes impaired FcγR effector functions

Blood ◽  
2004 ◽  
Vol 104 (9) ◽  
pp. 2825-2831 ◽  
Author(s):  
Wouter L. W. Hazenbos ◽  
Björn E. Clausen ◽  
Junji Takeda ◽  
Taroh Kinoshita
Keyword(s):  
Myeloid Cells ◽  
Dendritic Cell Maturation ◽  
Membrane Microdomains ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Effector Functions ◽  
Inflammatory Processes ◽  
Factor Α

Abstract Signaling by transmembrane immunoglobulin G (IgG)-Fc receptors (FcγRs) in response to ligand involves association with membrane microdomains that contain glycosyl phosphatidylinositol (GPI)-anchored proteins. Recent in vitro studies showed enhancement of FcγR signaling by forced monoclonal antibody-mediated cocrosslinking with various GPI-anchored proteins. Here, the possibility that GPI-anchored proteins are involved in normal physiologic FcγR effector functions in response to a model ligand was studied using myeloid-specific GPI-anchor-deficient mice, generated by Cre-loxP conditional targeting. GPI-anchor-deficient primary myeloid cells exhibited normal FcγR expression and binding or endocytosis of IgG-immune complexes (IgG-ICs). Strikingly, after stimulation with IgG-ICs, tumor necrosis factor-α release, dendritic cell maturation, and antigen presentation were strongly reduced by GPI-anchor deficiency. Tyrosine phosphorylation of the FcR γ-chain in response to IgG-IC was impaired in GPI-anchor-deficient cells. Myeloid GPI-anchor deficiency resulted in attenuated in vivo inflammatory processes during IgG-IC-mediated alveolitis. This study provides the first genetic evidence for an essential role of GPI-anchored proteins in physiologic FcγR effector functions in vitro and in vivo. (Blood. 2004;104:2825-2831)

Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin

1996 ◽  
Vol 271 (3) ◽  
pp. C887-C894 ◽  
Author(s):  
C. Mirre ◽  
L. Monlauzeur ◽  
M. Garcia ◽  
M. H. Delgrossi ◽  
A. Le Bivic
Keyword(s):  
Apical Membrane ◽  
Electron Microscopic Level ◽  
Membrane Microdomains ◽  
Morphological Aspect ◽  
Electron Microscopic ◽  
Western Blots ◽  
Glycosyl Phosphatidylinositol ◽  
Triton X ◽  
The Relationship ◽  
Detergent Resistant Membrane

In this study we analyzed the relationship between detergent-resistant microdomains and caveolae in Caco-2 cells. Caveolin was not detected on Western blots or Northern blots or by immunoprecipitation in these cells, in contrast to A 431 cells. Triton X-100-resistant membranes from Caco-2 and A 431 cells showed the same morphological aspect by electron microscopy and peaked at the same isopycnic density on sucrose gradients. Detergent-resistant microdomains from Caco-2 cells were enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins, in sucrase-isomaltase, an apical marker, and in most of the proteins found in caveolin-rich membranes such as src-like proteins, fimbrin, ezrin, and Gi alpha. Caveolae-like structures were present in A 431 but absent from Caco-2 cells at the electron microscopic level. Detergent-resistant microdomains from Caco-2 cells resemble caveolin-rich microdomains in their molecular composition but do not seem to derive from morphologically identified caveolae. Our results also indicate that caveolin is not necessary for sorting of GPI-linked proteins to the apical membrane of Caco-2 cells.

scholarly journals Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae.

1993 ◽  
Vol 123 (3) ◽  
pp. 595-604 ◽  
Author(s):  
M P Lisanti ◽  
Z L Tang ◽  
M Sargiacomo
Keyword(s):  
Cell Surface ◽  
Protein Complex ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Protein A ◽  
Cell Types ◽  
Apical Surface ◽  
Glycosyl Phosphatidylinositol ◽  
Surface Molecules ◽  
Oligomeric Protein

Glycosyl-phosphatidylinositol (GPI)-linked proteins are transported to the apical surface of epithelial cells where they undergo cholesterol-dependent clustering in membrane micro-invaginations, termed caveolae or plasmalemmal vesicles. However, the sorting machinery responsible for this caveolar-clustering mechanism remains unknown. Using transfected MDCK cells as a model system, we have identified a complex of cell surface molecules (80, 50, 40, 22-24, and 14 kD) that interact in a pH- and cholesterol-dependent fashion with an apical recombinant GPI-linked protein. A major component of this hetero-oligomeric protein complex is caveolin, a type II transmembrane protein. As this hetero-oligomeric caveolin complex is detectable almost immediately after caveolin synthesis, our results suggest that caveolae may assemble intracellularly during transport to the cell surface. As such, our studies have implications for understanding both the intracellular biogenesis of caveolae and their subsequent interactions with GPI-linked proteins in epithelia and other cell types.

scholarly journals In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1784-1792 ◽  
Author(s):  
Gianluca Civenni ◽  
Samuel T. Test ◽  
Urs Brodbeck ◽  
Peter Bütikofer
Keyword(s):  
Membrane Vesicle ◽  
Human Erythrocytes ◽  
Membrane Extraction ◽  
Membrane Microdomains ◽  
Membrane Insertion ◽  
Vesicle Formation ◽  
Calcium Loading ◽  
Lipid Moiety ◽  
Triton X

In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

Nonpolarized surface distribution and delivery of human CD7 in polarized MDCK cells

1993 ◽  
Vol 265 (4) ◽  
pp. C1069-C1079 ◽  
Author(s):  
C. Haller ◽  
S. L. Alper
Keyword(s):  
Membrane Proteins ◽  
Protein Targeting ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Surface Membrane ◽  
Experimental System ◽  
Surface Polarity ◽  
Epithelial Surface ◽  
Surface Distribution

Madin-Darby canine kidney (MDCK) cells grown on permeable supports have served as the most common experimental system for in vitro studies of the generation and maintenance of epithelial surface polarity. Protein targeting to the apical and basolateral plasmalemmal domains of these and other polarized epithelia has been suggested to rely on targeting sequences. Two simple sorting models for MDCK cells have proposed active sorting to a single domain, with "default" movement to the other domain. Examples of both apical and basal sorting signals have been found to support each hypothesis, but the idea of a default pathway has remained in question. Indeed, all endogenous and heterologous wild-type proteins so far studied in MDCK cells achieve polarized distributions at steady state. It is not known whether these selected proteins are representative of all surface membrane proteins or represent only a subset. We report here the apparent absence of sorting by MDCK cells of the transmembrane protein of T-cells, CD7. CD7 is expressed at similar density in apical and basolateral membranes of MDCK cells as assessed by both immunocytological and biochemical criteria. Furthermore, CD7 appears to be directly sorted to both surfaces at similar rates and turns over at both surfaces at similar rates. The nonpolarized distribution of CD7 appears independent of its level of expression. CD7 may identify a "bulk-flow" default pathway for plasma membrane proteins expressed in polarized MDCK cells.

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