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© The Rockefeller University Press,
0021-9525/1998//617 $5.00
The Journal of Cell Biology, Volume 140, Number 3,
, 1998 617-626
Article |
Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells
Margaret Dyson Vision Research Institute and Department of Cell Biology, Cornell University Medical College, New York 10021
Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The "raft" hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.
Abbreviations used in this paper: cav1, caveolin 1; CMV, cytomegalovirus; DAF, decay accelerating factor; FRT, Fischer rat thyroid; GPI, glycosylphosphatidylinositol; GSL, glycophospholipid; TIFF, Triton-insoluble floating fraction; TX-100, Triton X-100.
EPITHELIAL cells are characterized by the presence of polarized plasma membrane domains with different compositions of proteins and lipids (Rodriguez-Boulan and Powell, 1992; Eaton and Simons, 1995; Drubin and Nelson, 1996). In recent years, several sorting signals have been identified that mediate localization of proteins to apical or basolateral plasma membrane domains (Mostov et al., 1992; Matter and Mellman, 1994; Le Gall et al., 1995). Whereas basolateral signals are short, discrete sequences localized in the cytoplasmic domain of the protein, the best characterized apical signal is a glycophospholipid, glycosylphosphatidylinositol (GPI)1 (Lisanti and Rodriguez-Boulan, 1990), which is used by some proteins as an anchor to the membrane bilayer (Cross, 1987; Ferguson and Williams, 1988; Low and Saltiel, 1988; Low, 1989; Doering et al., 1990; Lisanti et al., 1990). GPI-anchored proteins are selectively localized to the apical membrane of most epithelial cells studied to date (Lisanti et al., 1988; Ali and Evans, 1990; Lisanti et al., 1990; Wilson et al., 1990). Furthermore, a GPI anchor is sufficient to target recombinant GPI-anchored proteins to the apical membrane of MDCK cells (Brown et al., 1989; Lisanti et al., 1989).
Attachment of the GPI moiety occurs in the luminal face of the endoplasmic reticulum by enzymatic replacement of COOH-terminal sequences that act as signals for GPI anchoring (for review see Englund, 1993; McConville and Ferguson, 1993; Vidugiriene and Menon, 1995). The newly synthesized GPI-anchored proteins are then transported to the cell surface, where they are exposed on the topologically equivalent extracytoplasmic face of the plasma membrane (Vidugiriene and Menon, 1994). Sorting of GPI-anchored proteins occurs after their carbohydrates are processed in the Golgi complex (Brown et al., 1989; Lisanti et al., 1989), presumably by incorporation into post-Golgi vesicles assembled in the TGN (Lisanti and Rodriguez-Boulan, 1990; Wandinger-Ness et al., 1990).
As they migrate through the proximal Golgi complex, GPI-anchored proteins undergo a dramatic change in their biophysical properties, reflected by their becoming insoluble in certain nonionic detergents, such as Triton X-100 (TX-100) (Brown and Rose, 1992; Garcia et al., 1993; Zurzolo et al., 1994). This appears to reflect the association of GPI-anchored proteins with glycosphingolipid–cholesterol clusters in the Golgi complex, which are also detergent insoluble (Thompson and Tillack, 1985). When purified by flotation in sucrose density gradients, these aggregates, denoted TIFF (Triton-insoluble floating fraction; Kurzchalia et al., 1995) or detergent-insoluble glycosphingolipid-enriched domains (DIG; Parton, 1996) can be shown to be rich in GPI-anchored proteins, sphingomyelin, glycosphingolipids (GSL)s and cholesterol (Brown and Rose, 1992; Garcia et al., 1993; Sargiacomo et al., 1993; Zurzolo et al., 1994). Fluorescence energy transfer experiments indicate that GPI-anchored proteins are still clustered when they arrive at the cell surface, but slowly disperse in the next few hours (Hannan et al., 1993).
The "raft hypothesis" postulates that clustering with GSLs is required for the sorting of apical proteins (Simons and Ikonen, 1997; van Meer and Simons, 1988). GSLs are sorted apically in MDCK (kidney) and Caco-2 (intestinal) epithelial cells, at least as indicated by experiments with short acyl chain fluorescent glycolipids (van Meer et al., 1987; van't Hof and van Meer, 1990). In its extended version, the raft includes transmembrane apical proteins, such as influenza hemagglutinin, which also becomes detergent insoluble as it traverses the Golgi complex (Skibbens et al., 1989). The mechanisms involved in the formation of the raft are still obscure. The length and saturation of the acyl chains of GSLs appear to be important in this process (Schroeder et al., 1994). A similar GSL–cholesterol enrichment as in the Golgi raft was reported for plasma membrane caveolae (Fra et al., 1994; Gorodinski et al., 1995; Kurzchalia et al., 1995; Mayor et al., 1995; Parton, 1996; Schnitzer et al., 1995b). Initial reports suggested that GPI-anchored proteins are also enriched in caveolae (Rothberg et al., 1990; Sargiacomo et al., 1993); however, recent evidence suggests that GPI-anchored proteins are distributed evenly throughout the plasma membrane, but redistribute to caveolar regions upon cross-linking, e.g., with antibodies (Mayor et al., 1994; Parton et al., 1994; Schnitzer et al., 1995a). Caveolae are coated on the cytoplasmic side by a coat that includes a phosphorylated cholesterol-binding protein of 21 kD, caveolin (Rothbergh et al., 1992; Murata et al., 1995). Recent transient transfection experiments in lymphocytes indicate that caveolin is required for the assembly of morphological caveolae (Fra et al., 1995). The observations that caveolin is identical to VIP21, a protein present in post-Golgi vesicles and the TGN (Wandinger-Ness et al., 1990; Kurzchalia et al., 1992), and that caveolin localizes into TIFF (Sargiacomo et al., 1993; Zurzolo et al., 1994; Kurzchalia et al., 1995), suggest a possible involvement of this protein in the formation of post-Golgi transport vesicles (Dupree et al., 1993). However, caveolae have not been identified in the TGN to date.
The polarized epithelial cell line Fischer rat thyroid (FRT) provides an excellent model to study the role of caveolin in both GPI-anchored protein sorting and caveolar assembly (Zurzolo et al., 1992; Nitsch et al., 1985). FRT cells do not express caveolin (Sargiacomo et al., 1993; Zurzolo et al., 1994) and do not form caveolae (this paper). Although FRT cells can sort transmembrane apical proteins to their correct plasma membrane domain (Zurzolo et al., 1992), they deliver endogenous and transfected GPI-anchored proteins and fluorescent GSLs to the basolateral membrane and fail to incorporate GPI proteins into TIFF (Zurzolo et al., 1993, 1994). In this paper, we report the results of experiments in which we transfected caveolin1 (cav1) into FRT cells to examine its effect on GPI-anchored protein sorting, clustering, and caveolar assembly. Although caveolin was sufficient to promote the assembly of morphologically normal caveolae in FRT cells, it failed to redistribute GPI proteins to the apical surface or to affect their clustering into TIFF.
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Cell Culture and Transfection
FRT cells stably expressing gD1–DAF (Zurzolo et al., 1993), and FRT cells expressing transfected cav1 were grown in F12 Coon's modified medium containing 5% FBS. MDCK cells were maintained in DME supplemented with 5% FBS. FRT cells were cotransfected as previously described using a modification of the calcium phosphate precipitation procedure (Zurzolo et al., 1993). The VIP21/cav1 cDNA from MDCK cells was a gift from P. Dupree (European Molecular Biology Laboratory, Heidelburg, Germany, [EMBL]) and was inserted into pCMV5, carrying the gene for resistance to G418, using the EcoRI and KpnI restriction sites. For some experiments we used myc–caveolin cDNA (from R.G.W. Anderson, University of Texas Health Science Center, Dallas, TX). The construct containing gD1–DAF under the Rous sarcoma virus promoter was a gift of I. Caras (Genentech Inc., South San Francisco, CA) and has been described elsewhere (Lisanti et al., 1989; Zurzolo et al., 1993).
Biotinylation Assays
Confluent monolayers on transwells were labeled overnight using 1 mCi/ml of [35S]met-cys (Amersham Corp., Arlington Heights, IL), and were biotinylated and processed for immunoprecipitation as previously described (Le Bivic et al., 1989; Sargiacomo et al., 1989; Zurzolo et al., 1993). To recover the immunoprecipitated biotinylated antigens, the beads were boiled in 10 ml of 10% SDS for 5 min, diluted with lysis buffer, and then centrifuged for 1 min at 14,000 rpm. Biotinylated antigens present in the supernatants were precipitated with streptavidin–agarose beads. Finally, the beads were boiled in Laemmli buffer (Laemmli, 1970) and analyzed by SDS-PAGE. Dried gels were processed as described (Zurzolo et al., 1992) using preflashed films; densitometry analysis was carried out within the linear range of the film. The steady-state distribution of endogenous GPI-anchored proteins was determined using domain-selective biotinylation and treatment with phospholipase C, as previously described (Lisanti et al., 1988; Zurzolo et al., 1993).
Pulse-chase and TX-100 Extraction
TX-100 extractability during pulse-chase experiments was assayed as previously described (Brown and Rose, 1992; Zurzolo et al., 1994). Briefly, cells that had just reached confluency in 35-mm dishes were starved of methionine and cysteine for 20 min, pulse labeled for 5 min with 100 ml of pulse medium containing 
500 mCi/ml of [35S]met-cys, and then incubated in chase medium (DME containing 10% FBS and 100X met and cys) for different times. After each time point, cells were washed twice with PBS containing 1 mM CaCl2 and 1 mM MgCl2 (PBS CM) on ice as described previously, and then lysed for 20 min on ice in 1 ml TNE/TX-100 buffer (Brown and Rose, 1992; Zurzolo et al., 1994). Lysates were collected and centrifuged at 13,000 rpm for 2 min at 4°C. Supernatants, representing the soluble material, were removed and then pellets were solubilized in 100 ml of solubilization buffer (50 mM Tris-HCl, pH 8.8, 5 mM EDTA, 1% SDS); DNA was sheared through a 22-g needle. Both soluble and insoluble materials were adjusted to 0.1% SDS before immunoprecipitation with specific antibodies as previously described (Le Bivic et al., 1989).
Sucrose Gradients
Sucrose gradient analysis of TX-100–insoluble residue was performed using previously published protocols (Brown and Rose, 1992; Zurzolo et al., 1994). Briefly, cells were grown to confluence in 100-mm dishes, labeled for 30 min with 500 mCi/ml of [35S]met-cys, and then incubated in chase medium for 3 h. Monolayers were then rinsed in PBS CM and lysed for 20 min in TNE/TX-100 buffer on ice. Lysates were scraped from the dish, brought to 40% sucrose, and then placed at the bottom of a centrifuge tube. A linear sucrose gradient (5–35% in TNE) was layered on top of the lysates and then the samples were centrifuged at 39,000 rpm for 18–20 h in a rotor (model SW41; Beckman Instrs., Fullerton, CA). 1-ml fractions were harvested from the top of the gradient. Immunoprecipitation of gD1–DAF and other antigens was performed on the different fractions, after bringing them to 20% sucrose and 1% TX-100. Samples were solubilized in Laemmli buffer and either boiled for 5 min, or left at 25°C for 30 min before running on SDS-PAGE, and were then analyzed by autoradiography.
Immunofluorescence and Electron Microscopy
Cells grown on coverslips to confluence were processed for immunofluorescence as described (Zurzolo et al., 1993). For EM experiments, cells were washed three times with PBS pH 7.4, containing 2.7 mM KCl, 1 mM CaCl2, and 1 mM MgCl2, and then fixed for 30 min at room temperature with 2.5% glutaraldehyde, 0.1% tannic acid in 0.1 M cacodylate buffer, pH 7.4. Cells were then rinsed three times in 0.1 M cacodylate buffer and postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 30–60 min at room temperature. Cells were further stained en bloc with 3% uranyl acetate in 50% ethanol for 20 min, dehydrated through graded ethanol, and finally embedded in Epon 812 (Polysciences, Inc., Warington, PA). Thin sections were cut parallel to the plane of the culture on a microtome (model MT 5000; Sorvall, Inc., Newtown, CT) and then stained with 1.5% uranyl acetate and 0.1% lead citrate (Venable et al., 1965). For the immunogold localization of gD1–DAF, cav1-FRT cells grown to subconfluence on coverslips were incubated on ice with the monoclonal antibody anti-gD1– DAF for 1 h. After three washes in PBS, bound antibody was detected with goat anti–mouse IgG 10-nm gold conjugates. After three washes in PBS, the cells were processed as described above through to dehydration in ethanol. In 70% ethanol, cells were scraped from the coverslips with a razor blade and then the pellets were collected in a microfuge tube before embedding in Epon 812. Specimens were examined and photographed in either a 100 C X II (JEOL USA, Inc., Peabody, MA) or electron microscope (model 400T; Philips Electron Optics, Eindhoven, The Netherlands).
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To determine whether caveolin was involved in either the apical sorting of gD1–DAF or in its clustering with GSLs, we stably transfected FRT cells with a cDNA encoding the cav1 protein. After initial attempts to transfect cav1 into FRT cells already expressing gD1–DAF failed, we simultaneously transfected a plasmid containing the gD1–DAF gene under control of the RSV promoter (Lisanti et al., 1989), and then a plasmid containing the cav1 coding sequence fused to the cytomegalovirus (CMV) promoter and the gene conferring resistance to neomycin (G418). By selection with G418, we isolated different clones expressing both proteins. Among many clones heterogeneous in their expression of caveolin and gD1–DAF proteins, as visualized by double immunofluorescence experiments (data not shown), we selected two clones (FRTcl1 and FRTcl2) that displayed a homogeneous distribution of both markers within all cells (Fig. 1, A and B). Both clones displayed caveolin as a punctate pattern at the plasma membrane and within the Golgi apparatus (Fig. 1 A). gD1– DAF was similarly enriched at the plasma membrane, albeit with a different, more diffuse, pattern (Fig. 1 B), but was also found intracellularly, as previously shown (Zurzolo et al., 1993).
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gD1–DAF Is Excluded from TIFF in FRT Cells Expressing Cav1
In MDCK cells, GPI-anchored proteins are incorporated into TX-100–insoluble aggregates, presumably reflecting their association with GSLs in the Golgi complex (Brown and Rose, 1992). In apparent agreement with the idea that this association may be required for their apical sorting (Van Meer and Simons, 1988; Lisanti and Rodriguez-Boulan, 1990), we previously demonstrated that basolaterally sorted gD1–DAF fails to be incorporated into TIFF in FRT cells, whereas it is apically sorted and becomes detergent insoluble in MDCK cells (Zurzolo et al., 1994). To determine whether transfection of cav1 promotes clustering of gD1–DAF with GSLs, we performed pulse-chase experiments in the two FRT clones stably expressing cav1, and then determined whether gD1–DAF was incorporated into the TX-100–insoluble fraction. We found that in both caveolin expressing clones, gD1–DAF was mainly soluble in TX-100 at all times of chase (Fig. 3), exactly as previously shown in nontransfected cells (Zurzolo et al., 1994).
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350, 300, and 200 kD, as well as 45-kD dimers (Fig. 6, top), similar to that formed by endogenous caveolin in MDCK cells (Fig. 6, bottom) as previously shown (Monier et al., 1995; Sargiacomo et al., 1995). Furthermore, these oligomers appeared to migrate to TIFF in both cell lines in similar amounts as the monomer (compare Figs. 5 and 6, top and bottom).
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Recent circumstantial evidence suggests that the caveolar coat protein caveolin might play a role in the clustering of GSLs and GPI-anchored proteins (Dupree et al., 1993; Sargiacomo et al., 1993; Zurzolo et al., 1994). Although TIFF exists in the absence of plasmalemmal caveolae, the lipid composition of caveolae is very similar to that of TIFF (Brown and Rose, 1992; Fra et al., 1995). Furthermore, it has been shown that caveolin, which is 90% enriched in plasma membrane caveolae, associates with TIFF (Sargiacomo et al., 1993; Kurzchalia et al., 1995). Although some controversy exists as to whether GPI-anchored proteins are enriched within caveolae (Rothberg et al., 1990; Anderson, 1993; Mayor et al., 1994), nonetheless, these proteins show affinity for caveolae after they have been cross-linked with antibodies. Finally, the presence of caveolin in the TGN and post-TGN vesicles (Kuzchalia et al., 1992), and the absence of caveolin in FRT cells, which exhibit defective apical targeting and clustering of GPI-anchored proteins, further underlines the need for studies that address the role of caveolin in intracellular sorting of proteins in the Golgi complex.
The approach we used in this report was to transfect the cav1 gene into FRT cells to determine its effect on the sorting of GPI-anchored proteins and its association with TIFF. In two FRT clones stably expressing caveolin at levels comparable with MDCK cells, we observed that although caveolin typically associated with TIFF and formed large oligomers similar to those assembled in MDCK cells, gD1–DAF remained unable to cluster with GSLs that independently fractionated into TIFF, and was still sorted to the basolateral membrane, as in wild-type cells. Furthermore, no changes were detected in the distribution of endogenous GPI- anchored proteins, which remained mainly basolateral or nonpolar. Taken together, our data suggest that cav1 does not mediate the association of GPI-anchored proteins with GSL-enriched microdomains in the Golgi complex, nor participates in apical sorting.
Another possible explanation for our results is that cav1 is, for some reason, nonfunctional in FRT cells and, therefore, fails to carry out the functions that it performs normally in other cells. For example, FRT cells might not be able to perform a critical posttranslational processing modification of cav1, or might fail to form the oligomers observed in MDCK cells. To test this hypothesis, we studied the effect of cav1 transfection on the assembly of plasma membrane caveolae. Indeed, we found that wild-type FRT cells do not form caveolae, but, upon transfection with cav1, formed large numbers of normal flask-shaped caveolae. Our results confirm and extend previous experiments in which caveolin transfection was shown to be necessary and sufficient to promote the assembly of caveolae in lymphocytes (Fra et al., 1995). FRT caveolae were indistinguishable from the caveolae endogenously present in other epithelial and endothelial cells; they frequently showed a classical septum at the neck and often associated into racemose clusters of five or six units (Fig. 8) (Palade and Bruns, 1968). We also found that gD1–DAF clustered into these newly formed caveolae upon cross-linking with antibodies. Furthermore, cav1 transfected into FRT cells forms high molecular weight oligomers as in cells that form caveolae, such as MDCK cells. Therefore, our results show that both cav1 and caveolae are functional in FRT cells, as judged by (a) the ability of cav1 to oligomerize and promote caveolar assembly, and (b) by the capacity of these organelles to interact with GPI-anchored proteins. This is the first report of caveolar assembly in cells permanently transfected with caveolin.
Although our data indicate that cav1 is functional and sufficient to promote the assembly of caveolae in FRT cells, they do not exclude the possibility that FRT cells lack additional factor(s) required for GPI-anchored protein clustering with GSLs, enriched microdomains, and/or apical sorting. For example, GPI-anchored proteins might have different intrinsic properties in FRT cells that prevent their clustering with GSLs. Alternatively, FRT cells might lack a clustering molecule that would be involved in partitioning GPI-anchored proteins into GSL clusters. However, some data already present in the literature do not favor a role for caveolin in these processes (Gorodinsky and Harris, 1995; Kurzchalia et al., 1995; Mirre et al., 1996). Most importantly, CaCo2 cells lack caveolin and are, nonetheless, able to form TX-100–insoluble GSLs/GPI-protein– enriched microdomains, and sort them to the apical plasma membrane (Mirre et al., 1996).
Because we did not check whether FRT cells also lack caveolin2 (Parton, 1996; Scherer et al., 1996), a possibility remains that this isoform of caveolin could have some role in the apical sorting of GPI-anchored proteins. However, considering that such a role was proposed originally for cav1, which was found to be enriched in the TGN and post-TGN sorting vesicles (Dupree et al., 1993; Kurzchalia et al., 1992), and considering that caveolin2 appears to be targeted largely to the basolateral plasma membrane in MDCK cells (Scheiffele et al., 1998), we believe this scenario is unlikely.
The data reported here support the hypothesis that caveolin oligomerization is the basis of caveolar coat assembly (Anderson, 1993; Fra et al., 1995; Monier et al., 1995) and clearly demonstrate that cav1 is necessary and sufficient to promote formation of these characteristic plasma membrane invaginations. Furthermore, because we found similar levels of TX-100–insoluble GSLs in FRT cells expressing or not expressing cav1 and caveolae, our data definitively rule out the suggested identity between caveolae and detergent-insoluble microdomains, and do not support a role for caveolae as carrier vesicles for the transport of GPI-anchored proteins to the apical plasma membrane.
An alternative model is that clustering with GSLs is indeed a prerequisite for apical sorting of GPI-anchored proteins, and that caveolin and caveolae are not involved in these events. Although it is not yet clear whether incorporation of a protein in these rafts would be sufficient for apical sorting, there could be different mechanisms mediating the association of GPI-anchored proteins with GSLs rafts. One could hypothesize that MDCK and other epithelial cells that sort GPI-anchored proteins to the apical surface possess a clustering factor, such as was originally suggested for caveolin, that could also be recognized as an apical targeting signal. An equivalent function could be performed by the membrane-spanning domains of apical transmembrane proteins (Kundu et al., 1996). Additionally, the ectodomain may also play a role in apical sorting since it has been shown that most of the ectodomains of GPI-anchored proteins are secreted selectively into the apical medium (Brown et al., 1989; Lisanti et al., 1989; Powell et al., 1991). Further experiments will be necessary to discriminate among these possibilites. Since the sorting machinery seems to be conserved also in nonpolarized cells (Musch et al., 1996; Yoshimori et al., 1996), we believe that the use of in vitro assays, together with comparative analyses between different epithelial cells displaying different sorting profiles of GPI-anchored proteins and GSLs, will help to address these possibilities.
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Acknowledgments |
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Submitted: 14 July 1997
Revised: 1 December 1997
Work in the Zurzolo lab was supported by Ministero Università e Ricerca Scientifica e Tecnologica. Work in the Rodriguez-Boulan lab was supported by a National Institutes of Health grant (GM41771).
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ali N & Evans WH. Priority targeting of glycosyl-phosphatidylinositol-anchored proteins to the bile canalicular (apical) plasma membrane of hepatocytes, Biochem J, 1990, 271, 193–199.[Medline]
Anderson RGW. Plasmalemmal caveolae and GPI-anchored membrane proteins, Curr Opin Cell Biol, 1993, 5, 647–652.[Medline]
Brown DA, Crise B & Rose JK. Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells, Science, 1989, 245, 1499–1501.
Brown DA & Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface, Cell, 1992, 68, 533–544.[Medline]
Cross GAM. Eukaryotic protein modification and membrane attachment via phosphatidylinositol, Cell, 1987, 48, 179–181.[Medline]
Doering TL, Masterson WJ, Englud PT & Hart GW. Biosynthesis of glycosylphosphatidylinositol membrane anchors, J Biol Chem, 1990, 265, 611–614.
Drubin DG & Nelson WJ. Origins of cell polarity, Science, 1996, 84, 335–344.
Dupree P, Parton RG, Raposo G, Kurzchalia TV & Simons K. Caveolae and sorting in Trans Golgi Network of epithelial cells, EMBO (Eur Mol Biol Organ) J, 1993, 12, 1597–1605.[Medline]
Eaton S & Simons K. Apical, basal, and lateral cues for epithelial polarization, Cell, 1995, 82, 5–8.[Medline]
Englund PT. The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors, Annu Rev Biochem, 1993, 62, 121–138.[Medline]
Ferguson MAJ & Williams AF. Cell-surface anchoring of proteins via glycosyl-phosphatidil structures, Annu Rev Biochem, 1988, 57, 285–320.[Medline]
Fra AM, Williamson E, Simons K & Parton RG. Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae, J Biol Chem, 1994, 269, 30745–30748.
Fra AM, Williamson E, Simons K & Parton RG. De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin, Proc Natl Acad Sci USA, 1995, 92, 8655–8659.
Garcia M, Mirre C, Quaroni A, Reggio H & Le Bivic A. GPI anchored proteins associate to form microdomains during their intracellular transport in CaCo2 cells, J Cell Sci, 1993, 104, 1281–1290.[Abstract]
Gorodinsky A & Harris DA. Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin, J Cell Biol, 1995, 129, 619–627.
Hannan LA, Lisanti MP, Rodriguez-Boulan E & Edidin M. Correctly sorted molecules of a GPI-anchored protein are clustered and immobile when they arrive at the apical surface of MDCK cells, J Cell Biol, 1993, 120, 353–358.
Kundu A, Avalos RT, Sanderson CM & Nayak DP. Transmembrane domain of influenza virus neuramidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells, J Virol, 1996, 70, 6508–6515.[Abstract]
Kurzchalia TV, Dupree P, Parton RG, Keliner R, Virta H, Lehnert M & Simons K. VIP 21, a 21-kD membrane protein is an integral component of trans-Golgi network–derived transport vescicles, J Cell Biol, 1992, 118, 1003–1014.
Kurzchalia TV, Hartmann E & Dupree P. Guilt by insolubility-does a protein's detergent-insolubility reflect a caveolar location? , Trends Cell Biol, 1995, 5, 187–189.[Medline]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4, Nature, 1970, 227, 680–685.[Medline]
Le Bivic A, Real FX & Rodriguez-Boulan E. Vectorial targeting of apical and basolateral plasma membrane protein in a human adenocarcinoma epithelial cells, Proc Natl Acad Sci USA, 1989, 86, 9313–9317.
Le Gall AH, Yeman C, Muesch A & Rodriguez-Boulan E. Epithelial cell polarity: new prospectives, Semin-Nephrol, 1995, 15, 272–284.[Medline]
Lisanti MP, Sargiacomo M, Graeve L, Saltiel A & Rodriguez-Boulan E. Polarized apical distribution of glycosylphosphatidyl inositol anchored proteins in a renal epithelial line, Proc Natl Acad Sci USA, 1988, 85, 9557–9561.
Lisanti MP, Caras IW, Davitz MA & Rodriguez-Boulan E. A glycosphingolipid membrane anchor acts as an apical targeting signal in polarized epithelial cells, J Cell Biol, 1989, 109, 2145–2156.
Lisanti M & Rodriguez-Boulan E. Glycosphingolipid membrane anchoring provides clues to the mechanism of protein sorting in polarized epithelial cells, Trends Biochem Sci, 1990, 15, 113–118.[Medline]
Lisanti MP, Rodriguez E, Boulan & Saltiel AR. Preferred apical distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype, J Membr Biol, 1990, 113, 155–167.[Medline]
Low M. The glycosyl-phosphatidylinositol anchor of membrane proteins, Biochim Biophys Acta, 1989, 988, 427–454.[Medline]
Low MG & Saltiel AR. Structural and functional roles of glycosyl-phosphatidylinositol in membranes, Science, 1988, 239, 268–275.
Matter K & Mellman I. Mechanisms of cell polarity: sorting and transport in epithelial cells, Curr Opin Cell Biol, 1994, 6, 545–554.[Medline]
Mayor S & Maxfield FR. Insolubility and redistribuition of GPI- anchored proteins at the cell surface after detergent tretment, Mol Biol Cell, 1995, 6, 929–944.[Abstract]
Mayor S, Rothberg KG & Maxfield FR. Sequestration of GPI- anchored proteins in caveolae triggered by cross-linking, Science, 1994, 264, 1948–1951.
McConville MJ & Ferguson MAJ. The structure, biosynthesis, and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes, Biochem J, 1993, 29, 305–324.
Mirre C, Monlauzer L, Garcia M, Delgrossi M-H & Le Bivic A. Detergent resistant membrane microdomains from Caco-2 cells do not contain caveolin, Am Physiol Soc, 1996, 271, C887–C894.
Monier S, Parton RG, Vogel F, Henske A & Kurzchalia TV. VIP21-caveolin, a membrane protein constituent of the caveolar coat, forms high molecular mass oligomers in vivo and in vitro, Mol Biol Cell, 1995, 6, 911–927.[Abstract]
Mostov KE, Apodaca G, Aroeti B & Okamoto C. Plasma membrane protein sorting in polarized epithelial cells, J Cell Biol, 1992, 116, 577–583.
Murata M, Peranen J, Schreiner R, Wieland F, Kurzchalia TV & Simons K. VIP21/caveolin is a cholesterol-binding protein, Proc Natl Acad Sci USA, 1995, 92, 10339–10343.
Musch A, Xu H, Shields D & Rodriguez-Boulan E. Transport of VSV G protein to the cell surface is signal mediated in polarized and nonpolarized cells, J Cell Biol, 1996, 133, 543–558.
Nitsch L, Tramontano D, Quarto N, Bonatti S & Ambesi-Impiombato FS. Morphological and functional polarity of an epithelial thyroid cell line, Eur J Cell Biol, 1985, 38, 57–66.[Medline]
Palade GE & Bruns RR. Structural modulations of plasmalemmmal vesicles, J Cell Biol, 1968, 37, 633–649.
Parton RG. Caveolae and caveolins, Curr Opin Cell Biol, 1996, 8, 542–548.[Medline]
Parton RG & Simons K. Digging into caveolae, Science, 1995, 269, 1398–1399.
Parton RG, Joggerst B & Simons K. Regulated internalization of caveolae, J Cell Biol, 1994, 127, 1199–1215.
Powell SK, Lisanti MP & Rodriguez-Boulan E. Thy-1 expresses two signals for apical localization in epitheliall cells, Am Physiol Soc, 1991, 260, C715–C720.
Rodriguez-Boulan E & Powell SK. Polarity of epithelial and neuronal cells, Annu Rev Cell Biol, 1992, 8, 395–427.
Rothberg KG, Ying Y-S, Kolhouse JF, Kamen BA & Anderson RGW. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway, J Cell Biol, 1990, 110, 637–649.
Rothberg KG, Heuser JK, Donzell WC, Ying Y-S, Glenney JR & Anderson RGW. Caveolin, a protein component of caveole membrane coats, Cell, 1992, 68, 673–682.[Medline]
Sargiacomo M, Lisanti M, Greve L, Le Bivic A & Rodriguez-Boulan E. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells, J Membr Biol, 1989, 107, 277–286.[Medline]
Sargiacomo M, Sudol M, Tang Z & Lisanti MP. Signal transducing molecules and GPI-linked proteins form a caveolin-rich insoluble complex in MDCK cells, J Cell Biol, 1993, 122, 789–807.
Sargiacomo M, Scherer PE, Tang Z, Kuebler E, Song KS, Sanders M & Lisanti MP. Oligomeric structure of caveolin: implications for caveolae membrane organization, Proc Natl Acad Sci USA, 1995, 92, 9407–9411.
Scheiffele, P., P. Verkade, A.M. Fra, H. Victa, K. Simons, and E. Ikonen. 1998. Caveolin-1 and -2 in the exocytic pathway of MDCK cells. J. Cell Biol. In press.
Scherer PE, Okamoto T, Chun M, Nishimoto I, Lodish HF & Lisanti MP. Identifaction, sequence, and expression of caveolin-2 defines a caveolin gene family, Proc Natl Acad Sci USA, 1996, 93, 131–135.
Schnitzer JE, Oh P, Jacobson BS & Dvorak AM. Caveolae from luminal plasmalemma of rat lung endothelium: microdomains enriched in caveolin, Ca2+-ATPase, and inositol triphosphate receptor, Proc Natl Acad Sci USA, 1995a, 92, 1759–1763.
Schnitzer JE, Mcintosh DP, Dvorak AM, Liu J & Oh P. Separation of caveolae from associated microdomains of GPI-anchored proteins, Science, 1995b, 269, 1435–1439.
Schroeder R, London E & Brown D. Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior, Proc Natl Acad Sci USA, 1994, 91, 12130–12134.
Simons K & Ikonen E. Functional rafts in cell membranes, Nature, 1997, 387, 569–572.[Medline]
Skibbens JE, Roth MG & Matlin KS. Differential extractability of influenza virus Hemagluttinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts, J Cell Biol, 1989, 108, 821–832.
Thompson TE & Tillack TW. Organization of glycosphingolipids in bilayers and plasma membranes of mammalian cells, Annu Rev Biophys Chem, 1985, 14, 361–386.[Medline]
van Meer G & Simons K. Lipid polarity and sorting in epithelial cells, J Cell Biochem, 1988, 36, 51–58.[Medline]
van Meer G, Stelzer EH, Wijnaendts van Resandt RW & Simons K. , J Cell Biol, 1987, 105, 1623–1635.
van't Hof W & van Meer G. Generation of lipid polarity in intestinal epithelial (Caco2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane, J Cell Biol, 1990, 111, 977–986.
Venable JH & Coggeshal R. A simplified lead citrate stain for use in electron microscopy, J Cell Biol, 1965, 25, 407–408.
Vidurgiriene J & Menon AK. The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum, J Cell Biol, 1994, 127, 333–341.
Vidurgiriene J & Menon AK. Soluble constituents of the ER lumen are required for GPI anchoring of a model protein, EMBO (Eur Mol Biol Organ) J, 1995, 14, 4686–4694.[Medline]
Wandinger-Ness A, Bennett MK, Antony C & Simons K. Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells, J Cell Biol, 1990, 111, 987–1000.
Wilson JM, Fasel N & Kraehenbuhl J-P. Polarity of endogenous glycosylphosphatidylinositol-anchored membrane proteins in Madin Darby Canine Kidney cells, J Cell Sci, 1990, 96, 143–149.
Yoshimori T, Keller P, Roth MG & Simons K. Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells, J Cell Biol, 1996, 133, 247–256.
Zurzolo C, Le Bivic A, Quaroni A, Nitsch L & Rodriguez-Boulan E. Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line, EMBO (Eur Mol Biol Organ) J, 1992, 11, 2337–2344.[Medline]
Zurzolo C & Rodriguez-Boulan E. Delivery of Na+, K+,-ATPase in polarized epithelial cells, Science, 1993, 260, 550–552.
Zurzolo C, Lisanti MP, Caras IW, Nitsch L & Rodriguez-Boulan E. Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer Rat Thyroid epithelial cells, J Cell Biol, 1993, 121, 1031–1039.
Zurzolo C, van't Hof W, van Meer G & Rodriguez-Boulan E. VIP 21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells, EMBO (Eur Mol Biol Organ) J, 1994, 13, 42–53.[Medline]
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