GPI membrane anchor is determinant in

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Journal of Cell Science 107, 2679-2689 (1994) Printed in Great Britain © The Company of Biologists Limited 1994

GPI membrane anchor is determinant in intracellular accumulation of apical plasma membrane proteins in the non-polarized human colon cancer cell line HT-29 18 F. Nicolas1, M.-C. Tiveron1, J. Davoust2 and H. Reggio3,* 1Laboratoire de Génétique et de Physiologie du Développement, UMR CNRS 9943, Case 907, Faculté des Sciences de Luminy, 163 Avenue de Luminy, 13288, Marseille, Cedex 9, France 2Centre d’Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, Faculté des Sciences de Luminy, 163 Avenue de Luminy, 13288, Marseille, Cedex 9, France 3Laboratoire de Dynamique Moléculaire des Interactions Membranaires, URA CNRS 1856, Université de Montpellier II, Case 107, Place Eugène Bataillon, 34095 Montpellier, Cedex 5, France

*Author for correspondence

SUMMARY We have compared the intracellular localization of plasma membrane proteins anchored either with a transmembrane segment or with a glycosylphosphatidylinositol moiety to estimate the effects of membrane anchor on protein segregation in the non-polarized form of the human colon cancer cell line HT-29 18. We have monitored two endogenous proteins: the carcinoembryonic antigen, a glycosylphosphatidylinositol protein and the transmembrane protein dipeptidyl peptidase IV, and two transfected proteins: the glycosylphosphatidylinositol protein Thy-1 and an engineered transmembrane form of Thy-1. Using immunocytochemistry on ultra-thin cryosections and confocal microscopy, we detected a carcinoembryonic antigen-rich vesicular compartment, excluding classical pre-lysosomal and lysosomal markers such as mannose 6-phosphate receptor, lamp-1 and cathepsin D. This compartment,

where carcinoembryonic antigen accumulated, excluded the transmembrane protein dipeptidyl peptidase IV and was reduced during the polarization of the cells. Moreover, the glycosylphosphatidylinositol form of Thy-1 also accumulated in the carcinoembryonic antigen-rich compartment whereas the transmembrane form of Thy-1 was excluded. We proposed that, in the non-polarized HT-29 18 cells, accumulation of glycosylphosphatidylinositol proteins independently of transmembrane proteins reveals different intracellular fates for proteins according to their anchor in the plasma membrane.

INTRODUCTION

membrane (TM) segment or a glycosylphosphatidylinositol (GPI) moiety, apical proteins can follow different intracellular routes in polarized cells. Transmembrane proteins can be transiently expressed on the basolateral membrane before being rerouted to the apex by transcytosis (Quaroni et al., 1979; Bartles and Hubbard, 1988; Bartles et al., 1987; Massey et al., 1987; Matter et al., 1990; Le Bivic et al., 1990). However, there is a discrepancy between the proportion of apical transmembrane proteins following this indirect route compared to the proportion reaching the apical surface directly. This may depend on the protein itself and/or the cell type (Hammerton et al., 1992; Mostov et al., 1992). Conversely, GPI-anchored proteins are routed directly to the apical membrane and are sorted intracellularly from the other class of proteins presumably by inclusion in glycosphingolipid patches (Lisanti et al., 1988, 1989; Lisanti and Rodriguez-Boulan, 1990; Brown and Rose, 1992; Brown et al., 1989; Garcia et al., 1993). These glycosphingolipid patches, sorted in the TGN, are supposed to be part of vesicles

The transepithelial transport and vectorial functions of epithelial tissues are related to the structural and functional polarity of individual epithelial cells. In particular, the apical and basolateral membrane domains of the cell surface have distinct protein and lipid compositions that are needed to achieve the asymmetric functions of epithelial cells. To establish and maintain this asymmetric distribution, an epithelial cellspecific machinery actively sorts and targets membrane components to their final destination (Simons and Fuller, 1985; Wollner and Nelson, 1992; Matlin, 1992; Louvard et al., 1992). Over the past few years considerable attention has been devoted to the vesicular transport of apical membrane proteins after they pass through the Golgi complex and the trans-Golgi network (TGN) (Griffiths and Simons, 1986; Simons and Wandiger-Ness, 1990; Rodriguez-Boulan and Powell, 1992). According to their anchor in the membrane, either a trans-

Key words: membrane protein, carcinoembryonic antigen, dipeptidylpeptidase IV, cell polarity, intestinal epithelium, cancer cell line

2680 F. Nicolas and others directly reaching the apical membrane (Simons and van Meer, 1988). The sorting and transport of apical membrane proteins have been extensively studied in polarized cell lines, but it is not known whether this mechanism is sufficient per se to induce cell polarization. We have decided to investigate the intracellular behaviour of differently anchored apical proteins before the cell polarization process takes place. We have chosen to use the human colon cancer cell line HT-29 18 because this cell line can be maintained in a non-polarized form or maintained in a polarized form, according to the tissue culture conditions (Pinto et al., 1982; Godefroy et al., 1988). We have followed the fates of several proteins that are anchored differently in the apical plasma membrane: GPIanchored proteins versus transmembrane proteins. Two of these proteins, carcinoembryonic antigen (CEA), a GPIanchored protein (Hefta et al., 1988; Jean et al., 1988; Sack et al., 1988; Takami et al., 1988) and dipeptidyl peptidase IV (DPP IV), a transmembrane protein (Hong and Doyle, 1990), have an apical localization in polarized HT-29 18 cells and are two of the rare apical membrane proteins significantly expressed in the non-polarized form of the cells (Le Bivic et al., 1987; Garcia et al., 1991). To address more precisely the role of membrane anchor, we expressed in HT-29 18 cells two differently anchored forms of the lymphocyte adhesion molecule Thy-1, a GPI-form and an engineered transmembrane form. The results indicate that GPI-anchored proteins accumulated intracellularly, in a specific compartment, independently from transmembrane proteins. MATERIALS AND METHODS Reagents Cell culture reagents were purchased from Flow (Irvine, Scotland). Foetal calf serum was purchased from Biosys (Compiègne, France). Glutaraldehyde was from Ladd Research Industries (Burlington, VT). Protein A was from Pharmacia (Saint Quentin, France). BSA-FITC was from Sigma (L’Isle d’Abeau, France). All others chemicals were reagent grade.

discarded after 15 passages. Polarized HT-29 18 cells were grown in the same conditions except that glucose was replaced by 5 mM galactose. Polarized cells were kept for ten days at confluence prior to use. Transfection of HT-29 18 cells Non-polarized HT-29 18 cells were transfected with DNA fragments coding for the GPI protein Thy 1.2 or for an engineered transmembrane form of Thy 1.2 (Tiveron et al., 1994). For this purpose 8×106 cells were electroporated at 250 V, 1500 pF with 50 µg of DNA and seeded in 25 cm2 plastic flasks. At 72 hours after electroporation, 0.3 mg/ml G418 was added and kept routinely for transfected cell culture. Two weeks later, the surviving cells were removed from the flasks with 5 mM EDTA in PBS and incubated for 1.5 hours in DMEM without FCS at 37°C with Dynabeads anti-rat IgG (Dynal, Oslo, Normay) coated overnight with monoclonal anti-Thy-1 antibody. After magnetic collection of the beads and several rinses in DMEM, cells were seeded in 10 cm Petri dishes. Individual colonies were isolated and expanded. Cell lines stably expressing high levels of either GPI-Thy-1 or TM-Thy-1 were kept for study. Semi-quantitative immunocytochemistry Cells (106) were seeded in 25 cm2 plastic flasks and used four days after confluence. Cells were fixed with 2% formaldehyde or 2% formaldehyde, 0.1% glutaraldehyde in PBS containing 10 mM Ca2+, 10 mM Mg2+ and were infiltrated with 2.0 M sucrose in the same buffer. Ultra-thin cryosections were prepared according to Tokuyasu (1973) on a RMC MT-7 cryoultramicrotome (Tucson, USA). They were labelled with antibodies to antigen 517 (1 µg/ml), CEA (1.2 µg/ml), cathepsin D (1:400), CI-MPR (1/500), G1/139 (supernatant 1:3), A3/775 (supernatant 1:50), antibodies to Thy-1 (1:25) and goldconjugated antibodies or gold-conjugated Protein A (used at A=0.15). Labelled sections were positively-negatively stained according to the method of Griffiths et al. (1983). For quantitative analysis, four independent sets of cells were processed and sections were doublelabelled with antibodies to CEA and antibodies to one of the three lysosomal proteins studied. At least two grids were examined from each experiment and four micrographs were made randomly from each grid at a magnification of ×25,000. We evaluated the number of immunogold particles revealing CEA in two locations: the vesicles where CEA was detected alone, and the vesicles where CEA was detected in association with a lysosomal protein. No intracellular CEA labelling was observed in these two locations.

Antibodies Monoclonal antibody (mAb) 517 was described previously (Le Bivic et al., 1987); rabbit anti-bovine cathepsin D and mAb anti-cationindependent mannose 6-phosphate receptor (CI-MPR) described by Griffiths et al. (1990) were gifts from Dr B. Hoflack (EMBL, Heidelberg, Germany); mAb G1/139 anti-Lamp-1 and mAb A3/775 antiDPP IV were gifts from Dr H.-P. Hauri (Biozentrum des Universität, Basel, Switzerland). Rat monoclonal antibody to mouse Thy-1.2 was from Boehringer Mannheim (Meylan, France). Polyclonal antibodies anti-CEA were from Dakopatts (Glostrup, Denmark); goat anti-mouse IgG coupled to horseradish peroxidase, FITC or TRITC were from Biosys (Compiègne, France); gold-conjugated antibodies were from Biocell (Cardiff, Wales); and gold-conjugated Protein A was prepared according to the method of Slot and Geuze (1985). Cell culture Non-polarized HT-29 18 cells (Godefroy et al., 1988) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum and 2 mM glutamine, in an atmosphere of 10% CO2/90% air at 37°C. To maintain the cell line, non-polarized culture conditions were standardized as follows: the medium was changed at least daily to avoid glucose exhaustion, which induces cell polarization; the cell culture was split every two days and 2×106 cells were seeded in a 25 cm2 plastic flask to avoid cyst formation. Cell cultures were

Fig. 1. Immunoblot characterization of the antigens recognized in non-polarized HT-29 cells, grown on glucose, by mAb 517, (lane A), by polyclonal antibodies to CEA (lane B). Size standards (in kDa) are shown at the left.

GPI-membrane anchor and intracellular accumulation of proteins 2681

Fig. 2. Comparative distribution of CEA and DPP IV in non-polarized HT-29 cells as shown in confocal microscopy. Top planes (a,d), middle planes (b,e) and bottom planes (c,f) are shown. Internal CEA (a,b,c) labelling was scattered throughout the cells and was more pronounced in perinuclear regions. DPP IV (d,e,f) labelling was observed intracellularly and on the plasma membrane.

Confocal microscopy Cells were seeded on 10-well microscope slides from CML (Nemours, France) at a density of 40,000 cells/cm2 (20,000 cells per well) and used four days after confluence. The fluorescence labelling was performed according to described procedures (Reggio et al., 1983)

except that the cells were permeabilized with 0.75% Triton X-100 and 0.1% Saponin in 2-[morpholino]ethane sulfonic acid (MES) buffer, pH 6.0, at room temperature for 30 minutes after fixation. Saponin (0.05%) was present throughout the experiment. The cells were mounted in Mowiol containing 2.5% 1,4-diazabicyclo-(2,2,2)-octane

2682 F. Nicolas and others

Fig. 3. Ultrastructural localization of CEA and lamp 1. Thin cryosections from nonpolarized HT-29 cells were double-labelled with polyclonal antibodies to CEA (gold 10 nm) and mAb to lamp-1 (gold 5 nm). Doublelabelling was observed in multi-vesicular bodies (mvb). Small vesicles were singlelabelled with anti-CEA. (A) Colocalization of CEA and lamp 1 in a mvb, (B) A CEA vesicle (arrow) in the vicinity of a mvb. (C,D) Details of CEA compartment. Bars, 0.2 µm. (DABCO) and observed in a Leitz confocal laser scanning microscope and analyzed according to the procedure of Rémy et al. (1990). Eight pictures were taken, from the top to the cell-substratum plane, with an interspace of 0.4 µm between each plane. Gel electrophoresis and immunoblotting SDS-PAGE was carried out as described by Laemmli (1970), and the proteins were transferred from the gel to nitrocellulose according to

Burnette (1981). The cells were lysed with Triton X-100 (1%) containing a cocktail of protease inhibitors (5 µg/ml leupeptin, 5 µg/ml pepstatin A, 75 mg/ml benzamidine and 5 mg/ml antipaine A). The lysate was centrifuged at 10,000 g for 10 minutes in an Eppendorf centrifuge and 25 µl of the supernatant were loaded without reduction on an 8% acrylamide-polyacrylamide gel. After electrophoresis and transfer to nitrocellulose, CEA was detected with the relevant antibodies and revealed by peroxidase-conjugated antibodies.

GPI-membrane anchor and intracellular accumulation of proteins 2683 ELISA measurement ELISA measurements (Engvall, 1980) of CEA were done as follows: for measurements of CEA on the cell surface, 18×106 cells in a plastic Petri dish were incubated for 30 minutes in PBS containing 0.2% gelatin and 0.03% NaN3 as metabolic inhibitor, and then for 90 minutes at 4°C with mAb 517 (2 µg/ml) in the same buffer. For total CEA measurements, the same procedure was used except that 0.05% Saponin was present. The cells were washed 6 times with incubation buffer, collected and homogenized by passing through a 25 Gauge syringe needle in a buffer containing 3 mM imidazole, 250 mM sucrose, 1 mM EDTA, 1% Triton X-100 at pH 7.4. After centrifugation for 10 minutes at 10,000 g, the supernatant was collected and assayed for CEA. Meanwhile, 96-well plates (Maxisorp, Nunc) were incubated overnight at 4°C with polyclonal antibodies anti-CEA (100 µl at 1.2 µg/ml) in a carbonate/bicarbonate 50 mM buffer, pH 9.6. The wells were washed 6 times and incubated for 60 minutes in PBS containing 0.1% haemoglobin and 0.05% Tween-20. The cell supernatants (100 µl) were incubated in the wells for 60 minutes at 37°C. The CEA-mAb 517 complexes were detected with peroxidase-conjugated polyclonal anti-mouse IgG for 60 minutes at 37°C. The assay was developed with 2.2 mM orthophenylenediamine 2HCl in a citrate/phosphate buffer (pH 6.5) for 30 minutes. The plates were read at 492 nm. The assay was standardized with a serum of known concentration for CEA kindly provided by Dr Choux, Sainte-Marguerite Hospital, Marseille.

RESULTS To characterise the CEA antigens recognized by different antibodies in non-polarized HT-29 18 cells, we have performed immunoblotting on whole-cell extracts (Fig. 1). mAb 517 and polyclonal antibodies to CEA recognized a single band with a Mr of 170. This indicates that the major component recognized by the antibodies corresponds to the fully glycosylated form of the protein in these cells (Garcia et al., 1991). No crossreactions with CEA-related molecules was observed. Carcinoembryonic antigen is accumulated intracellularly in HT-29 18 cells HT-29 18 cells were maintained in the non-polarized form by the presence of glucose and defined culture conditions (see Godefroy et al., 1988, and Material and Methods). Using confocal laser scanning microscopy on whole fixed and permeabilized cells, CEA was found in intracellular vesicles scattered within the cell cytoplasm (Fig. 2a,b,c). A small amount of CEA was occasionally observed along the plasma membrane in a limited number of cells. This surface staining was significantly increased when the cells were kept at confluence for more than four days or after more than 15 passages and so polarized. To quantitate the proportion of intracellular CEA, we have used an ELISA assay. In cells permeabilized with Saponin, the total CEA was 34.3±0.7 ng per 106 cells. When the assay was performed solely on the cell surface, CEA was 3.5±0.6 ng per 106 cells, which was about 10% of the total. We have investigated the intracellular localization of CEA with regard to post-Golgi degradation compartments. Ultrathin cryosections were double-labelled with antibodies to lamp 1, or cathepsin D or CI-MPR, present in lysosomes and prelysosomes and with antibodies to CEA. The locations of the antibodies on the sections were then revealed with gold-conjugated antibodies or gold-conjugated Protein A and the sections

Fig. 4. Ultrastructural localization of CEA and CI-MPR. Thin cryosections from non-polarized HT-29 cells were double-labelled with mAb to CEA (gold 10 nm) and polyclonal antibodies to CIMPR (arrowheads, gold 5 nm). (A) Double-labelling was observed in multi-vesicular bodies, (B) CEA vesicles (arrow) were not labelled with CI-MPR but CI-MPR-positive vesicles near the plasma membrane were not labelled with CEA (arrowhead). Bars, 0.1 µm.

contrasted for electron microscopy (Fig. 3). To quantitate the fraction of intracellular CEA present in the degradation pathway, the number of gold particles of CEA labelling in vesicles together with the lysosomal markers was scored and compared to the total intracellular particles of CEA labelling (Figs 3, 4 and Table 1). The results indicated that about 50% of the CEA was found in vesicles without any lysosomal proteins. Conversely, the other half of the CEA labelling was found in lysosomes and pre-lysosomes colocalised with the lysosomal markers. The CEA structures, free of lysosomal proteins, consisted of small vesicles (average size: 0.15 µm) that had no visible internal structure and were clearly distinguishable from lysosomes and pre-lysosomes (multi-vesicular bodies). It was important to examine whether the intracellular accumulation of CEA in a compartment that lacked lysosomal proteins was related to the endocytic pathway since the CEA molecules could result from endocytosis from the plasma membrane. Cells were incubated from 10 minutes to 60 minutes with BSA-FITC (1 mg/ml) or gold-conjugated BSA


Fig. 5. Double-fluorescence labelling of CEA and fluidphase marker as shown in confocal microscopy. Top (a), middle (b,c) and bottom (d) planes were shown. The cells were incubated with BSA-FITC for 30 minutes, then fixed and stained with polyclonal antibodies to CEA. BSA-FITC-containing early endosomes did not colocalize with CEA. Bar, 10 µm.

(A=0.2) as a fluid-phase endocytosis marker in the culture medium. After fixation and labelling with antibodies to CEA, confocal microscopy revealed that CEA and BSA did not colocalize (Fig. 5). No significant differences were observed for the different times of BSA incubation. At 60 minutes of incubation, electron microscopy revealed that gold-conjugated BSA was never observed in multi-vesicular bodies or lysosomes (not shown). Therefore, CEA was not significantly present in early endocytic compartments. Moreover, CEA labelling was never observed clustered at the cell surface in endocytic structures such as coated pits or caveolae (not shown). Accumulation of CEA molecules decreased in polarized HT-29 18 cells In HT-29 18 cells grown in the presence of glucose, less than 10% of the total cell population, at confluence, were polarized along intercellular lumens, called cysts (Le Bivic et al., 1987). The number of cysts can be increased by keeping the cells at confluence for more than 10 days. The cells engaged in cyst formation displayed little cytoplasmic labelling for CEA but intense labelling at the cyst membrane (Fig. 6A). Cells not involved in cyst formation still displayed intense intracellular CEA labelling. When HT-29 18 cells were fully polarized by culturing them in the presence of galactose instead of glucose, CEA was found at the apical membrane and little cytoplasmic labelling was detected (Le Bivic et al., 1987). By electron microscope quantitative analysis on ultrathin cryosections of fully polarized cells, surface labelling for CEA was 69% of the total. It was then of interest to compare the CEA intracellular distribution with that of lysosomal proteins as in non-polarized cells. Some molecules remained in lysosomal structures, but the intracellular CEA that accumulated in the compartment free of

Table 1. Comparison of CEA in polarized and nonpolarized cells (A) Colocalization between CEA and lysosomal proteins in non-polarized cells Relative distribution of CEA Lysosomal protein Lamp-1 Cathepsin D CI-MPR

Total CEA With a Alone in No of No of grains lysosomal vesicular experiments micrographs counted protein compartment 4 4 4

54 35 34

3767 805 1317

0.52 0.51 0.51

0.48 0.49 0.49

(B) Colocalization between CEA and lamp 1 in polarized cells Relative distribution of CEA Lysosomal protein Lamp-1

Total CEA With a Alone in No of No of grains lysosomal vesicular experiments micrographs counted protein compartment 4

42

2908

0.74

0.26

lamp 1 was greatly decreased (Table 1). Under these conditions only 26% of the intracellular CEA was in such structures. CEA is accumulated independently from dipeptidyl peptidase IV, an apical transmembrane protein in non-polarized HT-29 18 cells We have questioned whether CEA was segregated from transmembrane proteins of the plasma membrane in non-polarized cells. DPP IV was found on the plasma membrane and in intracellular perinuclear vesicles by confocal microscopy (Fig.


Fig. 6. Transformation of CEA distribution during polarization. After 10 days of confluency, the cells were fixed and stained with polyclonal antibodies to CEA antibodies. (A) Immunolabelling, (B) phase-contrast. Compared to the non-polarized cells, intracellular CEA was greatly reduced in cells engaged in the formation of intercellular cysts (arrows), but accumulated on the membrane of the cysts. Bars, 25 µm.

Fig. 7. Comparative distribution of CEA and DPP IV. (A,B) Thin cryosection from non-polarized HT-29 18 cells were double-labelled with polyclonal antibodies to CEA (gold 10 nm) and mAb to DPP IV (gold 5 nm). Double-labelling was observed in multi-vesicular bodies (mvb). The CEA vesicles were single-labelled (arrow). Bars, 0.2 µm

Fig. 8. Expression of transfected Thy-1 in HT-29 18 cells. Cryosections were labelled with a monoclonal antibody to Thy-1 then revealed with FITC-conjugated antibodies. (a,c,e) Immunofluorescence; (b,d,f) phase-contrast. (a,b) Control: untransfected HT-29 18 cells. (c,d) GPI-Thy-1transfected cells. (e,f) TM-Thy-1-transfected cells. Bar, 10 µm.


Fig. 9. Colocalization of CEA and GPIThy-1.2. Thin cryosections from nonpolarized HT-29 18 cells, transfected with GPI-Thy-1.2, were double-labelled with polyclonal antibodies to CEA (gold 10 nm) and mAb to Thy-1.2 (gold 5 nm). (A) Double-labelling was observed in multivesicular bodies (mvb). (B,C) Double-labelling in the vesicular compartment. Bars, 0.1 µm.

2d,e,f). Double-labelling performed by electron microscopy indicated that CEA and DPP IV were colocalized in multivesicular bodies related to the degradation pathway (Fig. 7), but the small vesicles where CEA was observed without lysosomal markers, were never labelled with antibodies to DPP IV.

Membrane anchor is determinant for Thy-1 intracellular accumulation We next tested whether the different sorting behaviour of the two apical proteins and the intracellular accumulation of CEA were due to the manner in which the proteins were anchored in the plasma membrane. We expressed two isoforms of the


Fig. 10. Different localization of CEA and TM-Thy-1.2. (A,B) Thin cryosections from non-polarized HT-29 18 cells, transfected with TM-Thy-1.2, were double-labelled with polyclonal antibodies to CEA (gold 10 nm) and mAb to Thy-1.2 (gold 5 nm). Thy-1 was found exclusively expressed at the plasma membrane (arrowheads) and was absent from the CEA vesicular compartment (arrows). Bars, 0.1 µm.

Thy-1 molecule in the non-polarized form of HT-29 18 cells. Native Thy-1 (GPI-Thy-1) DNA and an engineered transmembrane form of Thy-1 (TM-Thy-1) DNA (Tiveron et al., 1994) were transfected and cells that stably express high levels of GPI-Thy-1 or TM-Thy-1 were isolated. Immunofluores-

cence labelling on cryosections with a monoclonal antibody to Thy-1 confirmed the efficiency of transfection and showed very different localizations for GPI-Thy-1 (intracellular) and TM-Thy1.2 (plasma membrane) (Fig. 8). The intracellular localizations of GPI-Thy-1 and TM-Thy-1

2688 F. Nicolas and others were compared to that of endogenous CEA. Ultrathin cryosections were double-labelled with a monoclonal antibody to Thy1 and with polyclonal antibodies to CEA. GPI-Thy-1 was found localized with the CEA in multi-vesicular-bodies, in the vesicular compartment where CEA was previously found accumulated, and occasionally on the plasma membrane (Fig. 9). In contrast, TM-Thy-1 labelled exclusively the plasma membrane and was markedly excluded from the CEA containing vesicles (Fig. 10). We concluded that intracellular accumulation of CEA and GPI-Thy-1 were directly dependent on the type of membrane anchor. Therefore, we propose to call this vesicular compartment where CEA and GPI-Thy-1 accumulated the GPI compartment. DISCUSSION The differential sorting and transport pathways of apical membrane proteins anchored either with a glycosylphosphatidylinositol (GPI) moiety or with a hydrophobic peptide have been extensively studied in polarized epithelial cells. It is clear that the polarized phenotype is dependent on a sorting mechanism of membrane proteins, but it is not yet known whether this mechanism exists before cell polarization. Therefore, we used immunocytochemistry to study this question in a cloned cell line that can be maintained in a polarized or in a non-polarized form, HT-29 18. Our results indicate that GPI-anchored proteins were stored intracellularly in non-polarized cells within specific vesicles that excluded transmembrane proteins. Quantitative analysis on ultrathin cryosections doublelabelled with antibodies to the cation-independent mannose 6phosphate receptor, or lamp 1, or cathepsin D and with antibodies to CEA indicated that half of the intracellular CEA molecules were localized in the lysosomal pathway composed of multi-vesicular bodies, pre-lysosomes and lysosomes. The other half was present in small vesicles distinct from multivesicular bodies and dense lysosomes. This vesicular compartment is not related to the endocytic pathway since fluid-phase markers like BSA-FITC did not reach it after 30 minutes and since CEA-anti CEA antibodies complexes were not internalized from the cell surface (data not shown). Moreover, CEA was never observed in CI-MPR-positive vesicles near the plasma membrane nor clustered in coated pits or caveolae. Thus CEA seems to reach the lysosomal compartments without transit by the plasma membrane or by early endocytic compartments. On the other hand, the amount of CEA released from the plasma membrane by non-polarized HT-29 is very low as compared to that released by polarized cells (not shown and Fantini et al., 1986). The CEA molecules, detected on immunoblots with the antibodies used in this study, displayed a Mr of 170, which is that of the complex glycosylated form (Garcia et al., 1991). This suggests that the accumulation of CEA occurs after the proteins have transited through the Golgi complex. A drastic diminution of the amount of CEA in intracellular vesicles was observed in polarized HT-29 cells with a concommitant increase of the antigen in the membrane surrounding the intercellular cyst. To determine whether the accumulation of CEA molecules is also the fate for apical proteins before the cell polarization takes place, we have compared the intracellular localization of CEA with that of DPP IV, an apical transmembrane protein. DPP IV was detected at the plasma membrane and in

lysosomal organelles but was never found in the CEA-rich vesicles. Therefore, the accumulation of proteins in these vesicles is not a general feature of apically destined membrane proteins. It is known that in Caco-2 and LLC-PK1 cells, CEA and DPP IV do not follow the same intracellular route on their way to the apical membrane. CEA is targeted directly to the apical membrane whereas DPP IV and transmembrane proteins are inserted first, at least in part, into the basolateral membrane before being rerouted to their final destination (Low et al., 1991; Matter et al., 1990; Le Bivic et al., 1990). However, the route followed depends on the cell type under consideration. For instance, it has been shown that DPP IV, transfected into MDCK cells reached the apical surface directly (Casanova et al., 1991) whereas it follows exclusively a baso-lateral route in hepatocytes (Bartles et al., 1987). It was then important to examine the effects of membrane anchorage upon accumulation of molecules in non-polarized cells. In many cells, the GPI anchor has been considered as a signal for direct apical routing (Lisanti et al., 1988, 1989; Lisanti and Rodriguez-Boulan, 1990; Brown et al., 1989) and could be involved in the intracellular sorting machinery. This may imply hydrophobic interactions between fatty acid chains and hydrogen bonding between polar heads of specific glycosphingolipids, which are transported to the apical membrane (Simons and van Meer, 1988; vant’Hof and van Meer, 1990). In hepatocytes, however, a direct route to the apical membrane seems to be excluded and the GPI protein 5′ nucleotidase is transported via the basolateral route (Schell et al., 1992). In the non-polarized intestinal cell line HT-29 18, the role of membrane anchor in intracellular accumulation of apically destined proteins was addressed using two isoforms of Thy-1. This molecule has an intrinsic apical signal in its exoplasmic domain (Powell et al., 1991). GPI-Thy-1 and an engineered transmembrane form TMThy-1 (Tiveron et al., 1994) were expressed in non-polarized HT29 18 cells. GPI-Thy-1 was detected at the plasma membrane, in lysosomal organelles but was also found with the CEA in the vesicular compartment. In contrast, the transmembrane form, TM-Thy-1, was detected only at the plasma membrane. We propose that the vesicular compartment where CEA and GPI-Thy-1 accumulate could be relevant to all GPI proteins and therefore we propose to call it the GPI compartment. The GPI compartment could be an obligatory intermediate compartment needed for the export of proteins to the cell surface. CEA and GPI protein-containing vesicles could fuse very slowly with the plasma membrane in the absence of a differentiated apical domain and therefore accumulate intracellularly to form the GPI compartment. The accumulation of proteins in the GPI compartment depending on membrane anchor clearly indicates the presence of a sorting mechanism that is independent of the polarized phenotype. Whether this is due to a sorting mechanism or to a retention mechanism are interesting questions for future work. We thank Dr Bernard Hoflack for the gift of antibodies to CI-MPR and cathepsin D, Dr Hans-Peter Hauri for the gift of monoclonal antibody to DPP IV and lamp 1 and for valuable criticisms. We thank Dr Ann Hubbard for the critical reading of the manuscript and helpful suggestions. This work was supported by the CNRS, by the INSERM (CRE 900707), by the Association pour la Recherche sur le Cancer, by the Ligue Nationale Française contre le Cancer, and by the Groupement Français des Entreprises en Lutte contre le Cancer.

GPI-membrane anchor and intracellular accumulation of proteins 2689 REFERENCES Bartles, J. R., Ferracci, H. M., Steiger, B. and Hubbard, A. L. (1987). Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. J. Cell Biol. 105, 1241-1251. Bartles, J. R. and Hubbard, A. L. (1988). Plasma membrane protein sorting in epithelial cells: Do secretory pathways hold the key? Trends Biochem. Sci. 13, 181-184. Brown, D. A., Crise, B. and Rose, J. K. (1989). Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science 245, 1499-1501. Brown, D. A. and Rose, J. K. (1992). Sorting of the GPI-anchored proteins to glycolipid-enriched subdomains during transport to the apical cell surface. Cell 68, 533-544. Burnette, W. N. (1981). Electrophoretic transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radio-iodinated Protein A. Anal. Biochem. 112, 195-203. Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L. and Mostov, K. E. (1991). Direct apical sorting of rat liver dipeptidylpeptidase IV expressed in Madin-Darby Canine Kidney cells. J. Biol. Chem. 266, 24428-24432. Engvall E. (1980). Immunoassay methods ELISA and EMIT. Meth. Enzymol. 70, 419-439. Fantini, J., Abadie, B., Tirard, A., Remy, L., Ripert, J. P., El Battari A. and Marvaldi, J. (1986). Spontaneous and induced dome formation by two clonal cell populations derived from a human adenocarcinoma cell line HT29. J. Cell Sci. 83, 235-249. Garcia, M., Seigner, C., Bastid, C., Choux, R., Payan, M. J. and Reggio, H. (1991). Carcinoembryonic antigen has a different molecular weight in normal colon and cancer due to N-glycosylation differences. Cancer Res. 51, 5679-5686. Garcia, M., Mirre, C., Quaroni, A., Reggio, H. and Le Bivic, A. (1993). GPIanchored proteins associate into microdomains during their intracellular transport in Caco2 cells. J. Cell Sci. 104, 1281-1290. Godefroy, O., Huet, C., Blair, L., Sahuquillo-Merino, C. and Louvard, D. (1988). Differentiation of a clone isolated from the HT-29 cell line: polarized distribution of histocomptability antigens (HLA) and of transferrin receptors. Biol. Cell 63, 41-55. Griffiths, G., Simons, K., Warren, G. and Tokuyasu, T. K. (1983). Immunoelectron microscopy using thin frozen sections: applications to studies of the intracellular transport of Semliki forest virus spike glycoproteins. Meth. Enzymol. 96, 466-485. Griffiths, G. and Simons, K. (1986). The trans-Golgi-network: sorting at the exit site of the Golgi complex. Science 234, 4381-4443. Griffiths, G., Matteoni, R., Back, R. and Hoflack B. (1990). Characterization of the cation-independent mannose 6-phosphate receptor-enriched prelysosomal compartment in NRK cells. J. Cell Sci. 95, 441-461. Hammerton, R. W., Krzeminski, K. A., Mays, R. W., Ryan, R. A., Wollner, D. A. and Nelson, W. J. (1992). Mechanism for regulating cell surface distribution of Na+,K+-ATPase in polarized epithelial cells. Science 254, 847-850. Hefta, S. A., Hefta, L. J. F., Lee, T. D., Paxton, R. J. and Shively, J. E. (1988). Carcinoembryonic antigen is anchored to membranes by covalent attachment to a glycosylphosphatidylinositol moiety: identification of the ethanolamine linkage site. Proc. Nat. Acad. Sci. USA 85, 4648-4652. Hong, W. and Doyle, D. (1990). Molecular dissection of the NH2-terminal signal/anchor sequence of rat dipeptidylpeptidase IV. J. Cell Biol. 111, 323328. Jean, F., Malapert, P., Rougon, G. and Barbet, J. (1988). Cell membrane, but not circulating carcinoembryonic antigen is linked to a phosphatidylinositol containing hydrophobic domain. Biochem. Biophys. Res. Commun. 155, 794-800. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Le Bivic, A., Hirn, M. and Reggio, H. (1987). HT-29 cells are an in vitro model for the generation of cell polarity in epithelia during embryonic differentiation. Proc. Nat. Acad. Sci. USA 85, 136-140 Le Bivic, A., Quaroni, A., Nichols, B. and Rodriguez-Boulan, E. (1990). Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line. J. Cell Biol. 110, 1533-1539. Lisanti, M. P., Sargiacomo, M., Graeve, L., Saltiel, A. R. and Rodriguez-Boulan, E. (1988). Polarized apical distribution of glycosylphosphatidylinositol anchored proteins in a renal epithelial cell line. Proc. Nat. Acad. Sci. USA 85, 9557-9561.

Lisanti, M. P., Caras, I. W., Davitz, M. A. and Rodriguez-Boulan, E. (1989). A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J. Cell Biol. 109, 2145-2156. Lisanti, M. P. and Rodriguez-Boulan, E. (1990). Glycosphingolipids membrane anchoring provides clues to the mechanism of protein sorting in polarized epithelial cells. Trends Biochem. Sci. 15, 113-118. Louvard, D., Kedinger, M. and Hauri, H. P. (1992). The differentiating intestinal epithelial cell: establishment and maintenance of functions through interactions between cellular structures. Annu. Rev. Cell Biol. 8, 157-196. Low, S. H., Wong, S. H., Tang, B. L., Tan, P., Subramaniam, V. N. and Hong, W. (1991). Inhibition by Brefeldin A of protein secretion from the apical cell surface of Madin-Darby Canine Kidney cells. J. Biol. Chem. 266, 17729-17732. Massey, D., Ferracci, H., Gorvel, J. P., Rigal, A., Soulié J. M. and Maroux, S. (1987). Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes. J. Membr. Biol. 96, 19-25. Matlin, K. S. (1992). W(h)ither default? Sorting and polarization in epithelial cells. Curr. Opin. Cell Biol. 4, 623-628. Matter, K., Brauchbar, M., Bucher, E. R. and Hauri, H. P. (1990). Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco2). Cell 60, 429-437. Mostov, K., Apodaca, G., Aroeti B. and Okamoto, C. (1992). Plasma membrane protein sorting in polarized epithelial cells. J. Cell Biol. 116, 577583. Pinto, M., Appay, M. D., Simon-Assman, P., Chevalier, G., Dracopoli, N., Fogh J. and Zweibaum, A. (1982). Enterocytic differentiation of cultured human colon cancer cells by replacement of glucose by galactose in the medium. Biol. Cell 44, 193-196. Powell, S. K., Lisanti, M. P. and Rodriguez-Boulan, E. (1991). Thy-1 expresses two signals for apical localization in epithelial cells. Am. J. Physiol. 260, 715-720. Quaroni, A., Kirsch, K. and Weiser, M. M. (1979). Synthesis of membrane glycoproteins in rat small intestinal villus cells. Biochem. J. 182, 203-212. Reggio, H., Webster, P. and Louvard, D. (1983). Use of immunocytochemical techniques in studying the biogenesis of cell surfaces in polarised epithelia. Meth. Enzymol. 98, 379-395. Rémy, L., Gorvel, J. P., Jacquier, M. F., Rigal, A. and Davoust, J. (1990). Confocal microscopy as a tool to reveal the tridimensional organization of intracellular lumens and intercellular cysts in a human colon adenacarcinoma cell line. Biol. Cell 69, 129-138. Rodriguez-Boulan, E. and Powell, S. K. (1992). Polarity of epithelial and neuronal cells. Annu. Rev. Cell Biol. 8, 395-427. Sack, T. L., Gum, J. R., Low, M. G. and Kim, Y. S. (1988). Release of carcinoembryonic antigen from human colon cancer cells by phosphatidylinositol-specific phospholipase C. J. Clin. Invest. 82, 586-593. Schell, M. J., Maurice, M., Stieger, B. and Hubbard, A. R. (1992). 5′ Nucleotidase is sorted to the apical domain of hepatocytes via an indirect route. J. Cell Biol. 119, 1173-1182. Simons, K. and Fuller S. D. (1985). Cell surface polarity in epithelia. Annu. Rev. Cell Biol. 1, 243-288. Simons, K. and van Meer, G. (1988). Lipid sorting in epithelial cells. Biochemistry 27, 6197-6202. Simons, K. and Wandiger-Ness, A. (1990). Polarized sorting in epithelia. Cell 62, 207-210. Slot, J. W. and Geuze, H. J. (1985). A new method for preparing gold probes for multiple-labeling cytochemistry. Eur. J. Cell Biol. 38, 87-93. Takami, N., Misumi, Y., Kuroki, M., Matsuoka, Y. and Ikegara, Y. (1988). Evidence for carboxyl-terminal processing and glycolipid-anchoring of human carcinoembryonic antigen. J. Biol. Chem. 262, 12716-12720. Tiveron, M.-C., Nosten-Bertrand, M., Jani, H., Garnett, D., Hirst, E. M. A., Grosveld, F. and Morris, R. J. (1994). The mode of anchorage to the cell surface determines both the function and membrane location of Thy 1 glycoprotein. J. Cell Sci. 107 (in press). Tokuyasu, K. T. (1973). A technique for ultracryotomy of cell suspension and tissues. J. Cell Biol. 57, 551-565. vant’Hof, W. and van Meer, G. (1990). Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphigolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane. J. Cell Biol. 111, 977-986. Wollner, D. A. and Nelson, W. J. (1992). Establishing and maintaining epithelial cell polarity. J. Cell Sci. 105, 185-190. (Received 20 April 1994 - Accepted 21 June 1994)