GPI-anchored proteins associate to form

Journal of Cell Science 104, 1281-1290 (1993) Printed in Great Britain © The Company of Biologists Limited 1993

1281

GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells Martine Garcia1, Christian Mirre1, Andrea Quaroni2, Hubert Reggio1 and André Le Bivic1,* 1Biologie de la Différenciation Cellulaire, U.R.A. 179, Faculté des Sciences de Luminy, 13288 Marseille 2Section of Physiology, Division of Biological Science, Cornell University, Ithaca, NY 14853, USA

Cedex 09, France

*Author for correspondence

SUMMARY 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°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.

INTRODUCTION

along the transcytotic pathway as shown for the polyimmunoglobulin receptor (Mostov and Deitcher, 1986) while Caco-2 cells possess a direct pathway followed, for example, by sucrase-isomaltase (SI) (Le Bivic et al., 1990; Matter et al., 1990). Signals that engage proteins along the apical or the basolateral pathways are not well defined yet and nothing is known about the sorting machinery used to package plasma membrane proteins into different vesicles (Mostov et al., 1992). Several studies however have shown recently that the cytoplasmic tail of several basolateral proteins is necessary for basolateral targeting (for review see Mostov et al., 1992) and a correlation between endocytosis and basolateral targeting has been stressed in several cases (Ktistakis et al., 1990; Hunziker et al., 1991; Le Bivic et al., 1991). In contrast the cytoplasmic domain does not seem to play any role in apical targeting, as suggested by several reports (for review see Hopkins, 1991). To date the only defined signal for apical sorting is the glycosyl phosphatidylinositol (GPI) anchor, which links the carboxy terminus of certain proteins to the outer leaflet of the lipid bilayer (Low, 1989). In fact GPI-anchored proteins are preferentially found on the apical membrane of several epithelial cell lines (Lisanti et al., 1988,1990). Furthermore, transfer of the GPIanchor to a basolateral protein redirects it to the apical membrane, suggesting that the GPI-anchor is an

Epithelial cells have distinct morphological and functional features, and in order to achieve their polarized organization they have evolved precise mechanisms for sorting their plasma membrane proteins (for review see RodriguezBoulan and Nelson, 1989; Simons and Wandiger-Ness, 1990). Progress in understanding these mechanisms has been made possible by the use of in vitro models of epithelial cells such as the MDCK cell line (derived from kidney) (Mc Roberts et al., 1981) and the Caco-2 cell line (derived from colon carcinoma) (Pinto et al., 1983). In MDCK cells, newly synthesized plasma membrane proteins are directly transported from the Golgi complex to their final destination and are probably sorted at the level of the trans-Golgi network (TGN) (Griffiths and Simons, 1986). In contrast, in Caco-2 cells, apical proteins are sorted at the post-Golgi level and in the basolateral membrane (Le Bivic et al., 1990; Matter et al., 1990). Aminopeptidase N (APN) and dipeptidylpeptidase IV (DPPIV) are directly transported for the most part to the apical membrane in MDCK cells (Wessels et al., 1990; Casanova et al., 1991; Low et al., 1991) while they mainly travel via the basolateral membrane in Caco-2 cells (Le Bivic et al., 1990; Matter et al., 1990). So far there is no explanation for such a difference, since MDCK cells are able to address proteins

Key words: GPI-anchored proteins, epithelial cells, apical sorting

1282 M. Garcia and others autonomous signal (Brown et al., 1989; Lisanti et al., 1989), although this does not rule out the possibility that the absence of a cytoplasmic tail would be sufficient to sort basolateral proteins to the apical pathway. Simons and van Meer (1988) have proposed that glycosphingolipids (GSLs), which are preferentially delivered to the apical surface, participate in the sorting of apical membrane proteins. GSLs can associate to form patches in the membrane under physiological conditions (Thompson and Tillack, 1985) and GPI-anchored proteins could develop interactions with GSLs to form clusters in the TGN (Lisanti and Rodriguez-Boulan, 1990). These clusters would then be packaged into apical vesicles providing a self-association mechanism for sorting. Unfortunately, interactions between lipids and apical proteins have not been demonstrated directly. GPI-anchored proteins have been reported to be poorly solubilized by non-ionic detergents such as Triton X-100 (TX-100) while transmembrane proteins are solubilized in the same conditions (Hooper and Turner, 1988; Low, 1989). Since GPI-anchored proteins do not interact directly with the submembrane cytoskeleton, their resistance to TX-100 extraction might be due to association with GSLs, which are also insoluble in the same detergent (Hagman and Fishman, 1982). Hemagglutinin (HA) has also been shown to become insoluble in TX-100 at the post-Golgi level during its transport to the cell surface (Skibbens et al., 1989), but there is no evidence yet that it is associated with GPIanchored proteins. In this study we have examined the possibility that GPIanchored proteins are concentrated into membrane microdomains together with some apical transmembrane proteins in Caco-2 cells. Using a solubilization protocol that closely resembles the one recently described by Brown and Rose (1992), we isolated membrane patches enriched in three different endogenous GPI-anchored proteins (alkaline phosphatase (PLAP), carcinoembryonic antigen 180 (CEA180) and melanotransferrin (P97)) and containing some SI, an apical transmembrane hydrolase. These membrane microdomains were devoid of basolateral markers (antigen 525 (Ag525), transferrin receptor (TFR)) and other apical transmembrane proteins such as DPPIV and a transmembrane form of carcinoembryonic antigen (CEA110; Barnett et al., 1989). These results suggest that GPIanchored proteins are closely associated in membrane patches and that some apical transmembrane proteins (such as SI) may form part of these complexes whereas others do not, thus providing new insights into the apical sorting processes operating in intestinal cells.

Immunotech (Marseille, France). Polymyxin B sulfate, Nystatin, Digitonin, n-octyl-glucoside (OG), Triton X-100 (TX-100) and Triton X-114 were from Sigma (St Louis, MO, USA).

Antibodies Rabbit polyclonal antibodies against CEA and PLAP were from Dakopatts (Glostrup, Denmark). Alkaline phosphatase in Caco-2 cells is probably of the intestinal type but as it is not certain we chose to refer to it as PLAP, since the antibody was raised against placental alkaline phosphatase. Mouse monoclonal against TFR was from Boehringer-Mannheim (Mannheim, RFA). Mouse monoclonals against Ag525 (Le Bivic et al., 1988), P97 (Alemany et al., unpublished data), SI (Beaulieu et al., 1989) and DPPIV (Gilbert et al., 1991) have been described previously. Rabbit polyclonal anti-mouse IgG was from Biosys (Compiègne, France). Goat anti-mouse IgG and anti-rabbit IgG conjugated with colloidal gold were from Janssen (Olen, Belgium).

Cell culture Caco-2 cells (Pinto et al., 1983) were grown in DME supplemented with 10% foetal calf serum, non-essential amino acids (1%), penicillin (50 mi.u./ml), and streptomycin (50 µg/ml). For experiments, cells were grown on Transwells (Costar Data Packaging Corp., Cambridge, MA, USA) and were used between 15 and 20 days after confluency to ensure optimal differentiation of the cells.

Phosphatidyinositol-phospholipase C (PI-PLC) treatment PI-PLC treatment was performed according to Lisanti et al. (1988) after Triton X-114 extraction (Bordier, 1981) of Caco-2 cells. After PI-PLC treatment, resulting aqueous and detergent phases were subjected to immunoblot with relevant antibodies.

Pulse-chase experiments Cells grown on filters were incubated for 30 min in DME without cysteine and pulsed for 20 min with the same medium containing 1 mCi/ml of [35S]cysteine. After a rapid wash with DME, cells were chased at 37°C for different times in DME containing 5× cysteine and kept at 4°C in NaHCO3-free DME, 20 mM Hepes, 0.2% BSA before extraction. Chase periods varied as described in the figure legends. For inhibition of N-glycoconjugate processing by 1-deoxymannojyrimycin (dMM), all steps (preincubation, labeling and chase) were done in the presence of 1 mM dMM.

Post-nuclear supernatant preparation

MATERIALS AND METHODS

Monolayers of Caco-2 cells grown on Petri dishes were rinsed four times with cold PBS and scraped with a rubber policeman in 1 ml of lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 200 mM sucrose), supplemented with a cocktail of protease inhibitors (Lisanti et al., 1988) in the absence of detergent and at 4°C. Cells were broken by 20 passages through a 23 G needle. After centrifugation in a Sigma microfuge at 750 revs/min at 4°C for 10 min, the post-nuclear supernatant was collected and kept at 4°C prior to treatment with detergents.

Reagents

Solubilization and detection of antigens

Cell culture reagents were purchased from Seromed (Biochrom KG, Berlin, RFA). Protein A-Sepharose was from Pharmacia (Uppsala, Sweden). Foetal calf serum was from JBio (Les Ulis, France). 1-Deoxymannojyrimycin (dMM) was from BoehringerMannheim (Mannheim, RFA). L-[35S]cysteine (1,000 Ci/mmole) was from Amersham (Amersham, Buckinghamshire, United Kingdom). Phosphatidylinositol phospholipase C was from

Post-nuclear supernatants were incubated with TX-100 or OG at a final concentration of 1% for 5 min at 4°C. Soluble and insoluble materials were separated by ultracentrifugation for 1 h at 100,000 g and at 4°C in a Beckman 50 Ti rotor. Supernatant and pellet were collected, one volume of 2× sample buffer was added to the supernatant and two volumes of 1× sample buffer were added to the pellet. Lysates were boiled for 10 min and analysed

Sorting of apical proteins in Caco-2 cells 1283 by gel electrophoresis according to Laemmli (1970). After transfer to nitrocellulose according to Burnette (1981), soluble and insoluble antigens were detected with the relevant antibodies and 125 I-Protein A. Percentage of insolubility of each antigen was determinated after densitometric analysis of autoradiograms. Metabolically labeled cells were solubilized in lysis buffer containing 1% TX-100 or 1% OG, at 4°C for 5 min. Lysates were centrifuged at 13,000 g at 4°C for 20 min in a Sigma microfuge. Analysis of pellets by immunoblotting showed that about 30% of PLAP was insoluble in this conditions while less than 5% of Ag 525 was at steady state. Radiolabeled antigens were recovered from the supernatant by immunoprecipitation according to Le Bivic et al. (1989), and analysed by SDS-gel electrophoresis and fluorography.

Drug binding to cholesterol Cells were preincubated for 7 min with 0.1 mg/ml Nystatin or 4 mM Polymyxin B sulfate at 37°C or with 0.1 mg/ml Digitonin at 4°C before lysis (Rothberg et al., 1990). Extraction in the presence of these reagents was performed as described above.

pH variation Cells were preincubated for 30 min at 37°C in DMEM, 20 mM Pipes, pH 6.5, or in DMEM; 20 mM Hepes, pH 7.5, or in DMEM; 20 mM Tris, pH 8.5. After 3 rinses with cold buffer 20 mM Pipes, pH 6.5, 20 mM Hepes, pH 7.5, or 20 mM Tris, pH 8.5, cells were scraped and lysed with detergent at the same pH. Analysis of soluble and insoluble materials and determination of percentage of insolubility for each antigen was done as described above.

Sucrose gradient After homogenization of cells and lysis with or without detergent as described above, lysates were loaded on a step sucrose gradient (0.2 M to 1 M) made up in lysis buffer with 0.5% detergent. Gradients were centrifuged for 15 h at 35,000 revs/min at 4°C in a Beckman SW41 rotor. Fractions (1 ml each) were harvested from the top, and proteins were recovered by TCA precipitation. Pellets of the gradient (F7) and TCA pellets (F1-F6) were treated with sample buffer prior to analysis by SDS-PAGE and immunoblotting as described above. When gradients were performed after pulse-chase experiment, pellets were solubilized by 1% SDS. Fractions 1-7 were diluted to contain about 0.2 M sucrose and the concentrations of SDS and TX-100 were brought to 0.1% and 1%, respectively. Antigens were immunoprecipitated in each fraction and analysed by SDSgel electrophoresis and fluorography.

Thin sections were contrasted with uranyl acetate and lead citrate before observation in a Hitachi H600 electron microscope.

RESULTS GPI-anchored proteins are partially resistant to TX-100 extraction in Caco-2 cells Reports in the literature have suggested that GPI-anchored proteins are not fully solubilized by TX-100 (reviewed by Low, 1989). To test if this is the case in Caco-2 cells, we extracted confluent Caco-2 cells at 4°C in a buffer containing 1% TX-100. Cell lysates were separated into soluble and insoluble fractions by centrifugation at 100,000 g for 1 h at 4°C. Fractions were analysed by Western blotting and compared with cells extracted in a buffer containing 1% OG. OG solubilized efficiently both transmembrane (Ag525, SI and CEA110) and GPI-anchored proteins (PLAP, CEA180 and P97) while TX-100 failed to solubilize a sizeable fraction of all the GPI-anchored proteins tested (Fig. 1, Table 1). In contrast, Ag525, CEA110 (Fig. 1) and TFR (not shown) were highly soluble in these conditions and a minor fraction of SI was not solubilized by TX-100 buffer. Table 1 shows the percentage of insoluble fraction in TX-100 or OG buffer for the proteins tested. GPI-anchored proteins become insoluble during intracellular processing In a previous study on Caco-2 cells we observed that detection in the soluble fraction of newly synthesized PLAP was greatly reduced during intracellular maturation (Le Bivic et al., 1990). Since the cells were solubilized in TX-100 we investigated the possibility that GPI-anchored protein resistance to TX-100 extraction was taking place intracellularly during its transport to the cell surface. Caco-2 cells were

Electron microscopy Pellets of gradients from TX-100- and OG-treated cells were fixed with 3% glutaraldehyde in PBS and 2% OsO4, dehydrated in a graded series of acetone (30 to 100%) and embedded in Epon 812. For immunoelectron microscopy pellets were fixed with 2% paraformaldehyde in PBS for 25 min, rinsed with PBS (2×5 min) and treated for 15 min with 50 mM NH4Cl in PBS. After a brief wash in PBS, pellets were incubated with 10% FCS/0.1% saponin/PBS (incubation buffer) for 25 min. Primary antibodies diluted in the same buffer (1/50) were added to the pellets for 1 h, then washed 3 times in incubation buffer before secondary antibodies (anti-mouse IgG coupled to 10 nm gold and anti-rabbit IgG coupled to 5 nm gold) diluted (1/30) in the same buffer, were added. After 3 rinses in incubation buffer and 2 washes in PBS, pellets were post fixed for 15 min with the following mixture: 7% glutaraldehyde and 2% OsO4 in PBS (by vol.). Finally, pellets were embedded in Epon 812 after dehydratation in a graded series of acetone.

Fig. 1. GPI-anchored proteins are partially resistant to TX-100 extraction in Caco-2 cells. Cells were broken by passing through a 23 G needle. Post-nuclear supernatant was obtained by centrifugation (1,000 g, 10 min, 4°C). After 5 min of extraction with 1% OG or 1% TX-100, soluble (s) and insoluble (l) materials were separated by centrifugation (100,000 g, 1 h, 4°C). Immunodetection of antigens was performed by SDS-PAGE (6% to 15%) and Western blotting with 125I-Protein A. Black arrowhead indicates CEA180; white arrowhead indicates CEA110. GPI-anchored proteins (PLAP, CEA180) were fully solubilized in OG and only partially solubilized in TX-100.

1284 M. Garcia and others Table 1. Insolubility of membrane proteins in OG and TX-100 525 SI CEA110 CEA180 PLAP P 97

OG

TX

1.5 (1.3) 11.2 (5.5) 6.8 (2) 7.7 (4.6) 8.6 (6.9) 15 (10)

1.63 (1.28) 20 (9.2) 10.5 (4) 53.1 (10.7) 57.5 (8.8) 43.2 (16.7)

Soluble and insoluble materials were separated as described in Fig. 1. Autoradiograms were quantitated by densitometric scanning and the percentages of insolubility for each membrane protein was determined. n=6, ± s.d. (standard deviation).

min

Fig. 2. GPI-anchored proteins lose solubility during intracellular processing. Cells were pulsed for 20 min with [35S]cysteine, and chased for the indicated times. After 5 min of extraction with 1% OG or 1% TX-100, proteins were immunoprecipitated, analysed by SDS-PAGE (6% to 15%), and revealed by fluorography. White circle: precursor form of GPI-anchored proteins; black circle: mature form of GPI-anchored proteins. White arrowhead: precursor form of transmembrane CEA110; black arrowhead: mature form of transmembrane CEA110. Mature forms of GPIanchored proteins (PLAP, CEA180) became insoluble in 1% TX100, whereas mature forms of transmembrane proteins (CEA110, Ag525, SI) remained soluble in the same conditions. Molecular mass markers are, from top to bottom, 45 and 36 kDa for Ag525, 200 kDa for SI, 66 kDa for PLAP, 116 and 80 kDa for CEA.

pulsed during 20 min with [35S]cysteine, chased for 0, 90 or 180 min and lysed for 5 min with 1% TX-100 or 1% OG buffer at 4°C. Several apical and basolateral antigens were then immunoprecipitated from the soluble fraction and analysed by SDS-PAGE and fluorography. CEA110, Ag525 (Fig. 2), TFR, APN and DPPIV (not shown) were equally well solubilized in TX-100 or OG buffer at any time of chase. This was not the case for PLAP, CEA180 (Fig. 2) and P97 (not shown), the three GPI-anchored proteins studied. At time 0 immature forms of these proteins were fully extractable in TX-100 but became readily insoluble upon maturation (90 min) to complex glycosylated forms. About 50% of P97 (not shown) and 95% of PLAP and CEA180 were insoluble after maturation. The difference between GPI-anchored and transmembrane proteins was most striking for CEA. CEA180 (a GPI-anchored form of CEA) became fully insoluble by 180 min of chase as opposed to CEA110 (a transmembrane form of CEA that is also apical), which remained soluble by the same time although fully processed (Fig. 2). PLAP was still insoluble after a 45 min extraction in TX-100 at 4°C, indicating that this resistance

to extraction was stable over time (not shown). On the other hand a 5 min extraction was enough to solubilize efficiently transmembrane proteins, so we used this extraction time for all subsequent experiments. To examine transport and insolubilization of the various proteins in more detail, Caco-2 cells were pulsed as above, chased from 0 to 120 min and extracted for 5 min with TX100 or OG buffer at 4°C. PLAP was immunoprecipitated and detected after SDS-PAGE and fluorography (Fig. 3). In OG extracts a mature form of PLAP was observed as early as 40 to 60 min of chase. In TX-100 extracts no mature form could be observed at any time of chase, indicating that insolubility occurred after exit from the ER and before full glycosylation processing. This was confirmed by incubating Caco-2 cells at 20°C during the chase. Under these conditions PLAP remained fully soluble in TX-100 after 3 h of chase while it became insoluble at 37°C (not shown). We found that incubation of Caco-2 cells at 20°C prevents

min

Fig. 3. Kinetics of loss of solubility of PLAP. Cells were pulsed for 20 min with [35S]cysteine, and chased for the indicated times. After 5 min of extraction with 1% OG or 1% TX-100, PLAP was immunoprecipitated, analysed by SDS-PAGE (6% to 15%), and revealed by fluorography. Mature form of PLAP (black circle) appears after 40 min of chase in OG extracts. In contrast, no mature form could be detected in TX-100 extracts. White circle: precursor form of PLAP. Molecular mass markers are, from top to bottom, 97, 66 and 45 kDa.

Sorting of apical proteins in Caco-2 cells 1285 A

EFFECT OF PH VARIATIONS 100

processing as well as transport of PLAP to the cell surface, presumably by accumulating it in the endoplasmic reticulum (ER) or a cis-Golgi compartment, and not in the TGN as has been described for other cells (Matlin and Simons, 1983). Since insolubility was concomitant with carbohydrate processing, we investigated the possibility that N-glycosylation was responsible for such behavior. We treated Caco2 cells with a glycosylation inhibitor, 1-deoxymannojirimycin (dMM), that blocks ER mannosidase and mannosidase IA,B leaving high-mannose N-linked glycosylated side chains (Fuhrmann et al., 1985). Caco-2 cells were pulsed and chased for 0, 90 or 180 min in the presence of 1 mM dMM (Fig. 4). As expected, PLAP did not acquire complex N-glycoconjugates but became insoluble in TX-100 by 90 min, suggesting that PLAP insolubilization was not dependent on glycoconjugate processing but occurred in the same compartment. Proteins following the biosynthetic pathway are exposed to pH ranging from 8-8.5 in the ER to 6 in the TGN (for review see Huttner and Tooze, 1989). We tested the effect of pH during cell lysis to evaluate its role in a potential aggregation of GPI-anchored proteins and found no difference in the insolubility of these proteins in TX-100 between pH 6.5 and pH 8.5 (Fig. 5A). Cholesterol has been involved in the formation of caveolae and in the clustering of some GPI-anchored proteins on the cell surface (Rothberg et al., 1990). Using two cholesterol-binding products, Nystatin and Digitonin, that have been shown to disrupt cholesterol-induced folate receptor clusters, we complexed cholesterol by pretreating Caco-2 cells with these drugs at concentrations known to disrupt caveolae (Rothberg et al., 1992). Cells were then lysed in TX-100 or OG buffer and the solubility of GPI-anchored proteins was analysed as before (Fig. 5B). Neither Nystatin nor Digitonin helped to solubilize GPI-anchored proteins, suggesting that binding to cholesterol was not directly responsible for their insolubility. Polymixin B, which forms complexes with phospholipids in the membrane, had no effect either. Insoluble GPI-anchored proteins are enriched in a membrane fraction To determine wether GPI-anchored proteins were still asso-

% OF INSOLUBILITY

Fig. 4. Insolubility of PLAP is independent of N-glycoconjugate processing. Cells were pulse-chase labeled in the presence of 1 mM dMM, and PLAP was immunoprecipitated and analysed as described for Fig. 3. PLAP did not acquire complex Nglycoconjugates but became insoluble in TX-100 extract after 90 min of chase.

60 pH 6.5 pH 7.5 pH 8.5

40

20 0

B

% OF INSOLUBILITY

min

80

525

SI

CEA110 CEA180 PLAP

P 97

EFFECT OF CHOLESTEROL BINDING DRUGS 100

80

60

CT NYS DIG POL

40

20

0

525

SI

CEA110 CEA180 PLAP

P97

Fig. 5. (A) Effect of PH variation on GPI-anchored proteins insolubility. Cells were pretreated and processed as described for Fig. 1 at pH 6.5, pH 7.5 or pH 8.5. Immunodetection of antigens was performed in soluble and insoluble fractions after SDS-PAGE (6% to 15%) and Western blotting with 125I-Protein A. Percentage of insolubility was determined after quantification by densitometric scanning of autoradiogams. No significative difference in insolubility of GPI-anchored proteins could be detected at different pH values. (B) Effect of cholesterol-binding drugs on GPI-anchored proteins insolubility. Cells were pretreated for 7 min with 0.1 mg/ml Nystatin (NYS) or 4 mM Polymyxin B sulfate (POL) at 37°C, with 0.1 mg/ml Digitonin (DIG) at 4°C, or without drug (CT) and lysed as described for Fig. 1 in the presence of the different drugs. Immunodetection of antigens was performed on soluble and insoluble fractions after SDS-PAGE (6% to15%) and Western blotting with 125I-Protein A. Percentage of insolubility was determined after quantification by densitometric scanning of autoradiograms. Neither unclustering of cholesterol with Nystatin or Digitonin, or Polymixyn B sulfate interaction with phospholipids enhanced solubilization of GPIanchored proteins by TX-100. Note that Digitonin slightly increased the insolubility of transmembrane glycoproteins and PLAP.

ciated with membrane structures after TX-100 extraction we devised the following protocol. Confluent Caco-2 cells were scraped, disrupted and TX-100 or OG was added to the post-nuclear supernatant at a final concentration of 1% (recovery of plasma membrane markers in post-nuclear supernatant was between 40 and 60%). Supernatants were loaded on top of a sucrose gradient and centrifuged for 15 h at 100,000 g and at 4°C. Fractions collected from top to bottom were analysed by Western blotting (Fig. 6A). We

1286 M. Garcia and others

A

B

Fig. 6. Purification of a GPI-anchored protein-enriched fraction on sucrose density gradient. (A) Steady-state analysis: cells were broken by passing through a 23 G needle. Post-nuclear supernatant was obtained by centrifugation (1,000 g, 10 min, 4°C) and lysed with 1% OG or 1% TX-100. Extracts were loaded on a sucrose density gradient (0.2 M to 1 M) and centrifuged (35,000 revs/min, SW41, 15 h, 4°C); 1 ml fractions were collected from top to bottom (1-6) and pellets (7) were solubilized in SDS. Proteins of each fraction were analysed by immunoblot with 125I-Protein A. GPI-anchored proteins, PLAP, CEA180 (black arrowhead), but not Ag525 or CEA110 (white arrowhead), were detected in F7. Molecular mass markers are, from top to bottom, 45 and 36 kDa for Ag525, 200 and 116 kDa for SI, 97 and 66 kDa for PLAP, 200 and 116 kDa for CEA. (B) Pulse-chase analysis: cells were pulsed for 20 min with [35S]cysteine, chased for 30 or 180 min, and lysed in TX-100. Sucrose density gradient centrifugation was performed as described above. Proteins were recovered by immunoprecipitation from each fraction (1-7), analysed by SDS-PAGE (6% to 15%), and revealed by fluorography. SI and PLAP were detected in TX-100 pellet (fraction 7) after 180 min of chase but not after 30 min, indicating that SI insolubility occurred during intracellular transport.

found that a major fraction of PLAP, CEA180 (Fig. 6A) and P97 (not shown) migrated to the pellet of the gradient in TX-100 but not in OG. The density of these TX-100 pellets was greater than 1.11. TX-100 pellets contained no detectable Ag525, CEA110 (Fig. 6A) or TFR (not shown) but a sizeable fraction of SI, which varied between experiments. Some SI was also found in OG pellets but in lesser quantities and no other marker could be detected in these OG pellets. Because we always observed an intermediate solubilization of SI by TX-100 (20%) at steady state, we were concerned that SI was never entirely solubilized under our conditions. To test this possibility, Caco-2 cells were pulsed as described and chased for 30 or 180 min before treatment with TX-100 and sucrose gradient centrifugation. Proteins were immunoprecipitated from each fraction and analysed by SDS-PAGE (Fig. 6B). After a 30 min chase all the proteins tested were fully soluble and migrated in the upper part of the gradient. However, after 180 min of chase a fraction of PLAP and SI was detected in the pellet while Ag525 and DPPIV were still fully soluble. For each protein the shift of molecular mass between 30 and 180 min of chase

indicates processing by the Golgi complex. This showed that SI insolubility was not intrinsic to the protein, since it was not observed in the ER but occurred like PLAP, during intracellular processing. However, this resistance to TX-100 extraction was less pronounced than for any GPI-anchored proteins. The fraction of PLAP found in gradient pellets was less than that found in solubilization assays (Figs 1 and 2), probably due to the fact that TX-100 was present in every fraction of the gradient leading to prolonged exposure (~15 h) to the detergent during centrifugation. We next examined the TX-100 and OG pellets from sucrose gradients at the electron microscopic level. In the OG pellet, only fibrous and amorphous material could be seen (Fig. 7c), suggesting that no membrane structures survived the OG treatment. In the TX-100 pellet, layers of fibrous and amorphous material were found together with layers of membrane structures (Fig. 7a). We observed a heterogeneous population of vesicles (~0.2 µm) and membrane fragments clearly formed of a lipid bilayer. Most of the vesicles were likely to be derived from the plasma membrane, since they were open, as shown in Fig. 7b. Immunolocalization was performed on the TX-100-insol-

Sorting of apical proteins in Caco-2 cells 1287 uble fraction by incubating the pellets recovered from the sucrose gradient with antibodies against Ag525, DPPIV, SI, PLAP and P97, followed by gold-conjugated secondary antibodies before sectioning (Fig. 8). Using double labeling, PLAP was observed to be associated with membranes together with P97 and SI (Fig. 8c, d) but not with amorphous material. Little or no labeling was found using DPPIV or Ag525 (Fig. 8a, b) and no labeling was observed when the primary antibody was omitted (not shown). Not all the membranes were labeled and SI, PLAP and P97 appeared to have a patchy distribution. These findings were in good agreement with immunobiochemical analysis of the gradients. Labeling with P97 was weaker than with PLAP, a finding consistent with its weaker expression on Caco-2 cells by immunofluorescence (not shown). Since some SI was found by Western blot in OG pellets, immunodetection of SI was also performed on these pellets and we found that gold particles were not associated with any identifiable membrane structure, suggesting, rather, an aggregation of insoluble SI into amorphous material (not shown). DISCUSSION

Fig. 7. Electron microscopy of OG and TX-100-insoluble material. Cells were broken by passing through a 23 G needle. Post-nuclear supernatant was obtained by centrifugation (1,000 g, 10 min, 4°C) and lysed with 1% OG or 1%TX-100. Extracts were loaded onto a sucrose density gradient (0.2 M to 1 M) and centrifuged (35,000 revs/min, SW41, 15 h, 4°C). Insoluble material recovered in the pellets was prepared for electron microscopy as described in Materials and Methods. Bars, 0.2 µm, a,c ; 0.1 µm, b. In TX-100 pellet (a,b), layers of membrane structures (vesicles and fragments) can be observed together with fibrous and amorphous material. In the OG pellet (c), only fibrous and amorphous material can be seen.

In this study we report that three different endogenous GPIanchored proteins were partially insoluble in TX-100 at 4°C. Such a phenomenon was not observed for several transmembrane proteins, with the exception of SI, which had an intermediate solubility in TX-100. All three GPIanchored proteins, PLAP, CEA180 and P97, were equally well solubilized by OG. This finding that GPI-anchored proteins are poorly solubilized in TX-100 confirmed previous reports in different tissues (for review see Low, 1989). This insolubility was acquired during intracellular transport, as PLAP, CEA180 and P97 were fully soluble in TX-100 after biosynthesis and for the time of residence in the ER compartment. This suggests that insolubility is not a property inherent in GPI-anchored proteins but results from a change in their environment or in their post-translational modifications. We could rule out, however, a role for N-glycosylation trimming in this process, since blocking the trimming of high-mannose N-linked glycoconjugates by dMM did not prevent PLAP from becoming insoluble with the same kinetics. These kinetics suggest that insolubility takes place between the ER and the medial Golgi, as no complex form of PLAP could be transiently detected in pulse-chase experiments (Fig. 3). Thus, it seems likely that GPI-anchored proteins entering the Golgi complex encounter a new protein or lipid environment and interact with it. PLAP insolubility was higher during its intracellular transport than when expressed at the cell surface, as seen in steady-state experiments. This result suggests that after arrival at the cell surface part of the PLAP may disengage from the microdomains. This agrees in part with the model proposed by Simons and van Meer (1988) of an interaction between apical proteins and GSLs during sorting to the apical surface. In fact GSLs are preferentially delivered to the apical membrane of MDCK (van Meer et al., 1987) and Caco-2 cells (van’t Hof and van Meer, 1990). They can self-associate to form microdomains in the membrane (Thompson and Tillack,

1288 M. Garcia and others

Fig. 8. Immunogold detection of apical and basolateral markers in TX-100 pellet: double labeling with PLAP (5 nm) and (a) Ag525 (10 nm), (b) DPPIV (10 nm), (c) GP97 (10 nm), (d) SI (10 nm) was performed with gold-conjugated antibodies on TX-100 pellets recovered after sucrose density gradient centrifugation. Bar, 0.1 µm. PLAP was detected on membrane structures, associated with GP97 and SI (c,d). No labeling was detected using DPPIV or Ag525 antibodies (a,b).

1985). Like GPI-anchored proteins, GSLs are insoluble in TX-100 and their biosynthesis takes place in post-ER compartments (for review see Pagano, 1990). GSLs and GPIanchored protein properties suggest that they assemble in the early Golgi complex to form microdomains resistant to TX-100 extraction. Interactions between GPI-anchor and GSLs are likely to occur by hydrogen bonding and the fact that OG was very efficient in solubilizing GPI-anchored proteins is probably due to its glycolipid-like structure. As further evidence that GPI-anchored proteins are included in insoluble microdomains we were able to isolate, after TX-100 extraction at 4°C and sucrose gradient centrifugation, a membrane fraction not found in OGextracted cells. GPI-anchored proteins (PLAP, CEA180 and P97) were enriched in this fraction while no other transmembrane proteins except SI could be detected. This membrane fraction consisted of open vesicles and membrane sheets clearly consisting of a lipid bilayer (Fig. 7b). These membranes were very stable at 4°C as gradients were run

in the presence of TX-100 for 15 h. We found that PLAP and P97 were associated with these membranes while Ag525 and DPPIV were not. SI, however, was easily detected in these membranes (Fig. 8d). SI is a transmembrane protein (Hunziker et al., 1986) and like DPPIV is expressed on the apical membrane of Caco2 cells (Pinto et al., 1983; Hauri et al., 1985). Some SI insolubility (~20%) was always observed using TX-100, and to a lesser extent with OG (see Table 1). The appearance of SI in TX-100-resistant microdomains occurred during processing, since no SI could be detected in such stuctures after a 30 min chase, while 10% of it was present in them after a 180 min chase. However, this insolubility was not as marked as for PLAP and varied from experiment to experiment. The fact that SI was also found in OG pellets where no membrane could be detected suggests that SI has a tendency to aggregate and to become insoluble to some extent whatever the detergent used. However, in TX100 pellets, SI was clearly membrane associated, indicat-

Sorting of apical proteins in Caco-2 cells 1289 ing that at least if part of it was insoluble, it was not due to aggregation. Recently, membrane structures resistant to TX-100 extraction have been isolated, using a slighty different protocol, from MDCK cells (Brown and Rose, 1992). Brown and Rose have shown that these detergent-insoluble membranes are highly enriched in exogenously expressed PLAP and that their molecular ratio of cholesterol/glycerophospholipids/sphingophospholipids is about 1:1:1, sim ilar to the one found in the apical plasma membrane of intestinal cells (for review see Simons and van Meer, 1988). Although we did not analyse the lipid composition of our TX-100-insoluble membranes it is reasonable to assume that this membrane fraction is similar to the one described by Brown and Rose (1992). The density of our membrane fraction (>1.11) was higher than the one found by Brown and Rose (1.081) and might reflect some loss of lipid during its migration through the sucrose gradient. Our results also indicate that cholesterol trapping with Digitonin or Nystatin did not increase GPI-anchored protein solubility in TX-100, suggesting that clustering with cholesterol is probably not responsible for their insolubilization. Variations of pH between 6.5 and 8.5 were also ineffective in modifying their solubility in TX-100. It is likely that the membrane structures we isolated are largely derived from the apical plasma membrane, simply because Golgi PLAP- or CEA180-enriched domains would not account for 50% of the total amount of PLAP or CEA180 present in the cells. However, we think that a minor population of these membranes might be derived from the Golgi complex, since microdomain formation occurs early in the secretory pathway. In Caco-2 cells insertion of GPI-anchored proteins into microdomains depends on the presence of the GPI anchor. We found that CEA is expressed in these cells in at least two forms: CEA180 and CEA110. CEA180 was sensitive to PI-PLC treatment and CEA110 was not (data not shown), suggesting that CEA110 is the transmembrane form described by Barnett et al. (1989). These two forms differ only by the presence of a cytoplasmic domain and the number of IgG-like domains. However, no CEA110 could be found in TX-100-insoluble microdomains by immunoblotting while CEA180 was present in the same fraction. Formation of microdomains enriched in GPI-anchored proteins in both MDCK (Brown and Rose, 1992) and Caco2 cells (this study) might indicate that they play a role in protein sorting. GPI-anchored proteins have been found preferentially on the apical surface of several epithelial cell lines (reviewed by Lisanti and Rodriguez-Boulan, 1990) and it has been shown that the GPI anchor is a potential apical sorting signal (Brown et al., 1989; Lisanti et al., 1989). Together with the fact that GSLs are also preferentially delivered to the apical membrane, a model is emerging for apical sorting of GPI-anchored proteins. They would first cosegregate with GSLs in the Golgi apparatus and the cellular machinery would then recognise the apical sorting signals present in the ectodomains of some of the GPIanchored proteins (Brown et al., 1989; Lisanti et al., 1989) so as to package them into apical transport vesicles. This model for apical protein sorting by GSLs and GPIanchored proteins cannot apply to several transmembrane

apical proteins from Caco-2 cells. In fact a major fraction of APN and DPPIV follows an indirect pathway in Caco2 cells while GPI-anchored proteins and SI follow a direct pathway (Matter et al., 1990; Le Bivic et al., 1989, 1990). Although a fraction of DPPIV has been shown to follow a direct pathway, we could not detect DPPIV in TX-100insoluble microdomains. It is possible that several pathways to the apical membrane coexist in Caco-2 cells and that they use different mechanisms of sorting. Still, the possibility exists that DPPIV and APN indeed participate in these microdomains but are fully soluble in our extraction conditions. As shown by Matter et al. (1990) and Le Bivic et al. (1990), SI is mainly transported by a direct route from the Golgi to the apical membrane. Thus, association of SI with GPI-enriched microdomains might be responsible for its apical targeting. If this was confirmed it would indicate that SI has some affinity for GPI-anchored protein/glycolipid complexes. It has been shown in MDCK cells that influenza HA becomes insoluble during transport to the cell surface (Skibbens et al., 1989; Brown and Rose, 1992). These results suggest that HA coaggregates with GPIanchored proteins. While this has not been directly shown yet, the kinetics of appearance of the HA-insoluble form indicates that it is a post-Golgi event while GPI-anchored enriched microdomains form in an early Golgi compartment. Recently, Kurzchalia et al. (1992) used an other detergent, CHAPS, to isolate large aggregates containing HA from a total membrane fraction, although it is not known if these aggregates are related to GPI-anchored proteinenriched domains. Like GPI-anchored proteins, SIanchored proteins were soluble in an ER compartment but only a minor fraction became insoluble after processing in the Golgi complex. This suggest that the mechanisms of association of SI- and GPI-anchored proteins to microdomains might differ. Further investigations should show if GPI-anchored protein clustering acts as a nucleation center for apical vesicle sorting or if apical transmembrane proteins utilize a different mechanism. One possibility is that in CaCo-2 cells sorting to the direct apical pathway is mediated by microdomain formation, while sorting to the indirect apical pathway involves signals present in the cytoplasmic tail of proteins. Purification of apical exocytic transport vesicles should be helpful in clarifying this issue. We thank F.X. Real and R. Alemany for providing us with KF104 antibody against P97. We thank P. Weber for letting us using densitometric scanning facility. We also thank G. Rougon and E. Rodriguez-Boulan for helpful discussions and stimulating interest during this study. This work was supported by URA 179, CNRS, INSERM (CRE 900707) and ARC. M. Garcia was supported by a fellowship from ARC.

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