Detergent Insolubility of Alkaline Phosphatase during Biosynthetic ...

THEJ O U R N A L OF (c:

BIOLOGICAL CHEMISTRY

Voi. 268, No. 5, issue of February 15, pp. 3150-3155, 1993 Printed in U.S.A.

1993 by The American Society for Biochemistry and Molecular Biolow, inc

Detergent Insolubility of Alkaline Phosphatase during Biosynthetic Transport and Endocytosis ROLE OF CHOLESTEROL* (Received for publication, June 23, 1992)

Dirk P. Cerneus, Elles Ueffing, George Posthuma, Ger J. Strousz, and Arie van der Endeg From the Department of Cell Biology, Univerdy. of. Utrecht, Medical School, Heidelberglaan 100, H02.314, 3584 CX Utrecht, The Netherlands

Alkaline phosphatase is anchored to theouter leaflet of the plasma membraneby a covalently attached glycosyl-phosphatidylinositolanchor. We have studied the biosynthetic transportand endocytosis of alkaline phosphatasein the choriocarcinoma cell line BeWo, which endogenouslyexpresses this protein. It was demonstrated that the protein was synthesized as a Triton X-100-soluble precursor. During transport to the cell surface the enzyme was converted in a mature form, which was insoluble in Triton X-100 at 0 “ C . Once at the cell surface 85%of alkaline phosphatase remained in the detergent-insoluble form. Under steady state conditions 15% of alkaline phosphatase was endocytosed. Most interestingly, this fraction of internalized alkaline phosphatase was completely soluble in Triton X- 100 at 0 “C. After depletion of membranecholesterol by saponin, alkaline phosphatase became completely soluble in Triton X - 1 0 0 at 0 “C, suggesting that cholesterol plays a critical role in the formation and maintenance of Triton X- 100-resistant membrane domains.

sage through theGolgi complex, the N-linked“high mannose” oligosaccharides are “complex” glycosylated, and the “mature” protein is thendelivered to the plasma membrane (Takami et al., 1988). Less information is available concerning the fateof APase once arrived at the plasma membrane. It is believed that signal(s) within the cytoplasmic domain of transmembrane proteins promote their clustering intocoated pits and subsequent rapid clathrin-mediated endocytosis (Pearse, 1988; for a review see Hopkins, 1992). SinceGPI-anchored proteins are inserted into the out.er leaflet of the membrane bilayer, they lacka directcontact with cytosolic components. Consequently, GPI-anchored proteins are not trapped in coated pits, as is reported for Thy-1, 5’-nucleotidase and the folate receptor (Bretscher et al., 1980; Matsuura et al., 1984; Rothberg et al., 1990a), and this should affect their rate of endocytosis. The finding that GPI-anchored proteins are either slowly endocytosed (van den Bosch et al., 1988; Lisanti et al., 1990; Rothberg et al., 1990a; Keller et al., 1992), via a clathrinindependent pathway, or remain on the cell surface (Lemansky et al., 1990) is consistent with this notion. Observations made by immunocytochemistry suggest that APase is not randomly distributed at the plasma membrane, Severalintegral membraneproteinsareattachedtothe exoplasmatic side of the cell surface by a glycosyl-phosphati- but rather, is present in clusters (Jemmerson et al., 1985b). dylinositol (GPI)’ anchor (Low, 1989; Cross, 1990; Ferguson, The same observations were made with the folate receptor 1991).One of the best characterized examples of a GPI- (Rothberg et al., 1990b; Anderson et al., 1992) In addition, anchored proteinis alkaline phosphatase (APase; EC 3.1.3.1.), GPI-anchored proteins are resistant tosolubilization in nona homodimer, with a molecular mass of the subunits of 66 ionic detergents, such as Triton X-100 (TX-loo), and partikDa (McComb et al., 1979; Millan, 1990). APase has a well tionpredominantlyintothedetergent-insoluble fraction characterized enzymatic activity, i.e. it hydrolyses phosphate (Hooper and Turner,1988; Hooper and Bashir, 1991). Recent monoesters, however, its physiological role is still unclear. observations indicate, that the membrane lipid content may The GPI moiety is added to the nascent peptide chain after be responsible for the aggregated state of certain GPI-ancleavage of29 amino acidsfrom the hydrophobicCOOH chored proteins within microdomains on the plasma memterminus, shortly after its translocation into the lumen of the brane. For example, the distribution of folatereceptors in endoplasmic reticulum (Micanovic et al., 1988). During pas- dense clusters on thecell surface depends on thepresence of ________~~__ ~____ membrane cholesterol (Rothberg et al., 1990b). Brown and * The costs of publication of this article were defrayed in part by Rose (1992) have providedevidence, that placental APase the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 stably expressed in transfected MDCK cells, as well as other GPI-anchoredproteins, is associatedwithlarge vesicular solely to indicate this fact. $ T o whom correspondenceshould be addressedDept. of Cell membranestructures,containingequal molar amounts of Biology, University of Utrecht, Medical School, Heidelberglaan 100, cholesterol, phospholipids, and sphingolipids. Since some of H02.314, 3584 CX Utrecht,TheNetherlands. Tel.: 31-30-506476; the lipid components are insoluble in TX-100, it was conFax: 31-30-541797; Email [email protected]. § Present address: Dept. of Medical Microbiology, University of cluded that the glycophospholipid portion of the GPI anchor Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. is inserted into detergent-resistant microdomains within the I The abbreviations used are: GPI, glycosyl-phosphatidylinositol; lipid bilayer (Brown and Rose, 1992). TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; TGN, transIn the present investigation we have studied the biosynGolgi network; TX-100, Triton X-100; MEM,minimalessential thesis, transport to the plasma membrane, andendocytosis of medium; MEMH, MEM containing 20 mM Hepes, pH 7.3; PBS, APase. The use of the BeWo choriocarcinoma cell line was of phosphate-buffered saline; BSA, bovine serum albumin; PAGE,polyadvantage, since these cells express considerable acrylamide gel electrophoresis; PAC, proteinA-gold; ER, endoplasmic great amounts of APase. The APase synthesizedby BeWo cells is reticulum; APase, alkaline phosphatase.

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Insolubility of Alkaline Phosphatase during Transport and Endocytosis the product of the germ cell APase gene, and closely resembles the placental APase (Lowe and Strauss, 1990; Watanabe et al., 1989, 1991). Both isozymes can be releasedfrom the plasma membrane by PI-PLC by cleavage of the GPI moiety from the COOH terminus (Micanovic et al., 1988; Ogata et al., 1988; Lowe and Strauss, 1990). We report here that APase is synthesized as a detergentsoluble precursor,but becomes detergent-insolubleduring transport to thecell surface. It remains at the cell surface in a detergent-insoluble form, which undergoes limited endocytosis. Furthermore,we show that the detergentinsolubility is abolished upon pretreatment of the cells with saponin, suggesting a critical role for cholesterol in maintaining APase clustered in specialized membrane regions. EXPERIMENTALPROCEDURES

Materials Sulfosuccinimidobiotin and streptavidin-agarose beads were purchased from Pierce Chemical Co. All other reagents were obtained from Sigma. Polyclonal antibodies against APasewere obtained from Dakopatts (Glostrup, Denmark). Monoclonal antibodies against the transferrin receptor was kindlyprovidedby Dr. J. Hilkens(The Netherlands Cancer Institute, Amsterdam). Cell Culture The b24 strain, cloned from the parental BeWo cell line (van der Ende et al., 1987) was cultured in Eagle's minimal essential medium (MEM) containing 10% fetal calf serum, penicillin and streptomycin (MEM/FCS) as describedpreviously (Cerneus and van der Ende, 1991). For experiments, cells (3 X lo5) were plated in 60-mm dishes and cultured as monolayers for 3 days.

3151

procedure as described by Lisanti et al. (1988).Briefly,cells were washed with PBS-CM on ice and sulfosuccinimidobiotin was added (1 mg/ml in ice-cold PBS-CM) and kept on ice for 30 min. After labeling, the monolayers were washed four times with MEM, containing 20 mM Hepes, pH 7.3 (MEMH), and subsequently lysed in TX100 lysis buffer. The lysates were treated as described above. After immunoprecipitationthesamples were boiled in 50 p1 of sample buffer, subsequently diluted with 1 ml of immunomix, followed by extraction with streptavidin-agarose beads (Lisantiet al., 1988). Internalization Assay for Surface-labeled '251-APase Cell surface iodination was performed on cells grown in 60-mm dishes essentially as described (Cerneus and vander Ende, 1991). Briefly, after washing with ice-cold PBS-CM, cells were iodinated in 1 ml of labeling medium (PBS-CM containing 25 pg of lactoperoxidase, 12.5 pg of glucose oxidase, 250 pg of glucose, and 0.3 mCi of Nalz5I (Radiochemical Centre, Amersham, United Kingdom)) and kept on ice for 30 min. Thereafter, the cells were washed four times in MEMH, andwarmed to 37 "C inprewarmed MEMH containing0.2% BSA for various times and then chilled quickly by washing in icecold PBS containing5 mM EDTA. To distinguish between cell surface and intracellular 1251-APase,the cells were treated with bromelain (1 mg) in 1 ml of PBS-EDTA for 90 min on ice (Webb and Todd,1988). The proteolytic digestion was stopped by adding 1 ml of stop buffer (PBS-EDTA containing 1 mM phenylmethylsulfonyl fluoride, 1 mg/ ml TPCK, and 20 mM iodoacetamide), and the cells were collected by centrifugation (5 min at 500 X g). After three additional washes with stop buffer, the cells were lysed in 1 ml of TX-100 lysis buffer, supplemented with 20 mM iodoacetamide. Immunocytochemistry

Fixation-Petri dishes with confluent BeWo cells were rinsed twice with PBS solution. Subsequently, the cells were fixed at room temperature with 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. After 60-min fixation the cells were removed from the dishusing a cell scraper, transferred to anEppendorf Metabolic Labeling and Lysis of Cells vial, and centrifuged a t 10,000 X g for 30 s. The fixative was removed, Cells were washed twice withPBS,containingCa2+and Mg2+ and the cells were washed three times with PBS containing 0.15 M (PBS-CM), atroom temperature and preincubated in MEM lacking glycine (PBS/Gly). Finally 10% gelatin in PBS (37 "C) was added to methionine(GIBCO) for 30 min at 37 "C. In some experiments the pelleted cells, and the cells were resuspended and spun down, brefeldin A (5 pg/ml) was added during this preincubation in MEM whereafter the gelatin was allowed to solidify a t 0 "C. Ultracryotorny-Ultracryotomy was performed according to Toklacking methionine and throughout the experiment.Cells were then and Singer (1976) usingaslightly modified procedure. The labeled for 20 min with 2.2 MBq/ml [35S]methionine( T r a n ~ ~ ~ S - l a b e luyasu , 185 MBq/ml, 40 TBq/mmol, ICN,CA) and chasedfor variousperiods gelatin with cells was cut into small blocks and immersed in 2.3 M of time. Thereafter, cells were lysed for 30 min on ice in 1 ml of lysis sucrose in PBS for 2 h a t 4 "C. The blocks were mounted on copper buffer (20 mM Tris, pH 8, 150 mM NaCI, 1% (v/v) TX-100, 1 mM specimen holders and frozen in liquid nitrogen. Ultrathin sections phenylmethylsulfonyl fluoride, 1 mg/ml N-tosyl-L-phenylalanine (70-100 nm) were cut ona Reichert-Jung Ultracut S (Leica Ltd., Vienna, Ostria) with cryoattachment -95 at "C using a diamond knife chloromethylketone(TPCK)and 2 mM EDTA)eitherwithor without pretreatment of the cells with 0.2% saponin in PBS for 30 (Diatome Ltd., Bienne, Switzerland). The sections were picked up min at 4 "C. Lysates were split into two equal portions; one portion with a sucrose droplet (2.3 M sucrose in PBS), thawed, transferred to 2% gelatin in was kept on ice and the other portion was incubated for 30 min a t carbon-formvar-coated coppergrids, andstoredon 37 "C, after which the detergent-insoluble material was pelleted by PBS. Immunolabeling-The sections were immunolabeledwith anticentrifugation at 15,000 X g at 4 "C in a microcentrifuge. In some experiments, the pelletwas solubilized in 100 pl of buffer, containing APase and protein A-gold (PAG) (08 nm) by floating the grids on 20 mM Tris-HC1, pH 8, 150 mM NaCl, and 1% SDS (Skibbens etal., the next solutions: PBS/gly (5 min), anti-APase in PBS + 1% BSA 1989; Brown and Rose, 1992). DNA was sheared by passage through (30 min), PBS (10 rnin),PAG in PBS/BSA (30 min), PBS (10 min), a 22-gauge needle, and the solution was made up to 0.1% SDS by distilled water(20 rnin). Thesections were stainedwithuranyl acetate-oxalicacid, pH 7 (5 min), and finally transferred to 1.8% adding 900 p1 of extraction buffer. methyl cellulose, 0.3% uranyl acetate, pH 4, in water. Thegrids were picked upwithstainlesssteel wire loops, and the excess methyl Immunoprecipitation cellulose solution was removed with filter paper and air-dried. Immunoprecipitations were performed by adding 500 pl of immuQuantitation-In a JEOL 1200 EX electron microscope the secnomix (1%TX-100, 0.5% deoxycholate, 0.2% SDS, 1% BSA in Tris- tions were viewed a t 20,000 X. To measure the gold distribution we buffered saline, pH 8, supplemented with1mM phenylmethylsulfonyl selected 20 cells that displayedanucleus,apical, and basolateral fluoride and 5 mM EDTA and antibodies to500-pl TX-100 extracts. membrane. Gold particles were assigned to a membrane when the After an overnight incubation in a cold room, the antigen-antibody distance between membraneand gold particle was c 10 nm. To complexes were recovered by adding 50 pl of protein A-Sepharose investigate whether coated pits and vesicles played a role in APase from a 50% (v/v) slurry for 60 min. The precipitates were washed endocytosis, 100coated pitswere examined for the presence of APase three times with immunomix, once with PBS, and once with 10 times- labeling. diluted PBS. The precipitates were boiled in 50 p1 of SDS sample buffer (Laemmli, 1970) and analyzed in SDS-PAGE. After autoradiRESULTS ography protein bands on films were quantified using a laser densiAPase I s Present on the Plasma Membrane in a Detergenttometer. Surface Biotinylation BeWo cells were pulse-chased using [35S]methionine as described above. After various chase times, cells were surface-labeled using the

insoluble Form-BeWo cells express APase, which can be releasedfrom the plasma membrane by PI-PLC (data not shown; Micanovic et al., 1988; Ogata et al., 1988; Lowe and Strauss, 1990),showing that APase expressed by BeWo cells

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Insolubility of Alkaline Phosphatase during Transport and Endocytosis

is GPI-anchored.T o evaluate the solubility of APase in TX100, BeWo cells were pulse-labeledfor 20 min using ["SI methionine and chased for 3 h in order to get the labeled APase to the plasma membrane. The cells were lysed using TX-100, lysates were split, and one half was kept a t 4 "C, while the other half was incubated a t 37 "C for 30 min. The lysates were then centrifuged (15,000 x g) resultingin a detergent-insoluble fraction in the pellet and a soluble fraction in the supernatant. Aftersolubilization of the pellet fraction with SDS, APase was immunoprecipitated from both fractions and analyzed by SDS-PAGE and fluorography. As shown in Fig. 1, most of the APase was immunoprecipitated from the detergent-insolublepellet from lysates kept a t 4 "C (lune 2 ) , while little was found in the supernatant (lune 1). I n contrast, APase waseffectively solubilized after incubating t h e lysate a t 37 "C (lune 3 ) . Inthis case noAPase was recovered from the detergent-insoluble pellet (lune 4 ) . Centrifugation of the lysatesa t 100,000 x g gave identical results (data not shown) These results show that the solubilization of APase in TX-100 is temperature-dependent and are consistent with the resultsof Brown and Rose (1992). APare Becomes Detergent-insoluble during Transportto the Cell Surface-In order to determine time and placea t which APase becomes TX-100-insoluble on its way to the plasma membrane, BeWo cells were pulse-labeled and chased for various time intervals, after which the detergent solubility was determined a t 4 and 37 "C. As shown in Fig. 2 A , no temperature-dependent solubilization of the precursor form of APase was observed (compare lunes 1-6). The APase with

a slightly increased molecular weight, which appeared after 30 min of chase, is partlyTX-100-insoluble (Fig. 2 A , lunes 36). This form of APase, formed in the Golgi apparatus by complex glycosylation, is first detectable at theplasma membrane after 60 min. This was determined by a surface-biotinylation assay (according to Lisanti et ul. (1988)) in which surface-biotinylated APase was detected by immunoprecipitation followed by a precipitation with streptavidin-agarose beads (Fig. 2B, lunes 5 and 6). Longer chase timesresulted in an increased insolubility of mature APase (Fig.2, A and B, lunes 7-10). Theseresults suggest that APase is initially synthesized as a detergent-soluble precursor in the ER, but gradually becomes insoluble in TX-100 a t low temperature during transport to the cell surface. This was further investigated, when cells were treated with brefeldin A during the chase, known to prevent the exit of proteins out of the ER. In thepresence of brefeldin A, APase remains soluble in TX100 a t low temperature up to2 h of chase (Fig. 3, lunes 1-10). After 3 h some loss of APase (20-30%) is seen (Fig. 3, lune 12). Under these conditions, the secretory proteinhuman gonadotropin is not secreted (data not shown), thus confirming that transport from the ER to the cell surface is efficiently blocked by brefeldin A. The decrease in immunoprecipitable APase afterprolonged incubation with brefeldin Ais probably due to degradation in the ER. Indeed, turnover of several proteins have been reported to occur at this site (Klausner and Sitia, 1990). Together, the results indicate newly that synthesized APase gradually acquires detergent insolubility en route to the cell surface, whichis in support of previous observations by Brown and Rose (1992). 4 3 7 p-s s p Limited Endocytosis of APase-Immunocytochemistry revealed that 93% (percentage of 895 gold particles in 20 cells) - 69 m of the gold-labeled APase was localized and uniformly distributed at thecell surface (Fig. 4A).Only 5% of the gold-labeled 1 2 3 4 APase was found intracellular, while 2% was found on nuclei. FIG. 1. Temperature-dependent solubilization of APase in Only rarely, large clusters of labeled APase were observed in T-X100. Cells were pulse-labeled with ["S]methionine for 20 min or nearby caveolae (Fig. 4B).Moreover, less than 3% of the and chasedfor 3 h, after which the cells were lysed inTX-100 extraction buffer a t 4 "C. The lysateswere split in half and incubated coated pits were labeled for APase, suggesting that endocyat 4 "C (lanes I and 2 ) or a t 37 "C (lanes 3 and 4 ) . The lysates were tosis of APase is very limited. To study this in more detail a protease-protection assay, centrifuged, and APasewas immunoprecipitated from both the detergentin-solublepellet solubilized with SDS ( p ) and the detergentusing bromelain, was developed (Jemmerson et ul., 1985a). soluble supernatant (s) and analyzed by SDS-PAGE and fluorogra- Control experiments were performed to test thereliability of phy. the assay in discriminating between proteins present at the cell surface (bromelain-sensitive) and intracellularly (bromemin 0 30 R l 120 240 lain-resistant). First, both APase (Fig. 5B, lune 3 ) and the transferrin receptor (Fig. 5A, lune 2 ) , labeled by iodination of 437437437437437 the cell surface, were efficiently removed from the cell surface A - 69 by bromelain a t 4 "C. Upon incubation for 2 h at 37 "C most a " of the surface-labeled transferrin receptor (about 70%) was protease-resistant (Fig. 5A, lane 3 ) . Theseresultsarein B agreement with previous data, obtained by binding assays

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B F A - + - + - + - + - + - + 1 2 3 4 5 6 7 8 9 1 0 FIG.2. Solubility of newly synthesized APase in TX-100. Cells were labeled for 20 min with ["S]methionine, chased for the indicated time points, and subsequently surface-labeled with sulfosuccinimidobiotin (see "Experimental Procedures"). After lysis of the cells at 4 "C, the lysates were divided into two equal portions. One portion was furtherincubated a t 4 "C,while the other halfwas incubated a t 37 "C. APase was immunoprecipitated from the detergent-soluble supernatants ( A ) obtained from the lysates incubateda t 4 "C ( 4 ) or 37 "C (37). Surface-biotinylated APase was recovered after a subsequent precipitationwith streptavidin-agarose beads( B ) . Precipitates were analyzed by SDS-PAGE andfluorography.

- 69 1 2 3 4 5

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FIG. 3. Effect of brefeldin A on the solubility of APase during biosynthetic transport. Cells were preincubated with or without brefeldin A in medium without methionine for 30 min at 37 "C. Thereafter, cells were pulse-labeled with [%]methionine and chased during theindicated time intervals. Theinsolubility of APase a t 4 "C was determined as described in the legends of Fig. 1.

Insolubility of Alkaline Phosphatase during Transport and Endocytosis

3153

cells were washed extensively in buffer containing protease inhibitors and solubilized in TX-100 lysis buffer. The TX100 solubilityof APase was determined in the lysates a t 4 and 37"C as described above and analyzed in SDS-PAGE. As shown in Fig. 6A, lanes 1 and 2, the ratio of surface-labeled APase, solubilized in TX-100 a t either 4 or 37 "C, was 20:80 (see also Fig. 6B), thusconfirming our previous results. Upon incubating the cells a t 37 "C, about 10% became protected against proteolysis within 30 min of chase (Fig. 6A, lanes 7l o ) , indicating that APase is endocytosed to a very limited extent. Interestingly, the fraction of internalized APase was ., completely soluble in TX-100 at low temperature (Fig. 6A, .@.: compare lanes 1 and 2 with 7 and 8; Fig. 6B), which might indicate that APase converted is into a detergent-solubleform upon entering theendosomal system. Effect ofCholesterol Depletion on theDetergent Solubility of APase-Until now itisunclearwhat causes theTX-100 insolubility of APase. The resultsfrom Rothberg et al. (1990b) suggested that the GPI-anchoredfolate receptor may cluster . i in cholesterol-rich membrane domains. To determine whether Bk the insolubility of APase in TX-100 a t low temperature depends on the presence of cholesterol, cells were pretreated FIG. 4. APase localization in ultrathin cryosections of BeWo cells indirectly labeled with 8 nm of gold. ReWo cells were with saponin, a cholesterol-complexing agent (Schl6sser and prepared for electron microscopy as described under "Experimental Wulff, 1969), before solubilization with TX-100. Procedures." After labeling withanti-APase antibodies the specimens Cells were pulse-labeled for 20 min and chased for 3 h,after were labeled with protein A conjugated to 8 nm of gold. a, plasma which they were incubated with PBS, containing0.2% sapomembrane with microvilli; b, large cluster of labeled APase in the nin, for 30 min at 4 "C or in PBS alone. Then the cells were vicinity of a caveolus. The bar is 200 nm. lysed in TX-100 and the temperature-dependent detergent solubility of APase was determined as described. The results A B show that pretreatmentof the cells with saponin did not cause a loss of APase from the cells (compare lanes 2 and 4, Fig. 0 0 -92 - 69 7A). However, the temperature-dependent solubilization of .c.D APase in TX-100 (lunes 1 and 2 ) was abolished by pretreatment with saponin (lunes 3 and 4 ) . Identical results were 1 2 3 1 2 3 observedwhendigitonin (another cholesterol-complexing FIG. 5. Susceptibility of surface-iodinated APase and trans- agent) was used instead of saponin (data not shown). As a "

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ferrin receptors for bromelain. A, detection of internalized "'It ransferrin receptors after surface proteolysis. Surface-iodinated cells were kept on ice (lanes I and 2 ) or incubated for 2 h a t 37 "C (lane 3 ) , after which the cells were incubated with buffer containing 1 mg/ ml bromelain at 4 "C (lanes 2 and 3)or in buffer alone (lane I ). Then

cells were lysed in TX-100 a t 37 "C, and transferrin receptors were immunoprecipitated from the detergent-soluble supernatant. R, proteolysis of "'I-APase is blocked prior to solubilization in TX-100. Surface-iodinated cells were mock-treated (lanes I and 2 ) or treated with bromelain a t 4 "C (lane 3),followed by lysis in TX-100 a t 37 "C. Half of lysates from surface-labeled cells were mixed with lysates from nonlabeled cellsthat were either mock-treated(lane I ) or treated with 2 mg/ml bromelainon ice (lane 2). The mixed lysates were incubated for 30 min a t 37 "C, and APase was immunoprecipitated fromthe detergent-soluble supernatant.Immunoprecipitates were analyzed by SDS-PAGE and autoradiography.

(van der Ende et al., 1987), and consistent with the notion that the transferrinreceptor recycles rapidly between the cell surface and endosomes, reaching a steady state distribution within 30 min with 60-7096 of the transferrin receptorslocalized in endosomal compartments. Second, equal amounts of APase were immunoprecipitated from the lysate of surfaceiodinated cells when mixed with the lysate from unlabeled cells, which were either treated with 2 mg/ml bromelain or with buffer alone (Fig. 5B, lanes 1 and 2). Together, these results illustrate that the protease treatment was restricted to the cell surface and that proteolysis was blocked efficiently prior to cell lysis. The protease-protection assay was then used to study the endocytosis of APase. Cells were surface-labeled with '*'I and incubated at 37 "C at various times followed by proteolysis using 1 mg/ml bromelain at 4 "C. After protease treatment

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FIG.6. Endocytosis of surface-labeled APase. A , cells were surface-iodinated with '*'I on ice, recultured for the indicated times a t 37 "C to allow internalization of APase, and treated with 1 mg/ml bromelain on ice. Control cells were not treated andkept on ice (lanes 1 and 2). Cells were lysed in TX-100 as described in Fig. 2. The solubility of APase was determined a t 4 "C ( 4 ) and 37 "C (37) as described in Fig. 2. R, graphic representation of the datain A, showing the ratio (4/37) of APase, solubilized in TX-100 either at 4 or 37 "C.

Insolubility of Alkaline Phosphatase during Transport and Endocytosis

3154 sap-

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cholesterol-dependent, TX-100-insoluble complex of APase in BeWo cells starts after it leaves the ER, possibly in the TGN, en route to thecell surface. DISCUSSION

Although APasehas a well characterized enzymatic activity, 6 its function on thecell surface remains unclear. We therefore studied the biosynthetic transport and endocytosis of this D -92 protein in BeWo cells, which endogenously express APase. Both electron micrograph and biochemical data show that 90% of APase is at the cell surface, which is in a detergentC insolubleform.Moreover, the protein is converted from a - 69 0 " detergent-soluble precursor to a detergent-insoluble mature form during its biosynthetic transport. The results obtained in pulse-chase experiments with brefeldin A or at 22 "C sug1 2 3 4 gest that this conversion occurs during transport to the cell FIG. 7. Solubility of APase and transferrin receptors TXin 100 after treatment withsaponin. Cells were labeled for 20 min surface, probably prior to exit from the TGN, which is conand chased lor 3 h ( A and H ) or surface-labeled by iodination a t 4 "C sistent with recent observations made by Brown and Rose ( C ) .After treatment with 0.2% saponin in PBS a t 4 'C (+) or with (1992), using transfected Madin-Darby canine kidney cells, PBS alone (-), the TX-100 solubility of APase ( A and C ) and which stably express APase. The insolubility of APase may transferrin receptor ( R )was determined a t 4 "C ( 4 ) or 37 "C (37) as be a general phenomenon of GPI-anchored proteins, since described in Fig. 2. most, if not all, are resistant to solubilizationin TX-100 (HooperandTurner, 1988; fora review see Low (1989); s a p - + - + - + Hooper and Bashir, 1991); Brown and Rose, 1992). The TX-100 insolubility of GPI-anchored proteins might - 69 " . - % be a consequence of clustering of these proteins into certain membrane microdomains. However, our morphological data show an equal distribution of APase at the cell surface. Only 1 2 3 4 5 6 occasionally a cluster of APase was observed. These results FIG. 8. Effect of saponin pretreatment on the solubility of are in contrast with the results of Jemmerson et al. (1985b), at 22 "C. Cells were pulse- which observed aclustered APase during biosynthetic transport distribution of APase atthe labeled at 37 "C and chased for 3 h either a t 22 "C (lane 4 and 5 ) or plasma membrane in human placental tissue and cultured at 37 "C (Inno 5 and 6 ) . After the pulse (lane 1 and 2 ) or the chase cancer cells. The observed clustering might be a consequence the Triton X-I00 solubility of APase was determined with (+) or of the way APase is detected. A goat anti-rabbit antibody without (-) pretreatment with saponin. conjugated to gold was used to detect the rabbit anti-APase control, solubilization of the transferrin receptor in TX-100 antibody onprefixed cells. This techniqueby itself results in clusters: more than one goat anti-rabbit antibody can bind to was neither temperature-dependent nor dependent on pretreatment with saponin (Fig. 7B).Thesameresults were one rabbit anti-APase antibody (Slot et al., 1988). Alternaobtained when the cells were surface-labeled by iodination tively, this discrepancy might represent a real difference be(Fig. 7C). These results strongly suggest that cholesterol plays tween the proteins (BeWocells express the germ line APase) folate an important role in the insolubility of APase in TX-100 a t or thecells used. On the other hand, the GPI-anchored receptor is found indense clustersat thecell surface of MA104 low temperature. To study if cholesterol also plays a role in the insolubility cells (Rothberget al., 1990b). Moreover, Brown and Rose of APase duringbiosynthetictransport, cells were pulse- (1992) found that APase was associated with large vesicular labeled with ["'Slmethionine. After a chase of 3 h a t either 22 structures. The results of Rothberg et al. (1990b) show that or 37 "C, the cells were pretreated with saponin, and the TX- the clustered organization of GPI-anchored folate receptors 100 solubility of APase was determined a t 4 "C. The results on the plasma membrane depends on the presence of memshow that, when the cells were chased a t 37 "C, the complex brane cholesterol. This is in accordance with our results with glycosylated form of APase, is more readily solubilized in TX- pretreatment of the cells with saponin, a cholesterol complex100 at low temperature after saponin treatment(Fig. 8, lanes ing agent (Bangham and Horne, 1962; Schlbsser and Wulff, 5 and 6). In contrast, thesolubilization of APase precursor is 1969), and suggests an important role for cholesterol in the neither dependent on the presence of cholesterol (Fig. 8, lanes solubility APase. Nevertheless, other lipid components may 1 and 2), nor on temperature (Fig. 2 A ) . This conclusion was as well contribute to the detergent insolubility of GPI-anfurthersupported by experiments inwhich the cells were chored proteins. Notabiy, analysis of the lipid composition of the large vesicular structures, observed by Brown and Rose chased at 22 "C. Thistemperaturecauses a block in the transport of proteins from the trans-Golgi network (TGN) to (1992), to which GPI-anchored proteins are associatedrevealed an enrichment insphingolipids (eg. glycosphingolipids the plasma membrane(Sarasteetd., 1986). Underthese conditions no newly synthesized human gonadotropin was and sphingomyelin), comprising one-third of the total lipid content of the vesicles. Since sphingolipids are also resistant secreted into the chase medium (data not shown). After a to solubilization in TX-100, it was suggested that GPI-anchase of 3h at 22 "C the solubility of matureAPaseis dependent on pretreatmentwith saponin (Fig. 8, lanes 3 and chored proteins are inserted into detergent-resistant patches 4 ) . The newly synthesized matureAPase was protected formed by sphingolipids (Brown and Rose, 1992). However, these vesicles also show a 3-fold enrichment for cholesterol against bromelain treatment at 4 "C, showing that all the APase remain intracellular during the 3-h chase a t 22 "C in the detergent-resistantvesicles, as compared to whole cells. Pulse-chase experiments at 22 "C (a block for exit out of (data not shown). Together, these results indicate that the formation of a the TGN) suggest that complex glycosylated APase is con0 . ) -

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Insolubility of Alkaline Phosphatase during Transport and Endocytosis

3155

verted toa detergent-insoluble complex prior to arrival at thethe appearancein coated pits, perhapsby an increased lateral endocytic comcell surface, possibly in the TGN. Possibly, the 22 “C block mobility, thereby increasing the entry into may alter some of the properties of the TGN, perhaps by partments. Remarkably, both5”nucleotidase (Mescher et al., increasing the densitiesof sphingolipids and GPI-linked pro- 1981) and GPI-anchored gD-12 are only partly insoluble in teins. Consequently, this could leadto a premature conversion TX-100, which might suggest that the ability to be internalized is correlated to detergent insolubility. Experiments are of APase into a detergent-insoluble form, whichnormally occurs at the cell surface. However, the results with saponin under way to study this inmore detail. suggest that cholesterol plays an important role in this conREFERENCES version. This is consistent with the notion that cholesterol is Anderson, R. G. W., Kamen, B. A. , Rothberg, K. G., and Lacey, S. W. (1992) disproportionally distributed along the secretory pathway Science 255,410-411 A. D.,and Horne, R. W. (1962) Nature 196,952-953 with a substantial lower cholesterol content in the ER than Bangham, Bretscher. M. S.. Thomson. J. N.. and Pearse. B. M. F. (1980) . . Proc. Natl. Acad. intheplasmamembrane (Yeagle, 1985; vanMeer,1989). S C ~U. . s . A. 77,4156-4159 Brown, D. A,, and Rose, J. K. (1992) Cell 6 8 , 533-544 Cholesterol may directly interact with GPI-anchoredproteins, Cerneus, D. P., and van der Ende, A. (1991) J. Cell Biol. 114, 1149-1158 possibly in the cholesterol-rich trans-Golgi region (Orci et al., Cross, G. A. M. (1990) Annu. Reu. Cell Biol. 6 , 1-39 M. A. J. (1991) Curr. Opin. Struct. Biol. 1, 522-529 1981), leading to the formation of detergent-resistant micro- Ferguson, Hooper, N. M., and Turner, A. J. (1988).Biochem. J . 250,865-869 domains, which are then targeted to the plasma membrane. Hooper, N. M., and Bashlr,A. (1991) Brochem. J . 2 8 0 , 745-751 Hopkins, C. R. (1992) Trends Biochem. Sci. 17,27-32 Itisnotquite clear how cholesterol canmodulatethe Jemmerson, R., Millan, J. L., and Klier, W. H. (1985a) FEBS Lett. 1 7 9 , 316formation of these microdomains.Cholesterol, as well as 320 R., Klier, F. G., andFishman, W. H. (1985b) J . Nistochem. sphingomyelin, are thought toreside in the luminal aspectof Jemmerson, Cytochem. 3 3 , 1227-1234 the plasmamembraneand Golgi membranes(vanMeer, Keller, G. A., Siegel, M. W., and Caras,I. W. (1992) EMBO J . 11,863-874 R. D., and Sitia, R. (1990) Cell 6 2 , 611-614 1989). In model systems sphingomyelin appears to interact Klausner, Laemmli, U. K. (1970) Nature 227,680-685 more favorably with cholesterol than the other phospholipid Lange, Y., Swaisgood, M. H., Ramos, B. V., and Steck, T. I. (1989) J . Bid. Chem. 264.3786-3793 classes (Yeagle and Young, 1986; Lund-Katz et al., 1988). In Lange, Y.(1991) J. Lipid Res. 3 2 , 329-339 addition, interactionsbetween sphingomyelin and cholesterol Lemansky, P., Fatemi, S.,H., Gorian,B., Meyale, S., Rossero, R., and Tartakoff, J. Cell Brol. 110. 1525-1531 A. M. may accountfor the heterogeneous distribution of cholesterol Lisanti, (1990) M. P . , Sargiocomo, M:, Graeve, L., Saltiel, A. R., and RodriguezBoulan, E. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,9557-9561 in the plasma membrane of epithelial cells (Yandouzi andLe M. P., Caras, I. W., Gilbert, T., Hanzel, D., and RodriguezBoulan, E. Grimellec, 1992). About 90%of the cellularcholesterol is Lisanti, (1990) Proc. Natl. Acad. Sci. U. S. A. 8 7 , 7419-7423 associated with the plasma membrane, as is sphingomyelin Low, M. G. (1989) Biochim. Biophys. Acta 988,427-454 M. E., and Straus, A. W. (1990) Cancer Hes. 5 0 , 3956-3962 (Lange et al., 1989; Lange, 1991). In fact, the sphingomyelin Lowe, Lund-Katz, S., Laboda, H. M., McLean, L. R.,andPhillips,M.C. (1988) Biochemistry 2 7 , 3416-3423 content in the plasma membrane may determine its cholesMatsuura, S., Eto, S., Kato, K., and Tashiro, Y. (1984) J. Cell Biol. 9 9 , 166terol concentration (Slotte et al., 1990). Possibly, cholesterol 1 73 M k o m b , €2. B., Bowers, G. N., and Posen, S. (1979) Alkaline Phosphata,~rs, might be incorporatedinto sphingomyelinmicrodomains New York, Plenum within the Golgi complex, which are then sorted into carrier Micanovic, R., Bailey, C. A,, Brink, L., Gerber, L., Pan Y. C. E., Hulmes J. D., and Udenfriend, S. (1988) Proc. Natl. Acad. Sci. U.’S. A. 8 5 , 1398-1462 vesicles en route to the plasma membrane (van Meer,1989). J. L. (1990) in Isozymes: Structure, Function, and U,se in Biology and Glycosphingolipids may become associated with these sphin- Millan, Medicine, pp. 453-475, Wiley-Liss, Inc., New York S., Hayashi, Y., Takami, N., and Ikehara, Y. (1988) J . Biol. Chem. 2 6 3 , gomyelin/cholesterol-rich microdomains, due to their exten- Ogata, 10489-10494 sive hydrogen-bonding capacity (van Meer, 1989). If true, it Orci, L.,Montesano,R.,Media, M., Malaisse-LagaeF. Brown D. Perrelet, A., and Vassalli, P. (1981) Proc. Natl. Acad. Sci. U.’S. k.7 8 , 263-i97 is conceivable that the glycolipid moiety of GPI-anchored Pearse, B. M. (1988) EMBO J.7, 3331-3336 proteins have a similar fate. Thelipid content may affect the Rothberg, K. G., Ying, Y., Kolhouse, J. F., Kamen, B. A,, and Anderson, R. G. W. (1990a) J. Cell Biol. 1 1 0 , 637-649 rigidity of these microdomains, resulting in a decreased pen- Rothberg, K. G., Ying, Y. S., Kamen, B. A., and Anderson, R. G. (1990b) J . etration for TX-100 monomers a t low temperature, as was Cell Biol. 1 1 1,2931-2938 Saraste, J., Palade, G. E., and Farquhar, M. G. (1986) Proc. Natl. h a d . Sci. suggested by Brown and Rose (1992). It would be of interest U. S. A. 83,6425-6429 t o see whether depletion of cellularsphingomyelin has a Schlosser, E., and Wulff, G. (1969) Z . Naturforsch. B. 24, 1284-1290 J. E., Roth, M. G., and M a t h , K. S. (1989) J . Cell Bid. 108, 821similar effect as cholesterol on the solubility of APase, and Skibbens, 832 SlOi,-J. W., Geuze, H. J., and Weerkamp, A. J. (1988) Methods Enzymol. 2 0 , GPI-anchored proteins ingeneral, in TX-100. 211-236 Our morphological and biochemical data demonstrate that Slotte, J. P., Harmala, A. S., Jansson, C., and Porn. M. I. (1990) Biochim. APase is endocytosed to a very limited extent. Similar obser- Biophys. Acta 1030,251-257 Takami, N., Ogata, S., Oda, K., Misumi, Y., and Ikehara, Y. (1988) J. BirJl. vations were made for the also GPI-anchored Thy-1 (Leman- Chem. 2 6 3 , 3016-3021 sky et al., 1990). This is consistent with thegeneral idea that Tokuyasu, K. T., and Singer, S. J. (1976) J. Cell Biol. 71,894-906 van den Bosch, R., du Maine, A. P. M., Geuze, H. J., van der Ende, A,, and the motif for recognition by adaptor complexes of clathrinStrous, G. J. (1988) EMBO J. 7,3345-3351 coated domains resides in the cytoplasmic tail of recycling van der Ende, A,, du Maine A. Simmons C. F., Schwartz, A. L, and Strous, G. J. (1987) J. Biol. Chem.’ 262,8910-8616 receptors(Hopkins, 1992).However,5’-nucleotidase and van Meer, G. (1989) Annu. Reu. Cell Biol. 5 , 247-275 Watanabe, S., Watanahe, T., Li, W. B., Soong, B. W., and Chou, J. Y. (1989) GPI-anchored gD-1 do recycle between plasmamembrane J . Biol. Chem. 2 6 4 , 12611-12619 and endosomal compartments, albeit much slower than the Watanabe, T., Wada,N., Kim, E. E.,Wyckoff, H. W., and Chang,J . Y. (1991) J . Biol. Chem. 266,21174-21178 transferrin receptor, and equilibrates with an intracellular Webb, P. D., and Todd, J. (1988) Eur. J . Biochem. 172,647-652 pool within 60-120 min (van den Bosch et al., 1988; Lisanti Yandouzi, E. H. E., and Le Grimellec, C. (1992) Biochemistry 3 1 , 547-551 P. L. (1985) Biochim. Biophys. Acta 822,267-287 et al., 1990). Interestingly, our results suggest that the portion Yeagle, Yeagle, P. L., and Young, J. E. (1986) J . Biol. Chem. 261,8175-8181 of APase that is internalized has lost its TX-100insolubility. T h e conversion to a detergent-soluble form might enhance C. Zurzolo and E. Rodriguez-Boulan, personal communication.