Proc. Nati. Acad. Sci. USAVol. 85, pp. 1529-1533, March 1988Cell Biology

Isolation and characterization of the apical surface of polarizedMadin-Darby canine kidney epithelial cells

(plasma membrane/sialoglycoproteins/cel fractionation/protein sorting)

YULA SAMBUY AND ENRIQUE RODRIGUEZ-BOULAN*Department of Cell Biology and Anatomy, Cornell University Medical College, 1300 York Avenue, New York, NY 10021

Communicated by H. R. Kaback, October 16, 1987

ABSTRACT We have developed a fast and efficientmethod to isolate the apical surface of Madin-Darby caninekidney epithelial cells. After confluent cell monolayers werecoated with alternate layers of cationized colloidal silica and apolyanion, 60% of the apical surface was recovered as largemembrane sheets through the application of a polylysine-coated glass surface. Scanning electron microscopy of thecytoplasmic aspect of the apical surface revealed a honeycombpattern given by the cell borders fractured at or above the levelof the tight junctions. By transmission electron microscopy,the apical preparation appeared to be composed of plasmamembrane and a thin layer of cytoplasm. Enzyme assays andimmunoblots demonstrated a 6- to 7-fold enrichment of anapical marker and a low level of contamination by cytoplasmicand basolateral markers. After removal of cytosolic contami-nants and peripheral membrane proteins by alkaline extrac-tion, apical integral membrane proteins were characterized bysodium dodecyl sulfate/polyacrylamide gel electrophoresis(NaDodSO4/PAGE). Lectin blotting and [3H~glucosamine la-beling identified two major sialoglycoproteins of apparent Mr200,000 and 100,000. The apical membrane sheets here de-scribed provide a useful model for systematic characterizationof the molecular components of the membrane, for reconsti-tution of lipid and protein transport in cell-free systems, andfor study of the interactions of submembranous cytoskeletalproteins with the apical plasma membrane domain.

Recent studies with enveloped viruses in Madin-Darbycanine kidney (MDCK) cells have demonstrated that influ-enza hemagglutinin (HA) and vesicular stomatitis glycopro-tein G, respectively apically and basolaterally targeted gly-coproteins, are sorted at the level of the distal Golgi appa-ratus into different post-Golgi vesicles, which arc thentargeted to and fuse with the respective surface domain bymechanisms still completely unknown (1-3). The develop-ment of methods that result in isolated plasma membranefractions with exposed cytoplasmic aspects is therefore ofgreat importance to study the interactions of transportvesicles with this domain, also in view of the importancerecently attributed to the submembrane cytoskeleton inepithelial polarity (4).We report here a method to purify the apical plasma

membrane ofMDCK cells. The method is an adaptation of aprocedure used by Mason and Jacobson (5) to isolate frag-ments of fibroblast plasma membranes. As modified in thiswork, the procedure results in the fast (5-15 min) andefficient (60-70%) isolation of large open apical plasmamembrane sheets from confluent cultures of epithelial cells,with minimal contamination by basolateral membranes. Us-ing this procedure, we have started the structural and

biochemical characterization of the apical surface ofMDCKcells.

MATERIALS AND METHODSCell Culture and Isolation of the Apical Membrane. Con-

fluent MDCK monolayers (6, 7) grown on nitrocellulosefilters (Millipore; HATF, 0.45-,um pore size) were washedtwice with phosphate-buffered saline (PBS: 138 mMNaCl/13 mM Na2HPO4/1.5 mM KH2PO4/2.7 mM KCl/1mM MgCI2/0.1 mM CaC12) and twice with 130 mM NaCl/1mM MgCl2/0.1 mM CaC12/20 mM 2-(N-morpholino)ethane-sulfonic acid, pH 6.5 (Mes/saline) at 4°C. The monolayerswere treated for 10 sec with 1% cationized colloidal silica (8)in Mes/saline, washed with Mes/saline, treated for 10 secwith polyacrylic acid (Mr 50,000; Polysciences, Warrington,PA) at 1 mg/ml in Mes/saline, and washed extensively withMes/saline. The coating procedure was repeated once andthe cells on filters were overlaid with a glass coverslip orplate that had previously been coated with poly(L-lysine) (Mr> 300,000) at 1 mg/ml. The glass surface and the filter werepressed together with a marble rolling pin and the surfaceswere rapidly peeled apart and transferred to the appropriatemedium for further processing.Scanning and Transmission Electron Microscopy (SEM and

TEM). MDCK apical fractions were washed for 15 min with100 mM KCI/5 mM MgC12/2 mM EGTA/30 mM Hepes, pH7.0 (9), fixed with 1% glutaraldehyde in 0.1 M sodiumcacodylate (pH 7.4), and processed for SEM (10). Coatedand split cells were processed for TEM after fixation in 2.5%glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) contain-ing 4.5% (wt/vol) sucrose (7).

Fluorescence Microscopy. Procedures for immunofluores-cence were as previously described (7, 11). Influenza HAwas localized by indirect immunofluorescence on the apicalsurface of cells infected with a temperature-sensitive mutantvirus (ts6l) (11). Clathrin and the tight junctional polypeptideZO1 (12) were localized with antibodies kindly provided byS. Puszkin (Mount Sinai School of Medicine, New York) andby D. A. Goodenough (Harvard Medical School, Boston),respectively. Actin was stained on paraformaldehyde-fixedapical surfaces with fluoresceinisothiocyanate-conjugatedphalloidin (Molecular Probes, Eugene, OR) at 0.165 ,uM inPBS. Rhodamine-labeled wheat germ agglutinin (WGA) (100,ug/ml in PBS) was applied to unfixed cells for 15 min at4°C before coating and splitting. Nuclei were stained withBisbenzimid H33258 (Riedel De-Haen, Seelze-Hannover,F.R.G.) at 0.2 ,ug/ml in phosphate/citrate buffer (pH 5.6).Enzyme Assays. For enzyme assays, coated whole MDCK

cells, as well as apical and basolateral fractions, werescraped from the filters or glass plates into 150 mM NaCl/1%

Abbreviations: HA, hemagglutinin; SEM, scanning electron micros-copy; TEM, transmission electron microscopy; WGA, wheat germagglutinin.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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*1 1.1.1..I.1.++++++++++++

Coated MDCK cellsBasal

FIG. 1. Isolation of large apical membrane sheets from con-fluent MDCK cells. Confluent MDCK monolayers are grown on13-mm- or 137-mm-diameter nitrocellulose filters. The negativelycharged apical surface is covered with two alternate layers ofcationized colloidal silica (+) and polyanion (-) and removed witha polylysine-coated glass surface (+, coverslips or large plates).

(vol/vol) Triton X-100/10 mM Tris HCl, pH 7.4, at 4°C anddisrupted by sonication. Enzyme activities of y-glutamyltranspeptidase (13), NADPH-cytochrome c reductase (14),and N-acetyl-f3-glucosaminidase (15) were assayed by pub-lished procedures. Proteins were measured by the bicincho-ninic acid (BCA) method (16) (Pierce) using bovine serumalbumin as standard.NaDodSO4/PAGE, [3H]Glucosamine Labeling, Lectin

Blotting, and Immunoblotting. Apical and basolateral frac-tions were extracted with 100 mM sodium carbonate, pH11/1 mM phenylmethylsulfonyl fluoride at 4°C for 60 min(17) and the extracts were centrifuged at 100,000 x g for 2 hrat 4°C. Whole cells or membrane fractions after carbonateextraction were either solubilized in 1% Triton X-100/130mM NaCl/10 mM EDTA/1 mM phenylmethylsulfonyl fluo-ride/20 mM Tris-HCI, pH 7.5, and sonicated or directlycollected in NaDodSO4 buffer (2% NaDodSO4/5 mMEDTA/1 mM phenylmethylsulfonyl fluoride/1 mM iodoacet-amide/50 mM Tris HCl, pH 8.8) (18). Carbonate and TritonX-100 extracts were precipitated with 10% (wt/vol) tri-chloroacetic acid containing sodium deoxycholate at 125,ug/ml (19) and were redissolved in NaDodSO4 buffer.Following reduction and alkylation (18), the samples wereelectrophoresed in gradient (7.5-15%) polyacrylamide gels(20). Protein bands in the gels were visualized either byCoomassie blue or by silver staining (21). For metaboliclabeling, cells were incubated for 20 hr with D-[1,6(n)-3H]glucosamine hydrochloride (specific activity 1658 GBq/mmol; New England Nuclear) at 50 ,uCi/ml (1 ,uCi = 37kBq) in Dulbecco's modified Eagle's medium containingone-fifth the normal concentration of glucose and supple-mented with 0.2% bovine serum albumin. After electropho-resis, the gels were processed for fluorography using sodiumsalicylate as enhancer (22).

After electrophoretic transfer to nitrocellulose paper (23),the blotted proteins were probed either with 1251I-labeledWGA after sequential oxidation and reductive phenylamina-tion of the side chains of sialic acid residues (24) or with theprimary antiserum followed by 1251I-labeled protein A (23).Antibodies against the heavy chain of y-glutamyl transpep-tidase and the a subunit of Na',K+-ATPase were kindlydonated, respectively, by S. S. Tate and B. Rayson (CornellUniversity Medical College, New York).

RESULTS

Fig. 1 schematically shows the method employed to removelarge apical membrane sheets from confluent MDCK mono-layers grown on permeable nitrocellulose filters.Examination by SEM of the cytoplasmic aspect of the

isolated apical plasma membrane sheets revealed a typicalhoneycomb pattern given by the outlines of contiguous cellborders at the level of the fracture (Fig. 2B), as well asvesicles and filamentous structures (Fig. 2C).By SEM and TEM examination, the silica-polyanion coat

was seen to follow uniformly the apical microvilli (Figs. 2Aand 3A). The isolated apical membrane fraction was associ-ated with a thin layer of cytoplasm (Fig. 3B); cells that hadlost apical surface and part of their cytoplasm but hadretained relatively intact nuclei and basolateral membranesremained associated with the filter substrate (Fig. 3C). Theevaginations of the basal membrane into the pores of thefilter appeared to be essential for the splitting procedure;cells grown on glass or plastic tended to separate from thesubstrate and were removed attached to the polylysinesurface.The apical membrane fraction was further characterized

by immunofluorescence staining of integral and peripheralmembrane proteins. The coating procedure allowed normaltransport of HA to the cell surface in monolayers infectedwith the ts6l mutant and coated at the nonpermissivetemperature (Fig. 4A). The apical membrane could also befollowed during isolation by staining with rhodamine-labeledWGA (Fig. 4B). Actin and clathrin were detected associatedin large amounts with the cytoplasmic aspect of the apicalmembrane fraction (Fig. 4 C and D). Both proteins wereefficiently removed by alkaline extraction with carbonatebuffers at pH 11 (Fig. 4E); the same treatment failed toremove influenza HA (Fig. 4A), indicating that the carbonateextraction did not break the association of the membraneswith the polylysine-coated surface.Immunofluorescence localization of ZO1, an antigen as-

sociated with the cytoplasmic aspect of tight junctions (12),on the basal fraction showed positive staining for -90% ofthe cells (Fig. 4F), as confirmed by parallel nuclear staining(Fig. 4G). Less than 10% of the cells in the correspondingapical fraction displayed ZO1 (data not shown).The results of two radioimmunoassay experiments (Table

1) demonstrated that >60% of the apical HA was removed

FIG. 2. SEM of coatedMDCK monolayers and isolatedapical membrane sheets. (A)Apical view of coated MDCKcells. (Bar = 2 Am.) (B) Cyto-plasmic aspect of isolated apicalsheets. (Bar = 2 ,um.) (C) Detailof cytoplasmic aspect of iso-lated apical sheets. Note theborder between three neighbor-ing cells and the presence ofvesicles and cytoplasmic struc-tures associated with the termi-nal web. (Bar = 0.5 ,um.)

Proc. Nati. Acad. Sci. USA 85 (1988)

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FIG. 3. TEM of split MDCK cells. (A) Confluent MDCK monolayer coated with silica-polyanion. (Bar = 2 jim.) (B) Apical membranefraction removed with polylysine-coated nitrocellulose filter. (Bar = 1 Am.) (C) Basal portions of MDCK cells after removal of the apicalsurface. Nuclei and most cytoplasmic organelles distribute with this fraction. (Bar = 1 Am.)

with the apical membrane fraction. Determination in thisfraction of the enzyme activities of y-glutamyl transpepti-dase [an apical marker in kidney (25) and MDCK cells (datanot shown)], N-acetyl-,B-glucosaminidase (lysosomal mark-er) and NADPH-cytochrome c reductase (endoplasmic reti-culum marker) (Table 2) consistently showed a 6- to 7-foldenrichment of y-glutamyl transpeptidase, a depletion of theendoplasmic reticulum marker (0.4-fold enrichment), and noenrichment of the lysosomal marker (1.2-fold). Because of

difficulties in the measurement of basolateral marker en-zymes, the contamination with basolateral membranes wasassessed by immunoblot with antibodies against y-glutamyltranspeptidase and Na',K+-ATPase (Fig. 5). Quantitationof immunoblots indicated <10% ATPase in the apical frac-tion and 20-30% y-glutamyl transpeptidase in the basolateralfraction.The carbonate-extracted apical membrane fraction was

examined by NaDodSO4/PAGE and compared to a similarly

FIG. 4. Fluorescence micros-copy characterization of apical andbasal fractions obtained from splitMDCK monolayers. (A-E) Cyto-plasmic view of the apical mem-brane fraction. (Bars = 5 jim.) (A)Influenza HA after carbonate ex-traction. Cells infected with ts6l for5 hr at 400C were coated at 40C withsilica-polyanion and transferred tothe permissive temperature (320C)for 90 min, and HA was localizedon the isolated apical fraction byindirect immunofluorescence in thepresence of 0.05% saponin. (B)Rhodamine-WGA bound at 40C tothe apical surface prior to coating.(C) Actin filaments stained with flu-orescein isothiocyanate-conjugatedphalloidin. (D) Clathrin, indirectimmunofluorescence. (E) As in D,except for carbonate extraction be-fore staining. A similar result wasobserved for actin. (F and G) Basal

*portion of split MDCK monolayers.*(Bars = 5 jim.) (F) Indirect immu-

nofluorescence on unfixed split'cells with the tight-junction anti-

body ZOL (G) Nuclei stained withH33258.

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Table 1. Recovery of influenza HA in the apical fraction125i, cpm % apical recovery

Exp. Apical (A) Basal (B) [100A/(A + B)]1 10,376 ± 276 4961 ± 347 67 ± 22 3,837 ± 185 2641 ± 165 57 ± 2Confluent MDCK monolayers grown on Millicell HA nitrocellu-

lose filter chambers (Millipore) were infected with influenza WSNvirus for 7 hr, fixed with 2% paraformaldehyde, incubated with HAmouse monoclonal antibody followed by 1251-labeled goat anti-mouse IgG, and coated with silica-polyanion at 4TC. The apicalsurface was removed by use of a polylysine-coated coverslip and theradioactivity associated with the apical and basal fractions wasmeasured. Numbers are mean values ± SEM. Background valuesof 270 cpm (apical) and 700 cpm (apical plus basal), obtained frominfected cells treated with nonimmune IgG, were subtracted. Totalradioactivity recovered in the apical and basal fractions was 95% ofthe radioactivity measured in the unsplit monolayers. Eighteensamples were tested in each experiment.

treated basal membrane fraction. Although some differencescould be detected, the patterns were quite similar, perhapsdue to the 20-30o contamination of basal membrane frac-tions by apical fractions, as detected by radioimmunoassay(Table 1) and immunoblot (see above). Silver staining (Fig.6B) detected more differences than Coomassie blue staining(Fig. 6A). In particular, two major bands at apparent M,200,000 and 100,000 (Fig. 6B, lane 2), which did not stainwith Coomassie blue but bound "25I-labeled WGA (24) (Fig.6C, lanes 2 and 4), were clearly enriched in the apicalmembrane. That these were not adsorbed serum proteinspresent in the growth medium was attested by comparisonwith similarly stained parallel serum lanes (Fig. 6C, lanes 2and 4). The same two bands were the major Triton X-100-extractable glycoproteins labeled during a 20-hr incubationwith [3H]glucosamine (Fig. 6D, lanes 2 and 4). While themajor WGA-binding proteins were largely resistant to alka-line extraction (Fig. 6C, lanes 3 and 5), after metaboliclabeling the M, 200,000 band was also detected in the apical(and, to a lesser extent, in the basal) carbonate extract (Fig.6D, lanes 3 and 5), presumably representing newly synthe-sized glycoprotein in intracellular vesicles.

DISCUSSIONIn spite of the intense interest drawn by the MDCK cellsystem as a model for studying the biogenesis of epithelialcell surface polarity, we know of only one reported attempt(26), using the lactoperoxidase iodination procedure, tocharacterize the plasma membrane proteins of these cells.Here we report the presence of two major integral sialogly-coproteins, of M, 200,000 and 100,000, in the purified apicalsurface of confluent MDCK cells. Their identity as sialogly-coproteins was established by 125I-labeled-WGA blotting

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FIG. 5. Characterization of apical and basal fractions from splitMDCK monolayers by immunoblot localization of the heavy chainof y-glutamyl transpeptidase (A) and the a subunit of Na+,K+-ATPase (B). Protein (17 ,ug per lane) of apical membrane fraction(lanes 1) or basal membrane fraction (lanes 2) was electrophoresedafter carbonate extraction and solubilization in Triton X-100. Num-ber to the left of each autoradiograph indicates Mr x 1o-3. Areas ofthe filter corresponding to the proteins of interest were excised andthe radioactivity was quantitated in a y counter.

and [3H]glucosamine incorporation. The plasma membranesof other cells, such as hepatocytes (24) and erythrocytes(27), also show a restricted number of major sialoglycopro-teins. In MDCK cells the sialoglycoproteins here describedmay be part of the glycocalyx and play an important struc-tural role in the organization of the apical surface.

Standard homogenization-centrifugation procedures de-scribed for the purification of plasma membrane fractionsfrom epithelial cells in culture yield mostly right-side-outvesicles (28), useful for ion- and metabolite-transport studies(29) but inconvenient for characterizing the functions local-ized to the cytoplasmic aspect of these membranes. Solid-substrate fractionation procedures have been used to isolateplasma membrane fractions from erythrocytes (30), fibro-blasts (5), and macrophages (31). The method described inthis report allows the isolation of purified apical domainsfrom epithelial cells. The silica-polyanion coat confers rigid-ity to the apical surface, essential for its intact removal, butdoes not impair its functional properties since transport ofinfluenza HA proceeds normally in coated cells. Practically100% of the cells in the monolayer are split by this proce-dure. More than 60% of the apical surface is recovered, withminimal contamination from the basolateral membrane,since, in most cases, the fracture plane is above the tightjunctions. This represents a significant advantage over usingnitrocellulose filters on uncoated cells, since only smallfragments of apical membrane are removed by this proce-dure (32). The basal portions of the cells, complementary tothe removed apical surface, are also recovered relativelyintact, attached to the nitrocellulose filter.The accessibility of the cytoplasmic aspect of the apical

and basal plasma membrane fractions described in this studyprovides a unique opportunity to study various importantprocesses that take place at that level. For example, itshould be possible to carry out cell-free reconstitution ex-periments of protein and lipid transport between the Golgiapparatus and the plasma membrane. Furthermore, it pro-vides an ideal system to characterize the interactions of the

Table 2. Enzyme activities in split MDCK monolayers% recovery in

Activity or split monolayers Apical fractionmass in whole [100(A + Activity or % yield Specific activity, Relative

cells (W) B)/W] mass (A) (100A/W) nmol/(min-mg) enrichmentProtein 0.51 ± 0.1 37.2 ± 3.8 0.02 ± 0.004 3.6 ± 0.5y--Glutamyl transpeptidase 9.75 ± 1.0 61.2 ± 4.7 2.17 ± 0.23 22.8 ± 2.9 129.1 ± 24.7 6.4Cytochrome c reductase 27.7 ± 4.9 10.8 ± 0.5 1.52 ± 0.12 1.4 ± 0.2 21.5 ± 1.7 0.4N-Acetylglucosaminidase 139.0 ± 6.2 49.4 ± 4.8 7.18 ± 1.22 5.2 ± 1.0 329.9 ± 35.6 1.2W represents the protein mass (mg) and enzymatic activities (nmol/min) in 106 cells removed from the substrate after coating. After splitting,

the protein mass and enzymatic activities were measured in the apical (A) and basal (B) fractions. B is not shown but can be calculated fromthe data according to the formula B = (R-W/100) - A, where R is the recovery of enzyme (or protein) expressed as a percent of the activity(or mass) in whole cells (W). Relative enzyme enrichments were calculated as the ratio of specific activity in the apical fraction to the specificactivity in the whole-cell extract, with the latter normalized to 1. Results are expressed as mean values ± SEM, obtained from three differentexperiments.

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FIG. 6. Protein patterns after NaDodSO4/PAGE. (A) Coomas-sie blue stain (40 /Ag of protein per lane). (B) Silver stain (10 1Lg ofprotein per lane). (C) '251-labeled-WGA blotting (17 Zg of proteinper lane). (D) [3H]Glucosamine labeling (lane 1, 3 x 10' cpm; lanes2-5, 101 cpm per lane). Protein patterns shown in A and B are fromNaDodSO4-solubilized samples. Membrane fractions shown in Cand D were extracted in Triton X-100 and subsequently solubilizedin NaDodSO4. Lanes: 1, whole-cell extracts; 2, apical membranefractions after carbonate extraction; 3, carbonate extracts fromapical fractions; 4, basolateral membrane fractions after carbonateextraction; 5, carbonate extracts from basolateral fractions; 6, horseserum. Positions of two major glycoproteins (Mr 200,000 and100,000), which do not stain with Coomassie blue, are marked bywhite arrowheads in B, C, and D. Molecular weight markers weremyosin heavy chain (Mr 205,000), P-galactosidase (Mr 116,000),phosphorylase b (Mr 97,400), bovine serum albumin (Mr 66,000), eggalbumin (Mr 45,000), and carbonic anhydrase (Mr 29,000).

apical plasma membrane with the submembrane cytoskele-ton, which have been shown to play a crucial role in theorganization of the erythrocyte plasma membrane (27, 33)and, more recently, in the maintenance of epithelial surfacedomains (4). The procedure described in this report shouldtherefore prove a useful tool in the study of the mechanismsthat participate in the biogenesis of epithelial cell polarity.

We are grateful to Dr. Doris A. Wall and Dr. Suresh S. Tate forhelp and advice on the enzyme assays, to Dr. Bruce S. Jacobson forkindly providing the colloidal silica and for many useful suggestions,to Mr. Jim Dennis and Mr. Wayne Fryes for technical assistancewith the electron microscope, and to Ms. Lori Van Houten forexcellent photographic work. This work was supported by grantsfrom the National Institutes of Health (GM34107), the NationalScience Foundation (PCM-8217405), and the New York HeartAssociation. E.R.-B. was recipient of an Established InvestigatorAward from the American Heart Association.

1. Rodriguez-Boulan, E. (1983) Mod. Cell Biol. 1, 119-170.2. Simons, K. & Fuller, S. D. (1985) Annu. Rev. Cell Biol. 1,

243-288.3. Rodriguez-Boulan, E. & Sabatini, D. D. (1978) Proc. Natl.

Acad. Sci. USA 75, 5071-5075.4. Nelson, W. J. & Veshnok, P. J. (1986) J. Cell Biol. 103,

1751-1765.5. Mason, P. W. & Jacobson, B. S. (1985) Biochim. Biophys.

Acta 821, 264-276.6. Vega-Salas, D. E., Salas, P. J., Gundersen, D. & Rodriguez-

Boulan, E. (1987) J. Cell Biol. 104, 905-916.7. Rodriguez-Boulan, E. (1983) Methods Enzymol. 98, 486-501.8. Chaney, L. K. & Jacobson, B. S. (1983) J. Biol. Chem. 258,

10062-10072.9. Hirokawa, N. & Heuser, J. E. (1981) J. Cell Biol. 91, 399-409.

10. Peters, K. R. (1985) Scanning Electron Microsc. 4, 1519-1544.11. Rodriguez-Boulan, E., Paskiet, K. T., Salas, P. J. & Bard, E.

(1984) J. Cell Biol. 98, 308-319.12. Stevenson, B. R., Siliciano, J. D., Mooseker, M. S. & Good-

enough, D. A. (1986) J. Cell Biol. 103, 755-766.13. Meister, A., Tate, S. S. & Griffith, 0. W. (1981) Methods

Enzymol. 77, 237-253.14. Kreibich, G., Debey, P. & Sabatini, D. D. (1973) J. Cell Biol.

58, 436-462.15. Hubbard, A. L. & Cohn, Z. A. (1975) J. Cell Biol. 64, 461-479.16. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,

Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke,N. M., Olson, B. J. & Klenk, D. C. (1985) Anal. Biochem.150, 76-85.

17. Fujiki, Y., Hubbard, A. L., Fowler, S. & Lazarow, P. B.(1982) J. Cell Biol. 93, 97-102.

18. Matlin, K. S. & Simons, K. (1983) Cell 34, 233-243.19. Bensadoun, A. & Weinstein, D. (1976) Anal. Biochem. 70,

241-250.20. Laemmli, U. K. (1970) Nature (London) 227, 680-685.21. Morrissey, J. H. (1981) Anal. Biochem. 117, 307-310.22. Chamberlain, J. P. (1979) Anal. Biochem. 98, 132-135.23. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl.

Acad. Sci. USA 76, 4350-4354.24. Bartles, J. R. & Hubbard, A. L. (1984) Anal. Biochem. 140,

284-292.25. Marathe, G. V., Nash, B., Haschmeyer, R. H. & Tate, S. S.

(1979) FEBS Lett. 107, 436-440.26. Richardson, J. C. W. & Simmons, N. L. (1979) FEBS Lett.

105, 201-204.27. Bennett, V. (1985) Annu. Rev. Biochem. 54, 273-304.28. Lever, J. (1982) J. Biol. Chem. 257, 8680-8686.29. Murer, H. & Kinne, R. (1980) J. Membr. Biol. 55, 81-95.30. Jacobson, B. S. & Branton, D. (1977) Science 195, 302-304.31. Aggeler, J. & Werb, Z. (1982) J. Cell Biol. 94, 613-623.32. Simons, K. & Virta, H. (1987) EMBO J. 6, 2241-2247.33. Branton, D., Cohen, C. M. & Tyler, J. (1981) Cell 24, 24-32.

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