THE JOURNAL or BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 31, Issue of November 5, pp. 19170-19179,lW Printed in U.S.A.

Large Scale Purification and Immunolocalization of Bovine Uroplakins I, II, and III MOLECULAR MARKERS OF UROTHELIAL DIFFERENTIATION*

(Received for publication, June 6, 1990)

Xue-Ru Wu, Motomu Manabe, Jun Yu, and Tung-Tien Sun* From the Epithelial Biology Unit, Departments of Dermatology and Pharmacology, Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016.

The differentiation of mammalian urothelium cul- minates in the formation of asymmetrical unit mem- brane (AUM). Using gradient centrifugation and de- tergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major pro- teins (16,27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM- associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.- T. (1990) J. Cell Sol., 111,1207-1216). Peptide map- ping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa pro- teins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47- kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27- kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble com- plex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of uro- thelial differentiation.

The luminal surface of mammalian urothelium is covered with numerous angular, rigid-appearing plaques, which are thought to be important in stabilizing the apical urothelial surface. These plaques are also present in the cytoplasm in the form of fusiform vesicles (Porter and Bonneville, 1963; Hicks, 1965; Porter et al., 1967). It has been suggested that during bladder distention these cytoplasmic vesicles can insert into the luminal surface and are responsible for an expanded surface area (Porter and Bonneville, 1963; Hicks, 1966; Koss

* This work was supported by National Institutes of Health Grants DK39763 and AR39749 and a grant-in-aid from the New York Uni- versity Urology Department. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be sent: Department of Der- matology, New York University Medical Center, 560 First Ave., New York, NY 10016.

et aZ., 1969; Minsky and Chlapowski, 1978; Severs and Hicks, 1979; Lewis and Moura, 1984; Sarikas and Chlapowski, 1986). In cross-sections, these plaques display a profile of asymmet- ric unit membrane (AUM)’ (Hicks, 1965; Koss, 1969), the luminal leaflet (8 nm) being twice as thick as the cytoplasmic leaflet (4 nm). Negative staining as well as freeze-fracturing of isolated plaques have illustrated that their luminal surface is composed of densely packed, semicrystalline hexagonal arrays of particles with a lattice parameter of 16 nm (Hicks and Ketterer, 1969, 1970; Vergara et al., 1969; Warren and Hicks, 1970; Staehelin et aZ., 1972; Knutton and Robertson, 1976; Severs and Hicks, 1979; Robertson and Vergara, 1980). A detailed, three-dimensional model of the particle has been constructed based on high resolution electron microscopy coupled with image analysis (Brisson and Wade, 1983; Taylor and Robertson, 1984). According to this model, a particle can be divided into six dumbbell-shaped subunits, each of which can be further divided into an “outer” and an “inner” globular subdomain. Since existing data indicate that the proteins are limited exclusively to the luminal leaflet (Caruthers and Bonneville, 1980; Brisson and Wade, 1983; Taylor and Rob- ertson, 1984), it has been calculated that each dumbbell- shaped subunit can accommodate a total protein mass of about 49,000 daltons (Taylor and Robertson, 1984).

The protein composition of AUM has been studied mainly by two approaches. Several groups have “dissociated” AUMs from the underlying cytoskeleton with a reducing agent and then purified them by sucrose gradient centrifugation (Ket- terer et al., 1973; Vergara et al., 1974; Caruthers and Bonne- ville, 1977; Stubbs et al., 1979). Proteins ranging from 15 to 80 kDa have been seen in these AUM fractions (Ketterer et

al., 1973; Vergara et uZ., 1974; Caruthers and Bonneville, 1977; Stubbs et al., 1979). However, the protein patterns of these isolated AUMs, as analyzed by SDS-PAGE, vary significantly. Moreover, since no antibodies have yet been raised to any of these proteins, there is no evidence that any of them are actually AUM-associated in situ. Using an immunological approach, we have recently generated a mouse monoclonal antibody (AE31) against bovine AUM (Yu et al., 1990). This antibody recognizes a 27-kDa protein which can be localized to the apical surface of AUM by the immunogold technique. We have also identified a 15- and a (minor) 47-kDa protein which copurified, apparently through complex formation, with the 27-kDa protein during immunoaffinity chromatography. Whether these two copurified proteins are AUM associated is

1 The abbreviations used are: AUM, asymmetrical unit membrane; SDS, sodium dodecyl sulfate; PAGE, poiyacrylamide gel electropho- resis; PBS, phosphate-buffered saline; HEPES, 4-(2-hydroxyethyl)- l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylene- nitrilo)]tetraacetic acid.

Uroplakins and Asymmetrical Unit Membrane 19171

unknown. Even if they were, these immunological data alone cannot exclude the possible existence of additional AUM components. Finally, the relationship between the three pro- teins that we have identified using the AE31 antibody and those that might be present in gradient-purified AUMS has not yet been studied.

To address these issues, we have adopted and modified the sucrose gradient procedure of Caruthers and Bonneville (1977) for AUM preparation. We demonstrate here that, using this procedure, we can purify milligram quantities of AUMS that are morphologically pure; that these purified AUMs contain three major proteins that are extremely similar, if not identical, to the three proteins that we have previously iden- tified by immunoaffinity purification; and most importantly, that we have been able to generate rabbit-monospecific anti- bodies to each of these three gradient-purified proteins and to prove that all of them are indeed AUM associated in situ. These results provide the strongest evidence available so far that AUM contains three major protein subunits (15, 27, and 47 kDa). The urothelium specificity and differentiation-re- lated expression of these proteins firmly establish their being molecular markers for an advanced stage of mammalian uro- thelial differentiation. Moreover, our ultrastructural localiza- tion data raise the possibility that the 27- and 15-kDa proteins may form the 12nm apical protein particles of AUM, while the 47-kDa protein is a transmembranous component possibly involved in anchoring the plaques to an underlying cytoskel- eton.

MATERIALS AND METHODS Gradient Purification and Detergent Treatment of AUM-Fresh

bovine bladders were obtained within 2 h postmortem from a local slaughterhouse, where they were processed on site immediately. Blad- ders were inverted, rinsed with distilled water, and washed with ice- cold phosphate-buffered saline (PBS). The urothelium was scraped from the luminal surface with a blunt scalpel, and suspended in PBS. After centrifugation at 1,500 x g at 4 “C for 5 min, the cells were collected and transferred on dry ice to the laboratory. The subsequent steps were according to Vergara et al. (1974) and Car&hers and Bonneville (1977) with some modifications. The urothelial cells were homogenized in 10 mM HEPES buffer, pH 7.5, containing 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA and 1 pg/ ml each of protease inhibitors including chymostatin, leupeptin, pepstatin, and antipain (buffer A). In experiments designed to test the effect of a reducing agent, the buffer contained additional 10 mM dithiothreitol or 20 mM sodium thioglycolate. After centrifugation at 2,500 X g at 4 “C for 15 min, the pellet was resuspended and homog- enized in the same buffer. The homogenate was filtered through five layers of 100~pm mesh nylon net, and the filtrate was loaded onto a discontinuous sucrose density gradient constructed with, from bottom to top, 1.6,1.3,1.1, and 0.75 M sucrose in buffer A. The centrifugation was performed in a SW28 (Beckman) rotor at 26,000 rpm at 4 “C for 2 h. Membranes accumulated at each interface were collected, diluted with 10 volumes of buffer A, and centrifuged at 15,000 x g at 4 “C for 15 min.

The gradient-separated membrane fragments were treated with 2% deoxycholate in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride at room temperature for 30 min. The detergent-resistant membranes were recovered by centrifuging at 18,000 X g at 4 “C for 15 min, washed with 50 mM Tris-HCl, pH 7.4, dissolved in 2% SDS in 50 mM Tris-HCl, pH 7.4, cleared by centrifugation at 12,000 x g for 5 min, and analyzed by SDS-PAGE. BCA (Pierce Chemical Co.) reagent was used for protein quantitation in the presence of 1% SDS.

Affinity Purification of A UM-related Proteins-AE31 monoclonal antibody was conjugated to Sepharose 4B as described (Yu et al., 1990). To prepare the antigens, urothelial cells were extracted with 1% Triton X-100,50 mM Tris-HCl, pH 7.4, plus a mixture ofprotease inhibitors as in buffer A (buffer B). After centrifugation at 12,000 x g at 4 “C for 30 min, the supernatant was diluted with buffer B to about 1 mg of proteins/ml. Affinity purification was performed batch- wise using 0.5 ml of AE31-conjugated Sepharose beads (“immuno-

beads”) per 500 ml of urothelial extract (equivalent to six bovine bladders). After incubation at 4 “C overnight with gentle end-to-end mixing, the loaded immunobeads were washed three times with 50 mM Tris-HCl, pH 7.4, containing 0.5 M NaCl followed by three washes in PBS. The bound antigens were then eluted with 50 mM diethyla- mine, pH 11.5, containing 1% octyl glucoside.

Antibody Production-Gradient-purified AUM membranes (2 mg of total proteins) were mixed with Freund’s complete adjuvant (pri- mary immunization) or incomplete adjuvant (booster immunizations) and injected subcutaneously into rabbits and chickens. In addition, the 15-, 27-, and 47-kDa proteins (uroplakins) of gradient-purified AUMs were separated by SDS-PAGE and used as immunogens.

Antibodies monospecific for individual uroplakins were affinity purified as follows. AUM proteins were separated by preparative SDS-PAGE (17% acrylamide), transferred electrophoretically to ni- trocellulose paper, and stained with 2% Ponceau S in 1% acetic acid. After a brief destaining in 1% acetic acid, paper strips containing individual (protein) bands were cut out, blocked with 3% bovine serum albumin, and incubated with an antiserum to crude, total AUM membrane proteins at 37 “C for 1 h with agitation. The strips were washed extensively in PBS, and the bound antibodies were eluted with 50 mM diethylamine, pH 11.5, and immediately neutralized with 1 M Tris-HCl, pH 7.4. The eluted antibodies were concentrated with Centricon- (Amicon, Danvers, MA) and stored at -70 “C.

Electron Microscopy-Gradient-purified membrane fragments were collected by centrifugation, fixed in 2% glutaraldehyde, post- fixed with 2% OsO1, dehydrated in ethanol and acetone, and em- bedded in Epon (Manabe et al., 1981). For negative staining, the fixed membranes were stained with 2% uranyl acetate and examined using a transmission electron microscope (courtesy of Dr. Ueli Aebi, Bioz- entrum, Basel, Switzerland). Immunoelectron microscopy was carried out using affinity purified antibodies. Briefly, fresh bovine bladders were fixed with Zamboni’s solution (15% saturated picric acid and 2% paraformaldehyde in PBS), cut into 4-pm frozen sections, incu- bated with 3% bovine serum albumin in PBS at room temperature for 30 min, and reacted with primary antibodies at 37 “C for 1 h and 4 “C overnight. After washing in PBS for 1 h, the sections were treated with a secondary antibody conjugated with 5-nm colloidal gold particles at 37 ‘C for 1 h and 4 “C overnight. The specimens were then processed routinely as described (Amenta and Martinez- Hernandez, 1987; Martinez-Hernandez, 1987). For post-embedding immunoelectron microscopy, tissues were fixed with Zamboni’s solu- tion, dehydrated, and embedded in LR White (Polyscience; Warring- ton, PA). Ultra-thin sections were incubated with primary antibody, followed hy incubation with a secondary antibody conjugated with 15-nm colloidal gold particles (Life Sciences Products, Piscataway, NJ).

Cell Culture of Bovine Urothelium-Secondary cultures of bovine urothelial cells were grown in a 1:l mixture of Dulbecco’s modified Eagle’s Medium and Ham’s F-12 medium supplemented with 20% fetal calf serum and 0.5 pg/ml hydrocortisone, in the presence of mitomycin-treated 3T3 feeder cells (Sun et al., 1980; Wu et al., 1982).z

Immunofluorescence Staining-Tissues prefixed with Zamboni’s solution were cut into 5-am frozen sections and stained by indirect immunofluorescence (Sun and Green, 1978; O’Guin et al., 1986). Confluent urothelial cultures grown on 12-mm glass coverslips were fixed and permeabilized with cold methanol and acetone, and used for immunofluorescence staining.

Peptide Mapping-After SDS-PAGE and Coomassie Blue staining, pieces of the polyacrylamide gel containing individual proteins were cut out, washed with 70% formic acid, and treated with 5% cyanogen bromide in 70% formic acid for 2 h at room temperature in the dark (Lonsdale-Eccles et al., 1981). The gel pieces were washed with 10% acetic acid and equilibrated with 50 mM Tris-HCl, pH 7.4, containing 2% SDS for 2 h. Peptides in the gel pieces were then resolved using a Tricine SDS-PAGE system (16.5% total acrylamide/hisacrylamide and 6% bisacrylamide) for resolving small peptides (Schagger and VonJagow, 1987).

Gel Ekctrophoresis and Zmmunoblotting-Proteins were separated by SDS-PAGE according to Laemmli (1970) (acrylamide/bisacryl- amide = 12O:l). For immunoblotting analysis, proteins resolved by SDS-PAGE were electrophoretically transferred to nitrocellulose pa- per (Towbin et al., 1979) and stained by the peroxidase-anti-peroxi- dase technique (Woodcock-Mitchell et al., 1982; Eichner et al., 1984). Slot blottings were performed using a commercial mini-slot blotter (Bethesda Research Laboratories).

* B. Surya, J. Yu, M Manabe, and T.-T. Sun, unpublished data.

19172 Uroplakins and Asymmetrical Unit Membrane

Solubility of AUM Components-Isolated bovine AUMs were treated at room temperature for 30 min in one of the following conditions: 1 M NaCO.,. 0.5 M HCI. 1 M NaCl. 10 mM CHAPS. 2% Nonidet P-40, 2% Briton X-100, O.Oi% SDS, 0.65% SDS, 0.1% SDS, 2% octyl glucoside, 6 M guanidine chloride, 9 M urea, and 9 M urea plus 10% /3-mercaptoethanol. Except for the first two, all solutions also contain 50 mM Tris-HCl, pH 7.4. After centrifugation at 16,000 X g for 10 min, the insoluble material was dissolved in 2% SDS and analyzed by SDS-PAGE.

RESULTS

Isolation of Bovine A UMs by Sucrose Gradient Centrifuga- tion-To determine the relationship between the three pro- teins that we have previously identified by immunoaffinity purification and the proteins that might be present in isolated AUMs, we fractionated crude bovine urothelial membranes on a discontinuous sucrose density gradient basically accord- ing to Caruthers and Bonneville (1977). To localize the 27- kDa AUM subunit, we “slot-blotted” equal amounts of pro- teins from each gradient fraction and stained them immuno- chemically with the AE31 antibody (Fig. 1). The results indicated that this protein was concentrated in fractions 2 (interface between 0.75 and 1.1 M sucrose) and 3 (1.1 and 1.3 M sucrose).

Since the 27-kDa protein interacts strongly with a 15- and a 47-kDa protein during affinity purification (Yu et al., 1990), we wanted to know whether similar proteins also copurified with the 27-kDa protein during gradient separation of uro- thelial membranes. When membranes of various fractions were analyzed by SDS-PAGE, a large number of proteins were observed most likely due to contaminating, non-AUM mem- branes. These could be preferentially solubilized, however, with deoxycholate (DOC) which did not affect AUM mor- phology (Fig. 2b, the “supernatant” fractions; Caruthers and Bonneville, 1977). Analysis of the DOC-insoluble pellets re- vealed the selective enrichment, in fractions 2 and 3, of three major proteins, that were approximately 15, 27, and 47 kDa in size (Fig. 2c, the “precipitate” fractions).

If the 27-kDa protein is indeed AUM-associated (Yu et al., 1990), we should in theory see an enrichment of AUMs in fractions 2 and 3. Examination of the DOC-insoluble material by transmission electron microscopy showed that these two fractions indeed contained highly purified, rigid-appearing plaque structures, with no discernible contaminating organ-

a b C

FIG. 1. Fractionation of AUM by sucrose-gradient centrif- ugation. Panel a illustrates a four-step gradient constructed with, from bottom to top, 1.6, 1.3, 1.1, and 0.75 M sucrose. I-5 denote the five membrane fractions. Panel b, slot blot analyses using AE31 antibody. T denotes total proteins from urothelial membranes before gradient separation. Same amounts of total proteins from each mem- brane fraction were analyzed (5 rg for Fast Green staining (FG) and 50 ng for immunoblotting). Note that AE31 reactivity was observed weakly in fractions 2 (0.75/1.1 M) and strongly in fraction 3 (1.1/1.3 M), and that control antibody (P3) did not produce any staining. Panel c, slot blot analyses using a rabbit antiserum prepared against a purified, native AUM preparation (aAUM, see below). PI denotes preimmune serum.

G rad.-0%

a. b. sp c. PPt.

.- -47

.

d -27 .-

*i ‘- I, m - -15 . .

s 123 12 345 12345

FIG. 2. Selective enrichment of three deoxycholate-resist- ant proteins (15,27, and 47 kDa) in fractions 2 and 3 of the sucrose density gradient. Various membrane fractions from the gradient were resolved by SDS-PAGE and stained with Coomassie Blue. Panel a: lane s, molecular mass standards including, from top to bottom: bovine serum albumin, 66 kDa; egg albumin, 45 kDa; glyceraldehyde-3-phosphate dehydrogenase, 36 kDa; carbonic anhy- drase, 29 kDa; trypsinogen, 24 kDa; trypsin inhibitor, 20.1 kDa; and a-lactalbumin, 14.2 kDa. Lane I, total urothelial proteins solubilized by 2% SDS; lane 2, urothelial proteins soluble in buffer A (see “Materials and Methods”); lane 3, total proteins of urothelial mem- branes prior to gradient (Grad.) fractionation. Panel b shows the deoxycholate (DOC)-soluble membrane proteins from fractions 1 through 5 supernatant (Sup) fractions; 30 ,ug of protein/lane. Panel c shows the DOC-insoluble proteins from the corresponding fractions (in Ppt or pellets). The DOC-insoluble proteins from each fraction were dissolved in 2% SDS, and the same proportion of each fraction was analyzed by SDS-PAGE. Note the selective enrichment of three proteins (15, 27, and 47 kDa) in fractions 2 and 3 (compare with Fig. lb). The low molecular weight bands in he 5 (pellet) are probably nuclear histones.

elles (Fig. 3b, cf. Fig. 3a). At a higher magnification, these plaques displayed typical features of asymmetrical unit mem- brane, with an outer leaflet twice as thick as the inner one (Fig. 3c and its inset). Moreover, negative staining of these isolated plaques revealed highly regular hexagonal arrays of 12-nm particles (Fig. 3d). These results confirm Caruthers and Bonneville’s earlier finding (1977) that AUM can be isolated using a combination of sucrose gradient centrifuga- tion and detergent wash. Furthermore, our data indicate that such gradient-purified AUMs contain three well-defined pro- teins (Fig. 2c), and that the apparent molecular masses of these three proteins are similar to the three AE31-affinity purified proteins including the 27-kDa protein that we have shown to be AUM-associated (Yu et al., 1990; see below).

Optimization of the Purification Procedure-Although our gradient centrifugation is largely based on a procedure de- scribed by Caruthers and Bonneville (1977), we have intro- duced several modifications. First, it has been emphasized by earlier investigators that a reducing agent is required during the initial homogenization step for AUM isolation (Vergara et al., 1974; Caruthers and Bonneville, 1977). Presumably, the reducing agent breaks the disulfide bonds which anchor AUM plaques to an underlying keratin network, thereby releasing AUM and increasing the yield. We found, however, omission of the reducing agent under our experimental conditions affected neither the yield nor the purity of bovine AUM (data not shown). Second, to minimize proteolysis we have included in our extraction buffers a mixture of protease inhibitors (chymostatin, pepstatin, leupeptin, antipain, and phenyl- methylsulfonyl fluoride). This may explain why the 47- and 15-kDa proteins were present as major components in our AUM preparations but were either missing or were present only in trace amounts in some earlier preparations. Third, we

Uroplakins and Asymmetrical Unit Membrane 19173

1

I i ,r+ ’

’

FIG. 3. Ultrastructure of gradient-purified AUMs. Mem- brane fragments from fraction 3 of the gradient (shown in Fig. 2) were examined using a transmission electron microscope either before (a) or after (b and c) deoxycholate treatment. The DOC-resistant membranes were also examined after negative staining (d). Note in panel a the presence of a small amount of urothelial plaques among large quantities of contaminating membranes. Also note in panels b and c the selective removal of contaminating membranes by DOC treatment, and the preservation of rigid-looking, angular plaques characteristic of asymmetric unit membrane (panel c, inset; L, luminal side; C, cytoplasmic side, and H, the hinge area interconnecting neighboring plaques). Panel d shows semicrystalline arrays of hex- agonal 12.nm protein particles. Bars represent 1 pm in a and b, 250 nm in c, 150 nm in its inset, 100 nm in d, and 30 nm in its inset.

1 .!-I-*

-47

.

--E -v

c 0 -27

-15 .-

'Sl 2 3 1'3'

FIG. 4. Heat-induced aggregation of the 27-kDa protein. AUM proteins were dissolved in 2% SDS and incubated at room temperature for 30 min (lanes 1 and I’), 37 “C for 30 min (lane 2), or 100°C for 5 min (lanes 3 and 3’) before being resolved by SDS- PAGE. Samples in lanes Z-3 are not reduced, while those in lanes 1’ and 3’ were reduced with 10 mM dithiothreitol. Note in lanes 3 and 3’ the disappearance of the 27-kDa protein and the appearance of protein aggregates on top of the gel (larger asterisk). The small asterisk marks an unknown minor protein of -40 kDa present in greater amounts in reduced samples. S denotes molecular weight standards same as in Fig. 2.

found that the 27-kDa protein became aggregated, sometimes to the extent of its staying on top of the separating gel, if it was heated in SDS sample buffer before electrophoresis (Fig. 4). Such a heat-induced retardation of the 27-kDa protein occurs with or without a reducing agent and is therefore independent of disulfide bond formation (Fig. 4). Since most of the earlier investigators heated or even boiled their AUM-

proteins before SDS-PAGE analyses, this may explain some of the variations in the reported SDS-PAGE patterns (Ver- gara et al., 1974; Caruthers and Bonneville, 1977; Stubbs et al., 1979). A similar heat-induced reduction in the SDS-PAGE electrophoretic mobility has been reported for a number of bacterial proteins (Teather et al., 1978; Clarke et al., 1985), plant proteins (Gallagher and Leonard, 1987), as well as a 26- and a 21-kDa mouse gap junction protein (Henderson et al., 1979), presumably due to temperature-dependent protein ag- gregation.

With the above modifications and precautions, we were able to isolate approximately 1 mg of the three AUM-related (total) proteins/cow bladder, and the results are extremely reproducible.

Comparison of the Gradient- and Affinity-Purified AUM Proteins-To compare these gradient-purified proteins with the affinity purified ones, we needed to improve the yield of the latter which we had only minute amounts (Fig. 5a; Yu et al., 1990). Titration experiments established that under our adsorption conditions (4 “C, 16 h) 0.5 ml of AE31-conjugated immunoadsorbent beads (immunobeads) was sufficient to ad- sorb, from a 500-ml aliquot of urothelial extract (in 1% Triton X-100), a maximal amount of the 27-kDa subunit and its two associated proteins (Fig. 5b). To confirm that this saturating amount (0.5 ml) of immunobeads could indeed recover all the AE31-reactive antigens, we completed an adsorption cycle followed by four additional cycles, each time using fresh immunobeads. The antigens thus recovered from each cycle were compared semiquantitatively by SDS-PAGE. Unexpect- edly, we found that all five sequential cycles yielded identical amounts of the three proteins (Fig. 5~). These results indicate that under our experimental conditions the immunoadsorp tion of AE31 antigen proceeds extremely slowly and ineffi- ciently, possibly involving a rate-limiting step of solubilization and/or immunoadsorption of the membrane antigens (Pabst

a b C -_ .v

.

. IuB

-47

m --m -----w-15 .v

s123 123 12345

FIG. 5. Immunoaffinity purification of AUM-related pro- teins. Panel a shows urothelial proteins, at different stages of affinity purification, as resolved by SDS-PAGE (stained with silver nitrate). Lane 1, total urothelial proteins soluble in Triton X-100, the starting material for affinity purification; lanes 2 and 3, the flow-through and hound fractions, respectively, from an affinity purification experi- ment using AE31-conjugated Sepharose 4B heads. Note the one-step purification of three proteins (15, 27, and 47 kDa). S denotes molec- ular weight standards same as in Fig. 2. b, titration of AE31-conju- gated Sepharose heads: different amounts of immunobeads were incubated with 500-m] aliquots of urothelial extracts. Lanes l-3: 0.5, 1,2 ml. Note that maximum yield was achieved using 0.5 ml of AE31- heads. c, multiple cycles of immunoadsorption. After completing one cycle of affinity purification, 0.5 ml of fresh immunoheads were added to the “spent” urothelial extract, and the adsorption/elution cycle was repeated, for a total of five times. One-tenth of the purified proteins from each cycle was analyzed in lanes 1 through 5. The positions of the three urothelial proteins (15, 27, and 47 kDa) are marked. Note that an almost identical amount of AUM-related pro- teins was recovered from each cycle.

19174 Uroplakins and Asymmetrical Unit Membrane

a SL ‘m47 c . -_- . I

-

- O- ., a- 0-

.-15

I

1 - -15

-7.5 -6.0

* I ---15 0

512 s 12 12 3 4

FIG. 6. Comparison of gradient- and immunoaffinity puri- fied AUM proteins. Two-hundred ng of affinity and gradient- purified proteins were resolved by either a, Laemmli-type SDS-PAGE (17% T, 0.8% C), or b, a Tricine SDS-PAGE (16.5% T, 6% C). Lanes 1 and 2 are affinity and gradient-purified proteins, respectively. Note the coelectrophoresis of the three major proteins in the two samples. A minor 25-kDa band is present in both samples in varying quantities and may be a degradative product (lower filled circle in panel a). Another unknown minor protein, about 40 kDa in size, is present only in the gradient-purified sample (upper open circle). S in panel a denotes molecular weight standards as described in Fig. 2. The same molecular weight markers (filled circles) were used in lane S of panel b, which also contains additional small molecular mass markers (open circles) which are, from the top: myoglobin fragments, 17, 14.4, 8.2, 6.2, and 2.5 kDa. c, cyanogen bromide cleavage of the 15-kDa proteins isolated by immunoaffinity and by sucrose gradient. Lanes 1 and 3, the 15-kDa proteins purified by immunoaffinity and gradient, respec- tively. Lanes 2 and 4 show the CNBr-fragments of the corresponding samples. Note the generation of two identical fragments (7.5 and 6.0 kDa) from both samples. All gels were stained with silver nitrate.

et al., 1984). This explains why immunoaffinity chromatog- raphy suffers from an extremely low yield (about 1 pg of isolated antigens/cow bladder/adsorption cycle), although this can be improved by a few folds through repeated cycling.

Using two different SDS-PAGE systems (Laemmli type shown in Fig. 6a and Tricine type for better resolution of low molecular weight proteins shown in Fig. 6b), we found that the three gradient-purified proteins comigrated precisely with the 15-, 27-, and 47-kDa affinity purified proteins. In addition, peptide mapping data showed that the 15-kDa protein isolated by the two methods yielded identical CNBr fragments (7.5 and 6 kDa; Fig. 6~). N-terminal amino acid sequences of the 15-kDa proteins from the two sources were also found to be identical.” These results, plus the AE31 reactivity of the gradient-purified AUM fractions as shown in Fig. lb, strongly suggest that the three major proteins of the gradient-purified AUM preparation are closely related, if not identical, to the three proteins that we previously identified using a totally different approach, i.e. affinity purification using AE31 mono- clonal antibody (Yu et al., 1990).

Solubility Properties of the A UM-related Proteins-The availability of milligram quantities of the three gradient- purified, AUM-related proteins allowed us to study their solubility and other properties. Based on the results from Caruthers and Bonneville (1977) and some of our own prelim- inary testings, we have learned at an early stage of the investigation that AUM-structure is quite resistant to DOC. This unique property of the AUM greatly facilitated its puri- fication (Fig. 2).

These DOC-resistant AUMs remained insoluble in several other detergents including Nonidet P-40 and CHAPS (Fig. 7). They were also insoluble in 1 M Na2C03 (pH 12), 0.5 M HCl, or 1 M NaCl, thus adding support to our earlier sugges- tion that the major AUM proteins are integral membrane

‘I X.-R. Wu, unpublished observations.

-47

-15

8 9 10 11 12 13 14

FIG. 7. Solubility properties of AUM proteins. Equal amounts of deoxvcholate-insoluble material from fraction 3 were subjecmd t.o .__- __ various-treatments, centrifuged, and the pellets were dissolved in 2% SDS. The nroteins were resolved bv SDS-PAGE and stained with - . . . “ . .

Coomassie Blue. Lane I, the starting material; 2, same material after washing with 2% Nonidet P-40; 3, 10 mM CHAPS; 4, 0.01% SDS; 5, 0.05% SDS; 6, 0.1% SDS; 7, 2% octyl glucoside; 8, 2% Triton X-100; 9, 0.5 M HC1; 10, 0.1 M Na2C03, pH 12; 11, 1 M NaCl; 12, 9 M urea; 13, 9 M urea + 10% 3-mercaptoethanoh and 14, 6 M guanidine chloride. S denotes molecular weight standards (same as in Fig. 2). Note the remarkable insolubility of the three AUM proteins in most conditions except in octyl glucoside, SDS and, to some extent, Triton x-100.

proteins (Yu et al., 1990). The proteins were also insoluble in 9 M urea (with or without a reducing agent) and in 6 M guanidine chloride (Fig. 7), indicative of exceedingly strong protein-protein interactions that could be important in the formation and stabilization of AUM plaques (see below). That the three proteins could be effectively solubilized in SDS (Fig. 7) indicate that the stabilizing forces, however strong, were noncovalent. The three proteins were also soluble in 2% Triton X-100 (Fig. 7), the detergent that we used to solubilize the AE31 antigens for affinity purification (Yu et al., 1990). Although the three Triton-solubilized proteins remained in the “supernatant” after centrifugation, their copurification during affinity chromatography (Fig. 5; Yu et al., 1990) clearly indicated that they existed as a complex. Finally, the three proteins could be solubilized in 2% octyl glucoside (Fig. 7), which is unique among all the detergents that we have tested so far in that it can be removed by dialysis, a feature poten- tially useful for future membrane reconstitution.

The Three AUM-related Proteins Are Biochemically and Immunologically Distinct-To study the biochemical relation- ships among the three gradient-purified proteins, we resolved them by SDS-PAGE and digested them in gel pieces in situ with cyanogen bromide. Three distinct patterns were observed with no overlapping fragments (Fig. 8). Moreover, we have generated five to six stretches of N-terminal and internal amino acid sequences of the 15- and 27-kDa proteins, again finding no overlapping sequences.3 These results indicate that the three proteins are biochemically distinct.

Using the gradient-purified AUMs as immunogen, we have succeeded in raising several rabbit and chicken antisera to total AUM proteins (for the importance of using nondena- tured instead of SDS-denatured AUM as the immunogen, see Yu et al., 1990). Immunoblotting experiments revealed that these antisera reacted extremely strongly with the 47-kDa protein, reasonably strongly with the 27-kDa protein, and relatively weakly with the 15-kDa protein (see Fig. 9b, lane 2 for an example). Such a preferred immunoreactivity with the higher molecular weight proteins may reflect their higher antigenicity and/or more exposed configuration in native AUM antigens.

Uroplakins and Asymmetrical Unit Membrane 19175

.- .- a, b c . -F -47 .-

-27

a 0 t a D - -15

0 o-7.5 v-6.0

S 12 12 12

FIG. 8. Cyanogen-bromide cleavage of gradient-purified lb, 27-, and 47-kDa proteins. AUM proteins were resolved by SDS-PAGE, and gel pieces containing the three major proteins were treated with CNBr in situ. The resulting peptides were resolved by Tricine-type SDS-PAGE (16.5% T, 6% C) and stained with silver nitrate. a, lanes 1 and 2 are the intact 47-kDa proteins and its CNBr peptides, respectively. b, lanes I and 2 are the intact 27-kDa protein and its CNBr peptides, respectively. c, lanes 1 and 2 are the intact 15-kDa protein and its CNBr peptides, respectively. Note the distinct peptide patterns of the three proteins. S denotes molecular weight standards same as in Fig. 6b.

abcdefg

a ..d .- -47

0 -27

W e --15

12 12 12 12 12 12 12

affinity purified 15kDa protein. Note the immunological distinct ness of the three AUM-related proteins.

Fro 9. The 15-, 2’7-, and 47-kDa urothelial proteins are immunologically distinct. Lanes 1 and 2 show the SDS-PAGE patterns of 30 pg of total urothelial proteins and purified deoxycho- late-insoluble plaque proteins, respectively. These proteins were elec- trophoretically transferred to nitrocellulose sheet, and stained with Fast Green (a); b, a rabbit antiserum raised against DOC-insoluble AUM plaques (alum, the antibody used in Fig. lc); c, a rabbit antiserum raised against electrophoretically purified 47-kDa protein; d, antibodies to the 47-kDa protein affinity purified from aAUM (a- 47 kDa); e, antibodies to the 27-kDa protein similarly affinity purified from aAUM (a-27 kDa); f, affinity purified antibodies to the 15-kDa protein (a-15 kDa); g, a rabbit antiserum raised against a synthetic peptide corresponding to the N-terminal amino acid sequence of the

indirect immunofluorescent staining that all three proteins were urothelium specific and were limited to the superficial (umbrella) cells (27-kDa protein, Fig. 10, c and d; 15-kDa protein, e; and 47-kDa protein, f). By double immunofluores- cent staining, we showed that the distribution of the 27-kDa protein, as assessed by our new polyclonal rabbit antiserum (Fig. lOc), was identical to that revealed by our previously described AE31 monoclonal antibody (Fig. 10d; Yu et al., 1990). Taken together, these data confirm and extend our previous suggestion that the 15 and 27-kDa proteins are markers for an advanced stage of urothelial differentiation (Yu et al., 1990). These results also provide for the first time clear evidence that the 47-kDa protein coexpresses with the 27- and 15-kDa protein during a terminal stage of urothelial differentiation (Fig. lo/).

When we stained cultured bovine urothelial cells with these antibodies, we found some superficial squamous cells stained strongly indicating that such cells can express not only the 27-kDa protein, as we have shown earlier (Yu et al., 1990), but also the 15- and 47-kDa proteins (Fig. 11).

Using AE31 monoclonal antibody as a probe, we have previously shown by the immunogold technique that the 27- kDa protein is associated exclusively with the apical surface of mature, lumen-lining AUM (Yu et al., 1990). We also showed that in some small, cytoplasmic vesicles, presumably representing an earlier stage of AUM maturation, the immu- nogold particles are found mainly on the cytoplasmic side sometimes in small patches (Yu et al., 1990). We have now confirmed these observations using an independent rabbit polyclonal antibody highly specific for the 27-kDa protein (Fig. 12). These results support our earlier suggestion that parts or the entire molecule of this 27-kDa protein may translocate from the cytoplasmic side of the membrane to the luminal side during an advanced stage of AUM maturation (Yu et al., 1990).

The 15:kDa protein showed an almost identical ultrastruc- tural localization pattern, i.e. patchy staining of the cyto- plasmic side of immature AUM-containing vesicles, and ex-

t

m,'w c&+p?adE::

--_________------___----

C

I@--

d

c-H samna*- -y- fls-

__-- -_______- ----_ - ___--_- ---- __--

From these antisera to total AUM proteins, antibodies to .,m\ ,-..F r,k- p..~-a&i&&- -- *‘ individual proteins were affinity purified using the three proteinsgeneratedbypreparative SDS-PAGE(anti-47kDa,, __-------------------------- _____--------------

__--- --_.

Fig. 9d; anti-27 kDa, e; and anti-15kDa, f). In addition, an antiserum to the 47-kDa protein was raised by immunizing rabbits with electrophoretically purified 47-kDa component (Fig. 9c). Finally, a rabbit antiserum was produced against a synthetic peptide corresponding to the N-terminal amino acid sequence of the 15-kDa protein (Fig. 9g). The fact that all these antibodies turned out to be monospecific for their re- spective antigens (Fig. 9) provides persuasive evidence that the 15-, 27-, and 47-kDa molecules are immunologically dis- tinct.

Immunoloculizution of the Three AUM-related Proteins- With these monospecific antibodies, we were able to show by

sect&s (5 pm) of bovine bladder were prepared and stained by indirect immunofluorescence using a, a rabbit antiserum to total proteins of AUM (aA UM, cf. Figs. lc and 9b); b, the staining pattern of the same field produced by monoclonal antibodies AEl and AE3 to keratins (Woodcock-Mitchell et al., 1982); c, a-27 kDa; d, AE31 to the 27-kDa protein (Yu et al., 1990); e, a-15 kDa; and f, a-47 kDa. Note the selective staining of superficial umbrella cells by antibodies to all three AUM proteins. All pictures are of the same magnification; bar in a equals to 25 pm.

19176 Uroplakins and Asymmetrical Unit Membrane

FIG. 11. Expression of the 15-, 27-, and 47-kDa proteins in cultured bovine urothelial cells. Confluent cultures of bovine urothelial cells were fixed with acetone-methanol and stained with various antibodies by indirect immunofluorescence. a, the staining pattern generated by a-AUM; b, phase contrast view of the same field; c, a-27 kDa; d, a-15 kDa; e, double-staining of the same field using anti-keratin antibodies (AEl ~1~s AE3): f. a-47 kDa: and P. nhase contrast view of the same field. Note the’staining of some superficial cells by antibodies to the three AUM-related proteins. All pictures are of the same magnification; bar in g equals to 25 Wm.

elusive staining of the luminal side of mature, AUM-contain- ing, surface plaques (Fig. 13). This result indicates that the 15- and 27-kDa proteins not only coexpress (Fig. 10) but also undergo similar translocational events during AUM matura- tion.

The localization pattern of the 47-kDa protein was some- what different. In mature AUM plaques of the apical cell surface, this protein could be detected on both luminal and cytoplasmic sides (Fig. 14, a and b). The association of this protein with AUM was confirmed using a post-embedding method (Fig. 14, d-f). Such a transmembrane distribution of the 47-kDa protein in apical AUMs sets it apart from the 15- and 27-kDa proteins both of which are asymmetrically dis- tributed (detectable only on the luminal side; Figs. 12 and 13). This observation also proves that the cytoplasmic side of the apical AUM is readily available to antibody molecules, thus excluding the possibility that a lack of staining of this area by antibodies to the 27- and 15kDa molecules is simply due to its inaccessibility to the immunoglobulin molecules.

DISCUSSION

Protein Composition of AUM-To establish the protein composition of AUM, ideally one needs to fulfill the following three criteria: 1) to be able to isolate morphologically pure preparations of AUM, 2) to demonstrate that well-defined proteins are enriched in such preparations, and most impor- tantly, 3) to prove that all these proteins are AUM-associated in situ. We have now satisfied all these criteria. First, we have isolated large quantities of bovine AUMs that are morpholog- ically pure (Fig. 3). Although this procedure (discontinuous sucrose gradient coupled with DOC-treatment) was similar to that originally described by Caruthers and Bonneville (1977), we have introduced several modifications including the omis-

FIG. 12. Ultrastructural localization of the 27-kDa protein. Frozen sections of bovine urothelium were prepared, and preembed- ding immunoelectron microscopy was performed using either an affinity purified antibody to 27-kDa protein (a, b, and d), or our previously described AE31 monoclonal antibody (c and e; Yu et al., 1990). Note that both antibodies stain exclusively the luminal leaflet of apical membrane (a, b, and c). In the fusiform vesicles, however, the labeling is observed mainly on the cytoplasmic side. Also note that some small vesicles are stained in a patchy fashion (asterisks in d and e). Arrows in c point to the staining on both sides of the membrane in a maturing vesicle (also see Yu et al., 1990). L and V denote lumen and vesicles, respectively. Bars equal to 100 nm in a, 6, c, and e, and 200 nm in d.

sion of a reduction step, inclusion of protease inhibitors, and optimization of the SDS-PAGE characterization (Fig. 4). Second, we found that these purified AUM preparations are composed of three well-defined proteins (15, 27, and 47 kDa; Figs. 2 and 4). Significantly, these three proteins were found to be similar, if not identical, to the three proteins that we have identified independently using the AE31 monoclonal antibody which recognizes an AUM-associated 27-kDa uro- plakin I (Fig. 5). Although proteins in similar size ranges have been noted in some earlier AUM preparations (Vergara et al., 1974; Caruthers and Bonneville, 1977; Stubbs et al., 1979), the three protein bands that we have observed appear to be sharper and better defined, presumably due to reduced pro- teolysis. Finally, and most important, we have generated multiple antibodies specific for each of these three proteins and established that all of them are indeed AUM-associated in situ (Figs. 12-14). This is a significant step forward since, with the exception of the 27-kDa protein which we only recently immunolocalized on AUM (Yu et al., 1990), no data were available to establish that any of the proteins previously identified in various isolated AUM preparations were really AUM-associated in situ. Taken together, our data provide the strongest evidence available thus far that AUM contains three major protein subunits (15, 27, and 47 kDa, Fig. 6). The properties of these three urothelial plaque-associated proteins (uroplakins; Yu et al., 1990) will be discussed below.

Uroplukin Purification: Immunouffinity Versus Gradient Centrifugution-Although affinity purification has the advan- tage of being rapid (2 days), it suffers from an exceedingly low yield (about 1 Fg of total AUM-related proteins/cow bladder/adsorption cycle, Fig. 5). This severely limited our ability to further characterize the isolated proteins. In con- trast, gradient fractionation takes 3 to 4 days to complete,

Uroplakins and Asymmetrical Unit Membrane 19177

FIG. 13. Ultrastructural localization of the 15-kDa protein. An affinity purified antibody to the 15-kDa protein was used for preembedding immunoelectron microscopy. Note the exclusive stain- ing of the luminal side of apical surface (a), the sometimes patchy staining of the cytoplasmic side of immature AUM-containing vesi- cles (b-d), and the dense staining of mature vesicles (e). L and V denote the lumen and cytoplasmic vesicles, respectively. Bars in all panels equal to 200 nm.

..-: L :.. l .a: l .*... ::

:-. * .

* . a.- . l ;<

. l 4: 1

. . . . . I

. . . .

FIG. 14. Ultrastructural localization of the 47-kDa protein. An affinity purified antibody to the 47-kDa protein was used to stain bovine urothelium with either the preembedding (a-c) or the postembedding (d-n techniques (see “Materials and Methods”). Note the intense labeling of both luminal and cytoplasmic sides of apical surface suggesting that this is a transmembrane protein (arrows in a; also see b). Also note the intense staining of the cytoplasmic surface of many intracellular vesicles (c). L, V, and D denote lumen, vesicles, desmosomes, respectively. Bars in a and c equal to 200 nm; in b, e, and/, 100 nm; in d, 1 pm.

but the yield is several hundredfold higher (about 1 mg of total AUM proteins/bladder). The availability of milligram quantities of highly purified AUM and its subunit proteins played a crucial role in our being able to characterize these proteins (Figs. 4, 6-8), to generate monospecific antibodies to each of them (Figs. 9-14), and to obtain amino acid sequence data which should facilitate the molecular cloning of these molecules.3

Despite the different yields, both isolation procedures gave rise to highly purified fractions containing three major pro- teins of 27 kDa (uroplakin I), 15 kDa (uroplakin II), and 47 kDa (uroplakin III) (Figs. 2c, 5, and 6). Comparison of the 15- kDa protein isolated by the two procedures by CNBr peptide mapping and N-terminal amino acid sequencing clearly estab- lished their identity (Fig. 6~).~ Although no detailed biochem- ical data are available on the 27- and 47-kDa affinity purified proteins due to insufficient material, they comigrate precisely with those of the gradient-purified counterparts in two differ- ent SDS-PAGE systems (Fig. 6) and are most likely the same. However, we cannot rule out the possible existence of minor structural differences in the proteins isolated by the two methods, neither can we rule out the possibility that the two uroplakin preparations come from different pools, e.g. a small precursor pool uersus a much larger (and less soluble) pool of mature plaques.

The Three Uroplakins Represent Distinct Molecules-Some of the higher molecular mass proteins (55 kDa, Vergara et al., 1974; 30 kDa, Stubbs et al., 1979) previously identified in AUM preparations were thought to be dimers or complexes of the lower molecular mass proteins. In the mean time, relatively little attention was paid to some of the small mo- lecular weight proteins (515 kDa) because they were minor components which might be thought of as degradative prod- ucts. In our AUM preparations we reproducibly see three major protein components (e.g. Figs. 2c, 4, and 6). The three gradient-purified proteins have been compared in detail and found to be clearly distinguishable according to the following criteria. First, peptide mapping data showed that the three proteins share no common CNBr fragments (Fig. 8). Second, we have determined the N-terminal amino acid sequences of multiple CNBr and tryptic peptides of the 15- and 27-kDa proteins, and so far have found no common sequences between these two proteins (data not shown). Finally, we have pre- pared multiple antibodies to each of the three uroplakins (Fig. 9). Without exception, all these antibodies reacted exclusively with their respective antigens and showed no cross-reactivity toward other uroplakins (Fig. 9). These results provide ex- tremely strong evidence that the three uroplakins represent distinct molecules possibly encoded by different genes.

Uroplakins I (27 kDa) and II (15 kDa)-One of the three major components of the gradient-purified AUM is a 27-kDa protein (Figs. 2 and 4). Using both AE31 mouse monoclonal antibody and a newly prepared rabbit polyclonal antibody, we have shown that this protein is urothelium-specific and is restricted to the umbrella cells (Fig. 10); it is slightly acidic (p1 -5.3; Yu et al., 1990); it is first synthesized in cultured urothelial colonies as a 32-kDa precursor which is then proc- essed through a 30-kDa intermediate before it matures into the 27-kDa form (Yu et al., 1990); it partitions, along with a 15-kDa protein (uroplakin II, see below), exclusively into the detergent phase during Triton X-114 phase separation sug- gesting that it is an integral membrane protein (Yu et al., 1990); finally, it first appears on the cytoplasmic side of immature AUM-vesicles, but later becomes detectable exclu- sively on the apical side of AUM (Fig. 12; Yu et al., 1990) suggesting an unusual translocation event during later stages

19178 Uroplukins and Asymmetrical Unit Membrane

of AUM maturation (Yu et al., 1990). The properties of the 15kDa protein are remarkably similar

to those of the 27-kDa protein. This protein is also urothe- lium-specific and is restricted to the umbrella cells (Fig. 10); it copurifies in a roughly equimolar ratio with the 27-kDa uroplakin I during both immunoaffinity chromatography and gradient centrifugation (Figs. 2, 4, and 5; Yu et al., 1990); it is slightly basic (p1 -7.8) which may facilitate its interaction with the slightly acidic 27-kDa molecule (Yu et al., 1990); it partitions, along with uroplakin I, into the detergent phase during Triton X-114 phase separation (Yu et al., 1990); finally, it is first detected on the cytoplasmic side of immature AUM vesicles but is limited to the apical surface of luminal AUMs suggesting that this protein is, like the 27-kDa protein, trans- located during AUM maturation (Fig. 13).

kDa; Hertzberg and Johnson, 1988), antibodies to the former react specifically with AUM without cross-reacting with the gap junctions of urothelium, esophagus, kidney, and liver.

Taken together, these data indicate that the 15 and 27- kDa molecules undergo similar pathways of synthesis and assembly, and may play closely related roles in AUM struc- ture. It is not yet known how these two uroplakins contribute to the hexagonal protein particles seen on the apical surface of AUM. Ultrastructural studies have previously shown that these protein particles are “floating” on the apical surface, without penetrating to the cytoplasmic side (Brisson and Wade, 1983; Vergara and Chesnut, 1983; Taylor and Robert- son, 1984). The 27- and 15-kDa uroplakins, both of which were localized exclusively on the apical surface of mature plaques, may represent major building blocks of these apical protein particles. Interestingly, if one assumes that the 27- and 15kDa proteins correspond to the inner (larger) and outer (smaller) subdomains, respectively, of a dumbbell- shaped subunit (see Introduction), this will yield a total protein mass of 42,000 daltons, which is reasonably close to the calculated value of 49,000 daltons (Taylor and Robertson, 1984). Experiments are in progress to test this possibility.

Uroplakin III (47 kDa)-The third AUM component, a 47- kDa protein, is also urothelium specific, and its expression is also limited to the superficial umbrella cells (Figs. 10 and 14). Like the other two uroplakins, this protein can be detected on the cytoplasmic side of numerous intracellular vesicles. Interestingly, in mature, apical plaques, epitopes of this pro- tein are readily detected on both luminal and cytoplasmic sides of AUM (Fig. 14). These results suggest that the 47-kDa protein is a transmembrane component possibly playing a role in anchoring the AUM plaques through its interactions with an underlying cytoskeletal network (Chlapowski et al., 1972; Staehelin et al., 1972; Minsky and Chlapowski, 1978; Sarikas and Chlapowski, 1986, 1989). The detailed location of this 47-kDa protein within the hexagonal 12-nm protein particles remains unclear.

It has been suggested that keratin filaments interact with urothelial plaques (Chlapowski et al., 1972; Staehelin et al., 1972; Minsky and Chlapowski, 1978; Sarikas and Chlapowski, 1986, 1989). The identification of a transmembrane compo- nent of AUM (Fig. 14) should facilitate additional studies in this important area.

Relationship between Uroplukins and Gap Junctional Pro- teins-Gap junctions are composed of hexagonal protein par- ticles which bear some morphological similarities with those of AUM (Bennett and Spray, 1985; Revel et al., 1985). How- ever, unlike the AUM particles which are 12 nm in diameter (with 12 subdomains) with a center-to-center spacing of 16 nm, the gap junction particles are only 6.5 nm in diameter (six subdomains) with a center-to-center spacing of 10 nm (Staehelin et al., 1972; Robertson and Vergara, 1980; Bennett and Spray, 1985). Moreover, although the molecular mass of uroplakin I (27 kDa) is similar to that of connexin 32 (26

Conversely, an antiserum to rat liver connexin 32 (kindly provided by David Paul (Harvard Medical School); Gooden- ough et al., 1988) stains immunohistochemically the gap junc- tions of bovine liver and kidney, without reacting with bladder epithelium (data not shown). These data indicate that the protein subunits of AUM are immunologically distinct from those of the gap junctions.

Uroplakins as Biochemical Markers of Urothelial Differen- tiation-Although there is ample evidence in other differen- tiating systems that the expression of specific molecules ac- companies and underlies morphological and functional differ- entiation (see, e.g. Schermer et al., 1986; Maroux et al., 1988; Galvin et al., 1989; Grove, 1989; Piatigorsky, 1989), there has been no known molecular markers for urothelial differentia- tion. Our studies, reported here and in an earlier paper (Yu et al., 1990), provide the first evidence clearly establishing uroplakins as useful markers for studying urothelial differ- entiation. This is based on our observation that uroplakins are urothelium specific, being present in urothelial cells cov- ering renal pelvis, ureter, bladder, and urethra, but so far undetectable in any other epithelia (Fig. 10; Yu et al., 1990). Moreover, within the urothelium these proteins are detected only in superficial umbrella cells (Fig. 10). Although it is possible that uroplakins are present in lower cell layers but escaped detection due to “masking” of their epitopes, the large number of antibodies employed in our studies makes this highly unlikely. Together, our data indicate that the three uroplakins represent molecular markers for an advanced stage of urothelial differentiation. The biochemical basis underlying the urothelium specificity and the differentiation-related and apparently coordinated expression of these three markers is unknown and is the subject of ongoing molecular cloning experiments.

Acknowledgments-We thank Drs. Irwin M. Freedberg and Pablo Morales for their encouragements and their interests in this investi- gation, Dr. Ueli Aebi (Biozentrum, Basel, Switzerland) for performing negative staining of isolated bovine AUM plaques (Fig. 3d), Dr. David Paul (Harvard Medical School) for an antiserum to connexin 32, Drs. Babu Surya and John Provet for useful discussions, Yu Chen for technical assistance, and Maria Medina for typing this manuscript. Motomu Manabe is an Upjohn Fellow of the Dermatology Founda- tion.

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