J Biol Chem, Vol. 274, Issue 37, 26233-26239, September 10, 1999

Identification of Syntenin as a Protein of the Apical Early Endocytic Compartment in Madin-Darby Canine Kidney Cells^*

Irene Fialka^§, Peter Steinlein^§, Horst Ahorn^¶, Günther Böck, Peter D. Burbelo^**, Michaela Haberfellner, Friedrich Lottspeich, Karin Paiha, Christian Pasquali, and Lukas A. Huber^§§

From the  Research Institute of Molecular Pathology, Dr. Bohr Gasse 7, A-1030 Vienna, Austria, ^¶ Boehringer Ingelheim Austria, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria, the  Department of General and Experimental Pathology, University of Innsbruck, Fritz-Preglstrasse 3, A-6020 Innsbruck, Austria, the ^** Lombardi Cancer Center, Georgetown University Medical Center, Washington, D.C. 20007-2197, and the  Max Planck Institute of Biochemistry, Am Klopferspitz, D-82152 Martinsried, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after membrane internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene. Western blot analysis of sorted fractions showed enrichment of the early endosomal markers transferrin receptor and the small GTPase Rab5. Two-dimensional gel analysis indicated that the apical and basolateral early endosomes differed significantly in their protein composition. We found nine polypeptides to be specifically enriched in apical or basolateral endocytic vesicles. An apical protein identified by microsequencing was the adaptor molecule syntenin. This protein contains two PDZ domains (PSD-95, Dlg, and ZO-1 homology) that bind syndecan and ephrin-B2 cytoplasmic domains. In MDCK cells, transiently overexpressed Myc-tagged syntenin localized to both plasma membrane domains and to an intracellular vesicular compartment. Syntenin positive vesicles colocalized with internalized transferrin in the perinuclear region. In addition, syntenin colocalized in the apical supranuclear region with Rab5 and Rab11; the latter is a marker for the apical recycling endosomes in MDCK cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Endocytosis includes "cellular drinking" (the unselective uptake of fluids) and the selective uptake of nutrients, hormones, and growth factors by receptors. Endocytosis occurs from both apical and basolateral surfaces in epthelial cells (1, 2). Clathrin-coated vesicles derived from the apical or basolateral plasma membrane lose their coat and fuse with early endosomes. Endocytosed cell surface receptors and other membrane proteins, as well as fluid phase markers, therefore appear first in early endosomes. Early endosomes comprise at least two functionally distinct compartments (3). Internalized receptors and ligands first enter the peripheral sorting endosomes. In these compartments, some membrane proteins are sorted away from those proteins destined for degradation. In particular, some receptor-ligand complexes and the remaining solutes are transported to late endosomes and lysosomes to be degraded, whereas the transferrin receptor (Tf-R)¹ recycles back to the basolateral cell surface (4). Recycling back to the plasma membrane can occur directly from the so-called sorting endosome (fast cycle) or indirectly via the recycling endosome (5).

Work on endocytic compartments in epithelial MDCK cells has demonstrated that apical early endosomes are distributed below the apical microvilli above the ring of tight junctions and that basolateral early endosomes are found alongside the lateral as well as the basal membranes (6, 7). Both compartments form three-dimensional networks of tubular cisternal and vesicular structures (2). In vitro fusion assays with endosomes derived from the MDCK cell line have confirmed that there is a specific set of early endosomes associated with each plasma membrane domain. These endosomes show homotypic fusion but no heterotypic fusion activities with each other (7, 8). Recent evidence suggests that in epithelia, apical and basolateral endocytic pathways converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. Up to now, marker proteins for early endosomes, such as Rab5, were found on both apical and basolateral early endosomes (9). Other proteins, such as annexin II, show, in addition to miscellaneous subcellular localizations, an association with peripheral transferrin-labeled endosomes in epithelial cells (10), whereas Rab25 and Rab11a were found to be associated with the apical recycling system of polarized MDCK cells (11). In addition, it was shown that recycling of endocytosed transferrin and transferrin receptor through this pericentriolar endosomal compartment requires hydrolysis of GTP on Rab11 (12, 13).

The complexity of endocytic recycling and the limited number of marker proteins identified to date suggests that there are many yet unidentified molecules involved in regulating endocytic trafficking in polarized cells. One approach to gain more knowledge about the apical and basolateral specifities for endocytosis is to analyze the overall molecular composition and organization of apical and basolateral early endocytic vesicles. For this purpose, the lipophilic fluorescent probe trimethylamino-diphenylhexatriene (TMA-DPH) that intercalates in membrane structures has been used for studying endocytosis (14-20). TMA-DPH is a cationic analogue of DPH that has also proven useful as a probe for studying bulk-phase endocytosis (14-17, 20-22). In addition, the dye does not diffuse through tight junctions, and for this reason, it has been used successfully in anisotropy experiments to estimate in situ the lipid order of the plasma membrane of polarized MDCK II cells (23).

Here we combined the use of TMA-DPH with conventional density gradient centrifugation (24) with high speed organelle sorting in a flow cytometer (fluorescence-activated organelle sorting (FAOS)) to analyze the apical and basolateral endosomal proteins in MDCK cells (22, 25). Using high resolution 2D gel electrophoresis (2DE) and protein microsequencing, we identified the adaptor protein syntenin in apical endocytic vesicles. Syntenin contains a tandem repeat of two PDZ domains. PDZ domains mediate protein-protein interactions and typically bind to short amino acid motifs at the carboxyl terminus of interacting proteins, including certain ion channels and transmembrane receptors. As such, syntenin reacts with the FYA carboxyl-terminal amino acid sequence of syndecans (26) and with the carboxyl terminus of B-type ephrins (27). By combining a novel strategy for subcellular fractionation with biochemical analysis and transient overexpression of syntenin, we could for the first time demonstrate an association of this recently identified adaptor molecule with the apical recycling compartment of MDCK cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells, Media, and Antibodies-- MDCK I and II cells were cultured and seeded on filters (Costar; pore size, 0.4 µm) as described previously (10, 28). Before each experiment, the confluency of the monolayer was confirmed by measuring transepithelial electrical resistance. In vivo staining with TMA-DPH was carried out with a baby hamster kidney cell line (BHK-21) grown on coverslips (29).

Monoclonal anti-human Tf-R antibodies were obtained from Roche Molecular Biochemicals, and the monoclonal antibodies against E-cadherin and annexin II were from Transduction Laboratories. Polyclonal antibodies against Rab4, Rab5, and Rab7 were raised against synthetic peptides derived from the carboxyl terminus and prepared in our laboratory as outlined (30). Polyclonal antibodies against Rab11 (31) and monoclonal antibodies against Rab5 (9) were used as described previously. Myc-tagged syntenin was detected with an anti 9E10 monoclonal antibody or a polyclonal affinity-purified antibody generated in our laboratory against a Myc peptide (32). All secondary antibodies were obtained from Dianova.

In Vivo Labeling of Cells with TMA-DPH-- Lyophilized TMA-DPH was from Molecular Probes and reconstituted as a 20 mM stock solution in Me₂SO (Fluka).

BHK-21 cells were washed 2-3 times with prechilled PBS²⁺ (PBS, 1 mM CaCl₂, 1 mM MgCl₂) at 4 °C. Drops of TMA-DPH diluted to 100 µM in NaOAc transport buffer (250 mM HEPES, pH 7.4, 1.15 M NaOAc, 25 mM MgCl₂) were added on top of the monolayer. After incubation for 3-4 min on an ice-cold metal plate, the dye was removed by briefly washing with prechilled PBS²⁺. Internalization was carried out in an incubator at 37 °C for various time points. Double stainings with acridine orange were peformed as described (33).

Filter-grown MDCK II cells were washed twice in ice-cold PBS²⁺ for 5 min at 4 °C and once for 2 min in prechilled NaOAc buffer. TMA-DPH diluted to 50 µM in NaOAc transport buffer was added to the apical or basolateral cell surface and the cells incubating for 2 min at 4 °C. Immediately after TMA-DPH binding, the filters were incubated at 37 °C in a water bath for 2 min with prewarmed medium.

Subcellular Fractionation-- Following internalization, MDCK II cells were washed with ice-cold PBS and scraped, and a postnuclear supernatant (PNS) was prepared as described previously (22, 24, 34). The PNS from one and a half filter inserts was loaded on top of one SW60 tube (Beckman) containing a continuous sucrose gradient (10-40% sucrose). After centrifugation (100,000 × g at 4 °C for 16 h), 20 fractions were collected with an Auto Densi-Flow fraction collector (Labconco Corp.) and analyzed. To generate amounts suitable for preparative FACS sorting, up to 10 filter inserts were used.

Internalization of HRP as Fluid-Phase Marker-- Horseradish peroxidase (HRP) (5-10 mg/ml; Sigma) was internalized from fetal calf serum-free medium into early endosomes and further chased into late endosomes as described previously (24). HRP activity was assayed using o-dianisidine and peroxide as substrates as described (24). Specific activity of the fractions is given as ng of HRP/mg of protein.

Western Blotting-- Protein concentrations were measured using the Micro BCA protein assay reagent kit (Pierce) following the manufacturer's specifications. All protein precipitations were carried out with the CHCl₃/methanol method (35). Precipitated proteins were dissolved in SDS-sample buffer and separated by 10% SDS-polyacrylamide gel electrophoresis, followed by semidry electrophoretic transfer onto nitrocellulose membranes. Proteins were detected using specific antibodies as outlined previously (34).

FAOS of TMA-DPH-labeled Endosomes-- TMA-DPH was titrated by flow cytometry using labeled PNS to avoid complex quenching effects from the Förster-type fluorescence resonance autotransfer (18). The best compromise between the highest amount of sortable material and the minimum of false positive structures was obtained at 50 µM TMA-DPH (data not shown). Fractions enriched in TMA-DPH-labeled endosomes were diluted 1:100 in PBS and analyzed using a FACS Vantage Turbo Sort Option (Becton Dickinson, San Jose, CA) equipped with an Argon laser tuned to 40 MW multiline UV and 210 mW 488 nm output using a 4xx/44-nm bandpass filter and a 50-µm nozzle. System threshold was set on forward light scatter, and photomultiplier tube voltage was adjusted using unlabeled PNS. Fractions containing more than 10% of labeled vesicles were gated for preparative sorts. The concentration of these fractions was adjusted to give event rates of approximately 15,000 events/s at lowest possible sample differential pressure at a sheath pressure of 45 psi. Droplet formation frequency was adjusted to approximately 70 kHz. The large volumes (typically more than 20 ml) of sorted material was concentrated by several steps of sec-butanol extraction and finally CHCl₃/methanol precipitated (35).

2DE and Microsequencing-- 2DE was performed following in-gel sample reswelling as described (34, 36). The gels were stained with ammoniacal silver, scanned, and analyzed as described (34, 36, 37). Preparative amounts of total membrane fractions from MDCK II cells (34, 36) were precipitated, dissolved, and separated as outlined for analytical gels and stained with Coomassie (34, 36). Candidate proteins were identified in several gels and spots collected for microsequencing. Microsequencing was performed as described (34).

Transient Transfection, Transferrin Internalization, and Immunofluorescence-- A polymerase chain reaction fragment amplified with BamHI-XhoI linkers encoding the cDNA of syntenin (amino acids residues 2-298) was generated from a human syntenin cDNA clone obtained from the IMAGE Consortium (unique IMAGE Consortium identifier 33203 and GenBank^TM accession number R19118). This BamHI-XhoI fragment was subcloned in frame, into a BamHI-XhoI downstream of a Myc epitope in a pcDNA-3 expression vector (Invitrogen) containing a Kozak consensus sequence. Thus, an amino-terminally Myc-tagged human syntenin cDNA was created that could be recognized by the 9E10 anti-Myc antibody (32). DNA sequencing was performed to confirm the integrity of this pcDNAIII-Myc-syntenin construct.

Filter-grown MDCK I Rab5 cells were transfected with pcDNAIII-Myc-syntenin using a PEI/Ad5 system as described previously (38). For internalization of transferrin, the cells were washed with Hanks' balanced salts (Life Technologies, Inc.) and incubated in serum-free medium containing 50 µM desferoxamine mesylate (Sigma) for 3 h at 37 °C to deplete the cells from Fe²⁺ and enrich ligand-free transferrin receptor on the plasma membrane. Thereafter, the cells were washed with ice-cold PBS²⁺. The filters were then placed on a drop of prewarmed medium (on a piece of parafilm) containing 100 µg/ml transferrin-Alexa 488^TM (Molecular Probes), overlaid with prewarmed medium, and incubated at 37 °C for 15 min. Internalization was stopped by placing the filters in ice-cold PBS²⁺, followed by three washes to remove free transferrin. Subsequently, cells were extracted with 0.05% saponin in cytoskeleton buffer (10 mM PIPES, pH 6.8, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, 5 mM MgCl₂) and processed for immunofluorescence as described (28). The filter pieces were mounted in 50% glycerol in cytoskeleton buffer containing 4% n-propyl gallate (Sigma), and confocal microscopy images were obtained using a Leica TCS NT confocal microscope (Leica, Heidelberg, Germany). Images were processed using the Imaris and Colocalization software packages (Bitplane AG, Zürich, Switzerland) after deconvolution using measured point-spread functions with the Huygens software (Scientific Volume Imaging, Hilversum, Netherlands).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TMA-DPH Labeling and Sample Preparation for Flow Cytometry-- TMA-DPH has been shown to interact with living BHK-21 cells by instantaneous incorporation into the plasma membrane. To confirm the predicted properties of TMA-DPH as endocytic membrane tracer (14, 20, 21), we established an internalization protocol in BHK-21 cells, in which filling kinetics of endosomes are well characterized. For this purpose, we prebound TMA-DPH to the cell surface at 4 °C and internalized for 2 and 30 min by warming up the sample to 37 °C. The distribution of TMA-DPH in living cells was analyzed by direct fluorescence microscopy (Fig. 1). TMA-DPH bound to the plasma membrane at 4 °C (Fig. 1A) and could be internalized into an early endocytic compartment 2 min after raising the temperature to 37 °C (Fig. 1B). In addition, the perinuclear compartment (Fig. 1C) that was labeled after 30 min of TMA-DPH internalization was colocalized with acridine orange, a marker for acidic late endocytic/lysosomal compartments (Fig. 1D). This confirms that TMA-DPH is rapidly incorporated into the plasma membrane and follows the normal intracellular traffic of internalization, thus behaving as a suitable marker for membrane endocytosis under the applied conditions.

View larger version (107K): [in this window] [in a new window]   Fig. 1.   In vivo labeling of plasma membrane and various endocytic compartments in BHK-21 cells with TMA-DPH. BHK-21 cells were grown on coverslips, and TMA-DPH (50 µM) was bound to the cell surface at 4 °C. Subsequently, the dye was internalized for 2 min or 30 min at 37 °C. After 30 min of internalization, the coverslip was counterstained with the vital dye acridine orange, which labels acidic late endosomal/lysosomal compartments. Bar, 10 µm. A, TMA-DPH binds and labels the cell surface following incubation at 4 °C. B, bound TMA-DPH was internalized into early endosomes for 2 min at 37 °C. C, TMA-DPH labels a perinuclear late endocytic compartment after 30 min of internalization. D, double in vivo labeling of the same cells as in C with acridine orange.

Preenrichment of HRP and TMA-DPH Containing Endocytic Organelles-- We first compared the distribution of fluid phase internalized HRP with internalized TMA-DPH after subcellular fractionation of cells on sucrose gradients. We internalized HRP in filter-grown MDCK II cells through the apical or basolateral medium. Cells were homogenized, and the PNS was then loaded on top of a continuous sucrose gradient (10-40%) and centrifuged to equilibrium. A total of 20 fractions per gradient were collected and analyzed for their HRP activity and total amounts of protein (Fig. 2A). The apical and basolateral internalization profiles showed a peak area that spanned fractions 10-16, corresponding to 25-35% of sucrose concentration (1.104-1.151 g/cm³). We next examined the gradient distribution for TMA-DPH-labeled early endocytic structures. After binding TMA-DPH to the basolateral or apical plasma membrane for 2 min at 4 °C, the fluorescent dye was internalized for 2 min at 37 °C (Fig. 2B), and cells were fractionated as in Fig. 2A. When compared with the distribution profiles obtained by HRP labeling of apical and basolateral endocytic vesicles of MDCK II cells, TMA-DPH labeling revealed a largely overlapping peak area between fractions 14 and 16, corresponding to 30-35% concentration of sucrose.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Continuous sucrose gradients yield early and late endocytic fractions between 25 and 35% of sucrose and cannot separate apical from basolateral endosomes. A, profile of the HRP/protein content (ng/µg) of apical (filled symbols) and basolateral (open symbols) fractions obtained from a continuos sucrose gradient (10-40%), plotted against the fraction number. Cells were grown on permeable filter supports, and HRP (5 mg/ml) was internalized through the apical or basolateral medium for 10 min at 37 °C. The cells were scraped and homogenized, and the resulting PNS was loaded on top of a continuous sucrose gradient (10-40%). 20 fractions per gradient were collected, and the HRP/protein content determined. Both gradients showed a peak area covering fractions 10-16, corresponding to a sucrose concentration of 25-35%. B, TMA-DPH-labeled early endocytic vesicles (filled symbols, apical fractions; open symbols, basolateral fractions) were preenriched in a largely overlapping peak area with HRP-labeled endosomes (fraction 10-16, corresponding to 25-35% sucrose). MDCK II cells were grown on permeable filter supports and fractionated as in A, and fractions were analyzed by flow cytometry. The percentage of labeled vesicles per fraction was plotted against the fraction number. C, Western blot analysis of fractions collected from continuous sucrose gradients (A and B). Equal volumes of each fraction were precipitated and analyzed on immunoblots for Tf-R, Rab5, and Rab4 as early endocytic markers and Rab7 as a late endocytic marker. The figure indicates an overlap of early and late endocytic markers within the peak areas of internalized HRP/TMA-DPH (A and B).

Immunoblotting of these fractions revealed the presence of Tf-R, Rab5, and Rab4 as early endosomal markers (10, 39, 40) and Rab7 as a marker protein for late endosomes (41) in the overlapping peak areas (outlined in Fig. 2C). However, this sucrose concentration corresponded to the density of early endocytic organelles rather than to the reported density of late endocytic vesicles (42), emphasizing that conventional gradient fractionation is not sufficient to separate (a) early from late endocytic structures, or (b) apical from basolateral endosomes in MDCK II cells.

Western Blot Analysis of the FACS-sorted Fractions Confirms Enrichment of Endosomes-- Gradient fractions containing >10% TMA-DPH-labeled apical or basolateral vesicles, as referred to the PNS fraction, were pooled and subsequently sorted in a flow cytometer as described under "Experimental Procedures." Proteins of sorted fractions were concentrated and precipitated, and their protein content was determined. 5 µg of protein (equivalent to about 40 million sorted fluorescent events) derived from apical or basolateral sorts and 5 µg of protein precipitated from the starting fraction were separated by 10% Tris-glycine SDS-polyacrylamide gel electrophoresis. The gel was transferred onto a nitrocellulose membrane, stained with Ponceau-S to confirm equal loading of samples (Fig. 3A), and prepared for immunodetection. Apical as well as basolateral fractions revealed an enrichment of early endocytic markers after FACS sorting (Fig. 3B). Rab5a is a common component of the apical and basolateral endocytic machinery in polarized MDCK cells (9) and was strongly enriched in both fractions. However, Tf-R, a basolaterally endocytosed and recycling transmembrane protein (43, 44), was only enriched in basolateral fractions but was clearly present in the apical compartment. A marker protein for the plasma membrane, E-cadherin, was significantly decreased in the sorted fractions, as well as the late endosomal GTPase Rab7. Annexin II, a Ca²⁺-, phospholipid-, and actin-binding protein implicated in the regulation of vesicular traffic (10, 45, 46) was much less abundant when compared with the gradient purified starting fractions. Taken together, when compared with the gradient purified starting material, TMA-DPH-sorted fractions revealed substantial enrichment of early endocytic markers.

View larger version (82K): [in this window] [in a new window]   Fig. 3.   Western blot analysis of TMA-DPH-sorted endosomes. FACS-sorted fractions of apical and basolateral TMA-DPH-labeled early endosomes were separated on a 10% Tris-glycine polyacrylamide gel electrophoresis gel and transferred to nitrocellulose. Subsequent immunoblotting revealed enrichment of the sorted material in comparison to the input gradient fractions. A, scans of the Ponceau-S stained nitrocellulose membranes (5 µg of protein were loaded in all lanes). Lane 1, basolateral sorted fractions; lane 2, apical sorted material; lane 3, gradient fraction serving as a reference. B, antibody detection of the nitrocellulose membrane shown in A (E-cadherin, Tf-R, annexin II, Rab5, and Rab7).

2DE Illustrates Significant Differences in Overall Protein Content of FACS-sorted Fractions-- High resolution 2DE was used to analyze the complexity of the obtained samples and to characterize the protein composition of FACS-sorted fractions.

Subcellular fractionation of the PNS (Fig. 4A) revealed simplified protein patterns (Fig. 4B) and specific enrichment of proteins in the FACS-sorted fractions (Fig. 4, C and D). In order to identify some of the proteins by protein sequencing, we resolved preparative amounts of total MDCK II membrane fractions by high resolution 2D gels (34, 36). The analytical gels of basolateral or apical early endosomes were aligned with preparative gels of total membrane fractions, and some of the candidate proteins were found as Coomassie-stained spots (data not shown). Spots were excised and subsequently microsequenced after in-gel digestion (34, 36). First, we confirmed the presence of known organelle markers in the starting fractions (see Fig. 4A and Table I). The endoplasmic reticulum proteins calreticulin and BIP (GRP78) and VIP36, a lectin involved in biosynthetic transport, were clearly less abundant in the sorted fractions when compared with PNS and gradient-purified fractions. The association of the epithelial-specific cytokeratin 19 (intermediate filaments) and of cathepsin D (late endosomes/lysosomes) with membranes was not affected, whereas the 1- and 2-subunits of trimeric G proteins, as well as actin and annexin II (compare with Fig. 3), were significantly reduced in amounts after sorting. Taken together, the protein patterns of apical and basolateral early endocytic fractions differed remarkably (compare Fig. 4, C and D). Further experiments were aimed at identifying one of the apically enriched proteins and confirming its subcellular localization.

View larger version (124K): [in this window] [in a new window]   Fig. 4.   2DE of apical and basolateral endosomes of MDCK II cells. Equal amounts of protein (6 µg) were loaded in all gels. Gels were aligned, compared, and assigned with MELANIE. Numbers define either SWISS-PROT accession numbers of known proteins in the according PNS (e.g. P02570, -actin) or polypeptides specifically enriched in apical (e.g. ap1) and basolateral early endocytic fractions (e.g. bl1), respectively. A, PNS; B, gradient fraction; C, apical sorted fraction; D, basolateral sorted fraction.

Syntenin Is Associated with an Apical Endosomal Compartment-- We found six polypeptides specifically enriched in apical vesicles and three basolateral-specific ones (Fig. 4, C and D and Table I). Two peptides were sequenced out of the apical spot ap1, yielding two sequences of 6 and 18 amino acids (Table I), identical with amino acids 187-204 and 218-224 of a protein originally submitted as Pbp1 (human scaffold protein, GenBank^TM accession number U83463).² An identical sequence was submitted and characterized later as syntenin (GenBank^TM accession number AF000652, see Ref. 26). Syntenin was described as human scaffold/adaptor protein that can associate with the cytoplasmic leaflet of membranes by binding syndecan and B-class ephrin cytoplasmic domains (26, 27).

To confirm the association of syntenin with an apical endosomal compartment, we transiently transfected filter-grown MDCK I cells overexpressing Rab5 (10) with a pcDNAIII-Myc-syntenin construct. Indirect double immunofluorescence analysis by laser scanning confocal microscopy in saponin-permeabilized and PFA-fixed cells revealed a perinuclear colocalization of syntenin with an Alexa 488^TM-labeled transferrin after internalization through the basolateral medium, reminiscent of the pericentriolar recycling endosome (Fig. 5A). In support of this finding, syntenin partially co-localized with Rab5 containing apical early endosomes (Fig. 5B). Antibodies to Rab11 label an apical pericentriolar endosomal compartment that is accessible to membrane-bound markers internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome (11). Most importantly, a significant colocalization of syntenin with Rab11 in the apical recycling endosome of transfected cells was detected (Fig. 5C).

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Colocalization analysis of syntenin with Rab5, internalized transferrin, and Rab11. An MDCK I cell line stably overexpressing Rab5 was grown on permeable filter supports and transiently transfected with pcDNA-III-Myc-syntenin. Transferrin-containing compartments were labeled for 15 min by continuous internalization of transferrin-Alexa488TM. Red labeling shows Myc-syntenin (A-C); green labeling is transferrin-Alexa488TM (A), Rab5 (B), or Rab11 (C); and yellow indicates locations at which they are merged. Extended focus (summarizing staining in several stacks of x-y axis planes) and a vertical section image (x-z axis) taken by confocal microscopy and image processing are shown. Vertical section in C was recorded in the direction indicated by the white line. Myc-tagged syntenin partially co-localizes with internalized transferrin (A) and Rab5 (B) in the pericentriolar recycling compartment of MDCK cells. Significant overlap of syntenin with Rab11 is found in the apical supranuclear region (C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When grown on permeable filter supports, MDCK cells form a polarized monolayer with apical and basolateral compartments that are tightly sealed and separated from each other by tight junctions (47). Using this cell system and selective internalization of endocytic tracers, we have gained experimental access to apical and basolateral endocytic compartments (7). This was accomplished by internalizing TMA-DPH into early endosomes through the apical or basolateral medium for 2 min and then fractionating the MDCK homogenate using continuous sucrose gradients (22, 49). As expected, the TMA-DPH peak fraction contained early and late endocytic markers that co-migrated in sucrose gradients together with fluid phase-internalized HRP. These fractions served as starting material for the FAOS in a conventional FACS machine.

Basolateral fractions obtained after FAOS were enriched in endocytic markers, such as Tf-R and Rab5. However, although to a much lesser extent, there was also Tf-R found on vesicle populations isolated after apical membrane endocytosis, implying that after 2 min of membrane internalization, the lipophilic fluorescent probe TMA-DPH can reach an apical endosomal compartment containing Tf-R, Rab5, and Rab11. The significant simplification of protein patterns and the selective abatement of other organelle markers in the TMA-DPH-sorted fractions supports the potential of FAOS as an enrichment method for early endocytic vesicles (22, 25).

We mapped apical and basolateral fractions on high resolution 2DE and analyzed their overall protein composition. Among the six polypeptides specifically enriched in apical vesicles, we identified spot ap1 as syntenin by direct microsequencing from preparative 2D gels (34, 36). Syntenin is a 30-kDa protein that binds to the cytoplasmic domains of several transmembrane receptors. One target of syntenin is the family of syndecans, which are cell-surface heparan sulfate proteoglycans that participate in multiple cell functions (50). Syntenin binds via its tandem PDZ domains to the FYA carboxyl-terminal amino acid sequence of the syndecans (26). When syntenin is expressed as a green fluorescence protein fusion protein in Chinese hamster ovary cells, staining is observed at both the plasma membrane and uncharacterized intracellular vesicles (26). Interestingly, syntenin may have a general function as a scaffold protein, because another set of receptors, B-class ephrins, also bind syntenin through their carboxyl-terminal tail (27). B-class ephrins are transmembrane proteins and function as ligands for B-class ephrin receptor tyrosine kinases. Both receptors and ligands are postulated to possess an intrinsic signaling function and are involved in the hormone dependent morphogenesis of the mammary gland (27). Because syntenin itself has no obvious catalytic domain (26), it may function as an intracellular adaptor or scaffolding protein to link transmembrane receptors to signaling components or the submembranous cytoskeleton.

The correct recycling and localization of cell surface receptors after endocytosis is required for cellular integrity and in some cases may be linked to signaling events. For example, activated epidermal growth factor receptors are rapidly internalized into the endocytotic compartment and degraded in lysosomes after epidermal growth factor stimulation. The mechanism of epidermal growth factor receptor internalization occurs via its transient association with the SH3-SH2-SH3 adaptor protein GRB2, which is able to couple with the endocytic regulator dynamin (18). Likewise, syntenin may also function in rapid trafficking or recycling of transmembrane receptors, because our fractionation experiments demonstrated that endogenous syntenin protein was associated with an early endocytic vesicular compartment of apical origin. To further explore this possibility, we transiently expressed Myc-tagged syntenin in a MDCK I cell line stably transfected with Rab5 (9). Ectopically expressed syntenin was found to be uniformly distributed over both plasma membrane domains, as well as on intracellular Rab5 positive vesicles, as revealed by indirect immunofluorescence analysis. Interestingly, syntenin co-localized with internalized transferrin-Alexa 488^TM in the pericentriolar recycling compartment. Furthermore, apical syntenin vesicles showed substantial colocalization with Rab11. The small GTP-binding protein Rab11 is concentrated in the pericentriolar endosomal recycling compartment of cultured mammalian cells and plays a key role in the passage of recycling Tf-R through that compartment (13). The intracellular localization of syntenin as revealed by immunofluorescence analysis confirms the resolution obtained by the combined subcellular fractionation approach. Although these apical endocytic vesicles showed significant enrichment of syntenin after FAOS, the subcellular distribution of syntenin at the immunofluorescence level indicates that its passage through the apical recycling compartment in MDCK cells is quite dynamic, and only a small portion of cytoplasmic syntenin is associated with it at any time. The exact function of syntenin is still unclear. Syntenin has recently been shown to interact with the cytoplasmic tail of pro-transforming growth factor- and thereby participates in targeting it to the cell surface (48). In polarized epithelial cells, PDZ proteins, such as syntenin, may function in regulating the correct localization of transmembrane receptors via rerouting them through an apical endosomal compartment.

One of the major challenges of studying endocytosis in polarized epithelial cells has been the lack of proteins specific for apical endosomal compartments. In principle, flow cytometry can be used for purification of any organelle or cellular constituent that can be labeled with a fluorescent compound (25). The labeling and fractionation system that was developed here may allow the identification of additional proteins involved in apical and basolateral endocytosis.

	ACKNOWLEDGEMENTS

We thank Drs. Jola Glotzer and Jan-Michael Peters for carefully reading the manuscript and for helpful discussions. We acknowledge Drs. Angela Wandinger-Ness and Rob Parton for the supply with monoclonal antibodies against Rab5 and polyclonal sera against Rab11, respectively. We also thank Drs. Marino Zerial and Cecilia Bucci for MDCK I cell lines overexpressing Rab5A.

	FOOTNOTES

^* This work was supported by Austrian Industrial Research Promotion Fund Grant FFF 3/11504, Austrian Science Foundation Grant FWF P11446-MED, and a grant from the Johnson & Johnson Focused Giving Program (to L. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ The first two authors contributed equally to this work.

^§§ To whom correspondence should be addressed. Tel.: 43-1-79730-622; Fax: 43-1-7987153; E-mail: huber@nt.imp.univie.ac.at.

² P. D. Burbelo, unpublished data.

	ABBREVIATIONS

The abbreviations used are: Tf-R, transferrin receptor; TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene-sulfonate; FACS, fluorescence-activated cell sorter; FAOS, fluorescence-activated organelle sorting; MDCK, Madin-Darby canine kidney; BHK, baby hamster kidney; HRP, horseradish peroxidase; 2D, two-dimensional; 2DE, 2D gel electrophoresis; PDZ, PSD-95, Dlg, and ZO-1; PNS, postnuclear supernatant; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES