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J. Biol. Chem., Vol. 275, Issue 52, 41074-41081, December 29, 2000

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Thyroglobulin Is Selected as Luminal Protein Cargo for Apical Transport via Detergent-resistant Membranes in Epithelial Cells^*

Fernando Martin-Belmonte^§, Miguel A. Alonso, Xiaoqing Zhang^¶, and Peter Arvan^¶

From the  Centro de Biologia Molecular "Severo Ochoa," Universidad Autonoma de Madrid, Madrid 280-49, Spain and the ^¶ Department of Developmental and Molecular Biology, and Division of Endocrinology, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication, June 21, 2000, and in revised form, September 25, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Thyroid hormone synthesis by thyrocytes depends upon apical secretion of thyroglobulin (Tg), the glycoprotein prohormone. In stably transfected MDCK cells, recombinant Tg is also secreted apically. All secreted Tg has undergone Golgi carbohydrate modification, whereas most intracellular Tg (which is slow to exit the endoplasmic reticulum) is sensitive to digestion with endoglycosidase H. However, in MDCK cells and PC Cl3 thyrocytes, a subpopulation of newly synthesized recombinant and endogenous Tg, respectively, is recovered in a Triton X-100 insoluble, glycosphingolipid/cholesterol-enriched (GEM/raft) fraction, and this small subpopulation is overwhelmingly endoglycosidase H resistant. Upon apical secretion, Tg solubility is restored. Apical secretion of Tg is inhibited by cellular cholesterol depletion. In FRT cells, recombinant Tg becomes Triton X-100 insoluble within 60 min after synthesis and a portion is actually endoglycosidase H-sensitive, suggesting early Tg entry into GEMs/rafts. Interestingly in FRT cells, Tg remains associated with the apical plasma membrane upon exocytosis, and all surface Tg is GEM/raft-associated. Thus, Tg is the first secretory protein demonstrated to enter Triton X-100 insoluble membranes en route to the apical surface of epithelial cells. The data imply that Tg utilizes a cargo-selective mechanism for apical sorting.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The functional unit of the thyroid gland is the thyroid follicle, each follicle representing a simple epithelium in which a monolayer of thyroid epithelial cells surrounds a central (apical) lumen (1). Thyroglobulin (Tg),¹ the thyroid hormone precursor protein and predominant gene product of the thyroid, is a large, homodimeric glycoprotein that is secreted overwhelmingly to the apical (luminal) side of thyrocytes (2). A failure of Tg secretion is an established cause of hypothyroidism and goiter (3). Nevertheless, aside from the protein folding steps that are prerequisite for its ER-to-Golgi transport (4), the mechanism(s) responsible for apical Tg trafficking remain undefined.

In thyrocytes, it is clear that not only is the trans-Golgi network the site of polarized sorting of surface membrane proteins (5), but it is also the site of segregation of apically bound Tg away from basolaterally secreted proteins (such as thrombospondin 1, laminin, or tissue-type plasminogen activator (6-8)). Indeed, polarized sorting of secretory proteins shows similarities between thyrocytes and MDCK cells (9). Because in both cell types basolaterally as well as apically secreted proteins are highly polarized rather than exhibiting random distributions, the implication is that both basolateral (10) and apical secretory proteins (11) may exhibit cargo-selective entry into trans-Golgi network-derived export pathways (12). Although data supporting cargo-selective carrier mechanisms for apical transport have been presented for several model membrane proteins (13-15), thus far, no data have been shown that demonstrate any specific class of membranes or membrane binding sites for Golgi-derived apical secretory proteins. To understand apical Tg transport, including the possibility of luminal cargo selectivity in the process, we need to better define the nature of the apical pathways available for secretory proteins in simple epithelial cells.

In epithelial cells lacking a conventional regulated secretory pathway, one apical Golgi-to-surface transport pathway has been described for influenza virus hemagglutinin and other apical membrane proteins, which may involve segregation in detergent(Triton X-100)-insoluble glycosphingolipid/cholesterol-enriched membranes (GEMs) (16), also known as liquid-ordered domains (17) or rafts (18). The GEM/raft pathway, which is sensitive to perturbations of cellular cholesterol (18), has long been implicated in the surface delivery of glycosylphosphatidylinositol-anchored proteins (19) and has been speculated to serve as a route for apically secreted glycoproteins (11). Actually, Triton X-100-insoluble GEMs/rafts have never yet been shown to carry any apical secretory proteins. Indeed, both gp80/clusterin (the major endogenous apical secretory glycoprotein of MDCK cells) and a truncated, soluble form of placental alkaline phosphatase (that is apically secreted from FRT cells) have both been specifically investigated and not recovered in conventional Triton X-100-insoluble GEM/raft fractions, although such proteins might dissociate from components of GEM/raft microdomains upon permeabilization with detergent (20, 21).

Another apical secretory pathway typically seen in exocrine cells involves the regulated exocytosis of apical storage granules (22); in such cells this route might conceivably subserve a role in biogenesis of the apical plasma membrane (23, 24). Regulated apical secretion is particularly relevant to thyrocytes, which have an exocrine organization and an acute thyrotropin-stimulated exocytotic response (2). Indeed, we have recently described two heterologously regulated secretory proteins that can be secreted apically upon transient expression in filter-grown primary thyrocytes. From this and other studies, we have proposed important relationships between the apical trafficking of Tg and its sorting into the regulated secretory pathway (6, 9).

Recently, two differently regulated secretory protein markers, prohormone convertase 2 and carboxypeptidase E, have been found to associate with GEM/raft microdomains along the pathway of secretory granule biogenesis (25, 26). Thus, although additional mechanisms (16, 27-31) and additional trafficking routes (6, 32) may contribute to apical protein secretion (33), and although Tg secreted from dispersed thyroid cells is fully soluble in the presence or absence of cold Triton X-100 (34, 35),² we have investigated the possibility that Tg might associate with GEMs/rafts in the secretory pathway. We now report the targeting of newly synthesized recombinant Tg to Triton X-100 insoluble microdomains in MDCK and FRT cells, as well as endogenous Tg in PC Cl3 thyrocytes. From these studies, we can report that Tg is the first secretory protein positively identified to enter the GEM/raft pathway en route to the apical surface of epithelial cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The cDNA construct expressing bovine Tg was described previously (36). Rabbit polyclonal antibodies to Tg and to caveolin were obtained from Dr. P. R. Larsen (Brigham and Women's Hospital, Boston, MA) and Transduction Laboratories (Lexington, KY), respectively. The monoclonal antibodies against gp114 (37) or dipeptidylpeptidase IV (38) were obtained from the laboratory of Dr. E. Rodriguez-Boulan (Cornell Medical School, New York, NY). A monoclonal antibody to Golgi mannosidase II (originally made by Dr. B. Burke, U. of Calgary, Canada) was obtained from Dr. D. Shields, Albert Einstein College of Medicine). A monoclonal antibody against EEA1 was purchased from BC Transduction Laboratories (cat. No. E41120). Polyclonal rabbit antibodies against transferrin were from Dako (Carpinteria, CA; cat. No. A0061) and were biotinylated; these antibodies were applied to FRT cells that had been incubated with transferrin (1 mg/ml) for 30 min at 37 °C before fixation. Peroxidase-conjugated secondary anti-IgG antibodies, sulfo-N-hydroxyl-succinimido-biotin (sulfo-NHS-biotin), streptavidin-agarose, streptavidin-fluorescein isothiocyanate, and streptavidin-peroxidase were from Pierce. Fluorescein- and Texas Red-conjugated antibodies were from Southern Biotechnology (Birmingham, AL). Compactin was obtained from Fluka Chemie AG (Buchs, Switzerland). Triton X-100, methyl--cyclodextrin (CD), mevalonate, fumonisin B1 (FB₁), transferrin, and octylglucoside were purchased from Sigma.

Cell Culture and Transfection-- MDCK II cells were grown on Petri dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.), penicillin (50 units/ml), and streptomycin (50 mg/ml), at 37 °C in an atmosphere of 5% CO₂. FRT and PC Cl3 cells were grown in F12 Coon's medium (Sigma) plus 10% fetal bovine serum and antibiotics. In the case of PC Cl3 cells, the culture medium was supplemented with 10 micro-units/ml thyrotropin, 1 µg/ml insulin, 5 µg/ml transferrin, and 10 nM hydrocortisone.

MDCK II (2 × 105) cells were transfected with 1-2 µg of plasmid DNA using lipofectAMINE (Life Technologies, Inc.). FRT cell transfections were carried out by electroporation. Beginning at 48 h after transfection, stable transformants were selected by treatment with either 0.8 mg/ml (MDCK) or 0.5 mg/ml (FRT) G418 sulfate (Life Technologies, Inc.) for at least 4 weeks following transfection. Drug-resistant cells were selected and then screened either by immunoblotting or immunofluorescence analysis. Positive MDCK clones were routinely maintained in the presence of G418, whereas positive FRT clones were maintained in drug-free medium. After several passages, >95% of cells within selected positive clones retained expression of Tg.

Detergent Extraction Procedures-- GEMs/rafts were isolated by standard procedures (19). Cells grown to confluency in 100-mm dishes were rinsed with phosphate-buffered saline (PBS) and lysed for 20 min in 1 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100 at 4 °C. The lysate was scraped from the dishes with a cell lifter, the dishes rinsed with 1 ml of the same buffer at 4 °C, and the lysate homogenized by passing the sample through a 22-gauge needle. The extract was finally brought to 40% sucrose in a final volume of 4 ml beneath an 8-ml 5-30% linear sucrose gradient. Gradients were centrifuged for 18 h at 39,000 rpm at 4 °C in a Beckman SW41 rotor. Fractions of 1 ml were harvested from the bottom of the tube, and aliquots were subjected to immunoprecipitation or immunoblot analysis. For the experiment shown in Fig. 5C, a discontinuous sucrose gradient was used. In this protocol, the 40% sucrose load was overlaid with 6 ml of 30% sucrose and 2 ml of 5% sucrose. After centrifugation, the opalescent band at the 30-5% sucrose interface, containing GEMs/rafts, was collected as the Triton X-100 insoluble fraction, whereas the 40% sucrose layer containing the load was harvested as the Triton X-100 soluble fraction.

Immunoblotting and Immunoprecipitation-- For immunoblot analysis, samples were subjected to SDS-15% PAGE under reducing conditions and transferred to Immobilon-P membranes (Millipore, Bedford, MA). After blocking with 5% (w/v) nonfat dry milk plus 0.05% (v/v) Tween 20 in PBS, blots were incubated with the indicated primary antibody. After several washings, blots were incubated for 1 h with goat anti-mouse or anti-rat IgG antibodies coupled to horseradish peroxidase, washed extensively, and developed using an enhanced chemiluminescence Western blotting kit (ECL, Amersham Pharmacia Biotech).

For metabolic labeling, cells were starved in culture medium lacking methionine and cysteine for 30 min and incubated with 100-500 µCi of a [³⁵S]methionine/cysteine mixture (ICN, Costa Mesa, CA) for the indicated times at 37 °C. After labeling, the medium was removed and replaced with complete culture medium. For immunoprecipitation, antibodies were prebound overnight at 4 °C to protein A-Sepharose in 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Triton X-100. Cell extracts were precleared for 4 h at 4 °C with a control antibody bound to protein A-Sepharose, and the supernatant was immunoprecipitated by incubation for 4 h at 4 °C with the appropriate antibodies bound to protein A-Sepharose. After collection, the immunoprecipitates were washed six times with 1 ml of 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Triton X-100 and analyzed by SDS-PAGE under reducing conditions. To detect ³⁵S labeling, dried gels were finally exposed to Fujifilm imaging plates (Fuji Photo Film Co.). For analysis of endoglycosidase H resistance, immunoprecipitates were incubated overnight at 37 °C in the presence or in the absence of 0.05 units/ml endoglycosidase H (Roche Molecular Biochemicals) before analysis by SDS-PAGE. Precipitation with streptavidin-agarose used a protocol similar to that described for immunoprecipitation. Quantitative analyses were done using a computing densitometer.

Domain-selective Biotinylation-- For separate access to apical or basolateral domains, FRT or MDCK cells were seeded at confluent levels on 24-mm polyester tissue culture inserts of 0.4 mm pore size (Transwell-Clear, Costar Inc., Cambridge, MA). The integrity of the cell monolayer was monitored by measuring the transepithelial electrical resistance using the Millicell ERS apparatus (Millipore Corp.). For metabolic labeling of cells in filters, cells were starved in media lacking methionine and cysteine. After 30 min, 250 µCi of [³⁵S]methionine/cysteine were added to the basolateral compartment, and filters were incubated for 30 min at 37 °C. To analyze polarized secretion of Tg, apical and basolateral culture media were removed 4 h later and subjected to immunoprecipitation analysis with anti-Tg antibodies.

For surface labeling, cells were washed with ice-cold PBS containing 0.1 mM CaCl₂and 1 mM MgCl₂, and 0.5 mg/ml sulfo-NHS-biotin were added either to the apical or basolateral compartment of the filter chamber. After 30 min at 4 °C, the solution was removed and the remaining unreacted biotin quenched by incubation with ice-cold serum-free culture medium. Cell monolayers were finally washed with PBS and extracted with 0.5 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 60 mM octylglucoside for 30 min on ice (except in Fig. 7C, where octylglucoside was omitted). Tg was immunoprecipitated from the extracts, which were then further analyzed either by blotting with streptavidin-peroxidase or a by a second round of precipitation with streptavidin-agarose.

Confocal Immunofluorescence-- MDCK and FRT cells grown on tissue culture inserts were fixed in 4% formaldehyde for 15 min, rinsed, and then quenched for 5 min with 10 mM glycine. The cells were then permeabilized or not with 0.2% Triton X-100, rinsed, and incubated with 3% bovine serum albumin in phosphate-buffered saline for 15 min. Cells were then incubated for 1 h with the indicated primary antibodies, rinsed several times, and incubated for 1 h with specific fluorescent secondary antibodies. Images were obtained using a Bio-Rad Radiance 2000 confocal laser microscope. Controls to assess the specificity of the labeling included incubations with control antibodies or omission of primary antibodies.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In MDCK Cells and PC Cl3 Thyrocytes, Tg Is Secreted Apically and Is Recovered in the GEM/Raft Fraction-- Because of its large size, a full-length expressible Tg cDNA has become available only relatively recently (36), and the secretory polarity of the recombinant protein has never been examined. We therefore prepared stably transfected MDCK cells expressing Tg and selected positively expressing clones based on immunoblotting of conditioned media. Similar to endogenous Tg in primary thyrocytes (39), recombinant Tg secretion from MDCK cells appeared slow but efficient (exhibiting a t_1/2 of several hours; see below). Three clones were analyzed in detail, but all clones exhibited the same Tg secretion phenotype. After a pulse-chase with ³⁵S-labeled amino acids, by high resolution SDS-PAGE, intracellular Tg exhibited two very closely spaced bands: a dark faster-migrating species and a faint slower-migrating species (Fig. 1A). Previous studies have established that these bands reflect the state of carbohydrate processing and correspond to ER and Golgi/post-Golgi forms of Tg, respectively (39). Importantly, as in thyrocytes (7), recombinant Tg secreted from filter-polarized MDCK cells exhibited a marked apical polarity (Fig. 1A). Notably, all recombinant Tg recovered from the cell culture supernatant exhibited a mobility corresponding to the slower migrating, post-Golgi species (Fig. 1A) and was resistant to digestion with endoglycosidase H (Fig. 1B). By contrast, the pool of endoglycosidase H-resistant intracellular Tg, even at late chase times, was quite small (Fig. 1B), Thus, in MDCK cells as in CHO cells (36, 40), recombinant Tg transport from Golgi to surface is a relatively rapid step so that the cells maintain only a small intracellular Golgi/post-Golgi pool of Tg at any moment.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Polarized secretion of Tg in MDCK cells. Panel A, MDCK cells stably expressing recombinant Tg, grown on filters, were metabolically labeled with [35S]methionine/cysteine for 30 min. After a 4-h chase, media were recovered from both the apical ("A") and basolateral ("B") compartments and the cells were lysed (Lys). Tg was then immunoprecipitated and analyzed by SDS-PAGE and autoradiography. Note that Tg was secreted with apical predominance. B, MDCK cells grown in plastic dishes were labeled and chased as in panel A. The cell culture supernatant (Sup) and cell lysate (Lysate) was analyzed. After immunoprecipitation with anti-Tg, the samples were resuspended in denaturing buffer, split in two, and incubated either in the absence () or presence (+) of endoglycosidase H (Endo H). Note that secreted Tg was resistant (R), whereas intracellular Tg was sensitive (S) to endoglycosidase H digestion.

With this fact in mind, we proceeded to examine Tg solubility in cold Triton X-100 as analyzed by a flotation sucrose gradient in which proteins entering Triton X-100-insoluble lipid-enriched microdomains (GEMs/rafts) selectively float out of the load, whereas other Triton-insoluble proteins (such as cytoskeleton) or Triton-soluble proteins cannot float because of their higher buoyant density (19). As shown in Fig. 2A, the vast majority of intracellular Tg remained in the load fractions (1-4) at the bottom of the gradient. From a sample of this material (fraction 2) it was apparent that all of the load was composed of endoglycosidase H-sensitive Tg (Fig. 2B, first two lanes). However, a small portion of the total intracellular Tg was observed to float out of the load, into fractions enriched in GEMs/rafts (fractions 7-9), as indicated by the marker, VIP21/caveolin-1 (Fig. 2A). When a sample of this material (from fraction 8) was analyzed, all detectable Tg was endoglycosidase H-resistant (Fig. 2B, last two lanes). These data suggest that in MDCK cells, recombinant Tg enters the GEM/raft pathway on its way to the apical surface.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Incorporation of Tg into GEMs/rafts of MDCK cells. A, MDCK cells expressing Tg were metabolically labeled for 4 h with a mixture of [35S]methionine/cysteine, extracted with 1% Triton X-100 at 4 °C, and centrifuged to equilibrium in a continuous sucrose density gradient. Fractions of 1 ml were collected from the bottom of the tube. Fractions 1-4 are the 40% sucrose layer and contain the soluble material, whereas fractions 5-12 are the lower density fractions that include GEMs/rafts. Tg immunoprecipitated from each fraction was analyzed by SDS-PAGE and autoradiography (top panel). As a control, the distribution of VIP 21/caveolin on the gradient was analyzed by immunoblotting (bottom panel). B, samples of Tg from the Triton X-100-soluble and -insoluble (GEM/raft) fractions are sensitive (S) and resistant (R) to endoglycosidase H (Endo H) digestion, respectively. As indicated, Tg immunoprecipitated from fractions 2 and 8 of the gradient were analyzed by digestion in the absence () or presence (+) of endoglycosidase H.

The GEM/raft microdomain is known to be destabilized by cellular cholesterol depletion (18), and in MDCK cells, this leads to impaired apical delivery of proteins that use this pathway (41). We therefore examined the effect of cholesterol depletion (by treating cells with compactin/mevalonate plus methyl--cyclodextrin) on Tg secretion from MDCK cells. As a control, we also examined the secretion of endogenous gp80/clusterin, in which apical polarity is known to be disrupted by this treatment (41). Unlike untreated cells, which released a large fraction of Tg to the apical medium after cholesterol depletion, apical Tg secretion was profoundly inhibited (Fig. 3A). This behavior was paralleled by inhibition of the apical secretion of gp80/clusterin (Fig. 3B). These effects are quantitated in the panels at the bottom of Fig. 3. By contrast, fumonisin B1 (FB₁), an inhibitor of glycosphingolipid synthesis, which was recently reported to impair apical transport of placental alkaline phosphatase or chimeras thereof (21), had no effect on apical Tg or gp80 secretion.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Apical secretion of Tg in MDCK cells is sensitive to cholesterol depletion but not to FB1 treatment. MDCK cells grown in filter culture inserts were pretreated or not with 25 mM compactin, 200 mM mevalonate for 48 h or with FB1 (25 mg/ml) for 72 h. To increase the extent of cholesterol depletion, cultures pretreated with compactin/mevalonate were then treated with 10 mM methyl--cyclodextrin (CD) for 1 h at 37 °C. After these treatments, cells were metabolically labeled for 30 min with [35S]methionine/cysteine and chased in serum-free medium for 4 h at 37 °C. Chase media were collected from the apical ("A") and basolateral ("B") compartments. Panel A, immunoprecipitation of secreted Tg. Panel B, the same supernatants were precipitated with 10% trichloroacetic acid and analyzed by SDS-PAGE and autoradiography to detect gp80 without immunoprecipitation (because this is the major endogenous secretory protein of MDCK cells). The panels below, in A and B, show a quantitative analysis of the gels above.

Next, we turned our attention to endogenous Tg expressed in PC Cl3 thyrocytes, in which secreted Tg (as in normal thyrocytes) is predominantly apical (Fig. 4C) and exclusively endoglycosidase H-resistant.² Specifically, we examined the ability of newly synthesized Tg from PC Cl3 cells to partition into the Triton X-100-insoluble GEM/raft fraction, as analyzed by flotation sucrose gradient. The data shown in Fig. 4A, similar to that in MDCK cells, indicate the presence of a small population of newly synthesized Tg in the GEM/raft fraction. Further analysis of a sample of this material (from fraction 8) showed that, as for recombinant Tg in MDCK cells, this subpopulation of intracellular Tg was endoglycosidase H-resistant (Fig. 4B). Together, these data strongly suggest that in thyrocytes as well as MDCK cells, Tg en route to the apical cell surface enters GEM/raft microdomains.

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Incorporation of endogenous Tg into GEMs/rafts in PC Cl3 thyrocytes. A, PC Cl3 cells were metabolically labeled, extracted with 1% Triton X-100, and centrifuged to equilibrium in a continuous sucrose density gradient as described in the legend for Fig. 2A. Tg immunoprecipitated from each fraction was analyzed by SDS-PAGE and autoradiography. At a slightly longer exposure (not shown), labeled Tg was also detected in fraction 9. B, samples of Tg from the Triton X-100-soluble and -insoluble (GEM/raft) fractions are sensitive (S) and resistant (R) to endoglycosidase H (Endo H) digestion, respectively. As indicated, Tg immunoprecipitated from fractions 2 and 8 of the gradient, representative of Triton X-100-soluble and -insoluble fractions, respectively, were analyzed by digestion in the absence () or presence (+) of endoglycosidase H. C, PC Cl3 cells were grown on Transwell-clear filters. Apical ("A") and basal ("B") media bathing cells labeled continuously for 4 h were analyzed by Tg immunoprecipitation, SDS-PAGE, and fluorography.

In FRT Cells, Tg Enters the GEM/Raft Fraction and Is Delivered to the Apical Plasma Membrane-- FRT cells have gained attention over the past decade because, whereas the influenza hemagglutinin transmembrane protein is sorted apically (42), luminally anchored glycosylphosphatidylinositol (GPI)-linked proteins are preferentially delivered to the basolateral surface of these cells, unlike in MDCK cells (43). Apparently, this phenotype is not because GEMs/rafts fail to function as apical carriers, but rather is due to an inability of GPI-linked proteins to enter the Triton X-100 insoluble GEM/raft fraction (44). Thus, it was of obvious relevance to determine whether Tg could enter the GEM/raft pathway to the apical surface of FRT cells, especially since FRT cells are originally derived from thyroid cultures. However, FRT cells do not endogenously express any thyroid-specific differentiation markers; therefore, the cells were stably transfected with the Tg cDNA, and stable Tg-expressing clones were selected.

After 4 h of continuous labeling with ³⁵S-labeled amino acids, Tg-expressing FRT cells were extracted with ice-cold Triton X-100 and the extract analyzed by flotation during sucrose gradient centrifugation (Fig. 5A). Although a significant portion of labeled Tg was recovered in the load (fractions 1-4), a major portion of Tg floated with GEMs/rafts (fractions 7-9). Once again, a sampling of Tg from the load fraction was entirely endoglycosidase H-sensitive, whereas Tg recovered from the GEM/raft fraction was enriched (70%) in endoglycosidase H-resistant forms (Fig. 5B). However, a smaller portion of Tg recovered in the GEM/raft fraction (30%) was noted to be endoglycosidase H-sensitive (Fig. 5B). Recently, it has been suggested that association of exocytic proteins with the GEM/raft microdomain may start taking place shortly after their biosynthesis, increasing during transport along the secretory pathway (45). With this in mind, we performed pulse-chase experiments to look at the development of Triton X-100 insolubility in FRT cells. Within 60 min after a 30 min pulse-labeling of FRT cells, more than half of the newly synthesized Tg had become Triton X-100-insoluble (Fig. 5C), although a significant portion of this material was endoglycosidase H-sensitive (Fig. 5D); whereas at the end of the chase, all Triton X-100-insoluble Tg was endoglycosidase H-resistant (Fig. 5D). Thus, in the steady state, Triton X-100-insoluble Tg appears to be largely endoglycosidase H-resistant; however, the data suggest that at least a portion of newly synthesized Tg is likely to associate with GEMs/rafts even before its arrival in the medial Golgi compartment.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Tg rapidly enters the GEM/raft fraction of FRT cells. A, FRT cells stably expressing Tg were metabolically labeled, extracted with 1% Triton X-100, and centrifuged to equilibrium in a continuous sucrose density gradient as described in the legend for Fig. 2A. Tg immunoprecipitated from each fraction was analyzed by SDS-PAGE and autoradiography. B, as indicated, Tg immunoprecipitated from fractions 2 and 8 from the gradient, representative of Triton X-100-soluble and -insoluble (GEM/raft) fractions, respectively, were analyzed by digestion in the absence () or presence (+) of endoglycosidase H (Endo H). C, FRT cells were pulse-labeled for 30 min with [35S]methionine/cysteine and chased for the times indicated. Cells were then extracted with 1% Triton X-100 at 4 °C, and the soluble (S) and insoluble (I) fractions were separated by discontinuous sucrose gradient centrifugation as described under "Experimental Procedures." Tg immunoprecipitated from these fractions was analyzed by SDS-PAGE and autoradiography. D, Triton X-100-insoluble fractions from FRT cells prepared as described for panel C were digested in the absence () or presence (+) of endoglycosidase H.

We then proceeded to examine polarized Tg secretion from the stably transfected FRT cells. Surprisingly, no pulse-labeled Tg was recovered in either the apical or basolateral medium after 4 h of chase; instead, all labeled Tg remained cell-associated (Fig. 6A). We therefore extended our analysis to prolonged chase times of up to 2 days. Over this period in MDCK cells, labeled Tg converted from being quantitatively recovered in the cell lysate at the zero chase time to near quantitatively recovered in the culture supernatant (Fig. 6B). However, from FRT cells over 48 h, only very little Tg (<10%) was ever recovered in the culture supernatant. This behavior was observed in three independently selected clones of FRT cells expressing Tg (not shown). The fact that Tg in FRT cells progressed into a Triton X-100-insoluble fraction and acquired endoglycosidase H resistance (Fig. 5) rendered unlikely the possibility that the protein failed to undergo ER-to-Golgi transport. Moreover, unlike CHO cells (36, 40), COS cells (46), or MDCK cells (see above), as measured by immunofluorescence microscopy of permeabilized FRT cells, the predominant pool of recombinant Tg in the steady state was not in the ER but exhibited primarily a perinuclear pattern (Fig. 6C).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Tg is not secreted from FRT cells and accumulates in the juxtanuclear region. A, filter-grown FRT cells stably expressing recombinant Tg were metabolically labeled for 30 min and chased in complete medium for 4 h. Tg was immunoprecipitated from the apical ("A") and basolateral ("B") culture media as well as the cell lysate (Lys). The immunoprecipitates were then subjected to SDS-PAGE and autoradiographed. B, FRT and MDCK cells (grown in plastic dishes) were metabolically labeled for 30 min with [35S]methionine/cysteine and then chased for up to 2 days in complete medium. At each of the indicated chase times, Tg was immunoprecipitated from the cell lysate (L) and culture supernatant (S) and analyzed by SDS-PAGE and autoradiography. C, confocal immunofluorescence analysis of intracellular Tg in FRT cells. The cells were fixed, permeabilized, and labeled using anti-Tg antibodies followed by fluorescent secondary antibodies.

Interestingly, in FRT cells, caveolin-2 in the steady state fails to localize to the plasma membrane, yielding instead a perinuclear distribution. However, after FRT cells are transfected to express caveolin-1, caveolin-2 localizes to the plasma membrane (28). This could be taken to mean that in FRT cells, some proteins (especially those employing a caveolin-1-mediated pathway) may be impaired in transport to the cell surface (28). We also considered that in FRT cells, transport of Tg from the biosynthetic pathway to the plasmalemma might occur, but Tg release at the apical cell surface might be impaired (with Tg remaining adherent to the apical membrane or entering the endosomal system). To begin to investigate these possibilities, we asked if it was possible to tag Tg at the surface of FRT cells using a nonpermeant biotinylation reagent.

Filter-grown FRT or MDCK cells expressing Tg were metabolically labeled with ³⁵S-labeled amino acids. After a 4-h chase, the cells were biotinylated at either the apical or basolateral cell surfaces before cell lysis, Tg immunoprecipitation, SDS-PAGE, and electrotransfer. As shown in Fig. 7A (lower panel), cell-associated ³⁵S-Tg recovered from both sets of cells was equivalent. Finally, the samples were blotted with streptavidin-peroxidase to detect only surface-bound Tg. In MDCK cells, from which Tg is freely secreted (Figs. 1 and 6), essentially no Tg could be detected that was bound at the cell surface; however, in FRT cells, a strong apically biotinylated Tg signal was detected (Fig. 7A, upper panel). This experiment was then modified in two ways. First, FRT cells were labeled overnight with ³⁵S-labeled amino acids, biotinylated either apically or basolaterally, lysed, and immunoprecipitated with anti-Tg (Fig. 7B, first two lanes), and the immunoprecipitates were then reprecipitated with streptavidin-agarose (Fig. 7B, last two lanes). From this, we deduced that whereas the majority of cell-associated Tg was indeed intracellular, ~20% of cell-associated Tg was accessible to the biotinylation reagent at the apical plasmalemma (Fig. 7B). (This must be considered a minimum value, as the efficiency of cell surface biotinylation is almost certainly less than 100% (47).) Second, we repeated exactly the experiment of Fig. 7A, except without or with prior cholesterol depletion. Without cholesterol depletion, the apical:basolateral ratio of surface-tagged Tg was ~4.9, whereas after cholesterol depletion the ratio was only ~1.2.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Tg accumulates on the apical surface of FRT cells in GEMs/rafts. A, filter-grown FRT and MDCK cells were metabolically labeled for 30 min with [35S]methionine/cysteine and chased for 4 h. Cells were then surface biotinylated from either the apical ("A") or basolateral ("B") sides and lysed with radioimmune precipitation buffer. Tg immunoprecipitated from the cell lysates was then analyzed either by direct autoradiography (bottom panel) or by immunoblotting with streptavidin-peroxidase (top panel). B, FRT cells were labeled, chased, surface-biotinylated from either the apical or basolateral sides, and lysed as described in panel A. Tg immunoprecipitates from each sample were split in half. One half was saved for direct analysis (IP -Tg), and the second half was washed twice, eluted in the presence of boiling 1% SDS, reconstituted in radioimmune precipitation buffer, and then reprecipitated with streptavidin-agarose (IP St-Agar.). Quantitation of these data indicate that at least 20% of cell-associated Tg is accessible at the apical surface. C, biotinylated FRT cells (grown in plastic dishes) were lysed with cold 1% Triton X-100 and centrifuged to equilibrium in a continuous sucrose density gradient as in Fig. 2A. Tg was immunoprecipitated from each fraction and analyzed by SDS-PAGE and blotting with streptavidin-peroxidase. Note that all biotinylated Tg is recovered in the GEM/raft fraction.

Next, we examined nonpermeabilized FRT or MDCK cells expressing Tg by immunofluorescence with a polyclonal anti-Tg antibody. In nonpermeabilized MDCK cells, Tg immunofluorescence at the cell surface was extremely faint (Fig. 8, lower half) and by two-dimensional z-section reconstruction (bottom panels), most of the apical surface of MDCK cells was free of Tg immunoreactivity. However, in nonpermeabilized FRT cells, Tg showed a generally similar distribution to that of dipeptidylpeptidase IV, an established apical marker of these cells, with the majority of the apical surface positive for Tg immunofluorescence (Fig. 8, upper half). Together, the data in Figs. 7 and 8 indicate that in FRT cells, Tg exit from the Golgi is not blocked. Rather, apical Tg sorting appears similar to that found in MDCK and PC Cl3 cells, although Tg externalized by FRT cells remains adherent to the apical cell membrane. Importantly, when biotinylated FRT cells were subsequently lysed in cold Triton X-100, all surface-tagged Tg floated out of the load upon sucrose gradient centrifugation and was recovered in fractions enriched in GEM/raft microdomains (fractions 7-9, Fig. 7C). Thus, in FRT cells, Tg enters Triton X-100-insoluble microdomains during transit through the secretory pathway (within 60 min of biosynthesis; see Fig. 5C), thereafter appearing in GEMs/rafts at the apical cell surface.

View larger version (100K): [in this window] [in a new window]   Fig. 8.   Confocal analysis detects apical surface expression of Tg in nonpermeabilized FRT cells. FRT and MDCK cells grown in filter culture inserts were fixed and labeled using anti-Tg antibodies (panels on left). Sections in the x-y (larger image) and x-z (smaller image) dimensions are shown. As a control, the surface distributions of dipeptidyl peptidase IV and gp114, endogenous apical membrane markers of FRT and MDCK cells, respectively, were analyzed in parallel (panels on right).

Fate of Apically Biotinylated Tg in FRT Cells-- We then followed the fate of surface Tg using FRT cells that were apically biotinylated before being returned to routine cell culture. Although there was no loss of biotinylated Tg over the first 4 h of culture (not shown), thereafter biotinylated Tg slowly disappeared from the cells as measured in lysates prepared over a 2-day period (Fig. 9A). Importantly, however, no biotinylated Tg ever appeared in the culture supernatant (Fig. 9A). Instead, intracellular degradation of biotinylated Tg over 24 h was nearly completely blocked by treatment of the cells with 100 µM chloroquine (Fig. 9B). These data suggested the possibility that apically bound Tg might be internalized via the endocytic pathway and be slowly degraded in the endosome-lysosome system. To explore this possibility, we examined the intracellular immunofluorescent localization of Tg in FRT cells in conjunction either with mannosidase II (an established Golgi marker) or with EEA1 or transferrin, markers of early and recycling endosomes, respectively. As shown in Fig. 10, Tg colocalized negligibly with the first two markers (top panels) but showed partial overlap with transferrin (lower panels), consistent with the idea that some if not all Tg reaches the endosomal system in FRT cells.

View larger version (43K): [in this window] [in a new window]   Fig. 9.   Gradual degradation of surface Tg in FRT cells is chloroquine inhibitable. FRT cells grown in plastic dishes were surface-biotinylated and then returned to complete medium for the indicated times. A, Tg immunoprecipitated from the cell lysate (L) and culture supernatant (S) at each chase time was analyzed by SDS-PAGE and blotting with streptavidin-peroxidase. B, surface-biotinylated FRT cells were incubated in the absence or presence of 100 mM choroquine (Chloroq.) before Tg immunoprecipitation and blotting with streptavidin-peroxidase.

View larger version (60K): [in this window] [in a new window]   Fig. 10.   Immunofluorescence localization of intracellular Tg in permeabilized FRT cells. FRT cells expressing Tg were fixed and double-labeled using anti-Tg antibodies (these antibodies applied first; far left panels) and with antibodies against Golgi mannosidase II (top middle panel), EEA1 (center panel), or transferrin (lower middle panel; see "Experimental Procedures"). For mannosidase II or EEAI, secondary detection employed fluorescein isothiocyanate-conjugated antibodies, whereas transferrin was detected with fluorescein isothiocyanate-conjugated streptavidin. Merged images are shown in the panels on the right.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apical targeting of Tg to the lumen of thyroid follicles is essential for thyroid hormone synthesis (2). All studies to date indicate that pathophysiological conditions in which Tg is either delivered insufficiently to the follicle lumen (3) or delivered abnormally to the basolateral surface (48, 49) are associated with serious disorders of thyroid hormone production. To understand such disorders, it is first necessary to begin to understand the normal apical targeting of Tg, the mechanism(s) of which have never previously been explored. We now demonstrate that in MDCK cells, which lack a classical regulated secretory pathway, recombinant Tg is secreted apically (Fig. 1); it can be recovered in the GEM/raft fraction (Fig. 2); and its apical secretion is severely inhibited by cellular cholesterol depletion (Fig. 3). By contrast, Tg expressed endogenously in thyrocytes is packaged into the regulated secretory pathway (6, 7, 50, 51). Intriguingly, endogenously expressed Tg in PC Cl3 thyrocytes is also secreted apically and is recovered in the GEM/raft fraction (Fig. 4). Thus, whether in MDCK cells or thyrocytes, Tg is the first secretory protein demonstrated to associate with Triton X-100-insoluble membrane domains en route to the apical surface of epithelial cells. Superficially, the new findings in MDCK cells would seem to cast doubt on our previous postulate that the apical Tg targeting mechanism in thyrocytes is in some way related to, or dependent upon, sorting to the regulated secretory pathway (9). However, in light of very recent reports suggesting that two different secretory proteins may enter the regulated secretory pathway as a consequence of association with GEM/raft microdomains (25, 26), it now appears that, for some cases, there are indeed important similarities in the mechanisms of regulated secretory protein sorting and apical secretory protein sorting.

In PC Cl3 cells and MDCK cells, the detection of Triton X-100-insoluble Tg is limited to the Golgi/post-Golgi (endoglycosidase H-resistant) form (Figs. 1 and 2). Such detergent insolubility does not occur when the cells are lysed in octylglucoside,² a detergent that is known to solubilize the GEM/raft fraction. Further, because these cell types secrete rather than store post-Golgi Tg, only a small quantity of cellular Tg is ever recovered in Triton X-100-insoluble fractions (Fig. 2). Once secreted into the medium, Tg is completely soluble in cold Triton X-100.² Thus, we conclude that in MDCK and PC Cl3 cells, the detection of Triton X-100 insolubility represents a transient state of the protein that takes place during its biosynthetic transport to the plasma membrane.

Although Tg is also recovered in GEMs/rafts of FRT cells (Fig. 5A), the situation differs in two important ways. First, most Tg becomes Triton X-100-insoluble within 60 min of chase (Fig. 5C), i.e. with a half-time that is faster than the half-time of Golgi arrival (39), and indeed, a portion (albeit a minority) of the Tg recovered in the GEM/raft fraction is endoglycosidase H-sensitive (Fig. 5B). Indeed, it appears likely (Fig. 5D) that GEM/raft assembly may begin even in pre-Golgi or early Golgi compartments (45). Second, in a manner different from what has been observed in primary thyrocytes (6, 52), Tg is not effectively secreted from FRT cells into either the apical or basolateral medium (Fig. 6A) even at very prolonged chase times (Fig. 6B). Instead, Tg remains cell-associated and is distributed between two predominant regions of these cells.

One portion of Tg in FRT cells is extracellularly disposed on the apical plasma membrane (Fig. 8). Indeed, surface biotinylation of FRT cells indicates that apically bound surface Tg represents a significant minority of the total cellular pool (Fig. 7B). Importantly, all of this surface Tg is recovered in GEMs/rafts (Fig. 7C), and the apical predominance is blocked by cholesterol depletion. Thus, in FRT cells, although GPI-anchored proteins are unable to enter Triton X-100-insoluble GEMs/rafts (44), Tg is still able to do so. The situation is quite intriguing, as the extent and affinity of Tg association either with lipids or proteins endogenous to the GEM/raft pathway is dramatically greater in FRT than other cell types (Fig. 5). However, because FRT cells are not known to express any thyroid-specific gene products, and because cultured primary thyrocytes and PC Cl3 cells are known to secrete large quantities of Tg into the medium (Ref. 6 and this report), we must presume until further evidence becomes available that the absence of Tg secretion in FRT cells is atypical and not reflective of endogenous Tg trafficking in the secretory pathway of normal thyrocytes. A second portion of Tg in FRT cells is located intracellularly (Fig. 6C) in a perinuclear distribution. This seems consistent with Tg entry into the endosome/lysosome system, as suggested by the degradation of surface-biotinylated Tg in a chloroquine-inhibitable manner (Fig. 9). Interestingly, Tg entry into the endosomal system in FRT cells is reminiscent of such entry during hormonogenesis in the thyroid gland (1, 53, 54). Much of this endosomal Tg seems to be localized with transferrin in recycling endosomes rather than in early sorting endosomes (Fig. 10), although other endosomal compartments may also be involved in Tg trafficking in FRT cells. It will be of interest in future studies to examine whether expression of caveolin-1 in FRT cells could cause relocation of any portion of intracellular Tg from its perinuclear distribution to the cell surface (28).

Data from each of the three epithelial cell types investigated strongly suggest that Tg, the first documented member of what is certainly a larger class of apical secretory proteins, is specifically recruited into membrane carriers destined for the apical surface. The fact that newly synthesized Tg is insoluble in Triton X-100, under conditions in which some compartments are completely solubilized and most others are permeabilized, points strongly to the idea that Tg is specifically bound to one or more components of GEMs/rafts. To date, we do not know what these components are. A popular model to explain apical-selective transport is that one or more lectins incorporated into GEM/raft microdomains capture and deliver glycoprotein cargo (11, 14, 27, 55, 56). Although this model may be plausible for Tg, we do not wish to argue that this represents the sole mechanism for all apical secretory protein targeting. First, expression of at least two regulated secretory proteins, growth hormone and parathyroid hormone (neither of which is glycosylated), leads to apical delivery in primary thyrocytes (9). Second, certain glycoproteins, such as the extensively O-glycosylated human neurotrophin receptor, are delivered apically in thyroid-derived FRT cells but are not incorporated into the GEM/raft fraction (33). Further, apical pathways that are independent of glycosylation (57) or GEM/raft incorporation (58) have also been described in MDCK cells.

Thus, many possibilities for apical cargo delivery still exist. Nevertheless, it does appear that proteins that are recruited into GEM/raft microdomains (18) are dependent upon cellular cholesterol levels either at the level of cargo entry or at the level of delivery of the membrane carrier to the apical surface (41), and this is certainly true for Tg (Fig. 3). At present, we speculate that of these possibilities, cholesterol might be more important in the pathway of delivery of the apical membrane carrier, because cholesterol depletion does not appear to prevent Tg recruitment into the GEM/raft fraction.³

Prior to this report, two apical secretory proteins, a truncated form of alkaline phosphatase (21) and the endogenous gp80/clusterin of MDCK cells (20), were specifically tested and not recovered in Triton X-100-insoluble fractions. The secretory form of alkaline phosphatase appears to exhibit identical polarized sorting to its GPI-anchored, GEM/raft-associated counterpart in FRT cells (21). However, polarized trafficking of the latter form is unaffected by cholesterol depletion and instead is perturbed by FB₁, an inhibitor of glycosphingolipid synthesis (21). By contrast, we find that apical Tg trafficking in MDCK cells is largely blocked by cellular cholesterol depletion and is unaffected by FB₁ (Fig. 3). Interestingly, apical trafficking of the endogenous gp80 glycoprotein in MDCK cells is also largely blocked by cellular cholesterol treatment (41) and is unaffected by FB₁ (59). Thus, the lack of recovery of gp80 in GEM/raft microdomains may imply the existence of more than one kind of cholesterol-containing apical raft in MDCK cells, consistent with recent suggestions (60). Alternatively, the same GEM/raft pathway used by Tg may also carry gp80, but the association is insufficiently strong for gp80 to remain intact after Triton X-100 extraction (41).

In conclusion, we report that Tg is the first apical secretory protein demonstrated to associate with Triton X-100-insoluble GEMs/rafts, implying a cargo-selective process. Hopefully, these findings will promote further investigations to define both cargo-selective as well as possible cargo-nonselective secretory mechanisms.

	ACKNOWLEDGEMENT

We thank Dr. C. Sanchez for help with the confocal microscopy.

	FOOTNOTES

^* This work was supported by grants from the National Institutes of Health (DK40344 to P. A.), the Direccion General de Ensenanza Superior (PM99-0092 to M. A. A.), and the Comunidad de Madrid (08.3/0020/1998) and by an institutional grant from the Fundacion Ramon Areces to Centro de Biologia Molecular "Severo Ochoa."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.

^§ Recipient of a fellowship from the Comunidad de Madrid.

To whom correspondence should be addressed: Div. of Endocrinology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx NY 10461. Tel.: 718-430-8685; FAX: 718-430-8557; E-mail: arvan@aecom.yu.edu.

Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M005429200

² F. Martin-Belmonte, M. A. Alonso, X. Zhang, and P. Arvan, our unpublished observations.

³ F. Martin-Belmonte, M. A. Alonso, X. Zhang, and P. Arvan, our preliminary data.

	ABBREVIATIONS

The abbreviations used are: Tg, thyroglobulin; ER, endoplasmic reticulum; MDCK cells, Madin-Darby canine kidney cells; CHO cells, Chinese hamster ovary cells; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; GPI, glycosylphosphatidylinositol; FB₁, fumonisin B1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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