©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Polarized Distribution and Delivery of Plasma Membrane Proteins in Thyroid Follicular Epithelial Cells (*)

(Received for publication, September 22, 1994; and in revised form, November 2, 1994)

Regina Kuliawat (1) Michael P. Lisanti (3) Peter Arvan (1) (2)(§)

From the  (1)Division of Endocrinology, Beth Israel Hospital, Harvard Medical School and the (2)Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02215 and the (3)Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thyroid follicular cells coordinate several oppositely located surface enzyme activities. Recent studies have raised questions about the basic mechanisms used to achieve thyroid surface polarity. We investigated these mechanisms in primary thyroid epithelial monolayers cultured on porous filters. In the steady state, most Na/K-ATPase and aminopeptidase N were available for surface biotinylation, and these proteins exhibited physiological distributions (basolateral and apical, respectively). Glycosylphosphatidylinositol-anchored proteins were also apically distributed. By pulse-chase, newly synthesized transmembrane proteins exhibited polarized surface delivery that was oriented similarly to that observed at steady state. Little time elapsed between acquisition of Golgi-specific processing and cell surface arrival. Interestingly, when either newly synthesized or steady state-labeled thyroid peroxidase was similarly analyzed, only 30% of the enzyme was ever detected at the cell surface. Of this, the majority was localized apically. The data suggest that most thyroid peroxidase remains intracellular in these monolayers, consistent with the possibility of intracellular iodination activity in addition to apical extracellular iodination. Nevertheless, in filter-polarized thyrocytes, most newly synthesized plasma membrane proteins appear to be sorted in the Golgi complex for direct delivery to apical and basolateral domains.


INTRODUCTION

Like other epithelial cells, thyroid follicular cells maintain a highly polarized surface organization (Wollman, 1989). For example, the driving force for active uptake of iodide by the thyroid epithelium is provided by a basolateral Na gradient generated by Na/K-ATPase (Chow et al., 1986; Vilijn and Carrasco, 1989; Golstein et al., 1992). Upon transport to the opposite end of the follicular cell (Nakamura et al., 1990; Nilsson et al., 1992), iodide becomes available to the catalytic domain of thyroid peroxidase. In the presence of H(2)O(2) (Bjorkman and Ekholm, 1988), thyroid peroxidase catalyzes iodide organification predominantly in the apical extracellular space (Ofverholm and Ericson, 1984; Gruffat et al., 1991) where the substrate protein (thyroglobulin) is concentrated to extraordinary levels (Herzog et al., 1992). A smaller degree of initial iodination of thyroglobulin may also occur intracellularly (Matsukawa and Hosoya, 1979; Kuliawat and Arvan, 1994). Nevertheless, an asymmetric distribution of epithelial cell surface enzymes is thought to be required for thyroid hormonogenesis.

In some instances, epithelial surface asymmetry is regulated by intracellular sorting of newly synthesized membrane proteins at the level of the trans-Golgi network (Fuller et al., 1985; Rindler et al., 1985) (for review see Simons and Wandinger-Ness(1990)). Interestingly, the site of this sorting may vary for different protein-epithelial cell combinations (Pathak et al., 1990), since some proteins may be delivered bi-directionally (Haller and Alper, 1993) but may be selectively stabilized or degraded at one surface (Contreras et al., 1989; Hammerton et al., 1991) or may be relocated to the contralateral membrane surface (Bartles et al., 1987; Matter et al., 1990) (for reviews see Mostov et al. (1992) and Nelson(1992)). Moreover, the same cells may exhibit different surface protein distributions during the development versus subsequent maintenance of epithelial polarity (Balcarova-Stander et al., 1984; Herzlinger and Ojakian, 1984; Wollner et al., 1992) (for review see Rodriguez-Boulan and Powell(1992)).

It is well established that thyroid follicular cells in culture form highly polarized epithelial monolayers (Chambard et al., 1983; Gerard et al., 1985; Mauchamp et al., 1987). However, reports of the Fischer rat thyroid (FRT) (^1)cell line (Nitsch et al., 1985), a recently developed model of thyrocyte polarity (Zurzolo et al., 1992a), have described significant differences from many other epithelial cells such as the (apical) polarity of Semliki Forest virus budding and (basolateral) polarity of transfected CD8 lymphocyte antigen (Zurzolo et al., 1992b). In addition, despite the normal basolateral targeting of Na/K-ATPase (Zurzolo and Rodriguez-Boulan, 1993), FRT cells display a preferential distribution of glycosylphosphatidylinositol-anchored proteins (which in most epithelia reside apically) on the basolateral surface (Zurzolo et al., 1993). Such findings have been interpreted to suggest unique mechanisms for protein targeting in the thyroid (Zurzolo et al., 1992b). To investigate this question further, we have examined filter-polarized epithelial monolayers comprised of normal, primary thyrocytes, in which polarized sorting of secretory proteins has already been demonstrated (Arvan and Lee, 1991; Prabakaran et al., 1993). Using this cell culture system, a biotin tag has been introduced onto surface proteins in order to examine the distribution and delivery of three endogenous plasma membrane proteins: aminopeptidase N (APN, an apically polarized enzyme), Na/K-ATPase (NKA), and thyroid peroxidase (TPO), the latter two exhibiting basolateral and apical activities, respectively.


MATERIALS AND METHODS

Cell Culture

Primary porcine thyrocytes were isolated and seeded at high density onto Millicell HA (Millipore Corp., Bedford, MA) 0.4-µm pore filters and cultured in the presence of 5 milliunits/ml bovine TSH (Sigma) and 5% calf serum added basolaterally, as described previously (Kuliawat and Arvan, 1994). This approach allowed monolayers to achieve electrical resistances geq1000 ohm-cm^2. Experiments were carried out on day 7.

Cell Labeling, Biotinylation, and Lysis

Confluent monolayers were radiolabeled with ExpreSS (DuPont NEN) either for 30 min in Met/Cys-deficient medium (for pulse-chase analysis) or for 2 days in complete medium (for steady-state labeling). Before biotinylation, the cells were washed 3 times in ice-cold phosphate-buffered saline. Monolayers were then exposed either apically or basolaterally to 0.6 mg/ml sulfo-NHS-biotin (Pierce) for 30 min at 4 °C in a buffer containing 0.25 M sucrose, 2 mM CaCl(2), 0.5 mM MgCl(2), 10 mM triethanolamine, pH 8.5. The biotin reagent was quenched by subsequent incubation with 50 mM NH(4)Cl in phosphate-buffered saline for 10 min at 4 °C. The cells were again washed in phosphate-buffered saline and lysed either in boiling 1% SDS to inhibit proteolysis (Rolland and Lissitzky, 1976) or in 1 ml of immunoprecipitation buffer containing 0.15 M NaCl, 25 mM Tris, pH 7.5, 10 mM iodoacetamide, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 5 mM EDTA, and a mixture of protease inhibitors as described previously (Kim et al., 1992). Cells lysed directly in SDS were then diluted into immunoprecipitation buffer lacking SDS to yield the same final detergent concentrations prior to immunoprecipitation.

Antibodies, Precipitations, and Immunofluorescence

A rabbit antiserum to TPO was obtained through the laboratory of the late Dr. S. Ingbar (Beth Israel Hospital, Boston, MA). Antiserum to porcine aminopeptidase N (Hansen et al., 1987) was obtained from Drs. O. Noren and H. Sjostrom (Panum Institute, Copenhagen, Denmark). A rabbit antiserum to lamb kidney Na/K-ATPase, cross-reacting with the pig enzyme, was obtained from Dr. W. J. Ball (University of Cincinnati, OH). Immunoprecipitations were typically overnight at 4 °C, using protein A-agarose (Sigma) as a secondary reagent. Immunoprecipitates were treated with 1% SDS and then diluted 10-fold into immunoprecipitation buffer lacking SDS. UltraAvidin-agarose (Leinco) was then used for precipitation of surface-labeled proteins. Avidin-biotin conjugates were dissociated by boiling in SDS-gel sample buffer and analyzed by SDS-PAGE. After absorption of biotinylated proteins on immobilized avidin, nonbiotinylated proteins were concentrated by a 1:3 (v/v) addition of Pro-Cipitate according to the manufacturer (Affinity Technology) and were similarly analyzed by SDS-PAGE. Indirect immunofluorescence localization of aminopeptidase N was performed on nonpermeabilized filter-grown thyroid monolayers fixed in 2% formaldehyde prior to incubation with a fluorescein isothiocyanate-conjugated goat-antirabbit serum, using Bio-Rad confocal optical analysis coupled to a Zeiss Axiophot microscope.

Analysis of Surface Polarity of Glycophosphatidylinositol (GPI)-anchored Membrane Proteins

Sets of four thyrocyte cultures grown on porous filters were biotinylated from apical or basolateral sides before lysis, as above. Hydrophobic membrane proteins in the lysate were extracted by Triton X-114 phase partitioning (Bordier, 1981). The detergent phase was then either treated with phosphatidylinositol-specific phospholipase C (PI-PLC) to release the lipid anchor or mock-digested (Lisanti et al., 1988). Triton X-114 phase separation was then repeated for both the mock digests and PI-PLC-treated samples; proteins released into the aqueous phase were collected and run on SDS-PAGE. Proteins were then electrotransferred from the gel to nitrocellulose, and the filter was blotted with I-streptavidin before autoradiography.


RESULTS

Steady-state Distributions of APN and NKA

In physiologically polarized thyrocytes, APN (M(r) 160,000) resides nearly exclusively at the apical plasma membrane (Feracci et al., 1981; Barriere et al., 1986; Nilsson et al., 1987) while NKA (alpha-subunit, M(r) 100,000; beta-subunit M(r) 60,000) is predominantly localized to the basolateral surface (Gerard et al., 1985). We examined these distributions in confluent porcine thyroid epithelial monolayers cultured on porous filters. In monolayers radiolabeled to steady state with S-amino acids, surface proteins were tagged by vectorial biotinylation, using a reagent that reacts with the -amino groups of lysine residues that are extracytoplasmically disposed (see ``Materials and Methods''). The cells were then lysed and immunoprecipitated for specific antigens. After release from antibody by denaturation in SDS, biotinylated proteins were recovered by binding to avidin-agarose and were analyzed by SDS-PAGE and fluorography (Fig. 1). Finally, immunoprecipitable proteins that were not precipitated with avidin-agarose were recovered (see ``Materials and Methods''); presumably, these nonbiotinylated proteins were intracellular and hence inaccessible to the tagging reagent. However, as expected in the steady state, the preponderance of immunoprecipitable APN and NKA (as quantitated from the beta-subunit(^2)) were indeed at the cell surface. Further, APN was detected with markedly greater abundance apically, while NKA exhibited an opposite pattern (Fig. 2). Although other methods employed were not readily quantified, the apical localization of APN was visually confirmed by confocal immunofluorescence microscopy (Fig. 3), and the distributions of both plasmalemmal proteins were supported by blotting of tagged, unlabeled proteins with avidin-peroxidase, followed by enhanced chemiluminescence detection (not shown).


Figure 1: Surface distribution of thyrocyte surface proteins after metabolic labeling to steady state. Filter-polarized thyrocytes were labeled with S-amino acids for 2 days. The cells were then biotinylated at either the apical (A) or basolateral (B) cell surface, lysed, and immunoprecipitated for APN (M(r) 160,000), NKA (seen best for the beta-subunit,^2M(r) 60,000), or TPO (M(r) 105,000). The immunoprecipitates were solubilized as described under ``Materials and Methods,'' and the surface-tagged proteins were recovered by precipitation with avidin-agarose and analyzed by SDS-PAGE and fluorography.




Figure 2: Quantitation of the surface tagging of APN and NKA. Thyrocytes were labeled, biotinylated, lysed, immunoprecipitated, and reprecipitated with avidin-agarose as described in the legend to Fig. 1. Densitometric scanning of fluorographs was used to calculate apical:basolateral (APN) or basolateral:apical (NKA) distribution ratios, consistent with the known physiological distributions of these proteins. The data shown are from a representative experiment (of two).




Figure 3: Apical immunofluorescent staining of APN. Filter-grown thyrocytes were immunostained for APN according to ``Materials and Methods.'' The monolayers were optically divided into eight X-Y sections by confocal microscopy. The apical-most section is shown; lateral and basal layers, as well as the filter itself, revealed only background fluorescence (not shown).



Delivery of Newly Synthesized APN and NKA to the Follicular Cell Surface

Next, the biotinylation protocol was employed to measure surface delivery of newly synthesized proteins in pairs of thyrocyte monolayers tagged apically and basolaterally, respectively, at different chase times after pulse labeling with S-amino acids. At 5 min of chase, most APN was devoid of terminal carbohydrate and could not be detected at the cell surface (not shown). However, at 30 min of chase, some of the newly synthesized APN exhibited an electrophoretic mobility shift indicative of Golgi sugar processing; at this time, cell surface arrival was already under way (Fig. 4). As measured for NKA, the situation was quite similar (Fig. 4). Thus, it was apparent that cell surface delivery occurred shortly after arrival of newly synthesized proteins in the Golgi complex, and after 3 h a majority of these proteins were found at the cell surface (Fig. 5), approaching steady-state levels. Importantly, at all chase times tested, delivery of newly synthesized APN and NKA to the plasma membrane exhibited apical and basolateral predominance, respectively (Fig. 4). Thus, the data indicated that to a large extent, the proteins were already sorted as the newly synthesized molecules exited the Golgi complex.


Figure 4: Rapid arrival of newly synthesized plasma membrane proteins at the thyroid cell surface. Filter-polarized thyrocytes were pulse-labeled for 30 min with S-amino acids. At the different chase times shown, the monolayers were vectorially biotinylated and newly synthesized APN and NKA immunoprecipitated. The avidin-bound (Surface Tagged) and avidin unbound (Intracellular) fractions were analyzed by SDS-PAGE and fluorography. Note that at 30 min of chase, while considerable amounts of each newly synthesized enzyme had not yet received Golgi sugar processing as detected by electrophoretic mobility, cell surface arrival was nevertheless already under way. The data are representative of three experiments.




Figure 5: Quantitation of the surface arrival of newly synthesized APN, NKA, and TPO. After polarized cell surface biotinylation of pulse-labeled thyrocytes, APN, NKA, and TPO immunoprecipitates were reprecipitated with avidin-agarose. The avidin-bound fractions quantified by SDS-PAGE and fluorography were normalized to the full recovery of these antigens by immunoprecipitation in order to calculate the fraction of newly synthesized APN and NKA delivered to the cell surface over a 3-h chase. Each curve is representative of two independent experiments.



Distribution and Delivery of TPO

In thyrocytes cultured on porous filters, TPO (M(r) 105,000) exhibited important differences in distribution and delivery from the other surface proteins studied. After radiolabeling to steady state, reproducibly not more than 30% of immunoprecipitable TPO was detected at the cell surface. Of the surface-tagged TPO fraction (Fig. 1), the distribution was predominantly apical, with an A/B ratio of 4.4. When newly synthesized proteins were examined, surface delivery of TPO exhibited similar behavior, achieving a plateau with leq30% at the cell surface (Fig. 5). Since pulse radiolabeling was less extensive than steady-state labeling and the fraction of surface-tagged molecules was relatively low, it was difficult to state with confidence the precise polarity of new TPO delivery, although in all experiments the A/B ratio appeared roughly comparable with that observed for TPO at steady state (Fig. 6). Nevertheless, this did not appear quite as apically polarized as that observed for APN (e.g.Fig. 1).


Figure 6: Polarized surface delivery of a fraction of newly synthesized TPO. Filter-polarized thyrocytes were pulse-labeled for 30 min with S-amino acids. At the different chase times shown, the monolayers were vectorially biotinylated, and newly synthesized TPO was immunoprecipitated. The avidin-unbound (Intracellular) and avidin-bound (Surface Tagged) fractions were analyzed by SDS-PAGE and fluorography. Similar to steady state-labeled thyrocytes, the majority of newly made TPO at all chase times up to 4 h was inaccessible to surface biotinylation. Of the surface-tagged fraction, the majority exhibited an apically polarized delivery. The results from two experiments showing different levels of protein radiolabeling are shown.



Distribution of GPI-linked Proteins in Primary Thyroid Epithelial Monolayers

Clustering within membranes of the trans-Golgi network has been proposed to play an important role in the surface delivery of newly synthesized GPI-linked proteins, resulting in their apical polarity in many epithelia (Rodriguez-Boulan and Powell, 1992; Hannan et al., 1993). We wished to see if this behavior was characteristic of normal thyroid epithelial monolayers cultured on porous filters. For this, proteins from thyrocyte monolayers (biotinylated apically or basolaterally) extracted with Triton X-114 were either digested with PI-PLC or mock digested. These samples were then re-extracted to identify molecules that had lost their lipid anchor and now partitioned to the aqueous phase as a specific consequence of PI-PLC digestion (Fig. 7A). A number of proteins recovered in the aqueous phase were not specific to PI-PLC digestion; these nonspecific bands were recovered exclusively from cells biotinylated basolaterally (Fig. 7A, lane 3) where the degree of overall surface tagging was greater by manyfold (Fig. 7B). However, bands released selectively by PI-PLC digestion were found largely in the M(r) 30,000 range and were obviously apically polarized (Fig. 7A, lane 2). Thus, unlike the thyroid-derived FRT cell line, which displays a surface distribution of GPI-anchored proteins that is preferentially basolateral (Zurzolo et al., 1993), the distribution of such proteins in normal thyrocytes is predominantly apical.


Figure 7: Apically polarized distribution of GPI-anchored surface proteins in primary thyroid epithelial cells. Unlabeled filter-grown thyrocytes were vectorially biotinylated and probed by I-streptavidin as described under ``Materials and Methods.'' A, Triton X-114-extractable proteins from these monolayers were treated with or without PI-PLC as described in the text. Proteins released nonspecifically and as a specific consequence of PI-PLC cleavage are shown. Only two bands in the range of M(r) 30,000 were specifically released (asterisks); both bands were clearly apically polarized. B, profile of all vectorially biotinylated surface proteins reveals that overall tagging of surface proteins is heavily weighted toward the basolateral side; apically labeled bands were only detected upon further exposure (not shown).




DISCUSSION

There is little doubt that establishment and maintenance of epithelial surface polarity is essential to normal thyroid function (Mauchamp et al., 1987). However, to our knowledge, there are no previous studies examining polarized delivery of new thyroid plasma membrane proteins except in the FRT cell line, which has lost many features of differentiated thyroid function (Nitsch et al., 1985) and which behaves in a highly atypical manner (Zurzolo et al., 1992a, 1992b, 1993). For this reason, we have examined the well established system of primary thyroid epithelial monolayers cultured on porous filters (Arvan and Lee, 1991; Prabakaran et al., 1993; Kuliawat and Arvan, 1994).

We find that all three proteins studied in the reconstituted thyroid epithelium exhibit polarized surface distributions like that expected in vivo (Fig. 1). Specifically for APN and NKA, a large fraction of these enzymes arrives at the cell surface (Fig. 5) shortly after the acquisition of Golgi-specific posttranslational processing (Fig. 4), indicating direct delivery of these newly synthesized proteins predominantly to the apical and basolateral plasma membranes, respectively. While a modest fraction of newly synthesized proteins may be missorted or mistargeted to the contralateral epithelial surface and additional mechanisms (Nelson, 1992) may fine tune A/B ratios to those observed at steady state (Fig. 2), the data strongly support the idea that in normal thyroid follicular cells, epithelial surface asymmetry is regulated largely by intracellular sorting of newly synthesized membrane proteins at the level of the trans-Golgi network. Thus, the targeting mechanism is not unique for the thyroid as has been implied from studies of FRT cells (Zurzolo et al., 1992b) but is in fact consistent with conventional models of epithelial surface protein targeting such as in Madin-Darby canine kidney cells (Fuller et al., 1985; Rindler et al., 1985; Wessels et al., 1990). Moreover, the data suggest that, like most epithelia but unlike FRT cells, GPI-anchored proteins are heavily distributed to the apical surface of filter-polarized thyrocytes (Fig. 7). These data indicate that the behavior of the FRT cell line is not representative of normal thyroid epithelial cells. Such a conclusion is further supported by evidence indicating that FRT cells are deficient in caveolin (Zurzolo et al., 1994), a membrane protein implicated in the apically polarized distribution and delivery of GPI-anchored proteins (Lisanti et al., 1993), which is found in primary thyrocytes at levels comparable with other epithelial cells. (^3)

To our knowledge, the present report represents the first study of biosynthetic targeting of TPO. Surprisingly in filter-grown epithelial monolayers, a large fraction of immunoprecipitable TPO was inaccessible to surface labeling (e.g.Fig. 5and Fig. 6) and hence was presumably intracellular. Such an observation is very unlikely to represent a methodological artifact for the following reasons. First the ability to tag a high fraction of plasma membrane proteins was well preserved in these monolayers, as judged by the efficient biotinylation of other surface molecules (Fig. 2). Second, despite the fact that overall surface biotinylation was much greater basolaterally (Fig. 7B), there was no defect in apical vesicle trafficking or in apical-specific labeling in filter-grown thyrocytes, as judged by the apically polarized delivery and distribution of APN ( Fig. 1and Fig. 4), GPI-linked proteins (Fig. 7), and polarized delivery of thyroglobulin (Arvan and Lee, 1991; Prabakaran et al., 1993; Kuliawat and Arvan, 1994). Third, it is unlikely that intrinsic structural features prevent TPO from being efficiently biotinylated, since this type 1 membrane protein contains a large extracytoplasmic domain that includes 14 lysine residues (Magnusson et al., 1987). Indeed, it is not that TPO failed to be tagged or exhibited defective kinetics of surface delivery ( Fig. 1and 6); rather, only 30% of TPO could ever be detected at the plasma membrane (e.g.Fig. 5). Further, the enzymatic activity of TPO is well known to be responsible for the iodination of thyroglobulin, and in independent experiments, we have recently reported that filter-grown thyrocytes exhibit intracellular iodination activity in addition to that which is apically polarized (Kuliawat and Arvan, 1994). Since the N-linked glycans of porcine TPO undergo little or no modification by terminal sugars (Rawitch et al., 1992) and remain very sensitive to digestion with endoglycosidase H (Long et al., 1991), one might think that the intracellular TPO pool is in the endoplasmic reticulum. However, in filter-grown thyrocytes, intracellularly iodinated thyroglobulin is exclusively resistant to digestion with endoglycosidase H, suggesting that both the substrate and enzyme are contained in Golgi/post-Golgi compartments (Kuliawat and Arvan, 1994). This is compatible with a major intracellular distribution of TPO in the apical cytoplasm of thyroid epithelial cells in vivo, as determined by immunocytochemistry (Watanabe et al., 1991).

Unfortunately, the present data cannot distinguish whether only 30% of newly synthesized TPO ever arrives at the cell surface or whether TPO is efficiently delivered to the plasma membrane but is rapidly internalized, rendering it inaccessible to surface tagging. Regardless of which of these is the correct explanation, the surface delivery data are clearly concordant with the data regarding steady-state distribution of TPO. The large intracellular pool of TPO and the fact that surface TPO appears less apically polarized than APN indicate that thyrocytes handle these apical marker proteins differently. This is not surprising since there is already strong support for this fact, given that different apical membrane proteins reside in discrete apical subdomains (Barriere et al., 1986; Alquier et al., 1989). Thus, an important goal must be to try to identify the structural features among different apical membrane proteins that account for the rather substantial differences in their intracellular distributions.

Finally, it is tempting to speculate, as have others (Wessels et al., 1990), that the continuous mistargeting of a small fraction of TPO to the basolateral surface can account for a modest basolateral presence of TPO under steady-state conditions (Fig. 1). Moreover, this missorted fraction could contribute to the presentation of TPO to the circulating immune surveillance system and might help to explain the reproducible antigenicity of this protein in autoimmune thyroiditis (Czarnocka et al., 1986; Kotani et al., 1986). More work will be needed to investigate such a possibility.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 40344 (to P. A.), FIRST Award GM 50443 and the Whitehead Fellow's Program (to M. P. L.), as well as National Institutes of Health Training Grants DK07516 and AG08812 (to R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 617-735-4280; Fax: 617-735-2927.

(^1)
The abbreviations used are: FRT, Fischer rat thyroid; APN, aminopeptidase N; NKA, Na/K-ATPase; TPO, thyroid peroxidase; TSH, thyrotropin; PAGE, polyacrylamide gel electrophoresis; GPI, glycosylphosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C.

(^2)
As has been observed by others (Gottardi and Caplan, 1993), the NKA beta-subunit was biotinylated far more efficiently than the alpha-subunit.

(^3)
M. P. Lisanti, R. Kuliawat, and P. Arvan, unpublished data.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. W. Ball (University of Cincinnati) for the antiserum to NKA and Drs. O. Noren and H. Sjostrom (Panum Institute, Copenhagen, Denmark) for the antiserum to APN. We thank the animal facility of Beth Israel Hospital (Boston, MA) for assistance in access to fresh porcine tissue. We also thank members of the Arvan laboratory for helpful discussions during the course of this work.


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