(Received for publication, September 22, 1994; and in revised form, November 2, 1994)
From the
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.
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
O
(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) ()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.
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
160,000), NKA
(seen best for the
-subunit,
M
60,000), or TPO (M
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).
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.
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.
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
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).
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. ()
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.