(Received for publication, October 11, 1996)
From the Harvard Medical School, Thyroglobulin (Tg), the major protein secreted by
thyroid epithelial cells and precursor of thyroid hormones, is a large
dimeric glycoprotein with multiple disulfide bonds. The folding and
assembly of this complex molecule begins in the endoplasmic reticulum
(ER) and is likely to involve a variety of reactions catalyzed by
molecular chaperones (Kuznetsov, G., Chen, L. B., and Nigam, S. K. (1994) J. Biol. Chem. 269, 22990-22995). By
coimmunoprecipitation in rat thyroid cells, we were able to demonstrate
that BiP, grp94, ERp72, and grp170, four proteins believed to function
as specific molecular chaperones, complex with Tg during its
maturation. The same complex of the four putative chaperones with Tg
was observed in cells treated with tunicamycin, indicating that these
four ER chaperones stably associate with Tg when it is
misfolded/misassembled due to inhibition of its glycosylation. BiP,
grp94, and ERp72 were also found to associate with Tg in cells in which
misfolding was induced by perturbing ER calcium stores. To determine if
the assembly of a complex between the four chaperones and Tg under conditions of misglycosylation was unique to the maturation of this
particular secretory protein or a more general phenomenon, adenovirus-transformed rat thyroid cells that do not synthesize Tg were
analyzed. In these transformed cells, the only protein these same four
chaperones were found to complex with was a protein of approximately
200 kDa. This protein was subsequently identified as
thrombospondin, which, like Tg, is a large oligomeric secreted glycoprotein with multiple disulfide bonds. We therefore propose that
these ER chaperones complex together with a variety of large oligomeric
secretory glycoproteins as they fold and assemble in the ER.
The ER1 contains a number of lumenal
proteins, many of which have been proposed to function as molecular
chaperones in the folding and/or assembly of membrane and secretory
proteins (1-6). Molecular chaperones are postulated to bind
transiently to newly synthesized proteins, interact with the
polypeptides as they fold through sequential cycles of binding and
release, and finally release the proteins upon their folding and/or
assembly (7). Some of these ER chaperones are thought to bind to
exposed hydrophobic regions of proteins (grp78/BiP) (8, 9), whereas
others are thought to participate in disulfide isomerization (protein
disulfide isomerase, ERp72) (10, 11) and peptidyl-prolyl isomerization (FKBP13) (12, 13). Many of the ER chaperones have been shown to
associate with secretory proteins transiting the ER (examples include
BiP, grp94, ERp72, and calnexin) (6, 14-16), but it has been unclear
whether they work together as part of a large complex or separately.
One difficulty has been that it has often been necessary to resort to
cross-linking approaches to demonstrate associations of multiple ER
chaperones with secretory proteins (6, 16, 17). Whether stable
associations of multiple chaperones with secretory proteins occur
during their folding and assembly in the ER is a question of
considerable interest. Furthermore, it is unknown whether particular
sets of chaperones work together on certain classes of proteins or
whether the case for each protein is different.
In a previous study, we reported that several unrelated unfolded
protein substrates bind to the same set of resident ER proteins on
affinity columns (5). These ER proteins, which all exhibited characteristics of molecular chaperones (such as binding to denatured substrates and elution from them in an ATP-dependent
manner), included BiP, grp94, ERp72, protein disulfide isomerase, and
calreticulin. One of the substrates used in the study was thyroglobulin
(Tg), a protein precursor of thyroid hormones produced by thyroid
epithelial cells. In support of these in vitro findings, we
subsequently demonstrated by cross-linking and immunoprecipitation that
BiP, grp94, and ERp72 also associate with Tg in intact thyroid cells (6). Similar associations between Tg and these chaperones were observed
in a cell culture model for epithelial ischemia when ATP levels
declined to less than 10% of control (18). Nevertheless, it remained
unclear whether these associations exist in the absence of
cross-linking or severe ATP depletion.
Coordinate binding to substrates and coelution of a set of ER molecular
chaperones (5), as well as their coordinate induction under conditions
that perturb the internal environment of the ER (13), prompted us to
hypothesize the existence of large macromolecular chaperone-containing
complexes that are involved in maturation of certain classes of
secretory and membrane proteins. The association of multiple resident
ER proteins with Tg by cross-linking is consistent with this
hypothesis. In the present study we investigated the involvement of
multiple ER molecular chaperones in the maturation of large oligomeric
secreted glycoproteins under conditions of misfolding in the ER.
Thyroglobulin (19) typifies this class of proteins, made by many
specialized secretory cells. Like immunoglobulin chains (20, 21) and
thrombospondin (22), it is a large oligomeric glycoprotein with
multiple disulfide bonds. The size (Tg is a ~660-kDa homodimer
consisting of two subunits of ~330 kDa) and apparent complexity
(extensive glycosylation, disulfide bonding, and other
posttranslational modifications) of Tg suggest that its folding and
assembly may involve many reactions catalyzed by multiple ER
chaperones. Indeed, it appears as though Tg does, at least transiently,
associate with BiP, grp94, ERp72, and calnexin during its maturation
(6, 23); however, with the exception of BiP (6, 23, 24), stable
associations with Tg have not been observed. Since such associations
seemed more likely to occur when the ER transit time of Tg was
prolonged, we examined conditions that perturb the internal environment
of the ER and lead to accumulation of immature proteins in the ER
lumen. Here, we report that following treatment of cells with
tunicamycin, an agent that inhibits N-linked glycosylation
in the ER, the same set of four ER molecular chaperones (BiP, grp94,
ERp72, and grp170) stably complexes with two secreted proteins that
exhibit strikingly similar structural characteristics. We therefore
propose that this set of four ER chaperones is involved in a
dynamic "folding complex" for many large oligomeric
glycoproteins.
Rabbit polyclonal antiserum against BiP used for
immunoprecipitation was purchased from Affinity Bioreagents (Neshanic
Station, NJ). The mouse monoclonal antibody against BiP used for
Western immunoblot analysis and rat monoclonal antibody against grp94 were obtained from Stress-Gen (Victoria, British Columbia). Rabbit polyclonal antiserum against grp170 was a gift from Dr. J. Subjeck (Roswell Park Cancer Institute). Rabbit polyclonal antiserum against ERp72 was kindly provided by Dr. M. Green (St. Louis University). Rabbit polyclonal anti-thrombospondin antiserum used for
immunoprecipitation was a gift from Dr. J. Lawler (25) (Brigham and
Women's Hospital, Harvard Medical School), and the rabbit polyclonal
anti-thrombospondin antiserum used for Western immunoblot analysis was
kindly provided by Dr. V. M. Dixit (University of Michigan Medical
Center). Rabbit polyclonal antiserum against thyroglobulin was
purchased from DAKO Corp. (Carpinteria, CA). Secondary antisera were
obtained from Amersham Corp. The following materials were purchased
from Sigma: thyroid-stimulating hormone, insulin,
hydrocortisone, transferrin, somatostatin,
glycyl-L-histidyl-L-lysine acetate, leupeptin,
pepstatin A, antipain, and tunicamycin. DSP was obtained from Pierce.
A23187 and thapsigargin were from Calbiochem.
Three rat thyroid
epithelial cell lines were used in this study: FRTL-5 and PCCl3 (both
Tg-producing) and PCE1A (lacking the ability to synthesize Tg). FRTL-5
cell line was obtained from ATCC (Rockville, MD). PCCl3 and PCE1A
thyroid cells were kindly provided by Dr. M. T. Berlingieri (Universita
degli studi di Napoli, Italy). Thyroglobulin-producing cells, FRTL-5
(26) and PCCl3 (27), were maintained in Coon's modified Ham's F12
medium (Sigma) supplemented with 5% bovine calf serum
(Hyclone Laboratories, Logan, UT) and a six-hormone mixture consisting
of thyroid-stimulating hormone (1 mIU/ml), insulin (10 µg/ml),
hydrocortisone (10 nM), transferrin (5 µg/ml),
somatostatin (10 ng/ml), and
glycyl-L-histidyl-L-lysine acetate (10 ng/ml)
(28). PCE1A cells (lacking thyroglobulin) were grown in the same
culture medium but without the six-hormone supplement (27). Cells were
metabolically labeled for either 18-20 h in their respective growth
media or for 30 min in the serum-, methionine-, and cysteine-free
medium in the presence of [35S]methionine/cysteine (200 µCi/ml, Expre35S35S labeling mix, DuPont
NEN).
Cell monolayers were
washed twice with the phosphate-buffered saline (PBS) and suspended in
1 ml of PBS, pH 7.4, containing 5 mM EDTA and 5 mM EGTA (6). The cells were incubated with the
cross-linking agent, DSP (100 µg/ml), or vehicle (dimethyl sulfoxide,
final concentration of 1%) for 30 min at room temperature. The
cross-linking reaction was stopped by incubation with 100 mM Tris/HCl, pH 7.5, for 15 min. Cells were then lysed on
ice by addition of 1% Triton X-100 in the presence of protease
inhibitors (1 mM phenylmethylsulfonyl fluoride and 5 µg/ml each leupeptin, pepstatin A, and antipain). Prior to
immunoprecipitation, samples were incubated with protein A-Sepharose
CL-4B (Pharmacia Biotech Inc.) to remove any material that binds
nonspecifically to protein A. Samples were incubated with various
antisera for 16 h at 4 °C followed by incubation with protein
A-Sepharose for 1 h at 4 °C. The pellets were washed in PBS,
suspended in sample buffer (29) containing 50 mM
dithiothreitol, heated to 100 °C for 10 min and analyzed by SDS-PAGE
(29). Protein-associated radioactivity was analyzed on a PhosphorImager
using the Image-Quant software package (Molecular Dynamics). Images
were processed using the Adobe PhotoshopTM software and
printed with the Fujix Pictography 3000 color printer.
Electrophoresed proteins were
transferred to polyvinylidene fluoride membranes (Immobilon, Millipore
Corp.) by electroblotting at 30 V for 18 h at room temperature.
Membranes were incubated with various primary antisera followed by
incubation with secondary horseradish peroxidase-labeled
anti-immunoglobulin antisera as described (5). Immunoblots were
developed using the ECL kit (Amersham Corp). The proteins were detected
using Hyperfilm ECL (Amersham Corp). The exposed film was scanned using
the HP Scan Jet IIcx, and the images were processed using the Adobe
Photoshop software and printed with the Fujix Pictography 3000 color
printer.
Cell lysates were layered
on top of linear 5-20% sucrose gradients prepared in PBS, pH 7.4, containing 5 mM EDTA and 5 mM EGTA (6). The
gradients were centrifuged at 32,000 rpm for 24 h at 4 °C using
a Beckman SW40TI ultracentrifuge rotor. 15 fractions (0.8 ml) were
collected using a Buhler Instruments Auto Densi-Flow IIC gradient
fractionator. Trichloroacetic acid was added to fractions at a final
concentration of 10% to precipitate proteins. Trichloroacetic acid
precipitates were washed once with ice-cold acetone, dissolved in
sample buffer containing 50 mM dithiothreitol (DTT), heated
to 100 °C for 10 min, and analyzed by SDS-PAGE followed by Western
Immunoblotting with anti-Tg of anti-BiP antisera.
FRTL-5 cells grown on coverslips were
fixed in methanol ( Under conditions which perturb the folding and secretion of Tg,
existing interactions of this large dimeric secretory protein with ER
molecular chaperones would be expected to be prolonged and more stable,
and therefore easier to detect without the use of cross-linking
reagents. Inhibitors of glycosylation and agents that perturb ER
Ca2+ stores create such conditions, and we have employed
both types of agents to study the involvement of ER chaperones in Tg
maturation. The nucleoside antibiotic tunicamycin inhibits
N-linked glycosylation in the ER by blocking the formation
of the lipid-linked oligosaccharide donor through specific inhibition
of the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to dolichyl
phosphate (30). After tunicamycin treatment, misglycosylated proteins
accumulate in the ER (31), which is accompanied by the increased
synthesis of ER molecular chaperones (13, 36), including BiP, grp94,
and ERp72. Thus, since mature Tg is extensively glycosylated,
tunicamycin treatment would be expected to severely affect its
conformation and stability and to facilitate detection of interactions
of Tg with molecular chaperones of the ER without the use of
cross-linking agents. Two rat thyroid epithelial call lines that
synthesize and secrete Tg were used in our study: FRTL-5 and PCCl3. Our
findings in both cell lines were virtually identical; therefore, below
we present results obtained with only one type of cells.
A relatively low dose of tunicamycin (1 µg/ml) was chosen. A recently
published study (24) reported that as little as 0.5 µg/ml tunicamycin
results in a complete inhibition of N-linked glycosylation
of Tg. At the same time, a number of studies in recent years have shown
that concentrations of tunicamycin similar to the one we have chosen
result in virtually complete inhibition of N-linked
glycosylation without a significant effect on overall protein synthesis
(32, 33). When FRTL-5 cells were treated with 1 µg/ml tunicamycin, Tg
was found intracellularly in a lower molecular weight (unglycosylated)
form that could be clearly distinguished by SDS-PAGE from its
glycosylated counterpart (Fig. 1A,
lanes 10 and 12). Inhibition of
N-linked glycosylation of Tg persisted for up to 5 h
after the removal of tunicamycin from the culture medium (Fig.
1A, lane 11). Secretion of unglycosylated Tg was almost completely (>95%) blocked in tunicamycin-treated cells (Fig.
1A, lanes 3, 6, and 9) as
well as in cells pretreated with tunicamycin for 16 h and then
transferred to tunicamycin-free medium for up to 5 h (Fig.
1A, lanes 2, 5, and 8).
Thus, under the conditions used in our study, tunicamycin induces
complete inhibition of N-linked glycosylation of Tg and
completely blocks its secretion, and these two effects are not reversed
during the time periods employed in the experiments described below. By
immunofluorescence, the unglycosylated Tg localized largely to the ER
(Fig. 1B). Furthermore, both Tg and BiP appeared to
associate into larger macromolecular complexes when analyzed by sucrose
density gradient centrifugation (Fig. 1C). In control cells,
Tg sedimented largely in fractions 8-12 with a small amount of the
protein present in fractions 13-15 and in fraction P. Fraction P
represents material pelleted at the bottom of the gradient as a result
of transient association of Tg into large complexes in the course of
its normal maturation (34). The majority of intracellular BiP in
untreated cells was found in fractions 3-8, with a small amount
present in heavier fractions (fractions 9-12 and the pellet fraction,
P). Following treatment of cells with tunicamycin, both Tg and BiP have
shifted to heavier fractions of the gradient, and there was a marked
increase in the amounts of both proteins found in the pellet fraction
of the gradient. Together, these data suggested that tunicamycin treatment led to formation of large macromolecular complexes containing unglycosylated Tg and possibly ER molecular chaperones that were retained in the ER.
To ensure that the formation of Tg-containing macromolecular complexes
in tunicamycin-treated cells does not represent nonspecific protein
aggregation resulting from cell injury, we measured cell viability and
overall cellular secretory activity in both treated and untreated
cells. To assess cell viability, a trypan blue exclusion assay was
performed following treatment of FRTL-5 cells with 1 µg/ml
tunicamycin for 16 h. In both the untreated and
tunicamycin-treated cell preparations, less than 1% of cells were
non-viable (have incorporated the dye). The assay was performed in
triplicate (data not shown).
To estimate the effect of tunicamycin on the overall cellular secretory
activity, FRTL-5 cells were pretreated without or with tunicamycin (1 µg/ml) for 16 h, pulse-labeled with
[35S]methionine/cysteine, and chased in the presence or
absence of tunicamycin for 4 h (Fig. 2). Aliquots
of media were collected and subjected to SDS-PAGE followed by
autoradiography (Fig. 2A). Amounts of radioactivity
associated with Tg secreted into the culture medium as well as with
various other secretory products of FRTL-5 cells were quantified (Fig.
2, B and C). As shown in Fig. 2A, Tg
represents the major, although not the sole secretory protein product
of untreated thyroid cells (lane 1). Additional polypeptide
bands were present on the SDS gel, although by far the Tg band (~300
kDa) was the most prominent. In control cells, the 300-kDa band
represented approximately 75% of total labeled material secreted (Fig.
2B). Treatment of cells with tunicamycin reduced secretion
of all polypeptides, but to various degrees (Fig. 2A,
lanes 2 and 3). Secretion of three other
secretory products of FRTL-5 cells (denoted by arrows
designated p1, p2, and p3) was
compared to that of Tg. In tunicamycin-treated cells, the amount of Tg
present in culture medium was reduced by almost 200-fold (Fig.
2C). In contrast to Tg, secretion of polypeptides p1 (~112 kDa), p2 (~100 kDa), and p3 (~80 kDa) was reduced by only 1.7-2.5 fold (Fig. 2C). As shown in Fig. 2B, the
secretory profile of tunicamycin-treated cells is significantly
different from that of control preparations. Following treatment with
tunicamycin, Tg becomes a minor secretory product, representing only
~5% of total secreted protein, while other polypeptides (like bands
p1, p2, and p3) represent a substantial portion of total secretory material. Thus, different secretory proteins of thyroid cells exhibit
vastly different sensitivities to tunicamycin treatment. These findings
indicate that the inhibition of N-linked glycosylation by
tunicamycin results in selective inhibition of secretion rather than in
a nonspecific alteration of cellular function. Together with the lack
of toxicity of tunicamycin (as estimated by the trypan blue exclusion
assay), these findings suggest that the action of tunicamycin on
thyroid cells is specific and also support the validity of using
tunicamycin as a tool to induce stable associations of Tg with the ER
molecular chaperones.
The proteins associated with the unglycosylated Tg intermediate
retained in the ER of tunicamycin-treated cells were then analyzed by
coimmunoprecipitation with anti-Tg antibodies in the absence and
presence of a thiol-cleavable chemical cross-linking agent (DSP). For
initial identification of likely proteins associated with Tg in control
and tunicamycin-treated cells, coimmunoprecipitation of Tg from
metabolically labeled cells was performed. For subsequent positive
identification of the coimmunoprecipitated polypeptides, Tg was
immunoprecipitated from unlabeled cells, and the polypeptide bands were
analyzed by Western immunoblotting. Fig. 3 and
4, respectively, show coimmunoprecipitations from
metabolically labeled FRTL-5 cells and Western immunoblot analysis of
the immunoprecipitates. In the absence of tunicamycin, the association
of BiP, grp94, and ERp72 with Tg was seen after chemical cross-linking
(Fig. 3, lanes 2, 5, and 9; Fig. 4,
lanes 7, 11, and 15), as previously reported (6). It is important to note that, apart from these bands, no
others were consistently immunoprecipitated with anti-Tg antiserum,
suggesting that the associations we observed are highly specific and
authentic. Furthermore, to eliminate the possibility that
coprecipitation of BiP with anti-Tg results from binding of BiP
(otherwise known as immunoglobulin binding protein) to immunoglobulins
added during immunoprecipitation, we have conducted immunoprecipitation
of Tg in the thyroid cells that lack the ability to synthesize and
secrete Tg (Fig. 6, lanes 1-4) but contain normal amounts
of BiP. In these cells, neither Tg nor any other molecular chaperones
could be immunoprecipitated with anti-Tg antibodies. In Tg-containing
cells, following tunicamycin treatment, BiP, grp94, and ERp72 could be
seen even without cross-linkers (Fig. 3, lanes 3,
8, and 12; Fig. 4, lanes 8,
12, and 16), although the associations were
further enhanced by cross-linking (Fig. 3, lanes 4,
7, and 11; Fig. 4, lanes 9,
13, and 17). In addition, a band of approximately
150 kDa was coimmunoprecipitated with Tg in tunicamycin-treated cells
in the absence and presence of cross-linkers (Fig. 3, lanes
3 and 4). This band was identified as the putative ER
chaperone grp170 (Fig. 4B), a member of the glucose-regulated stress protein family that is thought to participate in folding and/or assembly of immunoglobulin chains (35). Thus, the
large aggregates of unglycosylated Tg in the ER appeared to stably
complex with four ER chaperones. No other polypeptides were present in
Tg immunoprecipitates from 35S-labeled cell lysates (Fig.
3). The same complex containing Tg, BiP, grp94, ERp72, and grp170 could
also be coimmunoprecipitated with anti-BiP antibodies in
tunicamycin-treated cells in the absence of cross-linkers (Fig. 3,
lane 8; Fig. 4, lane 12), though once again the
associations were enhanced by cross-linking (Fig. 3, lane 7;
Fig. 4, lane 13). Nevertheless, a stable complex of these four ER proteins with Tg appears to develop when normal Tg folding and/or assembly is perturbed by inhibition of its N-linked
glycosylation. In addition, in BiP and ERp72 immunoprecipitates, a
200-kDa band was consistently observed. This band was subsequently
identified (see below). The identity of an approximately 180-kDa
polypeptide coimmunoprecipitated with ERp72 only has not yet been
established (Fig. 3, lanes 9-12). It is possible that this
180-kDa polypeptide represents a secretory protein that specifically
associates with ERp72 in the ER.
Some interactions between Tg and molecular chaperones, already
increased after tunicamycin treatment, were further enhanced by
cross-linking more than others. For example, treatment of
tunicamycin-treated cells with cross-linker resulted in a marked
further increase in amount of grp94 coimmunoprecipitated with Tg and
BiP (Fig. 4C, lanes 8, 9,
12, 13) or in the amount of BiP coprecipitated with ERp72 (Fig. 4D, lanes 16, 17).
The amount of grp170 co-precipitated with either Tg or BiP was
unaffected by cross-linking (Fig. 4B, lanes 8,
9, 12, 13). Thus, the presumed
misfolding of the Tg caused by misglycosylation affects the stability
of interactions of the protein with molecular chaperones to different
degrees.
grp170 is a glycoprotein (35), and after tunicamycin treatment it was
indeed detected intracellularly as a lower molecular weight
(unglycosylated) form (Fig. 4B, lanes 4, 5 versus lanes 2, 3). We could not detect an association
of grp170 with either Tg or BiP in unstressed cells in the absence of
cross-linking (Fig. 4B, lanes 6 and
10). However, treatment of unstressed cells with the
cross-linking agent caused a small amount of the glycosylated form of
grp170 to coimmunoprecipitate with both Tg and BiP (Fig. 4B,
lanes 7 and 11). After treatment with
tunicamycin, a substantial amount of grp170 coimmunoprecipitated with
Tg and BiP (Fig. 4B, lanes 8 and 12),
and this was not further increased by cross-linking (Fig.
4B, lanes 9 and 13). However, by
Western immunoblot analysis, no grp170 was detected in any of the ERp72
immunoprecipitates (Fig. 4B, lanes 14-17),
although a trace amount of polypeptide of identical molecular weight to
grp170 was present in ERp72 immunoprecipitates from
35S-labeled, tunicamycin-treated cells (Fig. 3, lanes
11 and 12). Since grp170 and ERp72 were present in both
anti-Tg and anti-BiP immunoprecipitates from tunicamycin-treated cells,
failure to confirm by Western immunoblotting an association between
ERp72 and grp170 suggested by autoradiographic analysis was puzzling. It is possible, however, that the amounts of these two ER proteins associating with each other is extremely low, and Western blotting is
less sensitive than autoradiography in detecting these
associations.
A long-term treatment with tunicamycin (such as used in the above
described experiments) has been shown to induce the mRNA levels for
ER molecular chaperones (including BiP, grp94, and ERp72) in various
experimental systems (13, 36) as well as in rat thyroid epithelial
cells (18). It is currently believed that the accumulation of misfolded
and/or misassembled secretory proteins in the lumen of the ER serves as
the signal and/or the trigger for the induction (36-38). Stable
long-term associations of the ER molecular chaperones with their
misfolded/misassembled protein substrates may also be a determining
factor in signaling the induction of mRNA levels for these
chaperones. We have therefore analyzed the associations of Tg with ER
chaperones following treatment with other agents that induce their
mRNA levels (presumably through causing accumulation of misfolded
secretory proteins in the ER lumen): a Ca2+ ionophore
A23187 and an inhibitor of the ER Ca2+-ATPase thapsigargin
(39, 40). Both agents result in depletion of Ca2+ from the
ER (41, 42) and are not known to induce aggregation in the ER lumen; in
fact, it has been argued that calcium depletion may have the opposite
effect (43). Following treatment of FRTL-5 cells with A23187,
thapsigargin, or tunicamycin for 16 h, Tg was immunoprecipitated
from uncross-linked cell lysates, and the immunoprecipitates were
analyzed by Western immunoblotting with antisera against Tg (Fig.
5A) or ER molecular chaperones (Fig. 5,
B-D). Similar to tunicamycin, both
Ca2+-depleting agents resulted in coimmunoprecipitation of
BiP, grp94, and ERp72 with Tg (Fig. 5). grp170 was not
coimmunoprecipitated with Tg in cells treated with either A23187 or
thapsigargin (data not shown). Stable association of grp170 and Tg thus
appears to be a specific result of tunicamycin treatment, although both
treatments (inhibition of N-linked glycosylation and
Ca2+ depletion) have been shown to induce grp170 (35).
To determine if the assembly of a complex involving the four chaperones
was specific to Tg or represented a more general phenomenon, a group of
adenovirus-transformed rat thyroid cells, PCE1As (27), were studied.
These cells have lost their ability to synthesize and secrete Tg and no
longer depend on thyroid-stimulating hormone for growth following
transfection with the virus (27). As expected for these cells, no Tg
was detected by immunoprecipitation (Fig. 6, lanes
1-4) or Western immunoblotting (data not shown). However, in
[35S]methionine-labeled immunoprecipitates, the same
complex of BiP, grp94, ERp72, and grp170 was observed in association
with other bands (Fig. 6, lanes 8 and 12). An
unidentified 180-kDa band was only seen in ERp72 immunoprecipitates
(Fig. 6, lanes 9-12), but the most consistently observed
band seen in both BiP and ERp72 immunoprecipitates migrated at
approximately 200 kDa (Fig. 6, lanes 5-12). This band had
also been visualized in BiP and ERp72 immunoprecipitates of
[35S]methionine-labeled FRTL-5 cells that secrete Tg
(Fig. 3, lanes 5-12). The p200 might have represented a
resident ER protein that functions as a chaperone like BiP or ERp72.
Alternatively, p200 could be another secretory product of thyroid cells
that utilizes the same ER molecular chaperones in its maturation. The
latter seemed a more plausible hypothesis since we could not detect an association of p200 with Tg (data not shown). Like Tg, the 200-kDa protein only entered standard polyacrylamide resolving gels under reducing conditions, suggesting that it too was a highly
disulfide-bonded protein that possibly oligomerized under non-reducing
conditions (Fig. 7, lanes 1 versus 2). One
such protein made by thyroid and other cells is a member of the
thrombospondin protein family, thrombospondin-1 (TSP-1), a trimeric,
multiply disulfide-bonded glycoprotein (22, 44). Native TSP-1 is a
homotrimeric protein of 450 kDa, and its monomeric size of
approximately 190-200 kDa corresponds to the p200 polypeptide band
present on reducing gels. By coimmunoprecipitation and Western
blotting, the 200-kDa band was subsequently indeed identified as TSP-1
(Fig. 7, panels B and C).
To summarize, the existence in thyroid cells of complexes containing
these four ER chaperones was not dependent on Tg. In searching for
other proteins that might associate with the same four chaperones, it
was very striking to us that the only major protein detected turned to
have many characteristics similar to Tg, for TSP-1 is also a large
oligomeric secreted glycoprotein. Thus, TSP-1 would be expected to
require reactions very similar to Tg for proper folding and/or assembly
in the ER, consistent with our finding that the same set of ER
chaperones complexes with both secretory proteins. Based on these
results, we propose that BiP, grp94, ERp72, and possibly grp170
participate in complex(es) involved in the maturation of these two
secretory glycoproteins. On the basis of similarities between Tg and
TSP we hypothesize that, in general, large oligomeric glycoproteins may
follow the same pattern of interaction with these four molecular
chaperones as they pass through the ER. Two types of interactions are
possible: concurrent and sequential. In the case of thyroglobulin, one
would expect a heterogeneous population present in the ER lumen at all times, each subpopulation consisting of a certain intermediate in
post-translational processing of the protein. Each subpopulation would
then associate with a chaperone or a subset of the four chaperones. In
support of this hypothesis is the fact that coimmunoprecipitations with
different antibodies yielded different sets of proteins. For example,
while all four chaperones coprecipitated with Tg, grp170 did not
coprecipitate with ERp72. ERp72 and grp170 might therefore bind
different intermediates in Tg maturation that are present at different
time points along the maturation pathway, while binding of Tg to BiP
and to grp94 occupies a broader time period and overlaps with binding
to both ERp72 and grp170. Sequential action of chaperones like BiP and
grp94 has been suggested in maturation of immunoglobulin light chains
(17), and sequential action of calnexin and BiP has been suggested in
maturation of thyroglobulin (23). Our findings, and the notion that
chaperones recognize distinct features within polypeptides, are also
consistent with a model of interaction in which various chaperones or
sets of chaperones successively complex with various intermediates in
maturation of oligomeric glycoproteins.
We are grateful to Dr. Kevin T. Bush for
valuable help with this manuscript.
Division of Cellular
and Molecular Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
Materials
20 °C, 5 min), washed twice with PBS, pH 7.4, and incubated with rabbit polyclonal anti-Tg antiserum for 1 h at
37 °C in a humidified chamber. Cells were then washed in PBS three
times and incubated with rhodamine-conjugated anti-rabbit
immunoglobulin antiserum. Coverslips were mounted onto a glass slide in
glycerol-gelatin medium (Sigma). Staining was examined
using a confocal laser scanning microscope LSM 410 (Zeiss, Germany).
The images were processed using the Adobe Photoshop software and
printed with the Fujix Pictography 3000 color printer.
Fig. 1.
Inhibition of N-linked
glycosylation and secretion of thyroglobulin by tunicamycin.
A, PCCl3 cells were pretreated with (lanes 2,
3, 5, 6, 8, 9,
11, and 12) or without (lanes 1, 4, 7, and 10) 1 µg/ml tunicamycin
for 16 h. At the end of the pretreatment period, cells were washed
twice with fresh culture medium and incubated for additional 5 h
in the absence (lanes 1, 2, 4,
5, 7, 8, 10, and
11) or presence (lanes 3, 6,
9, and 12) of 1 µg/ml tunicamycin. Cell lysates
(lanes 10-12) and aliquots of culture medium (lanes
1-9) collected at various times were analyzed on SDS-PAGE
followed by Western immunoblotting with anti-Tg antiserum.
B, PCCl3 cells grown on coverslips were treated with 1 µg/ml tunicamycin for 16 h. At the end of the treatment period, the cells were fixed in methanol and analyzed for Tg immunofluorescence as described under "Experimental Procedures." Immunofluorescence was examined using a confocal laser scanning microscope. The untreated cells incubated with anti-Tg antiserum showed mostly a typical ER
staining (data not shown). C, PCCl3 cells were incubated
with or without 1 µg/ml tunicamycin for 16 h. At the end of the
treatment period, tunicamycin-treated and untreated cell lysates were
layered on top of linear 5-20% sucrose gradients and centrifuged to
equilibrium as described under "Experimental Procedures." 15 fractions were collected; fraction P represents material
pelleted at the bottom of each gradient. Fractions were analyzed on 5%
SDS-PAGE followed by Western immunoblotting with anti-Tg or anti-BiP
antisera.
[View Larger Version of this Image (66K GIF file)]
Fig. 2.
Effect of tunicamycin on the secretory
activity of thyroid cells. A, FRTL-5 cells were pretreated
with (lanes 2 and 3) or without (lane
1) 1 µg/ml tunicamycin for 16 h. The cells were
pulse-labeled with 200 µCi/ml [35S]methionine/cysteine
for 30 min in the presence (lanes 2 and 3) or
absence (lane 1) of 1 µg/ml tunicamycin and chased for
4 h in the presence of excess unlabeled methionine/cysteine and in
the presence (lanes 2 and 3) or absence
(lane 1) of tunicamycin (1 µg/ml). Aliquots of culture
media were subjected to SDS-PAGE followed by autoradiography.
Lane 3 is identical to lane 2 but overexposed to
help in visualizing polypeptide bands. Arrows denote positions of Tg and the three additional polypeptide bands (designated p1, p2, and p3) secreted by thyroid
cells. The amounts of radioactivity associated with Tg and bands p1,
p2, and p3 were quantified using Molecular Dynamics PhosphorImager and
the Image Quant software package and are presented in panels
B and C. B, altered secretory profile of
tunicamycin-treated cells. Open bars, control; closed bars, tunicamycin-treated. The relative amounts of four secreted proteins (Tg and bands 1, 2, and 3) are shown (expressed as percent of
total in each lane). C, the absolute amounts of
35S-labeled bands corresponding to Tg, and bands p1, p2,
and p3 in control (open bars) and tunicamycin-treated
(closed bars) are shown.
[View Larger Version of this Image (54K GIF file)]
Fig. 3.
Associations of Tg with the molecular
chaperones of the ER in stressed and unstressed thyroid cells.
Uncross-linked (lanes 1, 3, 6,
8, 10, and 12) and cross-linked
(lanes 2, 4, 5, 7,
9, and 11) lysates from
[35S]methionine-labeled, untreated (lanes 1,
2, 5, 6, 9, and
10) or tunicamycin-treated (lanes 3,
4, 7, 8, 11, and
12) FRTL-5 cells were subjected to immunoprecipitation with
anti-Tg (lanes 1-4), anti-BiP (lanes 5-8), or
anti-ERp72 (lanes 9-12) antisera. Note that lanes
5-12 containing cross-linked samples precede lanes containing
uncross-linked samples. Arrows denote positions of Tg, p200,
grp170, grp94, BiP, and ERp72; the asterisk denotes an
unidentified polypeptide band of approximately 180 kDa that was
immunoprecipitated with anti-ERp72 antiserum but not with the anti-Tg
or anti-BiP antisera.
[View Larger Version of this Image (74K GIF file)]
Fig. 4.
Identification of polypeptides
coimmunoprecipitated with Tg and ER molecular chaperones. FRTL-5
cells were incubated with or without 1 µg/ml tunicamycin for 18 h. Cells were then treated with or without cross-linking reagent,
lysed, and subjected to immunoprecipitation with anti-Tg (lanes
6-9), anti-BiP (lanes 10-13), or anti-ERp72
(lanes 14-17). Rat liver rough microsomes (lane
1), cell lysates (lanes 2-5), and immunoprecipitates
(lanes 6-17) were subjected to 5% SDS-PAGE followed by
Western immunoblotting with various antisera. A, anti-Tg;
B, anti-grp170; C, anti-grp94; D,
anti-BiP, E, anti-ERp72. Results of two separate experiments are combined in this figure.
[View Larger Version of this Image (98K GIF file)]
Fig. 6.
Associations between ER molecular chaperones
in thyroid cells lacking thyroglobulin. PCE1A cells were
metabolically labeled in the presence or absence of 1 µg/ml
tunicamycin for 16 h. Tg (lanes 1-4), BiP (lanes
5-8), and ERp72 (lanes 9-12) were immunoprecipitated
from cross-linked and uncross-linked cell lysates and subjected to 5%
SDS-PAGE. Protein-associated radioactivity was analyzed on
PhosphorImager using the Image-Quant software package (Molecular
Dynamics). Arrows denote positions of p200, grp170, grp94,
BiP, and ERp72.
[View Larger Version of this Image (67K GIF file)]
Fig. 5.
Associations of Tg with the ER molecular
chaperones following treatment with Ca2+-depleting
agents. FRTL-5 cells were treated without (lane 1) or
with 5 µM A23187 (lane 2), 50 nM
thapsigargin (lane 3), 300 nM thapsigargin
(lane 4), or 1 µg/ml tunicamycin (lane 5) for
16 h in complete culture medium. Tg was immunoprecipitated from
uncross-linked cell lysates. Immunoprecipitates were analyzed by
SDS-PAGE followed by Western immunoblotting with antisera against Tg
(panel A), grp94 (panel B), BiP (panel
C), or ERp72 (panel D).
[View Larger Version of this Image (48K GIF file)]
Fig. 7.
Identification of p200 as TSP-1.
A, BiP was immunoprecipitated from metabolically labeled,
uncross-linked PCCl3 cell lysates. Immunoprecipitates were resuspended
in sample buffer either containing 50 mM DTT (reducing
conditions, lane 1) or lacking DTT (non-reducing conditions,
lane 2). Reduced and non-reduced samples were resolved on
4% SDS-PAGE followed by autoradiography. B, TSP-1 was
immunoprecipitated from metabolically labeled, uncross-linked PCCl3 or
PCE1A cell lysates. Immunoprecipitates were resuspended in sample
buffer either containing (lanes 3 and 4) or
lacking (lanes 5 and 6) 50 mM DTT.
Reduced and non-reduced samples were analyzed on 5% SDS-PAGE followed
by autoradiography. C, BiP was immunoprecipitated from
unlabeled PCCl3 or PCE1A cell lysates treated with or without
cross-linking agent, DSP. Total PCCl3 or PCE1A cell lysates
(lanes 7 and 8) and BiP immunoprecipitates from
PCCl3 or PCE1A cells (lanes 9-12) were subjected to 5%
SDS-PAGE under reducing conditions followed by Western immunoblotting
with anti-TSP-1 antiserum.
[View Larger Version of this Image (47K GIF file)]
*
This work was done during the tenure of an American Heart
Association Established Investigatorship (to S. K. N). 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.
§
Supported by the Pharmaceutical Manufacturers Association
Foundation Postdoctoral Fellowship in Pharmacology-Morphology. Present address: Renal Division, Dept. of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115.
Supported by NIH RO1 Grants DK49517 and DK44503. To whom
correspondence should be addressed: Renal Division, Dept. of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St.,
Boston, MA 02115. Tel.: 617-278-0436; Fax: 617-732-6392.
1
The abbreviations used are: ER, endoplasmic
reticulum; BiP, immunoglobulin-binding protein; grp, glucose-regulated
protein; ERp, endoplasmic reticulum protein; FKBP, FK506-binding
protein; DSP, dithiobis(succinimidyl propionate); PBS,
phosphate-buffered saline; DTT, dithiothreitol; Tg, thyroglobulin; Tn,
tunicamycin; TSP, thrombospondin; PAGE, polyacrylamide gel
electrophoresis.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.