Enhanced Binding to the Molecular Chaperone BiP Slows Thyroglobulin Export from the Endoplasmic Reticulum
Zoia Muresan and
Peter Arvan
Program in Biological and Biomedical Sciences (Z.M.) Harvard
Medical School Boston, Massachusetts 02215
Division of
Endocrinology and Department of Developmental and Molecular Biology
(P.A.) Albert Einstein College of Medicine Bronx, New York
10461
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ABSTRACT
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To examine how binding of BiP (a molecular
chaperone of the hsp70 family that resides in the endoplasmic
reticulum) influences the conformational maturation of thyroglobulin
(Tg, the precursor for thyroid hormone synthesis), we have developed a
system of recombinant Tg stably expressed in wild-type Chinese hamster
ovary (CHO) cells and CHO-B cells genetically manipulated for
selectively increased BiP expression. The elevation of immunoreactive
BiP in CHO-B cells is comparable to that seen during the unfolded
protein response in the thyrocytes of certain human patients and
animals suffering from congenital hypothyroid goiter with defective Tg.
However, in CHO-B cells, we expressed Tg containing no mutations that
induce misfolding (i.e. no unfolded protein response), so
that levels of all other endoplasmic reticulum chaperones were normal.
Increased availability of BiP did not accelerate Tg secretion; rather,
the export of newly synthesized Tg was delayed. Tg detained
intracellularly was concentrated in the endoplasmic reticulum. By
coimmunoprecipitation, BiP exhibited enhanced binding to Tg in CHO-B
cells. Moreover, two-dimensional gel analysis showed that BiP
associated especially well with intracellular Tg containing mispaired
disulfide bonds, thought to represent early Tg folding intermediates.
An endoplasmic reticulum chaperone of the hsp90 family, GRP94, was also
associated in Tg-chaperone complexes. The results suggest that
increased binding of BiP to Tg leads to its delayed conformational
maturation in the endoplasmic reticulum.
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INTRODUCTION
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In the thyroid gland, T3 and T4 are
synthesized within the precursor protein, thyroglobulin (Tg), a 660-kDa
homodimeric secretory protein (1). Production of thyroid hormones is
significantly controlled at the level of synthesis and processing of
the Tg prohormone (2). Expression of Tg is physiologically regulated by
TSH (3), as is the regulated secretion of Tg into the lumen of thyroid
follicles (4), its uptake and delivery to lysosomes (5, 6), and finally
its degradation (7) that causes the release of T3 and
T4 (8). In addition to serving as a storage form of
organified iodine, Tg is unique in its ability to provide the correct
steric positioning of noncontiguous iodotyrosine residues such that
they undergo efficient coupling to iodothyronines. This behavior is
dependent upon conformational information in the protein precursor,
underscoring the necessity for correct folding and assembly of Tg in
the production of thyroid hormones (9). Indeed, Tg defective for
structural maturation has recently been elaborated as one of the
significant causes of congenital hypothyroid goiter (10). These
findings have emerged at a time of increasing general awareness of
defective protein folding as a basis for human disease (11).
Much of the conformational maturation of secretory proteins occurs in
the endoplasmic reticulum during cycles of association-dissociation
with molecular chaperones localized to this compartment (12). In
thyrocytes, before transport to the Golgi (13), newly synthesized Tg
proceeds through a series of discrete folding intermediates, including
protein aggregates (with and without interchain disulfide bonds),
unfolded free monomers, folded monomers, and finally dimers (14, 15).
This conformational maturation proceeds concomitant with the
establishment of
60 native intrachain disulfide bonds (16),
suggesting a role for protein disulfide isomerase in the folding
process (1). In addition, molecular chaperones, calnexin, calreticulin,
BiP (the endoplasmic reticulum member of the hsp70 class), GRP94 (the
endoplasmic reticulum member of the hsp90 class), ERp72, GRP170, and
ER60, have all been implicated in interaction with unfolded forms of Tg
(17, 18, 19, 20, 21).
In cog/cog mice, which suffer from congenital hypothyroid
goiter with defective Tg, an abnormally low fraction of newly
synthesized Tg is exported (22). Accumulation of the defective Tg
triggers the endoplasmic reticulum unfolded protein response, which
involves signaling from the endoplasmic reticulum to the nucleus
(23, 24, 25, 26). This results in induction of the synthesis of a group of
molecular chaperones (22), which is also seen in certain human kindreds
with congenital hypothyroid goiter (27). In thyrocytes of wild-type
animals, BiP associates only transiently with Tg during early stages of
its conformational maturation (14). By contrast, in cog/cog
thyrocytes, the level of immunoreactive BiP is elevated approximately
8-fold, and Tg is retained intracellularly, exhibiting enhanced and
prolonged association with BiP (and other chaperones) (22). Thus, the
increased retention of Tg in this disease a process known as
endoplasmic reticulum quality control (12) might result, at least in
part, from increased binding to molecular chaperones such as BiP
(28).
To attempt to examine possible consequences of increased BiP binding,
we have chosen to study the export of recombinant Tg containing no
mutations that induce misfolding, in a system that has been established
for selectively increased expression of BiP even in the absence of the
unfolded protein response (29). Using this system, we find enhanced and
prolonged association of BiP with Tg folding intermediates, resulting
in the delayed secretion of newly synthesized Tg, and leading to its
accumulation in the endoplasmic reticulum. These results are consistent
with a proposed role of BiP both during normal Tg folding and in
congenital hypothyroid goiter with defective Tg, as part of quality
control monitoring in the endoplasmic reticulum.
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RESULTS
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To explore the relationship between BiP binding and the folding
and export of Tg, we chose to express recombinant nonmutant Tg in
control Chinese hamster ovary (CHO) cells with a normal amount of
endoplasmic reticulum chaperones (parental, CHO-P cells) and CHO-B
cells that have been engineeered for increased expression of BiP at
both the mRNA and protein levels (29). As judged by Western blot of
cell extracts, the levels of other chaperones, calnexin, ER60,
calreticulin, PDI, ERp72, and GRP94, were not different between CHO-B
cells and controls (Fig. 1
, upper
panel). Also, the level of ribophorin II [an endoplasmic
reticulum membrane protein (30)] was unchanged, suggesting that the
amount of endoplasmic reticulum membrane was not different in the two
cell lines. However, BiP levels were indeed increased in CHO-B cells
(Fig. 1
, upper and lower panels). By quantitative
immunoblotting with [125I]Protein A we found that BiP was
5- to 10- fold more abundant in CHO-B cells, consistent with previous
reports (31). Thus, we find that elevated BiP levels represent a
selective chaperone increase in CHO-B cells. Additionally, previous
studies have established that this does not lead to any detectable
increase in BiP secretion (29).

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Figure 1. CHO-B Cells Exhibit Selectively Increased
Expression of BiP
Immunoblotting experiments were performed on CHO cell clones that
stably express Tg, used throughout the course of the studies described
in this report. Upper panel, Representative Western
blots with specific antibodies to various endoplasmic reticulum
proteins in cell extracts containing equal amounts of total protein
from CHO-B (B) and control CHO-P (P) cells. Lower panel,
Quantitative data showing a 5- to 10-fold increased expression of BiP
in CHO-B cells, from three different experiments. By contrast, the
relative expression levels of other endoplasmic reticulum resident
proteins varied by no more than 10%.
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In stably transfected CHO cells, Tg was detected as an immunoreactive
band by immunoblotting of cell extracts with polyclonal anti-Tg (Fig. 2A
). No immunoreactivity was detected in
nontransfected cells. In both CHO-B and controls, exogenously expressed
Tg was secreted. An easily identifiable band not present in the media
bathing untransfected CHO cells was observed upon
SDS-PAGE/autoradiographic analysis of media collected from transfected
cells; this band comigrated with authentic Tg and was
immunoprecipitated with anti-Tg (see below).

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Figure 2. Expression and Secretion of Tg in CHO-B and
Parental Control CHO-P Cells
A, Immunoblot of Tg in cell extracts (not normalized for protein) from
CHO-P, CHO-B, and nontransfected CHO cells (NT). The immunoreactive
approximately 330-kDa band migrating with identical mobility to an
authentic bovine Tg standard (indicated at left) is
undetectable in nontransfected CHO cells. B, Radiolabeled Tg contained
in chase media cumulatively collected from cultures of metabolically
labeled CHO-B and CHO-P cells for up to 10 h after pulse-labeling
was quantitated by SDS-PAGE/fluorography and scanning densitometry. The
total Tg collected over the 10-h period was defined as 100%. Note that
the secretion of newly synthesized Tg appears more slowly from CHO-B
cells.
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Reproducible from these experiments was the observation that CHO-B
cells secreted newly synthesized Tg more slowly than control CHO cells
(Fig. 2B
). This delayed Tg secretion suggested the possibility of
intracellular accumulation (i.e. an increase in steady-state
level) of Tg in CHO-B cells. To test this, Tg was immunoprecipitated
from control or CHO-B cells that had been metabolically labeled for a
long time (20 h, to try to reflect the steady state situation) or a
short time (20 min, to try to reflect the synthesis of new Tg). As
shown in Fig. 3
, although clones of
control CHO cells appeared to synthesize approximately twice as much Tg
per unit time, the CHO-B cells ultimately contained a significantly
larger pool of intracellular Tg (
4-fold). The data indicate that
increased availability of BiP strongly affects the residence time of
Tg, leading to its intracellular accumulation.

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Figure 3. Intracellular Accumulation of Tg in CHO-B Cells
Parental CHO (P) cells or CHO cells with augmented levels of BiP (B)
underwent long metabolic labeling with 35S- labeled amino
acids (20 h, upper panel) to try to approach steady
state labeling, or short labeling (20 min, lower panel)
to try to reflect the synthesis of new Tg. Duplicate clones of each
kind were examined. In both cases the cells were lysed,
immunoprecipitated for Tg, and analyzed by SDS-PAGE/fluorography. Note
that although approximately twice as much labeled Tg was synthesized
per 20 min in control CHO cells (lower panel), CHO cells
with increased expression of BiP ultimately accumulated approximately
4-fold more labeled intracellular Tg over a 20-h labeling (upper
panel).
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Next, to determine the intracellular compartment in which Tg is
retained, we examined its localization in transfected CHO-B and control
CHO cells by immunofluorescence microscopy. In contrast with
untransfected cells (Fig. 4A
), Tg
immunofluorescence in both sets of cells (Fig. 4B
, C) was distributed
over the cytoplasm in a fine reticular fashion, being more intense
around the nucleus and decreasing in brightness toward the cell
periphery. The same labeling pattern was obtained in CHO cells examined
for indirect immunofluorescence of the endoplasmic reticulum chaperone,
GRP94 (Fig. 4
, DF). These data indicated that intracellular Tg is
concentrated in the endoplasmic reticulum. Moreover, Tg fluorescence
intensity appeared greater in CHO-B cells (Fig. 4C
), consistent with
its greater intracellular accumulation in these cells (Fig. 3
).

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Figure 4. Immunofluorescence Localization of Tg in the
Endoplasmic Reticulum of CHO cells
Indirect immunofluorescence was performed with a polyclonal antibody
against Tg in nontransfected CHO cells (A), parental control cells (B),
or CHO-B cells (C). Immunoflurescence was also performed with a
polyclonal antibody to GRP94, a chaperone of the endoplasmic reticulum,
in nontransfected CHO cells (D), parental control cells (E), and CHO-B
cells (F). The Tg labeling pattern in both CHO-P and CHO-B cells is
characteristic for the endoplasmic reticulum, and the signal is clearly
stronger in CHO-B cells. Note the absence of labeling for Tg in
nontransfected cells. Bar, 10 µm.
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We also examined the status of intracellular Tg glycosylation by
digestion with endoglycosidase H (Endo H). Endo H digests high mannose
oligosaccharides on glycoproteins in the endoplasmic reticulum, but
does not cleave glycoprotein-bound oligosaccharides that have already
undergone processing within the medial Golgi compartment (32). For our
analysis, cells were extracted at various times after metabolic
labeling with 35S-labeled amino acids, digested with Endo
H, immunoprecipitated with anti-Tg, and analyzed by SDS-PAGE and
autoradiography. For both CHO-B and control CHO cells, intracellular Tg
was completely sensitive to digestion with Endo H at all chase times,
as measured by a mobility shift upon SDS-PAGE (Fig. 5
). Of course, labeled Tg drained more
rapidly from control CHO cells (Fig. 5
, upper panel),
consistent with relatively more rapid secretion of newly synthesized Tg
from these cells (Fig. 2B
). Nevertheless, even as control CHO cells
were secreting their labeled Tg at later chase times, the residual
fraction of intracellular Tg remained Endo H-sensitive (Fig. 5
). These
data indicate that the residence time of Tg molecules in
Golgi/post-Golgi compartments immediately before secretion is too brief
to allow any significant accumulation of this Endo H-resistant form.
Together, the data in Figs. 4
and 5
establish that the majority of
intracellular Tg is localized to the endoplasmic reticulum.

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Figure 5. Biochemical Localization of Intracellular Tg to a
pre-Golgi Compartment
Parental control (P cells) or CHO-B (B cells) were pulse-labeled with
35S-labeled amino acids for 10 min, extracted at the
various chase times indicated (in hours), digested (+) or mock-digested
(-) with Endo H, and immunoprecipitated with anti-Tg. The samples were
finally analyzed by SDS-PAGE/fluorography.
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One possible mechanism to explain the delayed export of Tg in cells
with elevated expression of BiP is increased accumulation of Tg folding
intermediates specifically associated with this chaperone. To begin to
examine this possibility, control CHO and CHO-B cells expressing Tg
were metabolically labeled for 20 h with
[35S]methionine, incubated or mock-incubated with the
dithiobis-succinimdylpropionate (DSP) cross-linker, and
immunoprecipitated with anti-Tg to recover nearest neighbor proteins
that had been cross-linked to Tg. The immunoprecipitates were reduced
with 2-mercaptoethanol and analyzed by SDS-PAGE and autoradiography.
Tg, as expected, was the predominant band recovered from samples that
had not been chemically cross-linked (Fig. 6
). However in cross-linked samples,
additional labeled bands1
were coprecipitated with anti-Tg, including a 78-kDa band that
comigrated with authentic BiP. Notably, this Tg-associated band was
selectively increased in CHO-B cells, and its recovery was to a large
extent dependent upon cross-linker, suggesting that it did not derive
from nonspecific precipitation with the immunoabsorbent (Fig. 6
, third
and fourth lanes). These data establish that there is an increased
association of a 78-kDa protein, presumably BiP, with nonmutant Tg
expressed in CHO-B cells.

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Figure 6. Increased Association of 78-kDa Band with
Intracellular Tg in CHO-B Cells
Control (CHO-P) and CHO-B cells were labeled for 20 h with pure
[35S]methionine (note that Tg has few methionine residues
and labels relatively poorly under these conditions). At the end of the
labeling period, cells were chemically cross-linked with DSP (+) or
mock-cross-linked () and extracted for immunoprecipitation with
anti-Tg. Immunoprecipitates were reduced (thereby breaking the
cross-links) and analyzed by SDS-PAGE/fluorography. The positions of
authentic Tg ( 330 kDa) and BiP ( 78 kDa) standards are indicated
at left. Note in CHO-B cells the increased Tg
association of a labeled 78-kDa band presumed to be BiP.
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Tg proceeding along the native folding pathway is known to associate
with GRP94 (another endoplasmic reticulum chaperone) as well as BiP
(22, 33). We therefore tested the effect of augmented BiP expression on
Tg association with GRP94. For this, complexes obtained after metabolic
labeling to approach steady state, followed by chemical cross-linking
and immunoprecipitation with polyclonal anti-Tg (as in Fig. 6
), were
reprecipitated with a polyclonal anti-GRP94 before analysis by SDS-PAGE
under reducing conditions (Fig. 7
). While
bands were not obtained in uncross-linked samples, GRP94-Tg-containing
complexes were clearly detected in the cross-linked samples (Fig. 7
, lanes 1 and 3). By quantification of band intensity in these samples,
the estimated molar ratio of GRP94:Tg in these complexes was found to
be quite similar in CHO-B cells (
2.6) and control CHO cells
(
3.1). Interestingly, sequential immunoprecipitation with anti-Tg
and anti-GRP94 coimmunoprecipitated a 78-kDa band that comigrated with
authentic BiP, which appeared greatly augmented in CHO-B cells. Thus,
although increased BiP availability increased BiP binding to Tg in the
endoplasmic reticulum, this did not significantly alter the
stoichiometry of interaction between Tg and GRP94.

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Figure 7. Isolation of GRP94-Tg Complexes
CHO-P (P cells) and CHO-B (B cells) were labeled with pure
[35S]methionine, cross-linked with DSP (+) or
mock-crosslinked (), extracted, and immunoprecipitated with anti-Tg
as in Fig. 6 . Subsequently, the immunoprecipitates were denatured with
SDS, diluted, and reimmunoprecipitated with a polyclonal anti-GRP94 to
isolate Tg complexes containing this chaperone. Finally, the samples
were reduced and analyzed by SDS-PAGE and the labeled bands were
quantified by phosphorimaging. The migration positions of authentic
GRP94 and BiP are indicated on the left.
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Finally, we investigated the folding state of intracellular Tg in CHO-B
and control CHO cells. Previous studies of thyrocytes have established
that during the progression of normal Tg folding en route to
the native state, high molecular weight Tg complexes containing
interchain disulfide bonds exist as a prevalent but transient
intermediate (13, 14, 15). To detect the possible existence of such forms,
we decided to couple a protocol of treating cells with the
thiol-cleavable cross-linker, DSP (34), with a two-dimensional SDS-PAGE
analysis designed to resolve disulfide-linked Tg complexes in a first,
nonreducing dimension at a low (4%) acrylamide concentration, followed
by dissociation of these complexes and separation of their components
using reducing SDS-6.5%-PAGE in a second dimension (33). As a control,
the position of native Tg in this two-dimensional gel system was
analyzed. Nonreducing SDS-4%-PAGE of immunoprecipitated Tg ran from
right to left, while the second dimensional reducing SDS-6.5%-PAGE ran
from top to bottom (Fig. 8
). The mobility
of radiolabeled Tg secreted from CHO cells (without cross-linking)
after the first dimensional SDS-PAGE (shown horizontally across
the top of Fig. 8
) clearly demarcated the position expected for
properly folded Tg upon two-dimensional SDS-PAGE analysis (bottom
panel of Fig. 8
).

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Figure 8. Migration of Native Tg by Two-Dimensional SDS-PAGE
CHO cells expressing Tg underwent long-term labeling with
35S-labeled amino acids as in Fig. 3 and were then rinsed
and chased for a further 6 h. The chase medium was collected and
immunoprecipitated with anti-Tg and then analyzed by nonreducing
SDS-4% PAGE in the first dimension from right to left, shown as a
single horizontal lane in the upper panel. Lanes excised
from the first dimensional gel were then run on reducing SDS-6.5% PAGE
from top to bottom, as shown in the lower panel. The
position of Tg and the migration positions of molecular weight
standards in the second dimension are indicated on the
right. Material running just above the dye front in the
first dimension contributes to radioactive background bands at the
left of the two-dimensional gel; such background bands
are also obtained after immunoprecipitation from cells that have not
been transfected with the Tg cDNA (33).
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In preparation for a similar analysis, CHO cells were metabolically
labeled for 20 h with [35S]cysteine/methionine
mixture (to try to approach steady state) and incubated with
cross-linker (Fig. 9
, right
panels) or mock incubated (left panels) before
immunoprecipitation with anti-Tg. In cells with increased availability
of BiP (Fig. 9
, bottom panels), a portion of intracellular
Tg ran at a high molecular weight (circled) position in the
first (nonreducing) dimension, indicating the presence of
disulfide-linked Tg complexes which, upon reduction, acquired the same
mobility in the second dimension. Importantly, even in uncross-linked
samples, a portion of intracellular Tg from CHO-B cells was still
recovered at a high molecular weight position (Fig. 9
, lower
left, circled band), indicating that intracellular Tg complexes
had not been formed artifactually by the cross-linker. Moreover, these
high molecular weight complexes specifically accumulated in cells with
increased availability of BiP, as they were below the limits of
detection in control CHO cells (Fig. 9
, upper panels).

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Figure 9. Tg Folding Intermediates Are Retained in the
Endoplasmic Reticulum in Association with BiP
Control CHO (P) or CHO-B (B) cells were labeled for 20 h with
[35S]cysteine and methionine, and either cross-linked
with DSP (right set of panels) or mock-cross-linked
(left set of panels). The cells were then extracted and
immunoprecipitated with polyclonal anti-Tg, before analysis by
nonreducing SDS-PAGE (first dimension, from right to
left) followed by reducing SDS-PAGE (second dimension, from
top to bottom). In both the absence and presence of
cross-linker, CHO-B cells accumulated a high molecular weight form of
Tg (encircled with dotted line) that was below detection
in parental control cells. After cross-linking, this form of Tg ran
directly above (and therefore was associated with) bands of 94 kDa and
78 kDa (arrows), attributed to GRP94 and BiP,
respectively. The presence of GRP94 in Tg complexes was confirmed by
experiments employing labeling with pure [35S]methionine,
as in Fig. 7 . The migration positions of molecular weight standards in
the second dimension are indicated at right of each
panel.
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Most remarkably, in chemically cross-linked samples of CHO-B cells
(Fig. 9
, lower right panel), high molecular weight
intracellular Tg complexes were found in the second dimension to be
associated with additional bands of 78 kDa and 94 kDa
(highlighted with arrows) that comigrated with authentic BiP
and GRP94, respectively. Taken together, these results suggest that the
most likely explanation for the kinetic delay in Tg export, and
accumulation of Tg in the endoplasmic reticulum as a consequence of
augmented expression of BiP in CHO cells, is the prolonged association
between Tg folding intermediates and this molecular chaperone. These
data highlight the important role of BiP in the quality control of Tg
folding and export (14).
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DISCUSSION
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Efficient production of T3 and T4 requires
secretion of the thyroid hormone precursor protein Tg, and one of the
important causes of congenital hypothyroid goiter is the presence of Tg
coding sequence mutations that limit Tg transport along the secretory
pathway (27). Part of the machinery providing recognition of unfolded
forms of Tg and other secretory proteins in the endoplasmic reticulum
of thyrocytes includes the molecular chaperones BiP and GRP94 (1). BiP,
in particular, has been linked to multiple different functions of the
endoplasmic reticulum, such as the translocation of secretory protein
nascent chains (35), secretory protein folding and oligomerization
(36), and possibly the degradation of misfolded secretory proteins
(37, 38, 39). Importantly, in thyrocytes of certain animals with congenital
hypothyroid goiter, defective Tg molecules are prevented from export,
and these same Tg molecules exhibit increased and prolonged association
with BiP (22). As a first step to directly test the effect of increased
BiP binding on Tg export in the absence of any drugs, toxins, or
inhibitor treatments, we have examined the fate of nonmutant,
recombinant Tg expressed in CHO cells that have selectively increased
levels of BiP, i.e. without a significant change in the
intracellular levels of other endoplasmic reticulum resident proteins
(Fig. 1
).
Just as in normal thyrocytes (22), our data indicate that in all stably
transfected CHO cells, nonmutant Tg forms complexes with BiP (Fig. 6
)
en route to secretion into the medium (Fig. 2
). These
results suggest that steps in the conformational maturation and export
of nonmutant Tg in CHO cells are likely to be similar to the respective
processes in thyrocytes. However, a selective 5- to 10-fold increase of
BiP expression in CHO cells results in increased retention of nonmutant
Tg in the endoplasmic reticulum (Figs. 4
and 5
). This is reflected in
at least two ways: retardation (i.e. diminished rate) of
secretion of newly synthesized Tg (Fig. 2B
) and accumulation of
intracellular Tg at conditions approaching steady state (Fig. 3
). This
increase in intracellular Tg occurred in spite of diminished formation
of newly synthesized Tg in the CHO-B cell clones studied. Thus, the
phenotype in CHO-B cells cannot readily be attributed to a proposed
BiP-mediated increase in the cotranslational translocation of nascent
secretory proteins across the endoplasmic reticulum membrane (40), but
rather appears to reflect events that occur during the chase period,
i.e. after the deposition of newly synthesized Tg in the
endoplasmic reticulum lumen.
Our data strongly suggest that increased retention of nonmutant Tg in
the endoplasmic reticulum is mediated by increased binding of BiP (Fig. 6
) and other chaperones (described further, below). These effects are
in some ways reminiscent of the behavior of mutant Tg protein in
certain forms of congenital hypothyroid goiter, described above (22),
in which levels of total intracellular BiP (and other chaperones) are
elevated, and Tg binding to BiP (and other chaperones) is enhanced and
prolonged. We therefore suggest the possibility that while some coding
sequence mutations might theoretically exert direct effects on Tg
hormonogenic domains, the primary pathophysiology in most cases (of
congenital hypothyroid goiter with defective Tg protein) is likely to
involve prevention of Tg export from the endoplasmic reticulum due to
increased association with certain molecular chaperones. If this can be
proved, then it follows that the intrinsic hormonogenic potential of
many Tg mutants may actually be normal or near-normal, and that the
dyshormonogenesis in many cases may be entirely an indirect consequence
of chaperone-mediated retention mechanisms, i.e. endoplasmic
reticulum quality control (12), resulting in a simple insufficiency of
Tg delivery to the site of iodination (1).
We emphasize that increased availability of the molecular chaperone BiP
did not accelerate Tg exit from the endoplasmic reticulum and instead
appeared to retard Tg export. This finding appears to contradict
observations in yeast, based on the behavior of certain peptide
binding-deficient mutants of yeast BiP, which has led to the conclusion
that BiP binding enhances advancement of certain proteins through the
export pathway (see published and unpublished studies cited in Ref.41). However, our findings are supported by previous investigations of
the effects of increased BiP expression on the secretion of a selective
subset of mammalian proteins (29, 31, 42). Most remarkable from these
earlier studies was the finding that increased availability of BiP did
not inhibit secretion of proteins that do not serve as suitable
substrates for BiP binding (29, 42). We have interpreted these findings
to mean that direct BiP binding to exportable proteins somehow causes
their retention. It was for this reason that we set about to explore
the underlying mechanism for how BiP binding might cause Tg retention
in the endoplasmic reticulum.
The results of our study indicate that, as a consequence of increased
BiP binding to Tg (Fig. 6
), an increased fraction of the secretory
protein accumulates in the form of folding intermediates (Fig. 9
). Such
an outcome is consistent with the widely held view that BiP (and other
molecular chaperones) tends to bind most strongly to incompletely
folded forms rather than mature native proteins (43). Thus, we
interpret the present data to mean that enhanced BiP binding retards
(but does not block) the advancement of Tg conformational maturation
through its normal sequence of progressively more folded forms
(13, 14, 15). Since BiP functions as a chaperone via cyclical on and off
associations coupled to the hydrolysis of ATP (44), our findings are
consistent with the notion that advancement in the folding of Tg
domains occurs primarily during the off periods, resulting in slower Tg
folding when the average BiP "on" time is prolonged.
Different molecular chaperones in the endoplasmic reticulum, such as
GRP94 (33), may also interact with folding intermediates of secretory
proteins, and these interactions can occur both concomitantly and
sequentially with BiP (45). The length and hydrophobicity of favored
peptide-binding sites for BiP are well described (46, 47, 48) whereas the
features favoring GRP94 binding have not yet been established. We
therefore considered the possibility that increased abundance of Tg
folding intermediates (Fig. 9
) caused by augmented BiP binding (Fig. 6
)
might also influence the association of other chaperones such as GRP94.
Indeed, the existence of ternary complexes containing Tg, BiP, and
GRP94 was strongly suggested by sequential immunoprecipitation
experiments (Fig. 7
). Interestingly, increased binding of BiP in CHO-B
cells did not significantly diminish the GRP94:Tg stoichiometry in
these complexes, suggesting the important possibility that binding
sites for BiP and GRP94 are largely nonoverlapping. We consider these
findings of great interest, as the binding of both hsp70 (BiP) and
hsp90 (GRP94) chaperone classes have been implicated in the quality
control of Tg folding and export from thyrocytes of control animals and
those with congenital hypothyroid goiter (22). Finally, possible
associations of such complexes with additional endoplasmic reticulum
chaperones, such as GRP1701 [which has been recently
reported to interact with Tg (21)], remain to be tested.
In conclusion, our results show that in CHO cells, increased binding of
BiP to Tg leads to delayed conformational maturation and prolonged Tg
retention in the endoplasmic reticulum. We believe that this is one of
several possible important mechanisms that may help to account for how
mutant Tg export is prevented, as part of an endoplasmic reticulum
quality control mechanism, in congenital hypothyroid goiter with
deficient Tg.
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MATERIALS AND METHODS
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Materials
The following vectors were employed: pCB6, a mammalian
expression vector carrying a neomycin resistance cassette and
cytomegalovirus promoter-driven insert (originally from Dr. M. Stinsky,
University of Iowa, Ames, IA); pBAT14, used as a shuttle vector, from
Dr. M. German (University of California, San Francisco CA); pBR322 was
purchased from Upstate Biotechnology Inc. (Lake Placid, NY). DSP was
purchased from Pierce Chemical Co. (Rockford, IL). A polyclonal rabbit
antiserum was raised against denatured Tg as described previously (49).
Antibody to ribophorin II was the kind gift of Dr. D. Meyer (University
of California, Los Angeles). An immunoprecipitating polyclonal
antiserum to GRP94 (50) was kindly provided by Dr. P. Srivastava
(Fordham University, Bronx, NY). A polyclonal antiserum to BiP was
purchased from StressGen (Victoria, Canada). Polyclonal antibodies to
protein disulfide isomerase and ER60 were from Dr. T. Wileman
(Pirbright Laboratories, Surrey, U.K.) and polyclonal antibodies to
calnexin, ERp72, and calreticulin were kindly provided by Dr. P. Kim
(University of Cincinnati, Cincinnati, OH). A rhodamine-conjugated,
affinity-isolated, goat anti-rabbit IgG was purchased from Tago, Inc.
(Burlingame, CA), and an alkaline phosphatase-conjugated goat
anti-rabbit IgG was from GIBCO BRL (Gaithersburg, MD).
[125I]Protein A was purchased from New England Nuclear
(Wilmington, DE). Zysorbin was from Zymed Laboratories (San Francisco,
CA). Other tissue culture reagents, restriction enzymes for molecular
biology, protease inhibitors, and stock chemicals were either from Life
Sciences (Gaithersburg, MD), New England Biolabs (Beverly, MA), or
Sigma (St. Louis, MO).
Construction of a Full-Length Tg cDNA
To generate a full-length Tg cDNA a strategy involving
subcloning of four contiguous partial cDNAs (51) (kindly provided by
Dr. G. Vassart, U. Libre, Brussels, Belgium) was used. These partial
cDNAs spanned nucleotides 519 to 8430 (i.e. from a conserved
NcoI site to the 3'-end) of the bovine Tg coding sequence.
Using the rat Tg-2 cDNA (52) obtained from Dr. P. Graves (Mt. Sinai
School of Medicine, New York, NY) the remaining Tg coding sequence
(i.e. from the extreme 5'-end to the conserved
NcoI site) was provided. Thus, the final full-length cDNA we
employed encodes for a polypeptide that exhibits overall 99.1%
identity to normal bovine Tg, with the remaining 0.9% being
conservative bovine
rat Tg substitutions contained within the first
155 amino acids.
For stepwise construction, a fragment of the rTg-2 cDNA extending from
an EcoRI site (17 bases upstream from the translational
start site) to the conserved NcoI site was ligated into the
shuttle vector pBAT14 along with a partial bovine Tg cDNA extending
from the NcoI site to a HindIII site at position
2826. The ligated insert was excised from pBAT14 using BamHI
from the polylinker and HindIII and then subcloned into the
BglII and HindIII sites in the polylinker of
pCB6. The resulting plasmid was digested with HindIII and
BamHI, and a partial Tg cDNA encoding the 3'-end (position
74468430) was directionally ligated. Independently,
HindIII-HindIII cDNA fragments extending from
positions 28264764 and 47647446, respectively, were ligated at the
HindIII site of pBR322, and appropriately oriented subclones
were selected from DNA minipreps. From this, the correctly ligated
4.6-kb insert was excised from pBR322 by partial digestion with
HindIII, and this gel-purified insert was ligated into the
HindIII-digested pCB6, which contained the rest of the Tg
cDNA. The size and orientation of the final full-length clone was
confirmed by identity to the known bovine Tg restriction map (51).
Cell Culture, Tg Transfection, and Selection of Stable
Tg-Expressing Clones
Two lines of CHO cells (prepared and graciously provided by Dr.
A. Dorner, Genetics Institute, Arlington MA) were used in this report.
The parental (CHO-P cell) line, containing endogenous levels of
endoplasmic reticulum chaperones, is a dihydrofolate
reductase-deficient line previously called DUKX-B11, from which pooled
transfectants overexpressing hamster BiP (CHO-B cells) were prepared as
a consequence of selection by coamplification of dihydrofolate
reductase (29). These CHO cells were maintained in media based on
-MEM containing 1% penicillin-streptomycin. The medium for CHO-P
cells also contained 10% FBS and 10 mg/liter each of adenosine,
deoxyadenosine, and thymidine. CHO-B cell medium contained 10%
dialyzed FBS, was without added ribonucleosides and
deoxyribonucleosides, and was supplemented with 10-7
M methotrexate.
For Tg transfection, subconfluent cell cultures were rinsed with PBS
and detached with trypsin-EDTA. A 0.25-ml suspension of 2 x
106 cells was transfected with the full-length Tg cDNA in
pCB6 (20 µg) in a 0.4-cm pass electroporation cuvette at 330 V and
250 µFarads (time constant
14 msec); cells were then diluted
400-fold and plated. After 2 days in culture, selection was started by
addition of 0.8 mg/ml geneticin. Colonies were picked and screened for
Tg expression by immunofluorescence; media and cell lysates from
positive clones were then analyzed by immunoblotting. At least two
Tg-expressing clones of each type were further studied; by initial
characterization, the results obtained with replicate clones were
essentially identical; thus the data presented in this report derive
from representative clones.
Immunofluorescence
Cells grown on coverslips were rinsed with PBS, fixed for 15 min
at room temperature with 4% formaldehyde, and permeabilized in PBS
containing 0.2% (vol/vol) Triton X-100 and 1 mg/ml BSA. Incubation
with primary antibody was carried out for 1 h at room temperature
in the permeabilization buffer. Cells were then rinsed with this buffer
and incubated for another hour with a 1:400 dilution of a
rhodamine-conjugated, affinity-isolated goat anti-rabbit IgG.
Chemical Cross-linking and Immunoprecipitation
For chemical cross-linking of intracellular Tg complexes, cells
were labeled for 20 h with 100 µCi/ml [35S]Express
in complete medium, rinsed with PBS, and then incubated for 30 min at
room temperature with 200 µM DSP in PBS. Uncross-linked
controls were incubated in parallel with PBS containing the carrier
(dimethylsulfoxide). The cross-linking reaction was terminated by lysis
of cells in 3% SDS in 62.5 mM Tris, pH 6.8, containing
protease inhibitors [1 µg/ml leupeptin, 1 µg/ml pepstatin, 5 mg/ml
EDTA, and 0.4 mg/ml 4-(2-aminoethyl) benzenesulfonylfluoride (ICN,
Cleveland, OH)]. The cell lysate was cleared by briefly spinning in a
microfuge, and aliquots from this supernate were diluted
20-fold in immunoprecipitation buffer (25 mM Tris buffer,
pH 7.5, containing 1% Triton X-100, 0.1% SDS, 0.2% deoxycholic acid,
10 mM EDTA, 100 mM NaCl). One milliter of the
diluted cell lysate was preabsorbed for 30 min at room temperature with
50 µl of a 10% suspension of fixed Staphylococcus aureus
(Zysorbin). The suspension was pelleted, and the supernate was
incubated for 16 h at 4 C with 10 µl of polyclonal rabbit
anti-Tg. Fifty microliters of a 10% suspension of Zysorbin were then
added, and immune complexes were allowed to adsorb for 1 h at 4 C.
Pellets of this suspension were washed once in immunoprecipitation
buffer, once in 0.5% Tween-20 in TBS (25 mM Tris, 150
mM NaCl, pH 7.4), once in TBS, and finally in water, before
boiling for 5 min in 2040 µl of 2x sample buffer. The two
dimensional SDS-PAGE analysis was done as described below.
For sequential immunoprecipitation, cell lines labeled to steady state
with 400 µCi/ml [35S]methionine and cross-linked with
DSP were first immunoprecipitated with anti-Tg as described above.
These immunoprecipitates were eluted from Zysorbin by incubation for
1 h at 60 C in 50 µl 1% SDS plus 62.5 mM Tris, pH
6.8. The supernate was then diluted to 1 ml in immunoprecipitation
buffer and mixed with 2 µl anti-GRP94. Final immunoprecipitates were
adsorbed to Zysorbin and eluted by boiling for 4 min in 30 µl sample
buffer containing ß-mercaptoethanol.
Digestion with Endo H
Cell lysates prepared in 0.5% SDS-1% ß-mercaptoethanol in 50
mM sodium citrate, pH 5.5, containing protease inhibitors
were boiled for 5 min and then either digested or mock digested for
1 h at 37 C as described (49). The samples were then diluted to 1
ml and immunoprecipitated with anti-Tg.
Immunoblotting
For immunoblot analysis, SDS gels were electrophoretically
transferred to nitrocellulose. The membrane was blocked for 1 h
with 3% gelatin in TBS plus 0.5% Tween-20, incubated for 1 h
with primary antibody in the same solution, and then washed three
times. The blot was then incubated for 1 h with a 1:3,000 dilution
of alkaline phosphatase-conjugated goat anti-rabbit IgG, rinsed several
times, and then reacted with 4-nitroblue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl phosphate. Otherwise, the secondary reagent
was radioiodinated protein A; in this case, bands were quantitated by
phosphorimaging.
Two-Dimensional SDS-PAGE
Tg immunoprecipitates were separated in the first dimension
under nonreducing conditions by SDS 4%-PAGE. For the second dimension,
the samples were reduced with 50 mM dithiothreitol and
resolved by SDS 6.5%-PAGE.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. A. Dorner (Genetics Institute, Arlington MA) who
graciously provided us with CHO-B and control CHO cells. We thank Drs.
P S. Kim (University of Cincinnati, Cincinnati, OH), P. Srivastava
(Fordham University, Bronx, NY), and T. Wileman (Pirbright
Laboratories, Surrey U.K.), D. Meyer (University of California, Los
Angeles, CA), for generously providing antibodies to endoplasmic
reticulum resident proteins used in these studies. We acknowledge Drs.
G. Vassart (U. Libre Brussels, Belgium) and P. Graves (Mt. Sinai School
of Medicine, New York, NY) for providing us with partial cDNAs encoding
Tg, and Drs. D. Prabakaran (Beth Israel Deaconness Hospital, Boston,
MA) and J. Deschler (Harvard Medical School, Boston, MA) for assistance
with constructing the full-length Tg cDNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Peter Arvan, Division of Endocrinology and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461.
This work was supported by NIH Grant 40344 (to P.A.) and an NRSA
Postdoctoral Fellowship (to Z.M.).
1 We note that a CHO cell protein of approximately
170 kDa, which is apparently methionine-rich, was also associated with
Tg-containing complexes (Figs. 6
and 7
), although the identity of this
protein currently remains unknown. 
Received for publication September 11, 1997.
Revision received November 11, 1997.
Accepted for publication November 24, 1997.
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