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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go, 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. 1Go, 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%.

 
In stably transfected CHO cells, Tg was detected as an immunoreactive band by immunoblotting of cell extracts with polyclonal anti-Tg (Fig. 2AGo). 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.

 
Reproducible from these experiments was the observation that CHO-B cells secreted newly synthesized Tg more slowly than control CHO cells (Fig. 2BGo). 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. 3Go, 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).

 
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. 4AGo), Tg immunofluorescence in both sets of cells (Fig. 4BGo, 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. 4Go, D–F). These data indicated that intracellular Tg is concentrated in the endoplasmic reticulum. Moreover, Tg fluorescence intensity appeared greater in CHO-B cells (Fig. 4CGo), consistent with its greater intracellular accumulation in these cells (Fig. 3Go).



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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.

 
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. 5Go). Of course, labeled Tg drained more rapidly from control CHO cells (Fig. 5Go, upper panel), consistent with relatively more rapid secretion of newly synthesized Tg from these cells (Fig. 2BGo). 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. 5Go). 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. 4Go and 5Go 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.

 
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. 6Go). 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. 6Go, 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.

 
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. 6Go), were reprecipitated with a polyclonal anti-GRP94 before analysis by SDS-PAGE under reducing conditions (Fig. 7Go). While bands were not obtained in uncross-linked samples, GRP94-Tg-containing complexes were clearly detected in the cross-linked samples (Fig. 7Go, 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. 6Go. 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.

 
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. 8Go). 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. 8Go) clearly demarcated the position expected for properly folded Tg upon two-dimensional SDS-PAGE analysis (bottom panel of Fig. 8Go).



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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. 3Go 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).

 
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. 9Go, right panels) or mock incubated (left panels) before immunoprecipitation with anti-Tg. In cells with increased availability of BiP (Fig. 9Go, 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. 9Go, 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. 9Go, 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. 7Go. The migration positions of molecular weight standards in the second dimension are indicated at right of each panel.

 
Most remarkably, in chemically cross-linked samples of CHO-B cells (Fig. 9Go, 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).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go).

Just as in normal thyrocytes (22), our data indicate that in all stably transfected CHO cells, nonmutant Tg forms complexes with BiP (Fig. 6Go) en route to secretion into the medium (Fig. 2Go). 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. 4Go and 5Go). This is reflected in at least two ways: retardation (i.e. diminished rate) of secretion of newly synthesized Tg (Fig. 2BGo) and accumulation of intracellular Tg at conditions approaching steady state (Fig. 3Go). 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. 6Go) 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. 6Go), an increased fraction of the secretory protein accumulates in the form of folding intermediates (Fig. 9Go). 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. 9Go) caused by augmented BiP binding (Fig. 6Go) 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. 7Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 7446–8430) was directionally ligated. Independently, HindIII-HindIII cDNA fragments extending from positions 2826–4764 and 4764–7446, 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 {alpha}-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 20–40 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. 6Go and 7Go), although the identity of this protein currently remains unknown. Back

Received for publication September 11, 1997. Revision received November 11, 1997. Accepted for publication November 24, 1997.


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