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- 10.1074/jbc.272.42.26095
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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 42, Issue of October 17, pp. 26095–26102, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. INVESTIGATION OF THE ROLE OF MOLECULAR CHAPERONE, GRP94, IN PROTEIN EXPORT FROM THE ENDOPLASMIC RETICULUM* (Received for publication, December 31, 1996, and in revised form, July 17, 1997) Zoia Muresan and Peter Arvan‡ From the Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02215 GRP94 serves as a molecular chaperone in the endo- group of hereditary illnesses originating from mutations in plasmic reticulum (ER). In normal thyrocytes, GRP94 secretory proteins or other exportable proteins that interfere interacts transiently with thyroglobulin (Tg), and in with protein folding and exit from the ER (7) . thyrocytes of animals suffering from congenital hypo- In both normal secretory protein export and in ER storage thyroid goiter with defective thyroglobulin, GRP94 and diseases, the functions of ER chaperones remain poorly under- thyroglobulin associate in a protracted fashion. In order stood. BiP, perhaps the most studied ER chaperone, interacts explore possible consequences of GRP94 binding, we transiently with certain wild-type exportable proteins and ex- have studied recombinant nonmutant thyroglobulin ex- hibits more prolonged interactions with certain mutant or un- pressed in control Chinese hamster ovary (CHO) cells in assembled exportable proteins (8 –18). Nevertheless, it has comparison to that produced in CHO cells genetically been difficult to clearly define a single post-translational role of manipulated for selectively increased GRP94 expres- BiP in the export of secretory proteins (see Introduction of sion. Levels of ER chaperones other than GRP94 did not Hendershot et al. (19)). Even less is known about the role detectably differ, and thyroglobulin achieved transport played by GRP94 in protein export, although its demonstrated competence in both kinds of CHO cells. However, in- ability to bind certain secretory proteins is also well established creased availability of GRP94 caused the residence time (20, 21). For instance, although it has been concluded that, in of Tg in the ER to be remarkably prolonged. This was accompanied by a major increase in Tg directly associ- conjunction with its chaperone function in the ER, GRP94 is a ated with GRP94 and an increase in the ER pool size of molecule capable of ATP binding (22), hydrolysis (23), and Tg. Importantly, co-immunoprecipitation analysis re- autophorphorylation activity (24), these conclusions have re- vealed disulfide-linked Tg complexes (previously re- cently been called into question by new experiments which ported as an early Tg-folding intermediate) especially suggest that peptide interaction with GPR94 is ATP- associated with GRP94. Indeed, non-native Tg, GRP94, independent (25). and a 78-kDa protein likely to be BiP, appeared in ter- We have been interested to know more about the effects of nary complexes. Under these conditions, GRP94 associ- binding of GRP94 on the folding and export of thyroglobulin ation appears directly involved in prolongation of Tg (Tg), the secretory protein precursor for thyroid hormone syn- folding and export, consistent with a role in quality thesis, and one of the reported “substrates” for GRP94 associ- control in the ER. ation in thyroid epithelial cells (18, 26, 27). Tg is a large (;330 kDa) glycoprotein that undergoes substantial initial folding, including the formation of nearly 60 intrachain disulfide bonds, Secretory proteins are translocated into the lumen of the before homodimerization, which normally occurs in the ER. endoplasmic reticulum (ER) in an unfolded form that is bio- Remarkably, even when great precaution is taken to prevent logically inactive and incompetent for intracellular transport. formation of artifactual disulfide bonds at the time of cell lysis, Prior to acquisition of native conformation, the nascent pro- immediately after translation in primary thyrocytes, newly teins are retained in the ER, where they associate with molec- synthesized nonmutant Tg is found in high molecular mass ular chaperones (1). Two of the better recognized ER chaper- complexes, many containing improper interchain disulfide ones are family members of the hsp90 (GRP94) (2) and hsp70 bonds (27–29). Subsequently the disulfide-linked Tg complexes classes (BiP) (3, 4), which are abundant proteins in the normal become undetectable while Tg is observed to advance toward ER (5) and are further induced under stress conditions (6). One more folded forms of individual monomers (14). During early physiologically relevant example of such stress occurs in endo- folding, nonmutant Tg dissociates from GRP94 with kinetics plasmic reticulum storage diseases, which include a large superimposable upon those of BiP (18). These kinetics precede Tg homodimerization and its vesicular egress from the ER (14). * This work was supported in part by National Institutes of Health In thyrocytes as in other cell types, GRP94 levels are phys- Grant DK40344 (to P. A.) and a National Research Service Award iologically regulated (30). Indeed, in humans suffering from postdoctoral fellowship (to Z. M.). The costs of publication of this article congenital goiter with mutant Tg, thyrocytes can achieve levels were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 of GRP94 that are elevated by $1 order of magnitude, which U.S.C. Section 1734 solely to indicate this fact. represents a greater increase than that observed for BiP (31). ‡ To whom correspondence should be addressed: Division of Endocri- Interestingly, in the cog/cog mouse (32–34), an animal model nology, Albert Einstein College of Medicine, 1300 Morris Park Ave., of this illness in which thyroid tissue is available for pulse- Bronx, NY 10461. Tel.: 718-430-8685; Fax: 718-430-8557; E-mail: [email protected]. chase analysis, the fraction of Tg molecules that exits the ER is The abbreviations used are: ER, endoplasmic reticulum; Tg, thyro- much lower than normal, while an increased fraction of Tg globulin; CHO, Chinese hamster ovary; CHO-P, CHO parental cells; interacts with GPR94 in a prolonged manner (18). CHO-G, GRP94-overexpressing cells; DSP, dithiobis(succinimdyl propi- Unfortunately, congenital hypothyroid goiter is not a suita- onate); PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis. ble model in which to independently examine the role of GRP94 This paper is available on line at http://www.jbc.org 26095 This is an Open Access article under the CC BY license. 26096 ER Residence Time of Thyroglobulin to a HindIII site at position 2826. The ligated insert was excised from pBAT14 using BamHI from the polylinker and HindIII, and then sub- cloned into the BglII and HindIII sites in the polylinker of pCB6, destroying the former site. The resulting plasmid was then digested with HindIII and the sole downstream BamHI site, and a partial Tg cDNA encoding the 39-end (position 7446 – 8430) was directionally li- gated (Fig. 1). Independently, HindIII-HindIII cDNA fragments extend- ing from positions 2826 – 4764 and 4764 –7446, respectively, were li- FIG.1. Diagram depicts construction of the full-length, ;8.4- gated together at the HindIII site of pBR322, and appropriately kilobase pair Tg cDNA. The arrows indicate the sites of ligation from oriented subclones were selected from DNA minipreps. From this, the existing partial cDNAs. correctly ligated 4.6-kilobase pair insert was excised from pBR322 by partial digestion with HindIII, and this gel-purified insert was ligated binding in Tg maturation, because it is difficult to resolve the into the HindIII-digested pCB6 which contained the rest of the Tg consequences of increased GRP94 binding from those related to cDNA. The size and orientation of the final full-length clone (Fig. 1) was confirmed by identity to the known bovine Tg restriction map (37). intrinsic alterations in the biophysical properties of mutant Tg. Cell Culture, Tg Transfection, and Selection of Stable Tg-expressing For this reason, in the present report we have examined the Clones—Two lines of CHO cells were graciously provided by Dr. A. conformational maturation and export of nonmutant Tg as a Dorner (Genetics Institute, Cambridge, MA) for use in the present consequence of manipulating the probability of GRP94 binding studies. The parental (“CHO-P” cell) line, containing endogenous levels to recombinant Tg in Chinese hamster ovary (CHO) cells engi- of ER chaperones, is a dihydrofolate reductase-deficient line, previously neered for wild-type or increased levels of GRP94 expression. called DUKX-B11 (39). The GRP94-overexpressing (“CHO-G” cell) line was prepared as pooled transfectants overexpressing murine GRP94 (2) Our results indicate that a major increase in the ER residence as a consequence of selection by co-amplification of adenosine time and expansion of the ER pool of Tg occurs when the deaminase (40). availability of GRP94 is increased, and this retention occurs as CHO cells were maintained in media based on a-minimum essential a direct consequence of complex formation between an appar- medium containing 1% penicillin-streptomycin. The medium for CHO-P ent Tg folding intermediate and this ER chaperone. cells also contained 10% fetal bovine serum and 10 mg/liter each of adenosine, deoxyadenosine, and thymidine, whereas CHO-G cell me- EXPERIMENTAL PROCEDURES dium was supplemented with 10% heat-inactivated fetal bovine serum, Materials—The following vectors were employed: pCB6, a mamma- 1mM uridine, 1.1 mM adenosine, 10 mg/liter each of deoxyadenosine lian expression vector carrying a neomycin resistance cassette and and thymidine, 1 mM pentostatin, and 0.05 mML-alanosine. Depending cytomegalovirus promoter-driven insert (originally from Dr. M. Stin- upon confluence, cells were fed every 2nd or 3rd day. sky, University of Iowa); pBAT14, used as a shuttle vector, was from Dr. For Tg transfection, subconfluent cell cultures were rinsed with PBS M. German (University of California, San Francisco); pBR322 was and detached with trypsin-EDTA. A 0.25-ml suspension of 2 3 10 cells purchased from Upstate Biotechnology Inc. Co-vidarabine (pentostatin, was transfected with the full-length Tg cDNA in pCB6 (20 mg) in a inhibitor of adenosine deaminase) was from Parke-Davis. L-Alanosine 0.4-cm pass electroporation cuvette at 330 V and 250 mF (time constant was obtained from the Drug Synthesis and Chemistry Branch, Devel- ;14 ms); cells were then diluted 400-fold and plated. After 2 days in opmental Therapeutics Program, Division of Cancer Treatment, NCI) culture, selection was started by addition of 0.8 mg/ml geneticin. Colo- as distributed by Ogden BioServices Corporation. Dithiobis(succinim- nies were picked and screened for Tg expression by immunofluores- dyl propionate) (DSP) was purchased from Pierce. Restriction enzymes, cence; media and cell lysates from positive clones were then analyzed by T4 DNA ligase, and recombinant endoglycosidase H were from New immunoblotting. At least two Tg-expressing clones of each type were England Biolabs (Beverly, MA). A polyclonal rabbit antiserum was further studied; by initial characterization, the results obtained with raised against denatured Tg as described previously (35). Antibody to replicate clones were essentially identical (e.g. see Fig. 4), thus the data ribophorin II was the kind gift of Dr. D. Meyer (University of California, presented in this report all derive from representative clones. Los Angeles). An immunoprecipitating polyclonal antiserum to GRP94 Immunofluorescence—Cells grown on coverslips were rinsed with (36) was kindly provided by Dr. P. Srivastava (Fordham University, PBS, fixed for 15 min at room temperature with 4% formaldehyde, and Bronx, NY). A polyclonal antiserum to BiP was purchased from Stress- permeabilized in PBS containing 0.2% (v/v) Triton X-100 and 1 mg/ml Gen (Victoria, Canada). Polyclonal antibodies to protein disulfide bovine serum albumin. Incubation with primary antibody was carried isomerase and ER60 were from Dr. T. Wileman (Pirbright Laboratories, outfor1hat room temperature in the permeabilization buffer. Cells Surrey, UK), and polyclonal antibodies to GRP94, calnexin, ERp72, and were then rinsed with this buffer and incubated for another hour with calreticulin (18) were also provided by Dr. P. Kim (Beth Israel Hospital, a 1:400 dilution of a rhodamine-conjugated, affinity-isolated goat anti- Boston, MA). A rhodamine-conjugated, affinity isolated, goat anti-rab- rabbit IgG. The rinsed and mounted specimens were examined with a bit IgG was purchased from Tago, Inc. (Burlingame, CA), and an alka- Zeiss microscope equipped with epifluorescence optics. line phosphatase-conjugated goat anti-rabbit IgG was from Life Tech- Metabolic Labeling—CHO cells were washed twice with Cys-free, nologies, Inc. The serine protease inhibitor 4-(2-aminoethyl)- Met-free medium, prior to pulse-labeling for either 10 or 20 min at 37 °C 125 35 benzenesulfonyl fluoride was from ICN (Costa Mesa, CA). I-labeled in the same medium containing 0.5 mCi/ml [ S]cysteine and methio- protein A was purchased from NEN Life Science Products. Zysorbin was nine. At the conclusion of the pulse labeling, the cells were washed and from Zymed Labs (San Francisco, CA). [ S]Cysteine/methionine chased in normal growth medium. For long term labeling to approach 35 35 35 (Expre S S) and pure [ S]methionine were from NEN. Other tissue steady state, the medium was not deficient (i.e. the medium contained culture reagents, protease inhibitors, and stock chemicals were either unlabeled cysteine and methionine, plus serum, at half the usual con- from Life Sciences or Sigma. centration of that in complete growth medium), and labeling was for Construction of Full-length Tg cDNA—Until now, no full-length Tg 20 –24 h. In preliminary experiments (not shown), results from exper- cDNA has ever been successfully prepared. For this reason, our strategy iments analyzed by two-dimensional SDS-PAGE (like those shown in involved subcloning four contiguous partial cDNAs (37), kindly pro- Figs. 7 and 8) were not significantly affected by continuous labeling vided by Dr. G. Vassart (University Libre, Brussels, Belgium), to create times ranging from 8 to 24 h, although labeled amino acid incorporation a cDNA corresponding to base pairs 519 – 8430 (i.e. from a conserved was proportional to the length of the labeling period. For the experi- NcoI site to the 39-end) of the bovine Tg coding sequence (Fig. 1). Using ment shown in Fig. 10, the continuous labeling employed 400 mCi/ml the rat Tg-2 cDNA (38) obtained from Dr. P. Graves (Mt. Sinai School of pure [ S]methionine. Medicine, New York), the remaining Tg coding sequence (i.e. from the Chemical Cross-linking and Immunoprecipitation—For chemical extreme 59-end to the conserved NcoI site) was provided. Thus, the final cross-linking, cells were rinsed with PBS and then incubated for 30 min full-length cDNA we employed encodes for a polypeptide that exhibits at room temperature with 200 mM DSP in PBS diluted from a solution overall 99.1% identity to normal bovine Tg, with the remaining 0.9% in Me SO (0.05% final concentration). Uncross-linked controls were being conservative bovine 3 rat Tg substitutions contained within the incubated in parallel with PBS containing the carrier only. The cross- first 155 amino acids. linking reaction was terminated by lysis of cells in 3% SDS in 62.5 mM For stepwise construction, a fragment of the rat Tg-2 cDNA extend- Tris, pH 6.8, containing protease inhibitors (1 mg/ml leupeptin, 1 mg/ml ing from an EcoRI site (17 bases upstream from the translational start pepstatin, 5 mg/ml EDTA, and 0.4 mg/ml 4-(2-aminoethyl)-benzenesul- site) to NcoI (Fig. 1) was ligated into the shuttle vector pBAT14 along fonyl fluoride) 6 50 mM iodoacetamide. No difference in results were with a partial bovine Tg cDNA extending from the conserved NcoI site obtained in either the presence or absence of the alkylating agent ER Residence Time of Thyroglobulin 26097 during lysis (indeed, previous detailed investigations of Tg folding have established that detection of high molecular mass disulfide-bonded Tg complexes, as shown in this report, is not due to the presence or absence of alkylating agent during cell lysis) (28, 29). The cell lysate was boiled for 3 min, passed 15 times through a 25-gauge needle, and then spun in a Microfuge for 10 min at 4 °C to remove debris. An aliquot from this supernatant was 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). 1 ml of the diluted cell lysate was preabsorbed for 30 min at room temperature with 50 mlofa 10% suspension of fixed Staphylococcus aureus (Zysorbin). The suspen- sion was pelleted in a Microfuge for 2 min at 3,000 rpm, and the supernatant was incubated for 16 h at 4 °C with 10 ml of polyclonal rabbit anti-Tg. 50 ml of a 10% suspension of Zysorbin was then added, and immune complexes were allowed to adsorb for1hat4 °C. Pellets of FIG.2. Levels of ER resident proteins in CHO cells genetically this suspension were washed once in immunoprecipitation buffer, once engineered for selectively increased expression of GRP94. A, in 0.5% Tween-20 in TBS (25 mM Tris, 150 mM NaCl, pH 7.4), once in equal amounts of total cellular protein from parental CHO cells (P) and TBS, and finally in water, before boiling for 5 min in 20 – 40 mlof2 3 CHO cells selected for amplified expression of GRP94 (G), were immu- sample buffer in the presence or absence of 10% b-mercaptoethanol. noblotted on nitrocellulose with antibodies either to GRP94 (M For quantitative determination of Tg by immunoprecipitation, the ;94,000) or BiP (M ;78,000), followed by I-labeled protein A. B, amount of cell lysate obtained from different clones was quantitated by quantitation of phosphorimages shows the amount of GRP94 or BiP DNA assay using bisbenzimide fluorescence with a Hoefer (San Fran- expressed relative to that in parental cells (error bars 5 S.D.). C, cisco, CA) Mini-Fluorometer, according to the protocol provided by the immunoblots of cell extracts similarly analyzed for lumenal ER resident manufacturer. proteins calreticulin (M ;58,000), protein disulfide isomerase (PDI, M r r For sequential immunoprecipitation, cell lines labeled to approach ;59,000), ERp72 (M ;72,000), ER60 (M ;60,000), and for ER mem- r r steady state and cross-linked with DSP, were first immunoprecipitated brane proteins ribophorin II (M ;65,000) and calnexin (M ;90,000). r r with anti-Tg as described above. These immunoprecipitates were eluted The levels of these proteins appear similar in CHO-P and CHO-G cells. from Zysorbin for1hat60 °Cin50 ml of 1% SDS plus 62.5 mM Tris, pH 6.8. The supernatant was then diluted to 1 ml in immunoprecipitation buffer containing 1 mg/ml unlabeled bovine Tg (Sigma) and mixed with GRP94 had no significant effect on the steady state level of BiP 2 ml of anti-GRP94. Final immunoprecipitates adsorbed to Zysorbin (Fig. 2, A and B). Further, compared with CHO-P cells, there were eluted by boiling for 4 min in 30 ml of sample buffer containing was no appreciable change in the total amount of rough ER in b-mercaptoethanol. CHO-G cells, based on the levels of ribophorin II and calnexin Digestion with Endoglycosidase H—Cell lysates prepared in 0.5% (Fig. 2C), which are among the membrane proteins that appear SDS, 1% b-mercaptoethanol in 50 mM sodium citrate, pH 5.5, plus protease inhibitors were boiled for 5 min and then either digested or to reflect the cellular quantity of rough ER (41, 42). Moreover, mock digested for 1 h at 37 °C as described previously (35). The samples increased expression of GRP94 did not alter the concentrations were then diluted to 1 ml and immunoprecipitated with anti-Tg. of any of the other ER lumenal resident proteins that were Immunoblotting—For immunoblot analysis, samples resolved on examined, calreticulin, PDI, ERp72, and ER60 (Fig. 2C). Thus, SDS-gels were electrophoretically transferred to nitrocellulose. The the levels of GRP94 appear selectively increased in CHO-G membrane was blocked for 1 h with 3% gelatin in TBS plus 0.5% cells. Tween-20, incubated for 1 h with primary antibody in the same solu- tion, and then washed three times. The blot was then incubated for 1 h Expression of Recombinant Tg in CHO Cell Lines—We next with a 1:3,000 dilution of alkaline phosphatase-conjugated goat anti- proceeded to constitutively express an 8.4-kilobase pair cDNA rabbit IgG, rinsed several times, and then reacted with 4-nitro blue encoding full-length, nonmutant Tg (Fig. 1, see “Experimental tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate. In the Procedures”). CHO-P and CHO-G cells were each stably trans- immunoblots of Fig. 2, the lanes were loaded based on total cellular fected, and clones were selected initially for neomycin resist- protein, and the secondary reagent employed was radioiodinated pro- ance and then screened for Tg expression by immunofluores- tein A; in this case, bands were quantitated by phosphorimaging. It is theoretically possible that normalizing to total cellular protein could cence and immunoblotting. As shown in Fig. 3A, in comparison unintentionally introduce small quantitative differences from that to mock-transfected cells, CHO-P and CHO-G cell lines each which might be obtained upon normalizing samples to cellular DNA, stably expressed the Tg polypeptide, although the relative Tg but we can estimate that any such error, if it existed, would be #10% pool sizes in these lines appeared to differ (see below). and would affect neither the outcome nor the conclusions obtained from In thyroid epithelial cells, most newly synthesized Tg follows these experiments. a high probability pathway that concludes with homodimeriza- Two-dimensional SDS-PAGE—Tg immunoprecipitates were sepa- rated in the first dimension under nonreducing conditions by 4% SDS- tion, arrival in the medial Golgi compartment, and secretion PAGE. For the second dimension, the samples were reduced with 10% from cells (28), while mutant Tg that exhibits defective ho- b-mercaptoethanol in sample buffer and resolved by 6.5% SDS-PAGE. modimer formation is quantitatively deficient in ER exit and Single dimensional gels used acrylamide concentrations that varied arrival in the extracellular space (18). This provides a useful depending upon the particular experiment. correlation between proper Tg folding/assembly and its subse- RESULTS quent secretion. With this in mind, when screening conditioned Characterization of CHO Cell Lines with Normal and In- media from Tg-transfected CHO-P and CHO-G cells, all clones creased Expression of GRP94 —To evaluate potential effects of were found to secrete a full-length polypeptide that could be GRP94 on Tg maturation and intracellular transport, we set detected by immunoprecipitation (Fig. 3B) and immunoblotting out to examine CHO cells that maintain either wild-type levels with anti-Tg (Fig. 3C, lanes 1–3). Even without immunoprecipi- of ER chaperones (parental CHO-P cells), or pooled transfor- tation, Tg was readily observed as an autoradiographic band mants achieving elevated levels of GRP94 (CHO-G). Using the from the SDS-PAGE analysis of secretion from labeled, trans- adenosine deaminase co-amplification system (40), GRP94 ex- fected cells (Fig. 3C, lanes 4 and 5). These results indicate that pression in CHO-G cells detected by immunoblotting (Fig. 2A) at least a fraction of recombinant Tg attains a conformation was increased ;13-fold, on average (see Fig. 2B). allowing for intracellular transport. Since increased abundance of GRP94 might indirectly affect Protracted Residence of Tg in the ER as a Consequence of Tg trafficking by displacing other ER resident proteins (56, 66, Increased Availability of GRP94 —To determine the extent to 67), we tested the levels of several other ER proteins by specific which increased availability of GRP94 might influence the immunoblotting (Fig. 2). Importantly, increased expression of intracellular residence of Tg, we compared Tg synthesis in the 26098 ER Residence Time of Thyroglobulin FIG.4. Relative synthesis and steady state level of Tg in dif- ferent CHO clones. A, two independent Tg-expressing clones of each type were metabolically labeled with S-labeled amino acids for either 20 min (pulse labeling) or 20 h (steady state labeling). Equal amounts of cell lysate (normalized to DNA content) were immunoprecipitated with anti-Tg, and analyzed by SDS-PAGE and phosphorimaging. B, quantitative comparison of intracellular Tg in CHO-P (P) and CHO-G (G) cells after pulse-labeling (open bars) or steady state labeling (closed bars). In each case, the labeled, immunoprecipitated Tg from CHO-P cells was arbitrarily assigned one unit (error bars 5 S.D.). As the specific radioactivity in the two different types of labeling experiments FIG.3. Stable expression and secretion of recombinant Tg in are markedly different, note that this graph cannot be used to compare CHO cells. A, immunoblot for Tg in lysates from mock transfected the absolute amount of newly made Tg protein to the amount of Tg CHO cells (lane 1), CHO-P (P, lane 2), or CHO-G (G, lane 3) cells stably protein contained within CHO cells at steady state. transfected with a cDNA encoding full-length Tg. The band shown has the identical electrophoretic mobility to that of purified bovine Tg. B, immunoprecipitation with anti-Tg from medium collected from CHO cells that had been mock-transfected (2, lane 1) or transfected with a Tg cDNA in CHO-P or CHO-G cells (lanes 2 and 3), after continuous labeling for 20 h with S-labeled amino acids. The immunoprecipitated recombinant protein is of the same mobility as purified bovine Tg (marked with arrow). C, CHO cells mock-transfected (lanes 1 and 4)or after Tg transfection into CHO-P cells (lanes 2 and 5) were labeled, and the media were collected as in B. The media were subjected to SDS- PAGE and electrophoretic transfer to nitrocellulose. The same transfer membrane was first autoradiographed (lanes 4 and 5) and then directly immunoblotted with anti-Tg and an alkaline phosphatase-coupled sec- ondary antibody (lanes 1 and 2). In lane 3, identically analyzed medium from a separate immunoblotting experiment using CHO-G cells is aligned for comparison. The mobilities of full-length bovine Tg and myosin molecular mass standards are shown at right. different clones (as reflected by S-labeled amino acid incor- poration into full-length Tg during a 20-min pulse) to the intracellular Tg accumulated at steady-state (as measured by FIG.5. Tg progression through the secretory pathway is re- S-Tg content of cells continuously radiolabeled for 20 h). Two tarded in cells with increased available GRP94. Clones of CHO-P independent lines of CHO-P and CHO-G cells were analyzed. and CHO-G cells at roughly comparable cell density were metabolically In each case, labeled intracellular Tg was recovered by immu- labeled with S-labeled amino acids for 10 min. After labeling, noncu- mulative hourly collections of media were analyzed by SDS-PAGE and noprecipitation from cell extracts, normalized for DNA content, phosphorimaging. One clone from each duplicate is shown in the half- and analyzed by SDS-PAGE and phosphorimaging (Fig. 4A). tone panels (at left). Identity of the Tg band was confirmed by immu- From this analysis, the Tg synthetic rate in CHO-G transfec- noprecipitation with anti-Tg (not shown). Relative band intensity of tants, on average, was found to be 1.7-fold compared with that labeled Tg secreted by a given clone type at each chase time is shown as the mean of duplicates (at right). No attempt was made to normalize the of parental cells (Fig. 4B, open bars). Remarkably, however, in data for cell number between different sets of clones; but within each CHO-G clones, intracellular Tg at steady state was elevated duplicate, variation in the total amount of labeled Tg protein secreted out of proportion to the synthetic rate, i.e. compared with over 10 h averaged ;9% for P cells and ;2% for G cells. Importantly, parental cells, CHO-G cells maintained 11.6-fold more intra- the maximum rate of newly synthesized Tg secretion by CHO-G cells (dashed line) appeared to be delayed ;2 h over that seen in CHO-P cellular Tg (Fig. 4B, closed bars). cells. The increase in intracellular Tg pool size as a consequence of increased availability of GRP94 suggested the possibility of slowed Tg egress from these cells. To test this point, we exam- cellular accumulation of Tg, immunofluorescence with anti-Tg ined the rate of newly synthesized Tg secretion by noncumula- was performed in stably transfected CHO cells. Unlike in un- tive hourly collections of chase medium after pulse labeling. In transfected CHO cells, a fine reticular fluorescence throughout CHO-G cells the maximum rate of labeled Tg release was the cytoplasm, characteristic of the ER, was observed both in delayed $2 h over that seen in CHO-P cells (Fig. 5). Thus, Tg-transfected CHO-P and CHO-G cells (Fig. 6A). This fluores- increased availability of GRP94 markedly prolonged the intra- cence pattern appeared essentially superimposable with that cellular residence of Tg. observed using antibodies to the ER chaperones, BiP and To identify the compartment containing the greatest intra- GRP94 (not shown). To confirm the Tg distribution, lysates of ER Residence Time of Thyroglobulin 26099 FIG.6. Intracellular Tg is contained in a pre-Golgi compart- ment. A, by indirect immunofluorescence, Tg in CHO-P and CHO-G cells show a fine, reticular labeling pattern characteristic for the ER. When primary antibody was omitted, no immunofluorescence signal was detected in these cells (not shown). To compensate for the increase in intracellular Tg (Fig. 4) in CHO-G cells for which film exposure was 30 s, the exposures shown for CHO-P cells not transfected (NT)or transfected with Tg (P) were lengthened (60 s) to increase signal in these cells. Bar 5 10 mm. B, CHO cell clones were labeled to approach steady state with S-labeled amino acids, extracted, and either mock- digested (2) or digested with endoglycosidase H (1), followed by immu- noprecipitation with anti-Tg and analysis by SDS-PAGE and autora- diography. After digestion, essentially all intracellular Tg shifted down upon SDS-PAGE, indicating that it is contained in a pre-Golgi compart- ment. By contrast, Tg molecules that passed through the Golgi complex FIG.7. Accumulation of disulfide-linked Tg complexes in CHO and were released into the medium bathing CHO-G cells (Med)or cells with increased availability of GRP94. Tg-expressing CHO CHO-P cells (not shown), were resistant to endoglycosidase H digestion. cells (right-sided panels) and control cells not expressing Tg (upper left panel) were labeled overnight with radioactive [ S]cysteine and methi- onine and lysed under SDS-denaturing, nonreducing conditions. Tg steady-state labeled cells were digested with endoglycosidase immunoprecipitates were then analyzed by two-dimensional SDS- H, immunoprecipitated for Tg, and analyzed by 4% SDS-PAGE PAGE as described in the text. Even in the absence of cross-linker, (Fig. 6B). By this assay, the fraction of intracellular Tg residing CHO-G (G) cells demonstrate increased abundance of disulfide-linked Tg complexes that run at a high molecular mass in the first (horizontal) in Golgi/post-Golgi compartments was undetectable, since vir- dimension (right of dashed line) and run broadly around the Tg position tually all was sensitive to digestion, although recombinant Tg in the second (vertical) dimension. The migration of immunoprecipi- secreted from CHO cells was entirely resistant to endoglycosi- table Tg by reducing 6.5% SDS-PAGE is shown (bottom left panel)ina dase H (Fig. 6B). Evidently, the transit time for recombinant single dimension lane. Note that the high molecular mass Tg-contain- ing complexes in CHO-G cells are not detected at the usual exposures of Tg through Golgi/post-Golgi compartments is sufficiently fast parental CHO-P (P) cells or untransfected control cells (upper left such that endoglycosidase H-resistant Tg does not accumulate panel). within the CHO cells. Thus, even though Tg spends most of its intracellular residence time in the ER of both CHO-P and CHO-G cells, the data in Figs. 4 – 6, taken together, indicate some of the Tg molecules were spread along the horizontal, that retention of Tg in the ER is substantially increased with indicating separation in the first, nonreducing dimension. The increased availability of GRP94. majority of recovered Tg appeared as the uncomplexed species Tg Retained in the ER Is Bound to GRP94 —One simple way (left of dashed line), and “trailing” of this form (just left of the to explain the prolonged Tg residence and expanded Tg pool in dashed line) suggested a range of Tg folding states with differ- CHO-G cells is chaperone-mediated retention of Tg in the ER. ent numbers of intrachain disulfide bonds. In addition, how- Support for such a model would require direct proof of trapping ever, in CHO-G cells, a fraction of immunoprecipitable Tg was Tg in complexes that include the chaperone whose availability broadly smeared in the high molecular mass region (right of is increased. Transient disulfide-linked Tg complexes associ- dashed line). The apparent molecular mass of this fraction of ated with ER chaperones have been found early in the matu- Tg is incompatible with uncomplexed Tg monomers or native ration of wild-type Tg; however, at a moment in time in the homodimers (which run as monomers upon SDS-PAGE). Per- steady state in normal thyrocytes, the quantity of these com- sistent band broadening of the high molecular mass fraction of plexes is at nearly undetectable levels (14, 28, 29, 43). We Tg in the second (vertical) dimension suggests incomplete di- therefore sought to determine whether such complexes might sulfide reduction after the first-dimensional gel. These results accumulate when recombinant nonmutant Tg was expressed in indicate that a meaningful fraction of immunoprecipitable Tg CHO cells with increased availability of GRP94. After labeling had accumulated in disulfide-linked complexes as a conse- overnight to approach steady state, the cells were extracted quence of increased abundance of GRP94. Interestingly, when under denaturing and nonreducing conditions, immunoprecipi- looking directly beneath this high molecular mass fraction of tated for Tg, and then analyzed in a first dimension by nonre- Tg on the two-dimensional gels of CHO-G cells, there was no ducing 4% SDS-PAGE, followed by a second dimensional 6.5% apparent detection of associated chaperones (Fig. 7). However, SDS-PAGE under reducing conditions. The intention of this such a result was not unexpected, since ER chaperones do not two-dimensional gel system was to segregate disulfide-linked remain associated with exportable proteins under SDS-PAGE Tg complexes from uncomplexed Tg in the first dimension, and conditions. then to dissociate disulfide-linked complexes in the second For this reason, the same protocol was followed in cells dimension. exposed to the membrane-permeant, thiol-cleavable cross-link- From this analysis (Fig. 7), Tg molecules were specifically ing reagent, DSP, which has been found to be required for obtained from both CHO-P and CHO-G cells, although the preserving protein associations with GRP94 (18, 21, 26, 27, 44). forms of Tg recovered in the two types of CHO cells were not Once again, radioactive background areas were detected in entirely identical. Radioactive background bands could be de- control CHO cells treated with the cross-linker that had not tected in control CHO cells that did not express Tg (Fig. 7, been transfected to express Tg (Fig. 8, upper left panel). In upper left panel) while the expected mobility of labeled Tg from CHO-P cells expressing Tg (upper right panel), the addition of CHO-G cells upon reducing 6.5% SDS-PAGE is indicated by a cross-linker did not shift Tg from the uncomplexed position (left sample run only in this dimension (Fig. 7, lower left panel). of dotted line) to the high molecular mass position (right of Interestingly, in the two-dimensional gels (Fig. 7, right panels), dotted line). Moreover, in CHO-G cells expressing Tg (lower 26100 ER Residence Time of Thyroglobulin FIG.9. A 78-kDa band cross-linked to immunoprecipitable Tg co-migrates with authentic BiP. CHO-G cells, metabolically labeled as in Fig. 7, were lysed and immunoprecipitated with anti-BiP (lane 2). Alternatively, the cells were cross-linked and immunoprecipitated with anti-Tg (lane 1), in which bands of 94 kDa (identified as GRP94, see Fig. 10) and 78 kDa were co-precipitated as in Fig. 8. In both cases, the samples were then analyzed by reducing 5% SDS-PAGE and phosphorimaging. FIG.8. In CHO-G cells, disulfide-linked Tg complexes include bound GRP94. After labeling with radioactive [ S]cysteine and me- thionine as in Fig. 7, Tg-expressing CHO cells (right-sided panels) and control cells not expressing Tg (upper left panel) were exposed to the membrane-permeant cross-linker, DSP, as described under “Experi- mental Procedures.” Tg immunoprecipitates were then analyzed by two-dimensional SDS-PAGE as in Fig. 7. In the second dimension, discrete bands of ;94 and ;78 kDa can be seen to underlie disulfide- linked Tg complexes. In the single dimension lane (bottom left panel), the migration of immunoprecipitable GRP94 by reducing 6.5% SDS- PAGE is shown, as well as a second band of 78 kDa, which could either represent a cross-reaction with BiP or recovery of protein complexes that include both proteins. Secondary immunodetection, described in Fig. 10, confirmed the identity of the 94-kDa protein as GRP94, while FIG. 10. Composition of TgzGRP94 complexes. Tg-expressing the 78-kDa protein co-migrates with authentic immunoprecipitable BiP CHO-P and CHO-G cells were metabolically labeled for 22 h with from CHO-G cells (see Fig. 9). 35 [ S]methionine. Cells were either cross-linked with DSP or incubated with carrier and lysed under denaturing, nonreducing conditions. Cell lysates were immunoprecipitated first with anti-Tg and then eluted and right panel) a significant fraction of intracellularly accumu- reimmunoprecipitated with anti-GRP94. A phosphorimage is shown of lated Tg again appeared broadly in the first (horizontal) dimen- the labeled TgzGRP94 complexes analyzed by SDS-PAGE under reduc- sion, including high molecular mass material, suggesting the ing conditions. Note that Tg is very poor (,1% abundance) in methio- presence of multiple different Tg-containing complexes. Thus, nine but contains 122 cysteine residues, while GRP94 has only 3 Cys the specific forms of Tg recovered after cross-linking (Fig. 8) residues but contains 18 methionines. Thus, unlike Fig. 8 in which most of the Tg signal is derived from labeling with [ S]cysteine, the ratio of were not fundamentally different from those detected in the GRP94 to Tg in TgzGRP94 complexes appears much higher after label- absence of cross-linking (Fig. 7). Importantly in Fig. 8, how- ing with pure [ S]methionine, which is used to precisely calculate a ever, the second dimensional reducing gel allowed for release of GRP94zTg stoichiometry in the isolated complexes (see text). co-precipitated, Tg-associated proteins (described below), upon hydrolysis of the thiol-cleavable cross-linker. tween recombinant nonmutant Tg and GRP94 in CHO-G cells. In a single dimensional analysis, specific immunoprecipita- However, only in highly overexposed autoradiograms did the tion from CHO-G cells with a polyclonal anti-GRP94 (Fig. 8, parental CHO-P sample reveal a small fraction of high molec- lower left panel) directly demonstrates the position (arrow)of ular mass Tg in association with these bands, indicating that this band (as well as a band of molecular mass ;78 kDa, this particular (un)folded form of Tg was specifically accumu- arrowhead). Interestingly, when examining two-dimensional lated as a consequence of increased abundance of GRP94 in the gels from CHO-G cells (Fig. 8, lower right panel), a labeled band ER. of the identical mobility (arrow) was specifically co-precipitated Stoichiometry of Immunoprecipitable of TgzGRP94 Com- with Tg and could be observed to run directly beneath Tg in plexes—If export of recombinant Tg from CHO cells requires high molecular mass Tg complexes, suggesting the presence of escape from GRP94-mediated binding in the ER, then it would GRP94 in these Tg complexes. Unequivocal proof of the identity be predicted that the accumulation of Tg complexes in CHO-G of GRP94 in these complexes was obtained by denaturation of cells would be accompanied by an increase in GRP94zTg stoi- these complexes with SDS (and subsequent detergent dilution), chiometry as a consequence of increased binding of GRP94. To followed by a secondary direct immunoprecipitation with anti- test this prediction, cross-linked or uncross-linked cells that GRP94 (see below). In parallel experiments, the 78-kDa band, had been labeled with pure [ S]methionine to approach steady which also co-precipitates with Tg (Fig. 9, lane 1, marked by state, were extracted under nonreducing conditions and then arrowhead) was found to co-migrate with BiP immunoprecipi- sequentially immunoprecipitated, first with anti-Tg, and then tated from the same cells without cross-linker (Fig. 9, lane 2), with anti-GRP94, before analysis by reducing SDS-PAGE. Us- strongly suggesting the presence of BiP in Tg complexes. Fi- ing this protocol, GRP94 could be recovered only from cross- nally, it must be emphasized that, in Fig. 8, the uncomplexed linked samples (Fig. 10). Tg molecules (recovered to the left of the dashed line in the Using the known numbers of methionine residues in both two-dimensional gels of CHO-P and CHO-G cells) were associ- GRP94 and Tg, the molar ratio of the two proteins associated in ated with GRP94 and the 78-kDa band (likely to be BiP) to a these sequentially immunoprecipitated complexes was calcu- much lesser degree than were the high molecular mass Tg lated. Importantly, this stoichiometry doubled (from 3.1 to 6.0) complexes (lower right panel, right of dashed line). in cells in which GRP94 abundance was increased. These data These data clearly establish the formation of complexes be- strongly support the idea that increased GRP94 availability ER Residence Time of Thyroglobulin 26101 leads to increased GRP94 interaction with Tg, a potential together, strongly support the notion that direct interaction “substrate.” with GRP94 mediates delayed Tg folding and exit from the ER. First, we report that disulfide-linked Tg complexes, previously DISCUSSION found in thyrocytes at an early stage in the Tg folding pathway It has been known for some years that each secretory protein (28, 29), are specifically accumulated in CHO-G cells (Fig. 7). species exits the ER with its own distinct rate (45, 46). The time More importantly, co-immunoprecipitation studies establish spent preparing newly synthesized secretory proteins for ex- that this particular form of Tg is directly associated with port from the ER is thought to be comprised of two kinds of GRP94 (Fig. 8). Finally, the stoichiometry of GRP94zTg in these activities. First, individual secretory proteins enter the ER complexes is increased as a consequence of increased chaper- lumen in an unfolded state and spend a characteristic period of one availability (Fig. 10). In this context, we conclude that the time converting their raw primary structure into a native or binding of GRP94 to Tg slows its advance through folding and near-native conformation. Second, differences in rates of entry ER export pathways. into anterograde transport vesicles may contribute to distinct There is reason to believe that GRP94 itself is largely re- exit rates for different secretory proteins, possibly as a conse- tained in the ER by mechanisms sensitive to lumenal calcium quence of their biophysical properties (47) or possibly based on levels (56). A natural consequence of this is the tendency of presentation or association with putative cargo receptors (48 – GRP94-associated proteins to be co-retained in the ER com- 51). Interaction with and dissociation from resident ER chap- partment. Second, persistent association of GRP94 is likely to erones are likely to strongly influence both kinds of activities, interfere with delivery of secretory proteins to ER export ves- and therefore are likely to be important factors influencing the icles which (by poorly understood mechanisms) exclude the ER exit rates of exportable proteins. entry of ER lumenal resident proteins (57, 58). For nonmutant secretory proteins, ER chaperone associa- Although the secretory phenotype consequent to manipula- tions are likely to be near-maximal during the earliest stages of tion of GRP94 expression has never been previously examined, folding when certain features, such as exposed hydrophobic such studies have been performed with BiP. Interestingly, it patches, are not yet buried in the tertiary/quaternary struc- has been established that, in cells with diminished levels of ture. A positive role for chaperone binding in the exit pathway BiP, increased secretion of mutant plasminogen activator is of exportable proteins has been described as largely circum- observed (59). Moreover, in cells with increased BiP levels, stantial (see Introduction), but has been suggested based on diminished secretion of factor VIII or von Willebrand factor has pharmacological manipulations that influence either chaper- been observed (39, 60). Importantly, selectively increased ER one availability (14) or post-translationally modified chaperone chaperone expression does not produce a generalized effect on binding sites (52), as well as by experiments expressing ER anterograde protein traffic, as export is slowed only for those chaperones in certain model systems (53). Nevertheless, the secretory proteins to which chaperone binding can be detected hypothesis of ER chaperones as quality controllers (54, 55) (39, 61). In the present study, Tg, a protein shown to bind suggests the possibility that the extent of chaperone involve- GRP94, exhibits similarly delayed export from cells with in- ment may be an independent factor influencing the ER resi- creased levels of GRP94. Thus the data suggest that enhanced dence time of many normal secretory proteins. binding of ER chaperones of the hsp90 class (GRP94), like that It is now established that GRP94 molecules interact with of the hsp70 class (BiP), can influence export dynamics by newly synthesized thyroidal Tg transiently after normal Tg retarding the progression of suitable secretory protein “sub- synthesis, and for a protracted period in thyrocytes of animals strates” through folding and ER exit pathways. suffering from congenital goiter with deficient Tg (18). The These findings appear consistent with our view (14) that thyrocytes of animals and humans with this illness exhibit the during conformational maturation of Tg (as well as other se- unfolded protein response (7), which causes total thyroidal cretory proteins), ER chaperones go through cycles of dissoci- levels of GRP94 to be elevated (31). Of course, in those thyro- ation-reassociation; we have proposed that this cyclical associ- cytes, much of the GRP94 binds to accumulated mutant Tg, ation is the very means by which ER chaperones ordinarily such that the level of free GRP94 in the ER, if it rises at all, monitor the progress of secretory protein folding. At appropri- almost certainly does not exhibit the same fold increase as the ate moments in the normal Tg folding pathway, different do- total elevation of this chaperone. By contrast, to examine the mains and subdomains within nascent Tg take advantage of consequences of increased GRP94 binding in a system uncom- the limited period of dissociation from GRP94, or BiP, to bury plicated by instrinsic Tg defects, in this report we have exam- individual binding sites. During successful folding, chaperone ined the folding and export of nonmutant Tg after genetic rebinding decreases progressively to zero as conformational manipulation to increase levels of free GRP94 in the ER of CHO maturation proceeds to completion. Of course in mutant Tg cells. The results of this study indicate that increased avail- molecules, structural defect(s) may drastically extend the pe- ability of GRP94 causes major prolongation of Tg residence in riod needed to bury certain hydrophobic patches, thus, chaper- the ER, and this results in a significant increase in the ER pool ones perpetually rebind to these regions, resulting in sustained size of Tg in the steady state that is disproportionate to the rate chaperone association and retention in the ER (18). Further- of Tg synthesis (Figs. 4 – 6). more, based on the evidence presented herein, we propose that How are these results to be interpreted in terms of mecha- the permissible length of time for burying features character- nism of action of GRP94 with respect to Tg? The possibility that istic of GRP94 binding is inversely related to the availability of the observed phenotype occurs by meaningfully diminishing this chaperone in the ER. Under conditions where chaperone the levels of BiP or other chaperones with pro-folding activity, availability is increased, chaperone “on” time increases and would appear to be excluded by our measurements establishing “off” time is diminished to the point where one or more Tg that intracellular levels of these ER resident proteins did not domains do not have enough time to complete the folding step. change (Fig. 2). Instead, three new pieces of evidence, taken This leads to a bottleneck in the conformational maturation pathway (Fig. 7), an increased concentration of complexes at Increased occupancy and consequent redistribution of the KDEL receptor can also be excluded as an explanation for the retarded Tg export phenotype in CHO-G cells, because even when redistribution of evidence for perturbed flow of exportable proteins through the antero- this receptor is induced by lysozyme-KDEL overexpression, there is no grade transport pathway (66). 26102 ER Residence Time of Thyroglobulin 11. Machamer, C. E., Doms, R. W., Bole, D. G., Helenius, A., and Rose, J. K. (1990) steady state (Figs. 8 –10), prolonged residence in the ER (Figs. J. Biol. Chem. 265, 6879 – 6883 5 and 6) and an increase in ER pool size (Fig. 4). 12. Singh, I., Doms, R. W., Wagner, K. R., and Helenius, A. (1990) EMBO J. 9, 631– 639 There are certainly differences in the peptide binding speci- 13. Watowich, S. S., Morimoto, R. I., and Lamb, R. A. (1991) J. Virol. 65, ficity of BiP (62– 64) from that of GRP94; this is suggested by 3590 –3597 sequential binding of these chaperones during immunoglobulin 14. Kim, P., Bole, D., and Arvan, P. (1992) J. Cell Biol. 118, 541–549 15. Knittler, M. R., and Haas, I. G. (1992) EMBO J. 11, 1573–1581 chain maturation (44). However, in the immediate post-trans- 16. Chessler, S. D., and Byers, P. H. (1993) J. Biol. Chem. 268, 18226 –18233 lational period, the large Tg protein must fold 20 or more 17. Schmitz, A., Maintz, M., Kehle, T., and Herzog, V. (1995) EMBO J. 14, 1091–1098 different domains (65). Thus, it is perhaps not surprising that, 18. Kim, P. S., Kwon, O.-Y., and Arvan, P. (1996) J. Cell Biol. 133, 517–527 in thyrocytes, Tg may form complexes simultaneously with 19. Hendershot, L., Wei, J., Gaut, J., Melnick, J., Aviel, S., and Argon, Y. (1996) both GRP94 and BiP (26, 27), while dissociation of these two Proc. Natl. Acad. Sci. U. S. A. 93, 5269 –5274 20. Navarro, D., Qadri, I., and Pereira, L. (1991) Virology 184, 253–264 ER chaperones from Tg also appears kinetically indistinguish- 21. Melnick, J., Aviel, S., and Argon, Y. (1992) J. Biol. Chem. 267, 21303–21306 able (18). Our present data in CHO cells suggest that high 22. Clairmont, C. A., De Maio, A., and Hirschberg, C. B. (1991) J. Biol. Chem. 267, 3983–3990 molecular mass complexes of recombinant Tg include both 23. Li, Z., and Srivastava, P. K. (1993) EMBO J. 12, 3143–3151 GRP94 and a 78-kDa protein (Fig. 8) likely to be BiP (Fig. 9); 24. Csermely, P., Miyata, Y., Schnaider, T., and Yahara, I. (1995) J. Biol. Chem. while sequential immunoprecipitation, first with anti-Tg, and 270, 6381– 6388 25. Wearsch, P. A., and Nicchitta, C. V. (1997) J. Biol. Chem. 272, 5152–5156 then with anti-GRP94, still co-precipitates the 78-kDa protein, 26. Kuznetsov, G., Chen, L. B., and Nigam, S. K. (1994) J. Biol. Chem. 269, pointing to the likelihood of ternary complexes that include BiP 22990 –22995 27. Kuznetsov, G., Chen, L. B., and Nigam, S. K. (1997) J. Biol. Chem. 272, (Fig. 10). Moreover, after steady state labeling of CHO cells 3057–3063 genetically manipulated for selectively increased expression of 28. Kim, P. S., and Arvan, P. (1991) J. Biol. Chem. 266, 12412–12418 29. Kim, P. S., Kim, K.-R., and Arvan, P. (1993) Am. J. Physiol. 265, C704 –C711 BiP (39) rather than GRP94, recombinant TgzGRP94 complexes 30. Kim, P. S., and Arvan, P. (1993) J. Biol. Chem. 268, 4873– 4879 analyzed precisely the same way as that shown in Figs. 8 and 31. Medeiros-Neto, G., Kim, P. S., Yoo, S. E., Vono, J., Targovnik, H., Camargo, R., 10 appear identical except for a selective increase in the 78-kDa Hossain, S. A., and Arvan, P. (1996) J. Clin. Invest. 98, 2838 –2844 32. Beamer, W. G., Maltais, L. J., DeBaets, M. H., and Eicher, E. M. (1987) band. 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Published: Oct 1, 1997