Thyroglobulin Transport along the Secretory Pathway
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26095
ISSN1083-351X
Autores Tópico(s)Ion channel regulation and function
ResumoGRP94 serves as a molecular chaperone in the endoplasmic reticulum (ER). In normal thyrocytes, GRP94 interacts transiently with thyroglobulin (Tg), and in thyrocytes of animals suffering from congenital hypothyroid goiter with defective thyroglobulin, GRP94 and thyroglobulin associate in a protracted fashion. In order explore possible consequences of GRP94 binding, we have studied recombinant nonmutant thyroglobulin expressed in control Chinese hamster ovary (CHO) cells in comparison to that produced in CHO cells genetically manipulated for selectively increased GRP94 expression. Levels of ER chaperones other than GRP94 did not detectably differ, and thyroglobulin achieved transport competence in both kinds of CHO cells. However, increased availability of GRP94 caused the residence time of Tg in the ER to be remarkably prolonged. This was accompanied by a major increase in Tg directly associated with GRP94 and an increase in the ER pool size of Tg. Importantly, co-immunoprecipitation analysis revealed disulfide-linked Tg complexes (previously reported as an early Tg-folding intermediate) especially associated with GRP94. Indeed, non-native Tg, GRP94, and a 78-kDa protein likely to be BiP, appeared in ternary complexes. Under these conditions, GRP94 association appears directly involved in prolongation of Tg folding and export, consistent with a role in quality control in the ER. GRP94 serves as a molecular chaperone in the endoplasmic reticulum (ER). In normal thyrocytes, GRP94 interacts transiently with thyroglobulin (Tg), and in thyrocytes of animals suffering from congenital hypothyroid goiter with defective thyroglobulin, GRP94 and thyroglobulin associate in a protracted fashion. In order explore possible consequences of GRP94 binding, we have studied recombinant nonmutant thyroglobulin expressed in control Chinese hamster ovary (CHO) cells in comparison to that produced in CHO cells genetically manipulated for selectively increased GRP94 expression. Levels of ER chaperones other than GRP94 did not detectably differ, and thyroglobulin achieved transport competence in both kinds of CHO cells. However, increased availability of GRP94 caused the residence time of Tg in the ER to be remarkably prolonged. This was accompanied by a major increase in Tg directly associated with GRP94 and an increase in the ER pool size of Tg. Importantly, co-immunoprecipitation analysis revealed disulfide-linked Tg complexes (previously reported as an early Tg-folding intermediate) especially associated with GRP94. Indeed, non-native Tg, GRP94, and a 78-kDa protein likely to be BiP, appeared in ternary complexes. Under these conditions, GRP94 association appears directly involved in prolongation of Tg folding and export, consistent with a role in quality control in the ER. Thyroglobulin transport along the secretory pathway. Investigation of the role of molecular chaperone, GRP94, in protein export from the endoplasmic reticulum.Journal of Biological ChemistryVol. 272Issue 48PreviewThe grant information was incomplete. The first sentence in the title footnote should read: This work was supported by National Institutes of Health Grant DK40344 (to P. A.) and by National Institutes of Health Postdoctoral Fellowship DK09411 (to Z. M.). Full-Text PDF Open Access Secretory proteins are translocated into the lumen of the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; Tg, thyroglobulin; CHO, Chinese hamster ovary; CHO-P, CHO parental cells; CHO-G, GRP94-overexpressing cells; DSP, dithiobis(succinimdyl propionate); PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis. in an unfolded form that is biologically inactive and incompetent for intracellular transport. Prior to acquisition of native conformation, the nascent proteins are retained in the ER, where they associate with molecular chaperones (1Helenius A. Marquardt T. Braakman I. Trends Cell Biol. 1992; 2: 227-231Abstract Full Text PDF PubMed Scopus (255) Google Scholar). Two of the better recognized ER chaperones are family members of the hsp90 (GRP94) (2Mazzarella R.A. Green M. J. Biol. Chem. 1987; 262: 8875-8883Abstract Full Text PDF PubMed Google Scholar) and hsp70 classes (BiP) (3Haas I.G. Wabl M. Nature. 1983; 306: 387-389Crossref PubMed Scopus (593) Google Scholar,4Bole D.G. Hendershot L.M. Kearney J.F. J. Cell Biol. 1986; 102: 1558-1566Crossref PubMed Scopus (567) Google Scholar), which are abundant proteins in the normal ER (5Marquardt T. Hebert D.N. Helenius A. J. Biol. Chem. 1993; 268: 19618-19625Abstract Full Text PDF PubMed Google Scholar) and are further induced under stress conditions (6Lee A.S. Curr. Opin. Cell Biol. 1992; 4: 267-273Crossref PubMed Scopus (395) Google Scholar). One physiologically relevant example of such stress occurs in endoplasmic reticulum storage diseases, which include a large group of hereditary illnesses originating from mutations in secretory proteins or other exportable proteins that interfere with protein folding and exit from the ER (7Arvan P. Kim P.S. Kuliawat R. Prabakaran D. Muresan Z. Yoo S.E. Hossain S.A. Thyroid. 1997; 7: 89-105Crossref PubMed Scopus (38) Google Scholar) . In both normal secretory protein export and in ER storage diseases, the functions of ER chaperones remain poorly understood. BiP, perhaps the most studied ER chaperone, interacts transiently with certain wild-type exportable proteins and exhibits more prolonged interactions with certain mutant or unassembled exportable proteins (8Dorner A.J. Bole D.G. Kaufman R.J. J. Cell Biol. 1987; 105: 2665-2674Crossref PubMed Scopus (241) Google Scholar, 9Hurtley S.M. Bole D.G. Hoover-Litty H. Helenius A. Copeland C.S. J. Cell Biol. 1989; 108: 2116-2126Crossref Scopus (222) Google Scholar, 10Hendershot L.M. J. Cell Biol. 1990; 111: 829-837Crossref PubMed Scopus (142) Google Scholar, 11Machamer C.E. Doms R.W. Bole D.G. Helenius A. Rose J.K. J. Biol. Chem. 1990; 265: 6879-6883Abstract Full Text PDF PubMed Google Scholar, 12Singh I. Doms R.W. Wagner K.R. Helenius A. EMBO J. 1990; 9: 631-639Crossref PubMed Scopus (81) Google Scholar, 13Watowich S.S. Morimoto R.I. Lamb R.A. J. Virol. 1991; 65: 3590-3597Crossref PubMed Google Scholar, 14Kim P. Bole D. Arvan P. J. Cell Biol. 1992; 118: 541-549Crossref PubMed Scopus (101) Google Scholar, 15Knittler M.R. Haas I.G. EMBO J. 1992; 11: 1573-1581Crossref PubMed Scopus (144) Google Scholar, 16Chessler S.D. Byers P.H. J. Biol. Chem. 1993; 268: 18226-18233Abstract Full Text PDF PubMed Google Scholar, 17Schmitz A. Maintz M. Kehle T. Herzog V. EMBO J. 1995; 14: 1091-1098Crossref PubMed Scopus (46) Google Scholar, 18Kim P.S. Kwon O.-Y. Arvan P. J. Cell Biol. 1996; 133: 517-527Crossref PubMed Scopus (99) Google Scholar). Nevertheless, it has been difficult to clearly define a single post-translational role of BiP in the export of secretory proteins (see Introduction of Hendershot et al. (19Hendershot L. Wei J. Gaut J. Melnick J. Aviel S. Argon Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5269-5274Crossref PubMed Scopus (131) Google Scholar)). Even less is known about the role played by GRP94 in protein export, although its demonstrated ability to bind certain secretory proteins is also well established (20Navarro D. Qadri I. Pereira L. Virology. 1991; 184: 253-264Crossref PubMed Scopus (44) Google Scholar, 21Melnick J. Aviel S. Argon Y. J. Biol. Chem. 1992; 267: 21303-21306Abstract Full Text PDF PubMed Google Scholar). For instance, although it has been concluded that, in conjunction with its chaperone function in the ER, GRP94 is a molecule capable of ATP binding (22Clairmont C.A. De Maio A. Hirschberg C.B. J. Biol. Chem. 1991; 267: 3983-3990Abstract Full Text PDF Google Scholar), hydrolysis (23Li Z. Srivastava P.K. EMBO J. 1993; 12: 3143-3151Crossref PubMed Scopus (249) Google Scholar), and autophorphorylation activity (24Csermely P. Miyata Y. Schnaider T. Yahara I. J. Biol. Chem. 1995; 270: 6381-6388Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), these conclusions have recently been called into question by new experiments which suggest that peptide interaction with GPR94 is ATPindependent (25Wearsch P.A. Nicchitta C.V. J. Biol. Chem. 1997; 272: 5152-5156Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). We have been interested to know more about the effects of binding of GRP94 on the folding and export of thyroglobulin (Tg), the secretory protein precursor for thyroid hormone synthesis, and one of the reported "substrates" for GRP94 association in thyroid epithelial cells (18Kim P.S. Kwon O.-Y. Arvan P. J. Cell Biol. 1996; 133: 517-527Crossref PubMed Scopus (99) Google Scholar, 26Kuznetsov G. Chen L.B. Nigam S.K. J. Biol. Chem. 1994; 269: 22990-22995Abstract Full Text PDF PubMed Google Scholar, 27Kuznetsov G. Chen L.B. Nigam S.K. J. Biol. Chem. 1997; 272: 3057-3063Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Tg is a large (∼330 kDa) glycoprotein that undergoes substantial initial folding, including the formation of nearly 60 intrachain disulfide bonds, before homodimerization, which normally occurs in the ER. Remarkably, even when great precaution is taken to prevent formation of artifactual disulfide bonds at the time of cell lysis, immediately after translation in primary thyrocytes, newly synthesized nonmutant Tg is found in high molecular mass complexes, many containing improper interchain disulfide bonds (27Kuznetsov G. Chen L.B. Nigam S.K. J. Biol. Chem. 1997; 272: 3057-3063Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 28Kim P.S. Arvan P. J. Biol. Chem. 1991; 266: 12412-12418Abstract Full Text PDF PubMed Google Scholar, 29Kim P.S. Kim K.-R. Arvan P. Am. J. Physiol. 1993; 265: C704-C711Crossref PubMed Google Scholar). Subsequently the disulfide-linked Tg complexes become undetectable while Tg is observed to advance toward more folded forms of individual monomers (14Kim P. Bole D. Arvan P. J. Cell Biol. 1992; 118: 541-549Crossref PubMed Scopus (101) Google Scholar). During early folding, nonmutant Tg dissociates from GRP94 with kinetics superimposable upon those of BiP (18Kim P.S. Kwon O.-Y. Arvan P. J. Cell Biol. 1996; 133: 517-527Crossref PubMed Scopus (99) Google Scholar). These kinetics precede Tg homodimerization and its vesicular egress from the ER (14Kim P. Bole D. Arvan P. J. Cell Biol. 1992; 118: 541-549Crossref PubMed Scopus (101) Google Scholar). In thyrocytes as in other cell types, GRP94 levels are physiologically regulated (30Kim P.S. Arvan P. J. Biol. Chem. 1993; 268: 4873-4879Abstract Full Text PDF PubMed Google Scholar). Indeed, in humans suffering from congenital goiter with mutant Tg, thyrocytes can achieve levels of GRP94 that are elevated by ≥1 order of magnitude, which represents a greater increase than that observed for BiP (31Medeiros-Neto G. Kim P.S. Yoo S.E. Vono J. Targovnik H. Camargo R. Hossain S.A. Arvan P. J. Clin. Invest. 1996; 98: 2838-2844Crossref PubMed Google Scholar). Interestingly, in the cog/cog mouse (32Beamer W.G. Maltais L.J. DeBaets M.H. Eicher E.M. Endocrinology. 1987; 120: 838-840Crossref Scopus (46) Google Scholar, 33Taylor B.A. Rowe L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1986-1990Crossref Scopus (42) Google Scholar, 34Mayerhofer A. Amador A.G. Beamer W.G. Bartke A. J. Hered. 1988; 79: 200-203Crossref Scopus (21) Google Scholar), an animal model of this illness in which thyroid tissue is available for pulse-chase analysis, the fraction of Tg molecules that exits the ER is much lower than normal, while an increased fraction of Tg interacts with GPR94 in a prolonged manner (18Kim P.S. Kwon O.-Y. Arvan P. J. Cell Biol. 1996; 133: 517-527Crossref PubMed Scopus (99) Google Scholar). Unfortunately, congenital hypothyroid goiter is not a suitable model in which to independently examine the role of GRP94 binding in Tg maturation, because it is difficult to resolve the consequences of increased GRP94 binding from those related to intrinsic alterations in the biophysical properties of mutant Tg. For this reason, in the present report we have examined the conformational maturation and export of nonmutant Tg as a consequence of manipulating the probability of GRP94 binding to recombinant Tg in Chinese hamster ovary (CHO) cells engineered for wild-type or increased levels of GRP94 expression. Our results indicate that a major increase in the ER residence time and expansion of the ER pool of Tg occurs when the availability of GRP94 is increased, and this retention occurs as a direct consequence of complex formation between an apparent Tg folding intermediate and this ER chaperone. 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); pBAT14, used as a shuttle vector, was from Dr. M. German (University of California, San Francisco); pBR322 was purchased from Upstate Biotechnology Inc. Co-vidarabine (pentostatin, inhibitor of adenosine deaminase) was from Parke-Davis. l-Alanosine was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, NCI) as distributed by Ogden BioServices Corporation. Dithiobis(succinimdyl propionate) (DSP) was purchased from Pierce. Restriction enzymes, T4 DNA ligase, and recombinant endoglycosidase H were from New England Biolabs (Beverly, MA). A polyclonal rabbit antiserum was raised against denatured Tg as described previously (35Arvan P. Lee J. J. Cell Biol. 1991; 112: 365-376Crossref PubMed Scopus (35) Google Scholar). Antibody to ribophorin II was the kind gift of Dr. D. Meyer (University of California, Los Angeles). An immunoprecipitating polyclonal antiserum to GRP94 (36Maki R.G. Old L.J. Srivastava P.K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5658-5662Crossref PubMed Scopus (115) Google Scholar) 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, UK), and polyclonal antibodies to GRP94, calnexin, ERp72, and calreticulin (18Kim P.S. Kwon O.-Y. Arvan P. J. Cell Biol. 1996; 133: 517-527Crossref PubMed Scopus (99) Google Scholar) were also provided by Dr. P. Kim (Beth Israel Hospital, Boston, MA). 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 Life Technologies, Inc. The serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride was from ICN (Costa Mesa, CA). 125I-labeled protein A was purchased from NEN Life Science Products. Zysorbin was from Zymed Labs (San Francisco, CA). [35S]Cysteine/methionine (Expre35S35S) and pure [35S]methionine were from NEN. Other tissue culture reagents, protease inhibitors, and stock chemicals were either from Life Sciences or Sigma. Until now, no full-length Tg cDNA has ever been successfully prepared. For this reason, our strategy involved subcloning four contiguous partial cDNAs (37Mercken L. Simons M.-J. Swillens S. Massaer M. Vassart G. Nature. 1985; 316: 647-651Crossref PubMed Scopus (217) Google Scholar), kindly provided by Dr. G. Vassart (University Libre, Brussels, Belgium), to create a cDNA corresponding to base pairs 519–8430 (i.e. from a conserved NcoI site to the 3′-end) of the bovine Tg coding sequence (Fig.1). Using the rat Tg-2 cDNA (38Graves P.N. Davies T.F. Mol. Endocinol. 1990; 4: 155-161Crossref PubMed Scopus (24) Google Scholar) obtained from Dr. P. Graves (Mt. Sinai School of Medicine, New York), 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 rat Tg-2 cDNA extending from an EcoRI site (17 bases upstream from the translational start site) to NcoI (Fig. 1) was ligated into the shuttle vector pBAT14 along with a partial bovine Tg cDNA extending from the conserved NcoI site to aHindIII site at position 2826. The ligated insert was excised from pBAT14 using BamHI from the polylinker andHindIII, and then subcloned into the BglII andHindIII sites in the polylinker of pCB6, destroying the former site. The resulting plasmid was then digested withHindIII and the sole downstream BamHI site, and a partial Tg cDNA encoding the 3′-end (position 7446–8430) was directionally ligated (Fig. 1). Independently,HindIII-HindIII cDNA fragments extending from positions 2826–4764 and 4764–7446, respectively, were ligated together at the HindIII site of pBR322, and appropriately oriented subclones were selected from DNA minipreps. From this, the correctly ligated 4.6-kilobase pair 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 (Fig. 1) was confirmed by identity to the known bovine Tg restriction map (37Mercken L. Simons M.-J. Swillens S. Massaer M. Vassart G. Nature. 1985; 316: 647-651Crossref PubMed Scopus (217) Google Scholar). Two lines of CHO cells were graciously provided by Dr. A. Dorner (Genetics Institute, Cambridge, MA) for use in the present studies. The parental ("CHO-P" cell) line, containing endogenous levels of ER chaperones, is a dihydrofolate reductase-deficient line, previously called DUKX-B11 (39Dorner A.J. Wasley L.C. Kaufman R.J. EMBO J. 1992; 11: 1563-1571Crossref PubMed Scopus (300) Google Scholar). The GRP94-overexpressing ("CHO-G" cell) line was prepared as pooled transfectants overexpressing murine GRP94 (2Mazzarella R.A. Green M. J. Biol. Chem. 1987; 262: 8875-8883Abstract Full Text PDF PubMed Google Scholar) as a consequence of selection by co-amplification of adenosine deaminase (40Kaufman R.J. Methods Enzymol. 1990; 185: 537-566Crossref PubMed Scopus (148) Google Scholar). CHO cells were maintained in media based on α-minimum essential medium containing 1% penicillin-streptomycin. The medium for CHO-P cells also contained 10% fetal bovine serum and 10 mg/liter each of adenosine, deoxyadenosine, and thymidine, whereas CHO-G cell medium was supplemented with 10% heat-inactivated fetal bovine serum, 1 mm uridine, 1.1 mm adenosine, 10 mg/liter each of deoxyadenosine and thymidine, 1 μm pentostatin, and 0.05 mml-alanosine. Depending upon confluence, cells were fed every 2nd or 3rd day. For Tg transfection, subconfluent cell cultures were rinsed with PBS and detached with trypsin-EDTA. A 0.25-ml suspension of 2 × 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 μF (time constant ∼14 ms); 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 (e.g. see Fig. 4), thus the data presented in this report all derive from representative clones. 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% (v/v) Triton X-100 and 1 mg/ml bovine serum albumin. 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. The rinsed and mounted specimens were examined with a Zeiss microscope equipped with epifluorescence optics. CHO cells were washed twice with Cys-free, Met-free medium, prior to pulse-labeling for either 10 or 20 min at 37 °C in the same medium containing 0.5 mCi/ml [35S]cysteine and methionine. At the conclusion of the pulse labeling, the cells were washed and chased in normal growth medium. For long term labeling to approach steady state, the medium was not deficient (i.e. the medium contained unlabeled cysteine and methionine, plus serum, at half the usual concentration of that in complete growth medium), and labeling was for 20–24 h. In preliminary experiments (not shown), results from experiments analyzed by two-dimensional SDS-PAGE (like those shown in Figs. 7 and 8) were not significantly affected by continuous labeling times ranging from 8 to 24 h, although labeled amino acid incorporation was proportional to the length of the labeling period. For the experiment shown in Fig.10, the continuous labeling employed 400 μCi/ml pure [35S]methionine.Figure 8In CHO-G cells, disulfide-linked Tg complexes include bound GRP94. After labeling with radioactive [35S]cysteine and methionine 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 "Experimental 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 the 78-kDa protein co-migrates with authentic immunoprecipitable BiP from CHO-G cells (see Fig. 9).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Composition of Tg·GRP94 complexes.Tg-expressing CHO-P and CHO-G cells were metabolically labeled for 22 h with [35S]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 reimmunoprecipitated with anti-GRP94. A phosphorimage is shown of the labeled Tg·GRP94 complexes analyzed by SDS-PAGE under reducing conditions. Note that Tg is very poor (<1% abundance) in methionine but contains 122 cysteine residues, while GRP94 has only 3 Cys residues but contains 18 methionines. Thus, unlike Fig. 8 in which most of the Tg signal is derived from labeling with [35S]cysteine, the ratio of GRP94 to Tg in Tg·GRP94 complexes appears much higher after labeling with pure [35S]methionine, which is used to precisely calculate a GRP94·Tg stoichiometry in the isolated complexes (see text).View Large Image Figure ViewerDownload Hi-res image Download (PPT) For chemical cross-linking, cells were rinsed with PBS and then incubated for 30 min at room temperature with 200 μm DSP in PBS diluted from a solution in Me2SO (0.05% final concentration). Uncross-linked controls were incubated in parallel with PBS containing the carrier only. 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)-benzenesulfonyl fluoride) ± 50 mm iodoacetamide. No difference in results were obtained in either the presence or absence of the alkylating agent 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) (28Kim P.S. Arvan P. J. Biol. Chem. 1991; 266: 12412-12418Abstract Full Text PDF PubMed Google Scholar, 29Kim P.S. Kim K.-R. Arvan P. Am. J. Physiol. 1993; 265: C704-C711Crossref PubMed Google Scholar). 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 μl of a 10% suspension of fixed Staphylococcus aureus (Zysorbin). The suspension 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 μl of polyclonal rabbit anti-Tg. 50 μl of a 10% suspension of Zysorbin was 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 2 × sample buffer in the presence or absence of 10% β-mercaptoethanol. For quantitative determination of Tg by immunoprecipitation, the amount of cell lysate obtained from different clones was quantitated by DNA assay using bisbenzimide fluorescence with a Hoefer (San Francisco, CA) Mini-Fluorometer, according to the protocol provided by the manufacturer. For sequential immunoprecipitation, cell lines labeled to approach steady state and cross-linked with DSP, were first immunoprecipitated with anti-Tg as described above. These immunoprecipitates were eluted from Zysorbin for 1 h at 60 °C in 50 μl 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 2 μl of anti-GRP94. Final immunoprecipitates adsorbed to Zysorbin were eluted by boiling for 4 min in 30 μl of sample buffer containing β-mercaptoethanol. Cell lysates prepared in 0.5% SDS, 1% β-mercaptoethanol in 50 mm sodium citrate, pH 5.5, plus protease inhibitors were boiled for 5 min and then either digested or mock digested for 1 h at 37 °C as described previously (35Arvan P. Lee J. J. Cell Biol. 1991; 112: 365-376Crossref PubMed Scopus (35) Google Scholar). The samples were then diluted to 1 ml and immunoprecipitated with anti-Tg. For immunoblot analysis, samples resolved on 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-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate. In the immunoblots of Fig. 2, the lanes were loaded based on total cellular protein, and the secondary reagent employed was radioiodinated protein A; in this case, bands were quantitated by phosphorimaging. It is theoretically possible that normalizing to total cellular protein could unintentionally introduce small quantitative differences from that which might be obtained upon normalizing samples to cellular DNA, but we can estimate that any such error, if it existed, would be ≤10% and would affect neither the outcome nor the conclusions obtained from these experiments. Tg immunoprecipitates were separated in the first dimension under nonreducing conditions by 4% SDS-PAGE. For the second dimension, the samples were reduced with 10% β-mercaptoethanol in sample buffer and resolved by 6.5% SDS-PAGE. Single dimensional gels used acrylamide concentrations that varied depending upon the particular experiment. To evaluate potential effects of GRP94 on Tg maturation and intracellular transport, we set out to examine CHO cells that maintain either wild-type levels of ER chaperones (parental CHO-P cells), or pooled transformants achieving elevated levels of GRP94 (CHO-G). Using the adenosine deaminase co-amplification system (40Kaufman R.J. Methods Enzymol. 1990; 185: 537-566Crossref PubMed Scopus (148) Google Scholar), GRP94 expression in CHO-G cells detected by immunoblotting (Fig.2 A) was increased ∼13-fold, on average (see Fig. 2 B). Since increased abundance of GRP94 might indirectly affect Tg trafficking by displacing other ER resident proteins (56Booth C. Koch G.L.E. Cell. 1989; 59: 729-737Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 66Lewis M.J. Pelham H.R.B. Cell. 1992; 68: 353-364Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 67Sonnichsen B. Fullekrug J. Nguyen Van P. Diekmann W. Robinson D.G. Mieskes G. J. Cell Sci. 1994; 107: 2705-2717Crossref PubMed Google Scholar), we tested the levels of several other ER proteins by specific immunoblotting (Fig. 2). Importantly, increased expression of GRP94 had no significant effect on the steady state level of BiP (Fig. 2,A and B). Further, compared with CHO-P cells, there was no appreciable change in the total amount of rough ER in CHO-G cells, based on the levels of ribophorin II and calnexin (Fig.2 C), which are a
Referência(s)