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• W2055242244 abstract "We have previously shown that only a fraction of the newly synthesized human δ opioid receptors is able to leave the endoplasmic reticulum (ER) and reach the cell surface (Petäjä-Repo, U. E, Hogue, M., Laperrière, A., Walker, P., and Bouvier, M. (2000) J. Biol. Chem. 275, 13727–13736). In the present study, we investigated the fate of those receptors that are retained intracellularly. Pulse-chase experiments revealed that the disappearance of the receptor precursor form (M r 45,000) and of two smaller species (M r 42,000 and 39,000) is inhibited by the proteasome blocker, lactacystin. The treatment also promoted accumulation of the mature receptor form (M r55,000), indicating that the ER quality control actively routes a significant proportion of rescuable receptors for proteasome degradation. In addition, degradation intermediates that included full-length deglycosylated (M r 39,000) and ubiquitinated forms of the receptor were found to accumulate in the cytosol upon inhibition of proteasome function. Finally, coimmunoprecipitation experiments with the β-subunit of the Sec61 translocon complex revealed that the receptor precursor and its deglycosylated degradation intermediates interact with the translocon. Taken together, these results support a model in which misfolded or incompletely folded receptors are transported to the cytoplasmic side of the ER membrane via the Sec61 translocon, deglycosylated and conjugated with ubiquitin prior to degradation by the cytoplasmic 26 S proteasomes. We have previously shown that only a fraction of the newly synthesized human δ opioid receptors is able to leave the endoplasmic reticulum (ER) and reach the cell surface (Petäjä-Repo, U. E, Hogue, M., Laperrière, A., Walker, P., and Bouvier, M. (2000) J. Biol. Chem. 275, 13727–13736). In the present study, we investigated the fate of those receptors that are retained intracellularly. Pulse-chase experiments revealed that the disappearance of the receptor precursor form (M r 45,000) and of two smaller species (M r 42,000 and 39,000) is inhibited by the proteasome blocker, lactacystin. The treatment also promoted accumulation of the mature receptor form (M r55,000), indicating that the ER quality control actively routes a significant proportion of rescuable receptors for proteasome degradation. In addition, degradation intermediates that included full-length deglycosylated (M r 39,000) and ubiquitinated forms of the receptor were found to accumulate in the cytosol upon inhibition of proteasome function. Finally, coimmunoprecipitation experiments with the β-subunit of the Sec61 translocon complex revealed that the receptor precursor and its deglycosylated degradation intermediates interact with the translocon. Taken together, these results support a model in which misfolded or incompletely folded receptors are transported to the cytoplasmic side of the ER membrane via the Sec61 translocon, deglycosylated and conjugated with ubiquitin prior to degradation by the cytoplasmic 26 S proteasomes. endoplasmic reticulum cystic fibrosis transmembrane conductance regulator n-dodecyl-β-d-maltoside Dulbecco's modified Eagle's medium G protein-coupled receptor human δ opioid receptor human embryonic kidney 293S heat shock protein 70 Z-Leu-Leu-Leu-CHO N-ethylmaleimide polyacrylamide gel electrophoresis phenylmethylsulfonyl fluoride peptide-N-glycosidase F Z-Ile-Glu(OtBu)-Ala-Leu-CHO soybean trypsin inhibitor The endoplasmic reticulum (ER)1 quality control scrutinizes newly synthesized proteins entering the secretory pathway and ensures that only correctly folded and, in the case of multimeric proteins, fully assembled complexes are deployed to distal cellular compartments (1Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1064) Google Scholar). Those that fail to fulfill these criteria are retained in the ER and subsequently degraded. Even minor changes in the protein primary structure can cause retention in the ER. This is exemplified in many diseases, including cystic fibrosis and cases of α1-antitrypsin deficiency and familial hypercholesterolemia, which are characterized by an “ER storage phenotype” (2Kim P.S. Arvan P. Endocr. Rev. 1998; 19: 173-202PubMed Google Scholar). Although less thoroughly characterized, numerous naturally occurring mutations leading to ER retention of G protein-coupled receptors (GPCRs) have also been evoked as the cause of inherited diseases. For example, ER-retained mutants of rhodopsin, the luteinizing hormone receptor, and the V2-vasopressin receptor have been implicated in some forms of retinitis pigmentosa, male pseudohermaphroditism, and nephrogenic diabetes insipidus, respectively (3Sung C.-H. Schneider B.G. Agarwal N. Papermaster D.S. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8840-8844Crossref PubMed Scopus (404) Google Scholar, 4Latronico A.C. Segaloff D.L. Am. J. Hum. Genet. 1999; 65: 949-958Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Oksche A. Rosenthal W. J. Mol. Med. 1998; 76: 326-337Crossref PubMed Scopus (115) Google Scholar, 6Morello J.-P. Salahpour A. Laperrière A. Bernier V. Arthus M.-F. Lonergan M. Petäjä-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Invest. 2000; 105: 887-895Crossref PubMed Scopus (477) Google Scholar). Emerging evidence indicates that degradation of aberrant ER proteins is mediated by the cytosolic multiprotease complex, the 26 S proteasome. Indeed, an increasing number of misfolded or unassembled yeast and eukaryotic integral membrane and secreted proteins have been shown to be substrates for this disposal mechanism (7–26). However, proteasome-mediated degradation is not restricted to misfolded or unassembled proteins, as evidenced by the observation that some resident ER proteins are substrates for this degradation pathway in response to cellular signals (27McGee T.P. Cheng H.H. Kumagai H. Omura S. Simoni R.D. J. Biol. Chem. 1996; 271: 25630-25638Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 28Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (121) Google Scholar, 29Roberts B.J. J. Biol. Chem. 1997; 272: 9771-9778Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). It can thus be hypothesized that this degradation pathway might have a role in regulating steady state levels of normal wild type proteins traversing the secretory pathway, as has been described for many cytosolic and nuclear regulatory molecules (for review, see Refs. 30Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6907) Google Scholar and 31Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1596) Google Scholar). Reported examples pointing to this phenomenon are, however, scarce, exceptions being apolipoprotein B100 (32Fisher E.A. Zhou M. Mitchell D.M. Wu X. Omura S. Wang H. Goldberg A.L. Ginsberg H.N. J. Biol. Chem. 1997; 272: 20427-20434Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 33Yeung S.J. Chen S.H. Chan L. Biochemistry. 1996; 35: 13843-13848Crossref PubMed Scopus (170) Google Scholar), apolipoprotein(a) (34White A.L. Guerra B. Wangand J. Lanford R.E. J. Lipid Res. 1999; 40: 275-286Abstract Full Text Full Text PDF PubMed Google Scholar), and tyrosinase (35Halaban R. Cheng E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar). In a recent report, Schubert and co-workers (36Schubert U. Antón L.C. Gibbs J. Norbury C.C. Yewdell J.W. Bennink J.R. Nature. 2000; 404: 770-774Crossref PubMed Scopus (1) Google Scholar) showed that at least 30% of newly synthesized proteins might be degraded by the proteasomes. Whether these degraded proteins represent defective ribosomal products resulting from errors in translation, as suggested, or might also include correctly translated but incompletely folded proteins remains to be determined. Proteasome-mediated ubiquitin-dependent degradation of cytosolic and nuclear proteins is a well described phenomenon (for review, see Refs. 30Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6907) Google Scholar and 31Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1596) Google Scholar), but the detailed mechanisms involved in the ER-associated proteasomal degradation remain unclear. Recent findings have led to the emergence of models suggesting that misfolded or incompletely folded ER proteins are retrotranslocated to the cytosolic side of the ER membrane through the Sec61 translocon complex and conjugated with ubiquitin before hydrolysis by the 26 S proteasomes (for review, see Refs. 37Kopito R.R. Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 38Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar, 39Römisch K. J. Cell Sci. 1999; 112: 4185-4191Crossref PubMed Google Scholar). We have previously shown that the human δ opioid receptor (hδOR) is an example of integral membrane proteins that are partially retained in the ER, possibly due to difficulties in the folding process (40Petäjä-Repo U.E Hogue M. Laperrière A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Another example is the cystic fibrosis transmembrane conductance regulator (CFTR) (for review, see Ref. 41Kopito R.R. Physiol. Rev. 1999; 79: S167-S173Crossref PubMed Scopus (375) Google Scholar). Only about 40% and 25% of the newly synthesized wild type hδOR and CFTR molecules, respectively, are able to leave the ER and reach the cell surface. We thus set out to determine whether the ER-retained hδORs are targeted for degradation by the 26 S proteasomes and, if so, to delineate the mechanisms involved in this process. The results presented here indicate that these receptors are transported back to the cytosol, in a process that involves the Sec61 translocon, and deglycosylated before being degraded by the proteasomes. Furthermore, inhibition of the proteasomal pathway led to the accumulation of ubiquitinated receptor forms, pointing to the fact that polyubiquitination may be a targeting signal for disposal of misfolded or incompletely folded hδOR molecules. EXPRE35S35S protein labeling mix (1175 Ci/mmol) and [9-3H]bremazocine (26.6 Ci/mmol) were purchased from PerkinElmer Life Sciences. Recombinant peptide-N-glycosidase F (PNGase F) of Flavobacterium meningosepticum, purified from Escherichia coli (EC3.5.1.52) was from Roche Molecular Biochemicals. The proteasome inhibitors lactacystin, Z-Leu-Leu-Leu-CHO (MG-132) and Z-Ile-Glu(OtBu)-Ala-Leu-CHO (PSI) were obtained from Calbiochem. Cell culture reagents were either from Life Technologies Inc. or Wisent. The anti-FLAG M2 monoclonal antibody, the anti-FLAG M2 affinity resin, and the FLAG peptide were products of Sigma. The anti-calnexin (SPA-860), the anti-heat shock protein 70 (Hsp70) (SPA-820), and the anti-ubiquitin (SPA-200) antibodies were from Stressgen BiotechnologiesCorp. and Protein G-Sepharose from Amersham Pharmacia Biotech. The anti-Sec61β antibody was a generous gift from Dr. Tom A. Rapoport (Harvard Medical School, Boston, MA). All the other reagents were of analytical grade and obtained from various commercial suppliers. Cells were cultured in either 25-cm2 or 75-cm2 culture flasks and grown to 80–90% confluence at 37 °C in a humidified atmosphere of 5% CO2. Human embryonic kidney 293S (HEK-293S) cells stably expressing the hδOR tagged at the C terminus with the FLAG epitope (DYKDDDDK) (40Petäjä-Repo U.E Hogue M. Laperrière A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum, 1000 units/ml penicillin, 1 mg/ml streptomycin, 1.5 μg/ml fungizone (complete DMEM) and 400 μg/ml Geneticin. A clone expressing 10 pmol of receptor/mg of membrane protein was chosen for this study. For [35S]methionine/cysteine labeling, cells were first preincubated in methionine and cysteine-free DMEM for 60 min at 37 °C, and the labeling was performed in fresh medium containing 150 μCi/ml [35S]methionine/cysteine. After an incubation of 60 min at 37 °C, the pulse was terminated by washing the cells twice with the chase medium (complete DMEM supplemented with 5 mmmethionine) and chased for different periods of time as specified in the figures. When labeling was performed in the presence of the proteasome inhibitors, the reagents were added 3 h before the pulse labeling at a final concentration of 10 μm(lactacystin) or 25 μm (MG-132 and PSI). After the labeling, cells were washed with and harvested in phosphate-buffered saline and, unless otherwise indicated, quick-frozen in liquid nitrogen and stored at −80 °C. Total cellular lysates were prepared in buffer A (0.5% n-dodecyl-β-d-maltoside (DDM) (w/v), 25 mm Tris-HCl, pH 7.4, 140 mmNaCl, 2 mm EDTA, 5 μg/ml leupeptin, 5 μg/ml soybean trypsin inhibitor (STI), 10 μg/ml benzamidine, 2 μg/ml aprotinin, 0.5 mm phenylmethylsulfonyl fluoride (PMSF), 2 mm 1,10-phenanthroline). Insoluble material was removed by centrifugation at 5000 × g for 30 min, and bovine serum albumin was added to a final concentration of 0.1% (w/v). The receptor was immunoprecipitated using the anti-FLAG M2 affinity resin as described (40Petäjä-Repo U.E Hogue M. Laperrière A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). For the Sec61β immunoprecipitation, DDM was replaced by digitonin while NaCl, EDTA, and 1,10-phenanthroline were omitted from the buffer. For the ubiquitin immunoprecipitation,N-ethylmaleimide (NEM) was included in all buffers to distinguish the high molecular weight ubiquitinated receptor forms from putative aggregated receptors. Before immunoprecipitation of calnexin, Hsp70, Sec61β, and ubiquitin, samples were precleared for 60 min with 15 μl of Protein G-Sepharose. The appropriate antibody (dilutions: 1:200, 10 μg/ml, 1:100, and 1:100, respectively) and 15 μl of Protein G-Sepharose were then added and incubated overnight at 4 °C with gentle agitation. Following washing of the resin as described (40Petäjä-Repo U.E Hogue M. Laperrière A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), the bound antigens were eluted in SDS-sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v) bromphenol blue) at 95 °C for 5 min. If two or more consecutive immunoprecipitation steps were performed, the antigens were eluted with 1% (w/v) SDS, 25 mm Tris-HCl, pH 7.4, at 95 °C for 5 min and the eluates diluted 10-fold with buffer A prior to the next immunoprecipitation. Cells were harvested and homogenized in buffer B (25 mm Tris-HCl, pH 7.4, 140 mm NaCl, 2 mm EDTA, 5 μg/ml leupeptin, 5 μg/ml STI, 10 μg/ml benzamidine, 2 μg/ml aprotinin, 0.5 mm PMSF, 2 mm 1,10-phenanthroline) with a Dounce homogenizer (Fig. 3,A–C). Alternatively (Figs. 3 (D and E) and 4B), cells were homogenized by freezing and thawing the cell suspension and passing it 10 times through a 26-gauge needle. Fractionation was performed as described (42Wiertz E.J.H.J. Jones T.R. Sun L. Bogyo M. Geuze H.J. Ploegh H.L. Cell. 1996; 84: 769-779Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar). Briefly, homogenates were first centrifuged at 1000 ×g for 10 min to pellet unbroken cells and nuclei. The supernatant was then centrifuged at 10,000 × g for 30 min to pellet the crude membranes, and the supernatant was further centrifuged at 100,000 × g for 60 min to clarify the cytosolic fraction. DDM was added to the soluble fraction to a final concentration of 0.5% (w/v) to decrease nonspecific binding of proteins to either the anti-FLAG M2 affinity resin or Protein G-Sepharose. Pellets were washed with buffer B, solubilized in buffer A, and insoluble material removed by centrifugation at 100,000 ×g for 60 min. Immunoprecipitations from the soluble fraction and the solubilized membranes were carried out as described above. The immunoprecipitated receptors were deglycosylated following elution from the anti-FLAG M2 affinity resin with 1% (w/v) SDS, 50 mmsodium phosphate, pH 7.5. Before the enzyme reaction, the eluates were diluted 10-fold with 0.5% (w/v) DDM, 50 mm sodium phosphate, pH 7.5, 50 mm EDTA, 0.5 mm PMSF, 2 mm 1,10-phenanthroline, 5 μg/ml leupeptin, 5 μg/ml STI, 10 μg/ml benzamidine, and 1% 2-mercaptoethanol. PNGase F was added at a final concentration of 0.01–10 units/ml, samples incubated at 37 °C for 16 h, and the reaction terminated by the addition of SDS-sample buffer. SDS-PAGE was performed as described (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar), using 4% stacking gels and 10% separating gels. Samples were heated at 95 °C for 2 min in the presence of 50 mm dithiothreitol and run on a Bio-Rad Mini-Protein II apparatus. Molecular weight markers (Bio-Rad) detected by staining with Coomassie Brilliant Blue (Bio-Rad) were used to calibrate the gels. For the detection of radioactivity, the gels were treated with EN3HANCE® (PerkinElmer Life Sciences) according to the manufacturer's instructions, dried, and exposed at −80 °C for 1–15 days using Biomax MR film and intensifying screens (Eastman Kodak Co.). The relative intensities of the labeled bands on the fluorographs were analyzed by densitometric scanning with an Agfa Arcus II laser scanner, and the data were quantified using the NIH image program, version 1.61, subtracting the local background from each lane. Total protein was measured using the Bio-Rad DC assay kit and bovine serum albumin as a standard. Binding assays were carried out using [3H]bremazocine, essentially as described (40Petäjä-Repo U.E Hogue M. Laperrière A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). We have previously shown that a large fraction of newly synthesized hδORs in HEK-293S cells remains in a pre-Golgi compartment as core-glycosylated M r45,000 precursors that are not transported to the cell surface (40Petäjä-Repo U.E Hogue M. Laperrière A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Now we set out to investigate the fate of these intracellularly retained receptors. To determine whether proteasomes are involved in the degradation of these receptor forms, cells were incubated with lactacystin, a specific inhibitor of the 26 S proteasome (44Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1501) Google Scholar, 45Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar). HEK-293S cells stably expressing the receptor carrying a C-terminal FLAG epitope (HEK-293S-hδOR-FLAG cells) were metabolically labeled with [35S]methionine/cysteine and after a chase in a medium containing an excess of unlabeled methionine, cell lysates were prepared and the receptors isolated by immunoprecipitation (Fig.1). A major band ofM r 45,000 and two minor ones ofM r 42,000 and 39,000 were apparent in untreated cells at the end of the pulse (Fig. 1 A). During the subsequent chase, label incorporated into these bands decreased and a slower, more diffusely migrating band of M r55,000 appeared, representing the fully mature receptor (40Petäjä-Repo U.E Hogue M. Laperrière A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). When the cells were treated with lactacystin, an increase in labeling of all receptor species was detected (Fig. 1 B). Furthermore, proteasome inhibition significantly retarded the disappearance of theM r 45,000, 42,000, and 39,000 receptor species, the half-life of the last one being the most dramatically affected (Table I). Similar findings were obtained using two other proteasome inhibitors, the peptide aldehydes MG-132 and PSI (45Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar) (data not shown). Taken together, these results suggest an active role of proteasomes in the elimination of newly synthesized hδORs. The fact that lactacystin promoted increase in receptor labeling, even within a shorter pulse period of 15 min, suggests that targeting to degradation begins very early, either cotranslationally or immediately after the receptor synthesis is complete. Inhibition of this rapid degradation is most likely responsible for the increase in the amount of mature receptors observed in pulse-chase labeling experiments (compare lanes 4 and 5 in Fig. 1, A and B). This increase was accompanied by a 38% increase in the number of [3H]bremazocine-binding sites detected. Our findings are not due to the presence of a FLAG-epitope tag at the C terminus of the receptor since the same results were also obtained using a receptor harboring the tag at its N terminus (data not shown). Unfortunately, the lack of a high affinity and selective antibody for the hδOR makes it impossible to carry out these experiments on the native untagged receptor.Table IHalf-lives of the hδOR species in the absence and presence of lactacystinReceptor speciesHalf-lifeUntreatedLactacystinMrmin45,000125 ± 11230 ± 49**42,00097 ± 7184 ± 29***39,00037 ± 2413 ± 88***Relative intensities of the labeled receptor species were quantified from fluorographs of pulse-chase experiments, an example of which is presented in Fig 1, by densitometric scanning. The half-lives of the different receptor species were determined using the GraphPad Prism program, version 2.01, and the data represent the mean ± S.E. of four independent experiments. Statistical significance of the differences was assessed using the two-tailed Student's t test. **, p < 0.01; ***, p < 0.001. Open table in a new tab Relative intensities of the labeled receptor species were quantified from fluorographs of pulse-chase experiments, an example of which is presented in Fig 1, by densitometric scanning. The half-lives of the different receptor species were determined using the GraphPad Prism program, version 2.01, and the data represent the mean ± S.E. of four independent experiments. Statistical significance of the differences was assessed using the two-tailed Student's t test. **, p < 0.01; ***, p < 0.001. It has been reported previously thatN-linked oligosaccharides of newly synthesized glycoproteins are removed before proteasomal degradation (13Wiertz E.J.H.J. Tortorella D. Bogyo M., Yu, J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (954) Google Scholar, 14Hughes E.A. Hammond C. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1896-1901Crossref PubMed Scopus (247) Google Scholar, 15Huppa J.B. Ploegh H.L. Immunity. 1997; 7: 113-122Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 16Yu H. Kaung G. Kobayashi S. Kopito R.R. J. Biol. Chem. 1997; 272: 20800-20804Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 17Bebök Z. Mazzochi C. King S.A. Hong J.S. Sorscher E.J. J. Biol. Chem. 1998; 273: 29873-29878Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 26Mancini R. Fagioli C. Fra A.M. Maggioni C. Sitia R. FASEB J. 2000; 14: 769-778Crossref PubMed Scopus (89) Google Scholar, 35Halaban R. Cheng E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6210-6215Crossref PubMed Scopus (232) Google Scholar, 42Wiertz E.J.H.J. Jones T.R. Sun L. Bogyo M. Geuze H.J. Ploegh H.L. Cell. 1996; 84: 769-779Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 46De Virgilio M. Weninger H. Ivessa N.E. J. Biol. Chem. 1998; 273: 9734-9743Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). We therefore assessed whether the smaller molecular weight receptor species of M r 42,000 and 39,000 represent deglycosylated receptors rather than proteolytic fragments. For this purpose, immunoprecipitated [35S]methionine/cysteine pulse-labeled samples were subjected to enzymatic deglycosylation using PNGase F to remove the two N-linked glycans from the receptor (40Petäjä-Repo U.E Hogue M. Laperrière A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). As can be seen in Fig. 2(lane 5), complete deglycosylation of theM r 45,000 receptor precursor led to the appearance of a single species migrating at M r39,000. The fact that the M r 42,000 species was found to be an intermediate in the stepwise deglycosylation of the receptor precursor suggests that this species represents a receptor carrying one N-linked glycan. The mobility of theM r 39,000 species that was stabilized in lactacystin-treated cells was indistinguishable from that of the PNGase F-digested core-glycosylated M r 45,000 receptor form (Fig. 2, compare lanes 5–7), confirming the fact that this species represents an intact receptor polypeptide with noN-linked glycans. Protein translocation into the ER membrane and core glycosylation are believed to occur cotranslationally in mammalian cells (47Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar). Thus, it is very likely that theM r 39,000 receptor species represents a previously core-glycosylated receptor that has been deglycosylated and is an intermediate in the degradation pathway. To further verify the nature and the subcellular localization of theM r 39,000 hδOR species, fractionation of cellular homogenates was performed after metabolic labeling (Fig.3). Receptors were immunoprecipitated from four fractions: 1000 × g pellet containing cellular debris, nuclei and trapped soluble proteins, 10,000 ×g pellet containing crude membranes, 100,000 ×g pellet containing residual microsomal membranes, and 100,000 × g supernatant containing cytosolic proteins. As a control, the ER membrane protein, calnexin, and the cytosolic protein Hsp70 were immunoprecipitated from aliquots of the same four fractions. Calnexin was detected in the membrane fraction (Fig.3 B, lanes 3 and 4) but not in the cytosolic one (lanes 7 and 8), whereas the opposite was true for Hsp70 (Fig. 3 C). As expected, both proteins were also detected in the 1000 ×g pellet (Fig. 3, B and C,lanes 1 and 2). As can be seen in Fig.3A, in addition to be found in the membrane fraction, theM r 39,000 receptor species was recovered from the cytosolic fraction of lactacystin-treated (lane 8) but not of untreated cells (lane 7). This is in agreement with the notion that it represents an intermediate in the degradation pathway that would normally be broken down by the cytosolic proteasomes. Higher molecular weight bands forming a regularly spaced ladder, of which the smallest had an apparent molecular weight of 45,000, were also found in the cytosolic fraction of lactacystin-treated cells (Fig. 3 A,lane 8). This M r 45,000 species does not represent the core-glycosylated receptor precursor seen in Fig. 3 A (lanes 1–4), because PNGase F was not able to decrease its molecular weight (Fig.3 D, lanes 3 and 4). It is more likely that the ladder and the accompanying high molecular weight smear comprise polyubiquitinated forms of the M r39,000 receptor (see below). The fact that inhibition of proteasomal degradation significantly increased the amount of the M r 39,000 hδOR species in the cytosolic fraction (Fig. 3 A, lanes 7 and 8), suggests that this species may dislocate from the ER membrane and accumulate in the cytosol prior to proteasomal degradation. To test this hypothesis, receptors were immunoprecipitated from the soluble fraction after pulse-chase labeling of lactacystin-treated cells. As seen in Fig. 3 E, a small amount of the M r 39,000 receptor species was present in the cytosol at the end of the pulse. However, the amount detected clearly increased during the chase, indicating that theM r 39,000 species originates from a pool of newly synthesized receptors that are retained in the ER and not from a failure or inefficiency of the translocation machinery. Thus, this species most likely results from deglycosylation of theM r 45,000 receptor precursor that is destined for degradation and eventually dislocates from the ER membrane. Removal of the N-linked glycans most likely occurs prior to retrotranslocation to the cytosol since the deglycosylated hδOR species was found both in the cytosolic and the membrane fractions (Fig. 3 A, lanes 8 and 4, respectively). To directly test the hypothesis that the hδOR is modified by ubiquitination, cellular lysates from [35S]methionine/cysteine pulse-labeled cells were subjected to sequential immunoprecipitation. First, the hδORs were immunoprecipitated with the anti-FLAG M2 antibody and the purified material was denatured by SDS. Proteins were then reimmunoprecipitated using an anti-ubiquitin antibody. All these immunoprecipitation steps (Fig. 4, A and B) were carried out in the presence of NEM to prevent inappropriate aggregation of proteins. In lactacystin-treated cells, the immunoprecipitated material migrated as a high molecular weight smear that extended upwards toward the top of the gel (Fig. 4 A,lane 4), a feature that is characteristic of ubiquitinated proteins (8Ward C." @default.
• W2055242244 created "2016-06-24" @default.
• W2055242244 creator A5007218841 @default.
• W2055242244 creator A5008779429 @default.
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• W2055242244 date "2001-02-01" @default.
• W2055242244 modified "2023-10-16" @default.
• W2055242244 title "Newly Synthesized Human δ Opioid Receptors Retained in the Endoplasmic Reticulum Are Retrotranslocated to the Cytosol, Deglycosylated, Ubiquitinated, and Degraded by the Proteasome" @default.
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