Underglycosylation of ATF6 as a Novel Sensing Mechanism for Activation of the Unfolded Protein Response
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m309804200
ISSN1083-351X
AutoresMin Hong, Shengzhan Luo, Peter Baumeister, Jen-Ming Huang, Raveen K. Gogia, Ming‐Qing Li, Amy S. Lee,
Tópico(s)RNA regulation and disease
ResumoATF6 is a key transcriptional activator of the unfolded protein response (UPR), which allows mammalian cells to maintain cellular homeostasis when they are subjected to a variety of environmental and physiological stresses that target the endoplasmic reticulum (ER). ATF6, a 90-kDa ER transmembrane protein, contains three evolutionarily conserved N-linked glycosylation sites within its carboxyl luminal domain. Although it is well established that p90ATF6 activation requires transit from the ER to the Golgi, where it is cleaved by the S1P/S2P protease system to generate a nuclear form p60ATF6 that acts as a transcriptional activator, the functional significance of p90ATF6 N-linked glycosylation is unknown. Here we show that ER Ca2+ depletion stress, a triggering mechanism for the UPR, induces the formation of ATF6(f), which represents de novo partial glycosylation of newly synthesized p90ATF6. By mutating a single amino acid within the N-linked glycosylation site closest to the carboxyl terminus of p90ATF6, we recreated ATF6(f). This mutation sharply reduces p90ATF6 association with calreticulin, a major Ca2+-binding chaperone for N-glycoprotein. We further determined that ATF6(f) exhibits a faster rate of constitutive transport to the Golgi, resulting in a higher level of p60ATF6 in the nucleus and stronger transactivating activity in the absence of ER stress. Additional analysis of p90ATF6 mutants targeting single or multiple N-glycosylation sites also showed higher constitutive transactivating activity than wild type ATF6. Because accumulation of underglycosylated proteins in the ER is a potent inducer for the UPR, these studies uncover a novel mechanism whereby the glycosylation status of p90ATF6 can serve as a sensor for ER homeostasis, resulting in ATF6 activation to trigger the UPR. ATF6 is a key transcriptional activator of the unfolded protein response (UPR), which allows mammalian cells to maintain cellular homeostasis when they are subjected to a variety of environmental and physiological stresses that target the endoplasmic reticulum (ER). ATF6, a 90-kDa ER transmembrane protein, contains three evolutionarily conserved N-linked glycosylation sites within its carboxyl luminal domain. Although it is well established that p90ATF6 activation requires transit from the ER to the Golgi, where it is cleaved by the S1P/S2P protease system to generate a nuclear form p60ATF6 that acts as a transcriptional activator, the functional significance of p90ATF6 N-linked glycosylation is unknown. Here we show that ER Ca2+ depletion stress, a triggering mechanism for the UPR, induces the formation of ATF6(f), which represents de novo partial glycosylation of newly synthesized p90ATF6. By mutating a single amino acid within the N-linked glycosylation site closest to the carboxyl terminus of p90ATF6, we recreated ATF6(f). This mutation sharply reduces p90ATF6 association with calreticulin, a major Ca2+-binding chaperone for N-glycoprotein. We further determined that ATF6(f) exhibits a faster rate of constitutive transport to the Golgi, resulting in a higher level of p60ATF6 in the nucleus and stronger transactivating activity in the absence of ER stress. Additional analysis of p90ATF6 mutants targeting single or multiple N-glycosylation sites also showed higher constitutive transactivating activity than wild type ATF6. Because accumulation of underglycosylated proteins in the ER is a potent inducer for the UPR, these studies uncover a novel mechanism whereby the glycosylation status of p90ATF6 can serve as a sensor for ER homeostasis, resulting in ATF6 activation to trigger the UPR. The discovery that ATF6 is a key transcriptional activator of endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; CHX, cycloheximide; CNX, calnexin; CRT, calreticulin; DTT, dithiothreitol; HA, hemagglutinin; HSV, herpes simplex virus; Luc, luciferase; Tg, thapsigargin; RT, reverse transcription; TK, thymidine kinase; Tu, tunicamycin; UPR, unfolded protein response. -resident molecular chaperones and folding enzymes in the unfolded protein response (UPR) has revealed novel molecular mechanisms employed by mammalian cells to respond to stresses that perturb ER homeostasis. ATF6, a 670-amino acid glycoprotein with the electrophoretic mobility of a 90-kDa protein (p90ATF6), is constitutively expressed in a variety of mammalian cells (1Zhu C. Johansen F.E. Prywes R. Mol. Cell. Biol. 1997; 17: 4957-4966Crossref PubMed Scopus (140) Google Scholar). It contains a single transmembrane domain with 272 amino acids oriented in the ER lumen. ER stress induces proteolysis of the membrane-bound p90ATF6, releasing the soluble amino portion of p60ATF6, which relocates to the nucleus and activates the transcription of a wide variety of ER stress-inducible promoters, of which Grp78/BiP is the most well characterized (2Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1562) Google Scholar, 3Li M. Baumeister P. Roy B. Phan T. Foti D. Luo S. Lee A.S. Mol. Cell. Biol. 2000; 20: 5096-5106Crossref PubMed Scopus (275) Google Scholar, 4Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1378) Google Scholar). Conservation of protease cleavage sites led to the discovery that Golgi-localized S1P/S2P proteases that process sterol response element binding proteins in response to cholesterol deprivation also process ATF6 in response to ER stress (4Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1378) Google Scholar). Specific luminal domains of p90ATF6 have been mapped which are required for translocation to the Golgi (5Chen X. Shen J. Prywes R. J. Biol. Chem. 2002; 277: 13045-13052Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). It was further determined that GRP78 retains p90ATF6 in the ER by inhibiting its Golgi localization signals (6Shen J. Chen X. Hendershot L. Prywes R. Dev. Cell. 2002; 3: 99-111Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar). However, whether other proteins also contribute to the ER retention of p90ATF6 and how ER stress causes ATF6 transit to the Golgi remain to be determined. p90ATF6 exists constitutively as a glycosylated protein. The consensus sequence for N-linked glycosylation sites consists of the Asn-X-Ser/Thr, where X denotes any amino acid except proline (7Bause E. Biochem. J. 1983; 209: 331-336Crossref PubMed Scopus (521) Google Scholar). The ER luminal domain of human p90ATF6 contains three such sites at 472, 584, and 643 amino acids, and the three sites are also conserved in mouse p90ATF6. The fully glycosylated form of ATF6, a 670-amino acid protein, exhibits an electrophoretic mobility of 90 kDa in denaturing SDS-gels in part because of the glycosylation modifications. N-Linked glycans have been shown to play a pivotal role in protein folding, sorting, and transport (8Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1999) Google Scholar). Thus far, the functional significance of N-linked glycosylation of p90ATF6 is not understood. Interestingly, blockage of N-linked protein glycosylation by treating cells with reagents such as tunicamycin (Tu) results in malfolded protein formation in the ER, ATF6 activation, and the onset of UPR. In Tu-treated cells, p90ATF6 is completely nonglycosylated (2Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1562) Google Scholar). In cells treated with DTT, a small fraction of p90ATF6 becomes O-linked glycosylated in the Golgi (6Shen J. Chen X. Hendershot L. Prywes R. Dev. Cell. 2002; 3: 99-111Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar). In cells treated with thapsigargin (Tg), which depletes the ER Ca2+ store by specific inhibition of the ER Ca2+-ATPase (9Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3010) Google Scholar), we reported earlier that in kinetics in parallel with activation of the UPR, p90ATF6 showed an additional appearance of a faster migrating form distinct from the completely nonglycosylated form ATF6 (3Li M. Baumeister P. Roy B. Phan T. Foti D. Luo S. Lee A.S. Mol. Cell. Biol. 2000; 20: 5096-5106Crossref PubMed Scopus (275) Google Scholar). While searching for the nature and functional significance of this new form of p90ATF6, we discovered that ER Ca2+ depletion stress induces the formation of a newly synthesized, partially glycosylated form of p90ATF6, referred to below as ATF6(f). This new form can be generated by mutation of a single amino acid at residue 645 within the N-linked glycosylation site closest to the carboxyl terminus of p90ATF6. We discovered that this mutation reduces sharply the association between p90ATF6 and calreticulin (CRT), a multifunctional ER luminal protein, which acts as a Ca2+-binding protein as well as a molecular chaperone for ER glycoproteins (8Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1999) Google Scholar, 10Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (374) Google Scholar). ATF6(f) exhibits a faster rate of constitutive transport to the Golgi, resulting in a higher level of p60ATF6 in the nucleus and higher transactivating activity toward the Grp78 promoter, a major target of the UPR. Additional analysis of p90ATF6 mutants targeting single or multiple N-glycosylation sites also showed higher constitutive transactivating activity than wild type ATF6. Because accumulation of underglycosylated proteins in the ER is a potent inducer for the UPR, these studies uncover a novel mechanism whereby the glycosylation status of p90ATF6 can serve as a sensor for ER homeostasis, resulting in ATF6 activation to trigger the UPR. Cell Culture Conditions—COS-7, NIH3T3, and 293T cells were maintained in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycinneomycin antibiotics at 37 °C. The CHO-7 and S2P-defective M19 cells were generously provided by Dr. Joseph L. Goldstein (University of Texas, Southwestern Medical Center, Dallas) and have been described (4Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1378) Google Scholar). For stress induction, cells were grown to 80% confluence and treated with 300 nm Tg or 1.5 μg/ml Tu for various time intervals as indicated. Tg, Tu, and cycloheximide (CHX) were purchased from Sigma. Endoglycosidase H was purchased from Roche Applied Science. Plasmids—The plasmid pCGN-ATF6 containing hemagglutinin epitope (HA)-tagged full-length human ATF6 driven by the cytomegalovirus promoter was provided by Dr. Ron Prywes (Department of Biological Science, Columbia University), and its construction has been described previously (1Zhu C. Johansen F.E. Prywes R. Mol. Cell. Biol. 1997; 17: 4957-4966Crossref PubMed Scopus (140) Google Scholar). pTK-HSV-ATF6 was provided by Dr. Joseph L. Goldstein, and its construction has been described previously (4Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1378) Google Scholar). The site-specific mutants pCGN-ATF6(T645I), pCGN-ATF6(T586I), pCGN-ATF6(T474I), pCGN-ATF6(DoubleM), pCGN-ATF6(TripleM), and pTK-HSV-ATF6(T645I) were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutated base in the construct was confirmed by DNA sequencing. The construction of the -169/Luc reporter gene, which spans from -169 to -29 of the rat Grp78 promoter with all three endoplasmic reticulum stress elements, has been described previously (11Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (216) Google Scholar, 12Luo S. Lee A.S. Biochem. J. 2002; 366: 787-795Crossref PubMed Google Scholar). Transient Transfection and Assay of Reporter Gene Activity—COS-7, 293T, CHO-7, or M19 cells were grown to 60–80% confluence. One μg of the -169/Luc reporter gene was cotransfected with various amounts of pCGN-ATF6 or pTK-HSV-ATF6 plasmids by using PolyFect or SuperFect reagents (Qiagen, Valencia, CA). Empty vector was added to adjust the total amount of plasmids to be the same, and the β-galactosidase reporter gene driven by the cytomegalovirus promoter was used as internal control for transfection efficiency as described previously (3Li M. Baumeister P. Roy B. Phan T. Foti D. Luo S. Lee A.S. Mol. Cell. Biol. 2000; 20: 5096-5106Crossref PubMed Scopus (275) Google Scholar). The transfected cells were either grown in normal culture conditions or subjected to Tg and Tu treatment. After drug treatment, cell lysates were prepared for Western blots or luciferase assays, which were performed using the Luciferase Assay System (Promega, Madison, WI). Each transfection was performed in duplicate and repeated three or four times. Western Blotting—Conditions for Western blot analysis of endogenous ATF6 using a rabbit polyclonal ATF6 antibody, HA-ATF6, and β-actin have been described previously (3Li M. Baumeister P. Roy B. Phan T. Foti D. Luo S. Lee A.S. Mol. Cell. Biol. 2000; 20: 5096-5106Crossref PubMed Scopus (275) Google Scholar). For the detection of endogenous ATF6 using a mouse monoclonal antibody against the amino-terminal 273 amino acids of ATF6 (Imgenex, San Diego), the primary antibody was diluted at 1:500 in 5% milk and incubated with the transferred membrane at 4 °C overnight. The secondary antibody used was horseradish peroxidase-conjugated goat anti-mouse (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:3,000. For the detection of HSV-ATF6, the mouse monoclonal anti-HSV tag (Novagen, Madison, WI) antibody was used as the primary antibody at a dilution of 1:10,000. For detection of GRP78, the anti-KDEL mouse monoclonal antibody (StressGen, Victoria, BC, Canada) was used as the primary antibody at a dilution of 1:1,500. For detection of CRT, the anti-CRT mouse monoclonal antibody (Calbiochem) was used at a dilution of 1:1,000. For detection of calnexin (CNX), the anti-CNX rabbit polyclonal antibody (StressGen) was used at a dilution of 1:2,000. Biochemical Fractionation—293T cells grown in 150-mm-diameter dishes to 70% confluence were transfected with various pTK-HSV-ATF6 expression plasmids. The conditions for the preparation of soluble nuclear protein have been described previously (3Li M. Baumeister P. Roy B. Phan T. Foti D. Luo S. Lee A.S. Mol. Cell. Biol. 2000; 20: 5096-5106Crossref PubMed Scopus (275) Google Scholar). For the isolation of microsomes, 293T cells transfected with pCGN-ATF6 or pCGN-ATF6(T645I) expression plasmids were processed for preparation of microsomes were as described (13Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Coimmunoprecipitation Assays—The conditions for coimmunoprecipitation have been described previously (3Li M. Baumeister P. Roy B. Phan T. Foti D. Luo S. Lee A.S. Mol. Cell. Biol. 2000; 20: 5096-5106Crossref PubMed Scopus (275) Google Scholar). 200 μg of microsome extract from each sample were incubated with protein A-Sepharose beads (Sigma) and 10 μl of mouse anti-HA monoclonal antibody (Santa Cruz Biotechnology) or 10 μl of rabbit anti-CRT polyclonal antibody (StressGen). After the incubation, the beads were washed four times. The immunoprecipitates were resolved by SDS-PAGE and then subjected to silver staining with a SilverQuest silver staining kit (Invitrogen) or Western blotting. Northern Blotting—The methods for total cellular RNA extraction and Northern blot hybridizations have been described (14Cao X. Zhou Y. Lee A.S. J. Biol. Chem. 1995; 270: 494-502Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The cDNA probes used for the detection of Grp78 and glyceraldehyde-3-phosphate dehydrogenase have been described previously (15Zhou Y. Lee A.S. J. Natl. Cancer Inst. 1998; 90: 381-388Crossref PubMed Scopus (133) Google Scholar). RT-PCR Conditions—RT-PCR was performed using the SuperScript™ Preamplification System (Invitrogen). For each reaction, 5 μg of total RNA and 1 μl of 0.5 μg/μl oligo(dT) were incubated in sterilized ddH2O for total volume of 12 μl at 70 °C for 10 min, then put on ice for 5 min, and spun down. Four μl of 5× first stand buffer, 2 μl of 0.1 m DTT, 1 μl of 10 mm dNTP were added subsequently and incubated in a 42 °C water bath for 2 min. 200 units of SuperScript reverse transcriptase were added. The reactants were incubated at 42 °C for 1 h and then inactivated at 70 °C for 15 min. Two μl of RT product was subjected to PCR using AmpliTaq Gold™ (PerkinElmer Life Sciences) at a final magnesium concentration of 1.5 mm. The PCRs were first heated for 3 min at 94 °C. The PCR cycle was 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C. After the completion of 45 PCR cycles, the mixture was incubated for 7 min at 72 °C. The PCRs were performed using GeneAmp PCR system 2400 (PerkinElmer Life Sciences). The primers used were: 5′-ATF-TM, 5′-ATGAAGTTGTGTCAGAGAAAC-3′ (base 1121–1141 of the ATF6 coding sequence); 3′-ATF-TM, 5′-CTCTTTAGCAGCAGAAAATCCTAG-3′ (base 1297–1317 of the ATF6 coding sequence); 5′-HindIII-hATF, 5′-AACCACAAGCTTAGGGAGCCGGCTGGGGTT-3′ (base 4–21 of the ATF6 coding sequence with the addition of the HindIII site at the 5′ end); 3′-BamHI-hATF, 5′-GTTGGATCCAGCTGCGGGTGCTATTGTAATGACTCA-3′ (base 1986–2010 of the ATF6 coding sequence with the addition of the BamHI site at the 5′ end). Immunofluorescence Staining—COS-7 cells were plated in 8-well Tek-II slides (Nalge-Nunc, Naperfield, IL) at 40% confluence and transfected with either HA-ATF6 or HA-ATF6(T645I) using Polyfect reagent. 36 h after transfection, the cells were treated with either 10 mm DTT (Roche Applied Science) or 2.5 μg/ml brefeldin A (Sigma) and fixed in 4% paraformaldehyde. The cells were incubated with anti-HA rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:500 dilution in TBS-Tween buffer with 1% bovine serum albumin, followed by incubation in 4 μg/ml in PBS of anti-rabbit polyclonal Texas Red-conjugated secondary antibody (Vector Laboratories, Burlingame, CA). Golgi staining was accomplished by incubating the fixed cells with a 1:500 dilution of fluorescence-conjugated anti-Golgi antibody (BD Biosciences). The cells were visualized on a Zeiss confocal microscope at 400× magnification. Five images of each time point containing at least 250 cells were taken and numbered to facilitate a blind controlled observation. Cells containing Golgi-localized HA-ATF6 were counted and confirmed by two separate investigators. The experiment was repeated twice. ER Ca2+ Depletion Induces a New Form of Full-length ATF6 —Upon treatment of NIH3T3 cells with Tg, the level of Grp78 mRNA remained low during the first 2 h and gradually increased to a high level that was sustained up to 16 h (Fig. 1A). The steady-state level of endogenous p90ATF6 as detected by a polyclonal antibody showed a transient reduction during the first 2 h of Tg treatment; however, the level of p90ATF6 was replenished by 4 h and by 8 h, the amount of ATF6 was elevated instead, correlating with sustained high level induction of Grp78 mRNA (Fig. 1B). In addition, the replenished form of p90ATF6 appeared to consist of a doublet band, referred to below as ATF6 and ATF6(f). Longer electrophoresis time further resolved the p90ATF6 doublet into two distinct bands, and the doublet was sustained throughout Tg treatment (Fig. 1C). The formation of ATF6(f) is not cell type-specific because it was also observed in other cell lines such as hamster CHO-7 after 2.5 h of Tg treatment and could be detected using a monoclonal antibody against the first 273 amino acids of ATF6 (Fig. 1D). It is noted that in these Western blots the S1P/S2P cleavage product p60ATF6 was not readily detectable after Tg treatment unless the amount of cell lysates applied was 5–10 times higher; under such conditions, the amount of p90ATF6 is usually saturated, obscuring the new form (data not shown). The formation of ATF6(f) is not restricted to endogenous p90ATF6, as the doublet formation was clearly evident for exogenously expressed, HA-tagged ATF6 following 4 h of Tg treatment (Fig. 1E). Thus, a large fraction of the full-length, transmembrane form of p90ATF6 remains uncleaved after ER Ca2+ depletion and is undergoing changes giving rise to a new form of ATF6. ER Ca2+ Depletion Does Not Induce Alternative Processing of the ATF6 Transmembrane Domain—We next investigated the molecular basis for the p90ATF6 doublet formation. One mechanism could be Tg-induced alternative processing of ATF6 mRNA. To test whether Tg treatment changes the size of the ATF6 mRNA, in particular, whether the transmembrane domain is altered by mRNA splicing, RT-PCR was performed using RNA extracted from nontreated NIH3T3 cells and cells treated with Tg for 2, 8, and 16 h. To scan for size alterations, four pairs of primers were designed (Fig. 2A). For all four primers, the sizes of the RT-PCR products were as predicted (2,007, 1,314, 890, and 197 bp). No new band was detected after Tg treatment, suggesting no change in ATF6 mRNA size (Fig. 2B). Thus, alteration of the transmembrane domain of p90ATF6 through mRNA splicing is unlikely to account for the new form of ATF6. Newly Synthesized ATF6 Becomes Partial Glycosylated Upon ER Ca2+ Deprivation—p90ATF6 contains three evolutionarily conserved N-linked glycosylation sites within its ER luminal domain, and in nonstressed cells p90ATF6 is a glycosylated protein (Fig. 3A). One mechanism for ATF6(f) formation is that Tg treatment results in partial glycosylation of a fraction of p90ATF6. In this scenario, ATF6(f) should exhibit an electrophoretic mobility faster than the fully glycosylated form but slower than the fully nonglycosylated form. If the new form of p90ATF6 is localized in the ER or cis-Golgi, it should be sensitive to endoglycosidase H, which removes simple, high mannose N-linked oligosaccharide characteristic of ER localization but cannot remove N-linked glycans that have been processed in the trans-Golgi (16Maley F. Trimble R.B. Tarentino A.L. Plummer Jr., T.H. Anal. Biochem. 1989; 180: 195-204Crossref PubMed Scopus (646) Google Scholar). Alternatively, if Tg results in a subpopulation of p90ATF6 relocated to the trans-Golgi where it undergoes further glycosylation modification, ATF6(f) should be endoglycosidase H-resistant. In both cases, blockage of p90ATF6 N-glycosylation by treatment of cells with Tu should eliminate ATF6(f). If the new form of p90ATF6 is caused by other mechanisms unrelated to N-glycosylation, it should persist upon simultaneous treatment with Tg and Tu. To test these different mechanisms, HA-tagged ATF6 was transfected into COS-7 cells followed by treatment of Tg or Tu alone or a combination of both Tg and Tu. Four lines of evidence support the partial N-linked glycosylation mechanism in the ER. First, ATF6(f) exhibited an electrophoretic mobility faster than the fully glycosylated form in nonstressed cells but slower than the nonglycosylated form of p90ATF6 generated by Tu, referred to as ATF6(Ψ-) (Fig. 3B). Second, simultaneous treatment of Tg and Tu reduced the p90ATF6 doublet to a single band with electrophoretic mobility identical to ATF6(Ψ-), which indicates that the critical mechanism for ATF6(f) formation is N-linked glycosylation inhibition. Third, both the fully glycosylated form and ATF6(f) were reduced to the nonglycosylated form upon endoglycosidase H treatment (Fig. 3B), thus ATF6(f) is not caused by altered glycosylation in the trans-Golgi. Lastly, we generated ATF6(f) by site-specific mutation of the glycosylation site closest to the carboxyl end. This site was chosen because progressive deletion of the carboxyl end of p90ATF6 showed that the new form of p90ATF6 was largely eliminated when this site was trimmed, whereas the sites at amino acids 472 and 584 remained intact (data not shown). Mutant T645I was created where the threonine residue at 645 adjacent to the asparagine residue was mutated to isoleucine (Fig. 3A). N-Glycosylation occurs on the ER luminal side of the membrane and requires the protein recognition sequence Asn-X-Ser/Thr. An asparagine residue not followed by a serine or threonine as shown will not be recognized by the oligosaccharyl transferase, a key enzyme in N-linked glycosylation mechanism, and therefore will not be glycosylated (17Wormald M.R. Dwek R.A. Structure Fold Des. 1999; 7: R155-R160Abstract Full Text Full Text PDF Scopus (233) Google Scholar). The wild type or the T645I mutant was transfected into COS-7 cells, and the cell lysates were prepared after Tg treatment. As predicted, in nonstressed cells, the electrophoretic mobility of T645I was faster than the fully glycosylated HA-ATF6 (Fig. 3C, compare lanes 1 and 3) but identical to HA-ATF6(f) generated by Tg treatment (compare lanes 2 and 3). Upon Tg treatment, T645I remained largely as one band (Fig. 3C, lane 4). As in the case for wild type p90ATF6, treatment of T645I with Tu alone or Tu in combination with Tg resulted in the fully nonglycosylated form (Fig. 3D). To address further whether ATF(f) is formed by partial deglycosylation of full glycosylated p90ATF6 or de novo partial glycosylation of newly synthesized p90ATF6, CHX was used to inhibit protein synthesis in control and Tg-treated cells. As showed in Fig. 3E, Tg-induced formation of ATF6(f) was blocked by CHX pretreatment, suggesting that ATF6(f) represents de novo partial glycosylation of newly synthesized p90ATF6 rather than partial deglycosylation of fully glycosylated p90ATF6, which should not be affected by CHX treatment. T645I Mutation Results in a Higher Constitutive Rate of Transport to the Golgi—To examine the functional consequence of partial glycosylation of p90ATF6, we first compared the cellular distribution of the mutant and wild type protein through confocal microscopy. COS-7 cells were transfected with either HA-ATF6 (wild type) or T645I. In both cases, immunofluorescence of the HA tag showed that although the majority of both proteins were localized in the perinuclear region consistent of the ER, constitutive relocalization to the Golgi, as revealed by colocalization with a specific Golgi marker, could also be detected. Examples of HA-ATF6 scored as ER and Golgi staining in the transfected cells are shown in Fig. 4, A and B. In nonstressed cells, Golgi location was detected in about 2% of the cells transfected with wild type ATF6. Cells transfected with the T645I mutant showed about 5.5% of ATF6 localized in the Golgi, representing about a 2.5-fold increase compared with wild type ATF6 (Fig. 4C). Upon treatment with DTT, a potent UPR inducer used previously to demonstrate ATF6 relocalization to the Golgi (6Shen J. Chen X. Hendershot L. Prywes R. Dev. Cell. 2002; 3: 99-111Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar), both wild type and the T645I mutant showed increased transit to the Golgi; by 30 min of DTT treatment, the difference between the mutant and the wild type protein was marginal (Fig. 4C). As expected, the relocalization of p90ATF6 from the ER to Golgi was suppressed substantially in cells treated with brefeldin A, which causes redistribution of the Golgi to the ER (Fig. 4D). CRT Association with ATF6 Is Sharply Reduced in the T645I Mutant—To examine possible contributing factors for the higher constitutive translocation to the Golgi for the T645I mutant, wild type HA-ATF6 or the T645I mutant was transfected into 293T cells, and microsome fractions were prepared. Coimmunoprecipitation followed by silver staining was used to identify protein partners that showed differential interaction between the wild type and mutant HA-ATF6 (Fig. 5A). Although the signal was weak, this approach yielded a candidate protein around 65 kDa that associated with the wild type ATF6 but was hardly detectable with the T645I mutant. The common band at 90 kDa was confirmed to be HA-ATF6 by Western blot, and the common band at 78 kDa showed electrophoretic mobility identical to that of GRP78 (Fig. 5A and data not shown). Although the amount of the p65 band was insufficient for direct sequencing, its electrophoretic mobility is similar to CRT, a 63-kDa Ca2+-binding protein that also serves as chaperone for glycoproteins in the ER lumen. To test directly the interaction between CRT and the two forms of p90ATF6, microsomal fractions were prepared from 293T cells that were transfected with either HA-ATF6 or T645I. Using anti-HA antibody as the immunoprecipitating antibody followed by Western blot with anti-CRT antibody, we observed a substantial decrease in the amount of CRT interacting with T645I compared with wild type ATF6 (Fig. 5B). Thus, CRT exhibits the same binding property as the 65 kDa band identified by silver staining. Reprobing the same filter with anti-HA antibody confirmed that equivalent amounts of wild type and mutant HA-ATF6 were immunoprecipitated. Further, Western blot analysis with microsome extracts confirmed that the endogenous level of CRT was also equivalent in cells expressing the wild type and the mutant ATF6 (Fig. 5B). The reduction in binding to CRT is specific because ATF6 binding to CNX, another ER chaperone with affinity for newly synthesized monomeric glycoproteins (18Bergeron J.J. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (465) Google Sch
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