The Luminal Domain of ATF6 Senses Endoplasmic Reticulum (ER) Stress and Causes Translocation of ATF6 from the ER to the Golgi
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m110636200
ISSN1083-351X
AutoresXi Chen, Jingshi Shen, Ron Prywes,
Tópico(s)RNA regulation and disease
ResumoATF6 is an endoplasmic reticulum (ER) transmembrane transcription factor that is activated by the ER stress/unfolded protein response by cleavage of its N-terminal half from the membrane. We find that ER stress induces ATF6 to move from the ER to the Golgi, where it is cut in its luminal domain by site 1 protease. ATF6 contains a single transmembrane domain with 272 amino acids oriented in the lumen of the ER. We found that this luminal domain is required for the translocation of ATF6 to the Golgi and its subsequent cleavage, and we have mapped regions required for these properties. These results suggest that the conserved CD1 region is required for translocation, whereas the CD2 region is required for site 1 protease cleavage. We also find that ATF6's luminal domain is sufficient to sense ER stress and cause translocation to the Golgi when fused to LZIP, another ER transmembrane protein. These results show that ATF6 has a mechanism to sense ER stress and respond by translocation to the Golgi. ATF6 is an endoplasmic reticulum (ER) transmembrane transcription factor that is activated by the ER stress/unfolded protein response by cleavage of its N-terminal half from the membrane. We find that ER stress induces ATF6 to move from the ER to the Golgi, where it is cut in its luminal domain by site 1 protease. ATF6 contains a single transmembrane domain with 272 amino acids oriented in the lumen of the ER. We found that this luminal domain is required for the translocation of ATF6 to the Golgi and its subsequent cleavage, and we have mapped regions required for these properties. These results suggest that the conserved CD1 region is required for translocation, whereas the CD2 region is required for site 1 protease cleavage. We also find that ATF6's luminal domain is sufficient to sense ER stress and cause translocation to the Golgi when fused to LZIP, another ER transmembrane protein. These results show that ATF6 has a mechanism to sense ER stress and respond by translocation to the Golgi. endoplasmic reticulum ER stress response element cAMP-response element-binding protein sterol response element-binding protein site 1 and site 2 protease, respectively cytomegalovirus amino acid(s) hemagglutinin dithiothreitol green fluorescent protein basic leucine zipper serum response factor The endoplasmic reticulum (ER)1 is a critical cellular compartment responsible for the proper localization and folding of transmembrane and secreted proteins. Eukaryotic cells, from yeast to humans, have therefore developed mechanisms to ensure proper quality control and folding of proteins in this compartment (1.Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1934) Google Scholar). The cellular responses to unfolded proteins have been termed the ER stress or unfolded protein response. Nutrient deprivation and drugs that cause unfolded proteins in the ER can activate the ER stress response and allow the cell to survive the insult (1.Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1934) Google Scholar). One part of the ER stress response is to activate new gene expression in the nucleus (2.Mori K. Cell. 2000; 101: 451-454Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar). Genes encoding ER chaperones, such as GRP78/BiP and GRP94, are transcriptionally activated such that their gene products are elevated in the ER and decrease the level of unfolded proteins (1.Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1934) Google Scholar). The analysis of promoters of several target genes has identified a consensus ER stress response element (ERSE) that is necessary and sufficient to mediate the activation of reporter genes by ER stress inducers (3.Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 4.Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (216) Google Scholar). The ERSE is bound constitutively by NF-Y, which is required for the inducible binding of ATF6 to the ERSE (5.Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell. Biol. 2000; 20: 6755-6767Crossref PubMed Scopus (793) Google Scholar, 6.Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell. Biol. 2001; 21: 1239-1248Crossref PubMed Scopus (260) Google Scholar). Both the ERSE and solo ATF6 binding sites can mediate ER stress induction of reporter genes (3.Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 4.Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (216) Google Scholar, 7.Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar). ATF6 is an unusual basic leucine zipper (bZIP) factor in that it contains a central transmembrane domain and is localized to ER membranes (8.Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1552) Google Scholar). It is activated by ER stress by cleavage from the membrane, freeing the N-terminal bZIP and transcriptional activation domains to move to the nucleus and activate transcription (3.Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 8.Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1552) Google Scholar). Dominant negative ATF6 constructs inhibited induction of target genes, suggesting that ATF6 is required for ER stress induction in vivo (5.Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell. Biol. 2000; 20: 6755-6767Crossref PubMed Scopus (793) Google Scholar, 7.Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar). ATF6 is similar to the CREB-RP/G13 gene (recently renamed ATF6β) in the bZIP and transmembrane domains as well as in two regions in the lumen, termed CD1 and CD2 here (Fig. 1A) (8.Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1552) Google Scholar, 9.Zhu C. Johansen F.E. Prywes R. Mol. Cell. Biol. 1997; 17: 4957-4966Crossref PubMed Scopus (139) Google Scholar). The role of CREB-RP is unclear; it appears to be activated similarly to ATF6, but its overexpression inhibited ER stress induction of the GRP78 promoter (3.Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 6.Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell. Biol. 2001; 21: 1239-1248Crossref PubMed Scopus (260) Google Scholar, 10.Haze K. Okada T. Yoshida H. Yanagi H. Yura T. Negishi M. Mori K. Biochem. J. 2001; 355: 19-28Crossref PubMed Scopus (200) Google Scholar). Sterol Response Element-binding Protein (SRE-BP) is a transcription factor that controls genes involved in maintaining cholesterol levels and is activated when cells are grown in low cholesterol (11.Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1105) Google Scholar). SRE-BP is similar to ATF6 in that it is also an ER transmembrane protein that is activated by cleavage from the membrane. SRE-BP is cleaved in a two-step process, first by site 1 protease (S1P) in the luminal loop of SRE-BP and second by site 2 protease (S2P) near the cytoplasmic end of the transmembrane domain (11.Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1105) Google Scholar). ATF6 is similarly regulated by S1P and S2P cleavage. An S1P-like site in ATF6's luminal domain and the S2P gene were required for ER stress-induced ATF6 cleavage (12.Ye 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 (1367) Google Scholar). A cell line lacking S1P was partially defective for ATF6 activation, suggesting that it participates in ATF6 regulation but that there is another protease that can cleave the S1P site (12.Ye 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 (1367) Google Scholar). S1P is localized to the Golgi, and SRE-BP is activated to move from the ER to the Golgi in low cholesterol media (13.DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 14.Nohturfft A. DeBose-Boyd R.A. Scheek S. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11235-11240Crossref PubMed Scopus (191) Google Scholar). This suggested that ER stress may induce ATF6 translocation to the Golgi, where it can be cleaved by S1P. We tested this hypothesis here and have mapped regions of ATF6 required for its activation. These results suggest that the luminal domain of ATF6 has an integral role in sensing ER stress leading to ATF6 cleavage in the Golgi and activation of target gene expression. The HA-GAL4-ATF6 expression series were constructed to contain SV40 or CMV promoters and an N-terminal HA epitope tag. They encode the N-terminal 147 residues of yeast GAL4 fused to the indicated regions of human ATF6. The S1P site mutation contains a change of amino acids 415 and 416 from RR to AA. The HA-ATF6 series was constructed in pCGN (15.Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar) and contains a CMV promoter, an N-terminal HA epitope tag, and the indicated regions of human ATF6. The M2-ATF6 plasmid was constructed in pcDNA3.1 (Invitrogen) and encodes the human ATF6 with a FLAG tag at its N terminus. The 3×FLAG-ATF6 plasmids were constructed in p3×FLAG-CMV7.1 (Sigma) and encode the indicated regions of human ATF6 with three copies of the FLAG epitope tag at the N terminus. The GFP-ATF6 plasmids were constructed in pEGFP-C3 (CLONTECH) and encode green fluorescence protein N-terminal to the indicated regions of ATF6. GFP-LZIP was constructed in pEGFP-C3 and encodes GFP fused to the N terminus of LZIP (full-length or aa 1–280). GFP-LZIP-(1–280)-ATF6-(430–670) encodes GFP fused in frame to LZIP-(1–280) and ATF6-(430–670). The LZIP cDNA was kindly provided by Dr. Angus Wilson (New York University). The Golgi marker DS1-GT was kindly provided by Dr. Karin Schwab (DKFZ, Heidelberg) and encodes red fluorescent protein fused to the transmembrane region of galactosyl transferase. S1P-KDEL and S1P-KDAS were as described (13.DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Reporter plasmids p5×GAL4-E1b-luc and pRL-SV40P were described previously (7.Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar, 16.Chen X. Prywes R. Mol. Cell. Biol. 1999; 19: 4695-4702Crossref PubMed Scopus (69) Google Scholar). A retrovirus expression vector for ATF6, pBABE-puro-ATF6, was constructed with an HA epitope tag at its N terminus in pBABEpuro (17.Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1900) Google Scholar). HeLa and NIH3T3 cells were grown in Dulbecco's modified Eagle medium with 10% newborn calf serum. NIH3T3 cells stably expressing HA-tagged ATF6 were prepared by retroviral infection and selection with 1.5 μg/ml puromycin. The virus expressing ATF6 was generated by transfection of 293 cells with pBABE-puro-ATF6 and a packaging site-defective Moloney murine leukemia virus construct. HeLa cells were set at 1 × 105 cells/35-mm diameter culture dish. After 24 h, the cells were transfected by the calcium phosphate-DNA coprecipitation method as described (18.Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Each SV40-driven HA-GAL4-ATF6 fusion protein plasmid was transfected together with 0.5 μg of a firefly luciferase reporter construct, p5×GAL4-E1b-luc, which has five GAL4 DNA binding sites and 50 ng of the Renilla luciferase reporter pRL-SV40P as an internal control for transfection efficiency. Plasmid pcDNA3.1 was used to bring the total amount of DNA to 5 μg/dish. The cells were incubated with the transfection mixture for 20 h and then grown in Dulbecco's modified Eagle's medium with 10% newborn calf serum for 12 h. The cells were then either left untreated or treated with 2 μg/ml tunicamycin for 12 h. Cells were then lysed and assayed for firefly and Renillaluciferase activities as described (7.Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar). All experiments were performed with duplicate plates for each point, and data are shown as the average of three or more independent experiments ± S.E. HeLa cells were transfected, incubated for 2 days, and then either left untreated or treated with 10 mmDTT or 1.5 μm thapsigargin (Calbiochem) for the times indicated 2 days after transfection. Cells were lysed in SDS-PAGE sample buffer (2% SDS, 60 mm Tris-HCl (pH 6.8), 10% glycerol, 0.001% bromphenol blue, 0.33% β-mercaptoethanol) and boiled for 5 min. The lysates were analyzed by immunoblotting using a 1:2000 dilution of anti-HA monoclonal antibody (Babco) or a 1:1000 dilution of anti-FLAG M2 monoclonal antibody (Sigma). Horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma) was used as a secondary antibody at a 1:4000 dilution, and the signal was visualized using the ECL chemiluminescence kit (Amersham Biosciences). NIH cells stably expressing HA-tagged ATF6 were either left untreated or treated with 10 mm DTT for 1.5 h. Cells were fractionated at 4 °C into membrane and nuclear extract fractions. Cells from two 100-mm plates were washed twice, scraped into ice-cold PBS, and centrifuged at 800 ×g for 3 min, and the pellets were suspended in 5 volumes of hypotonic buffer (10 mm HEPES, pH 7.6, 1.5 mmMgCl2, 10 mm KCl, 0.5 mm DTT, 1 mm EDTA, 1 mm EGTA, supplemented with protease inhibitor mixture (Sigma)). The cells were allowed to swell on ice for 30 min, disrupted with 50 strokes of a type B Dounce homogenizer, and centrifuged at 3000 rpm (1000 × g) for 10 min in a J-6B Beckman centrifuge. The resulting nuclear pellet was extracted with an equal volume of extraction buffer (20 mm HEPES, pH 7.6, 25% glycerol, 0.5 m NaCl, 1.5 mmMgCl2, 1 mm EDTA, 1 mm EGTA, supplemented with protease inhibitor mixture (Sigma)) for 1 h and centrifuged at 15,000 × g for 10 min in a microcentrifuge. This supernatant was the nuclear extract. The supernatant from the initial low speed centrifugation (1000 ×g) was further centrifuged at 100,000 × gfor 30 min. The pellet was the membrane fraction and was resuspended in SDS-PAGE loading buffer. The fractions were immunoblotted with anti-HA or anti-SRF serum. Polyclonal SRF serum was generated in rabbits against human SRF purified from Escherichia coli. For detection of GFP-ATF6, HeLa cells were grown on uncoated 22 × 22-mm number 1 coverslips (Fisher) in a 60-mm dish (Figs. 4, 5, and 7) at 2 × 105 cells/dish. After 24 h, cells were transfected with 20 μg of GFP-ATF6 constructs alone or together with 4 μg of Golgi marker construct DS1-GT. 20 h later, cells were washed with PBS and grown in fresh Dulbecco's modified Eagle's medium with 10% newborn calf serum for 4 h. A slide-coverslip chamber was prepared as described (19.Dai J. Sheetz M.P. Cell. 1995; 83: 693-701Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Briefly, two strips of double adhesive tape (1.5 × 30 mm) were placed on both edges of the slide, and then the coverslip, on which HeLa cells were growing, was pressed onto the adhesive tape with the cells facing downward into the chamber. Rapid solution exchanges were affected using a wick system as follows. A drop of Dulbecco's modified Eagle's medium plus 10% newborn calf serum (with 25 mm HEPES, pH 7.3), with or without 10 mm DTT, was placed at the end of one open edge of the slide-coverslip chamber, and tapered strips of filter paper were placed in contact with the medium at the other open edge. The medium-exchanged chamber was sealed with low melting point wax around the four edges. The assembly was mounted on the microscope stage's slide holder. The temperature was maintained at 37 °C as described (19.Dai J. Sheetz M.P. Cell. 1995; 83: 693-701Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Fluorescence microscopy was carried out on an Optiphot-2 microscope at ×600 magnification with appropriate filters for fluorescence detection. Pictures were taken with a Hamamatsu digital camera with Metamorph software.Figure 5The luminal domain of ATF6 is sufficient for ER stress-induced Golgi localization. HeLa cells were transfected with GFP-LZIP-(1–280) or with the GFP-LZIP-(1–280)-ATF6-(430–670) fusion construct and treated with or without DTT for 1 h, and GFP fluorescence was observed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7The CD1 domain is required for translocation of ATF6 to the Golgi. HeLa cells were transfected with GFP-ATF6 constructs containing the indicated residues (1–670 for wild type (WT)) or the indicated internal deletions in full-length ATF6. The cells were treated with or without 10 mm DTT for 1 h. Typical images of the GFP fluorescence are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For immunofluorescence, HeLa cells were transfected onto coverslips in 35-mm dishes with 0.5 μg of FLAG-ATF6 and, where indicated, 0.5 μg of DS1-GT as a Golgi marker. 2 days after transfection, the cells were treated with or without 5 mm DTT for the indicated times, and the cells were fixed in 4% paraformaldehyde for 10 min at room temperature, washed with PBS for 5 min, and permeabilized and blocked with blocking buffer (0.1% Triton X-100 and 1% bovine serum albumin in PBS) for 30 min at room temperature. The cells were then incubated with anti-HA (1:200) or anti-FLAG (1:1000) monoclonal antibodies in blocking buffer for 1 h at room temperature. The cells were washed with PBS for 10 min and incubated with fluorescein isothiocyanate-linked goat anti-mouse IgG (Pierce; 1:100) in blocking buffer for 60 min at room temperature. The cells were then washed twice with PBS at room temperature for 10 min each. The coverslips were mounted on microscope slides, and fluorescence microscopy was carried out on Nikon Diaphot 300 at ×400 magnification with appropriate filters for fluorescence detection. Pictures were taken with a Hamamatsu digital camera with Adobe Photoshop software. ATF6 is activated and cleaved in response to inducers of the ER stress response such as tunicamycin (an inhibitor of glycosylation), thapsigargin (an inhibitor of an ER Ca2+-ATPase), and DTT (a strong reducing agent) (1.Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1934) Google Scholar). We have found that ER stress induction of ATF6 can be reproduced with a GAL4-ATF6 fusion construct (7.Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar). We therefore mutagenized GAL4-ATF6 to test for the requirement of different domains. We first tested the importance of the S1P site in the functional activation of ATF6. The S1P site in SRE-BP requires the sequence R XXL (20.Duncan E.A. Brown M.S. Goldstein J.L. Sakai J. J. Biol. Chem. 1997; 272: 12778-12785Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). We therefore mutated a similar sequence in ATF6, RRHLL (aa 415–419), to AAHLL. The GAL4-ATF6 (aa 1–670) constructs were transfected into HeLa cells with a GAL4 site reporter gene. Wild type ATF6 mediated strong tunicamycin induction of the reporter gene, whereas mutation of the S1P site completely abolished induction (Fig. 1B). This regulation of GAL4-ATF6 requires low level expression such that we have used the relatively weak SV40 promoter without an enhancer to express GAL4-ATF6. Higher expression resulted in a low level of cleavage of ATF6, which is sufficient to activate the reporter gene in uninduced cells (data not shown). However, it was difficult to detect GAL4-ATF6 in immunoblots using the SV40 promoter. To compare expression and stability of the HA-tagged GAL4-ATF6 variants, we expressed the same variants with a CMV promoter. In this way, we found that both GAL4-ATF6 and GAL4-ATF6(S1P−) were expressed at similar levels (Fig. 1C). We previously had difficulty detecting cleavage of ATF6 in response to inducers of ER stress such as tunicamycin (7.Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar). Possible reasons are that cleavage occurs at low levels and is not synchronous in response to tunicamycin. We used DTT as a stronger and more synchronous inducer of unfolded proteins in the ER (8.Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1552) Google Scholar, 21.Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Nat. Cell Biol. 2000; 2: 326-332Crossref PubMed Scopus (2105) Google Scholar). We found that DTT was able to induce cleavage of GAL4-ATF6 after transfection into HeLa cells and that the S1P site mutations blocked this cleavage (Fig. 1C). Only a small amount of ATF6 cleavage was observed compared with full-length ATF6 after overexpression of GAL4-ATF6 in HeLa cells. To test whether lower levels of ATF6 would be more completely cleaved, we generated a stable cell line using a retroviral vector expressing ATF6 with an N-terminal HA epitope tag. In these cells, ATF6 was effectively cleaved in response to DTT with 80–90% of ATF6 cleaved after 1 h of treatment (Fig. 1D). Cleavage was also activated by another inducer of the ER stress response, thapsigargin, although the kinetics were slower (Fig. 1D). Two cleavage bands of ATF6 were observed after DTT treatment (Fig. 1D). This is consistent with the two-step cleavage by S1P and then S2P (12.Ye 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 (1367) Google Scholar). Cleavage by S1P would be predicted to result in a membrane-associated protein, while cleavage by both S1P and S2P frees the product to move to the nucleus. We tested whether the cleavage products conformed to this model by fractionation of the cell extracts. The upper cleavage product (labeled I) was in the membrane fraction, whereas the second product (N) was in both the membrane and nuclear extract fraction (Fig. 1E). As a control for the fractionation, we found that full-length ATF6 was exclusively in the membrane fraction, whereas a nuclear protein, SRF, was only in the nuclear extract fraction (Fig. 1E). It is not clear why the lower ATF6 product is in both the nuclear and membrane fractions. One possibility is that in some cases only a single monomer of the ATF6 homodimer is cleaved such that the cleaved monomer remains associated with the membrane through its dimerization with full-length or S1P-cleaved ATF6 (band I). It is also notable in Fig. 1D that there was not a clear precursor product relationship between the upper and lower ATF6 cleavage products. One explanation is that at least some of the S2P cleavage occurs quickly after S1P cleavage. One possible model for ATF6 activation is that its luminal domain senses ER stress. A prediction of this model is that deletions of this domain that do not affect the S1P cleavage site will affect activation. Possible significant domains of ATF6 are the CD1 (aa 467–506) and CD2 (aa 550–640) regions that are conserved with CREB-RP (9.Zhu C. Johansen F.E. Prywes R. Mol. Cell. Biol. 1997; 17: 4957-4966Crossref PubMed Scopus (139) Google Scholar). We made a series of C-terminal deletions of GAL4-ATF6 and tested for their activation by tunicamycin (Fig. 2A). Deletion to aa 640 had no effect, but deletion into the CD2 region abolished activation. An internal deletion spanning the CD1 region (Δ430–550) also strongly reduced induction. The basal level of luciferase expression with each of these deletion constructs varied but was always in the low range (data not shown). Immunoblot analysis of the GAL4-ATF6 variants showed that they were expressed at similar levels (Fig. 2B and data not shown). We tested whether the C-terminal domain was required for ATF6 cleavage. Transfected HeLa cells were treated with or without DTT for 1 h. While a cleavage product of full-length ATF6 was detected in immunoblots, no cleavage product was observed for GAL4-ATF6-(1–600) or -(1–550) (Fig. 2B). These results further correlate activation and cleavage of ATF6 and suggest that the CD2 domain is required for S1P cleavage of ATF6. S1P is localized in the Golgi, and SRE-BP must move from the ER to the Golgi in response to low cholesterol in order to be cleaved (13.DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 14.Nohturfft A. DeBose-Boyd R.A. Scheek S. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11235-11240Crossref PubMed Scopus (191) Google Scholar). The role of S1P in ATF6 cleavage therefore suggests that ATF6 also moves out of the ER in response to ER stress. We tested this prediction with a FLAG-tagged ATF6 construct that was transfected into HeLa cells. We detected FLAG-ATF6 by immunofluorescence in the perinuclear and cytoplasmic regions in a manner consistent with ER localization (Fig. 3A). DTT treatment for 30 min caused the movement of FLAG-ATF6 to a perinuclear spot that is similar to Golgi staining. To test this, we coexpressed GFP-ATF6 with a Golgi marker, galactosyl transferase, fused to red fluorescent protein (DS1-GT). There was a strong overlap of the signals as shown by the merged images in Fig. 3B, suggesting that FLAG-ATF6 moved from the ER to the Golgi in response to DTT. At 60 min after DTT treatment of transfected cells, we found that FLAG-ATF6 had moved to the nucleus as expected for cleavage of ATF6 by S1P and S2P (Fig. 3A). The localization to the nucleus was confirmed by colocalization with a nuclear red fluorescent protein marker (data not shown). The transient localization to the Golgi should be prolonged if S1P cleavage is blocked. We tested this by using FLAG-tagged ATF6 with mutations at the S1P site. This form of ATF6 moved to the Golgi similar to wild type ATF6 but was retained there even after 60 min of DTT treatment (Fig. 3C). To provide further evidence that ATF6 moves to the Golgi, we used brefeldin A, which causes the relocation of Golgi proteins into the ER (22.Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar, 23.Fujiwara T. Oda K. Yokota S. Takatsuki A. Ikehara Y. J. Biol. Chem. 1988; 263: 18545-18552Abstract Full Text PDF PubMed Google Scholar, 24.Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1312) Google Scholar). For this purpose, we used a GFP-ATF6 construct with the S1P site mutated. The GFP fusion was used for ease of detection. Although it requires a higher level of expression to observe, we similarly found that GFP-ATF6 translocated to a perinuclear focus that colocalized with the Golgi-targeted DS1-GT (data not shown). The S1P site mutation was used, since, as with FLAG-ATF6, this caused a longer retention of GFP-ATF6 in a Golgi-like staining pattern after DTT treatment (data not shown). HeLa cells transfected with GFP-ATF6(S1P−) were treated with DTT for 1 h and then with or without brefeldin A (Fig. 4). DTT caused GFP-ATF6(S1P−) to move to the Golgi-like pattern. Brefeldin A then caused the pattern to disperse and return to a more ER-like pattern. This is consistent with a Golgi localization for ATF6 in DTT-treated cells. Another method to test for Golgi localization is to probe for a change in glycosylation with endoglycosidase H. N-Linked oligosacharides in the ER are sensitive to endoglycosidase H cleavage. They become resistant to endoglycosidase H digestion in the Golgi after they are modified by α-mannosidase II, which is localized in the medial and/or trans-Golgi compartment in most cell types (25.Maley F. Trimble R.B. Tarentino A.L. Plummer Jr., T.H. Anal. Biochem. 1989; 180: 195-204Crossref PubMed Scopus (643) Google Scholar, 26.Velasco A. Hendricks L. Moremen K.W. Tulsiani D.R. Touster O. Farquhar M.G. J. Cell Biol. 1993; 122: 39-51Crossref PubMed Scopus (281) Google Scholar). We did not detect clear resistance to endoglycosidase H digestion after DTT treatment (data not shown). While this does not show that ATF6 moves to the Golgi, it is consistent with movement to the cis-Golgi or an early compartment that does not contain α-mannosidase II. W
Referência(s)