Stable binding of ATF6 to BiP in the endoplasmic reticulum stress response

Abstract

Endoplasmic reticulum (ER) stress-induced activation of ATF6, an ER membrane-bound transcription factor, requires a dissociation step from its inhibitory regulator, BiP. It has been generally postulated that dissociation of the BiP-ATF6 complex is a result of the competitive binding of misfolded proteins generated during ER stress. Here we present evidence against this model and for an active regulatory mechanism for dissociation of the complex. Contradictory to the competition model that is based on dynamic binding of BiP to ATF6, our data reveal relatively stable binding. First, the complex was easily isolated, in contrast to many chaperone complexes that require chemical cross-linking. Second, ATF6 bound at similar levels to wild-type BiP and a BiP mutant form that binds substrates stably because of a defect in its ATPase activity. Third, ER stress specifically induced the dissociation of BiP from ER stress transducers while the competition model would predict dissociation from any specific substrate. Fourth, the ATF6-BiP complex was resistant to ATP-induced dissociation in vitro when isolated without detergents, suggesting that cofactors stabilize the complex. In favor of an active dissociation model, one specific region within the ATF6 lumenal domain was identified as a specific ER stress-responsive sequence required for ER stress-triggered BiP release. Together, our results do not support a model in which competitive binding of misfolded proteins causes dissociation of the BiP-ATF6 complex in stressed cells. We propose that stable BiP binding is essential for ATF6 regulation and that ER stress dissociates BiP from ATF6 by actively restarting the BiP ATPase cycle.

FIG. 1.

ATF6 is a chaperone substrate of BiP. (A) Structural model of the peptide-binding domain of DnaK (50). The corresponding positions of proline 495 in human BiP and the peptide substrate are highlighted with arrows. (B) Diagram of constructs encoding wt BiP and myc-tagged BiP. The positions of threonine 37 and proline 495, which were mutated in our studies, are indicated. (C) Binding of wt and mutant BiP to Ig HC and ATF6. Ig HC (top two parts) or 3 × FLAG-tagged ATF6 (bottom two parts) was transiently expressed in HeLa cells along with either wt or P495L mutant BiP-myc. Ig HC and 3 × FLAG-ATF6 were precipitated with either protein A-Sepharose or anti-FLAG antibodies plus protein A-Sepharose, respectively. The precipitated complexes were resolved by SDS-PAGE and detected by immunoblotting with the indicated antibodies. (D) Immunoblots showing the expression levels of endogenous BiP and transfected BiP-myc. wt and mutant BiP-myc were transiently expressed in HeLa cells, and cell lysates were blotted with anti-BiP antibodies. (E) Ig HC (top) or 3 × FLAG-ATF6 (bottom) was transiently expressed in HeLa cells along with either wt (left) or T37G mutant (right) BiP-myc. Ig HC and 3 × FLAG-ATF6 were immunoprecipitated, and the immunocomplexes were incubated with or without 2 mM ATP for 30 min at 25°C. The immunocomplexes were then immunoblotted for BiP, Ig HC, or 3 × FLAG-ATF6 as indicated.

FIG. 2.

Stability of BiP-ATF6 complexes. (A) ATF6-BiP complexes were isolated by immunoaffinity purification from NIH 3T3 cells stably expressing 3× FLAG-ATF6 as described in Materials and Methods. The complexes were separated by SDS-PAGE and stained with Coomassie blue. The identities of ATF6 and BiP in the protein complexes were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis (data not shown). Asterisks indicate nonspecific bands. (B) COS cells transiently expressing 3× FLAG-ATF6 and hamster BiP (wt, T37G mutant, or both) were labeled with [³⁵S]methionine-cysteine for 10 min. Cells were then lysed, and 3× FLAG-ATF6 was immunoprecipitated with anti-FLAG antibodies (lanes 1 to 3). After ATF6 was immunoprecipitated from one of the samples (lane 3), half of the remaining sample was immunoprecipitated with polyclonal antibodies specific to hamster BiP (lane 4). (C) COS cells transfected with or without Ig HC and both wt and T37G mutant hamster BiP were labeled with [³⁵S]methionine-cysteine for 10 min. Cells were lysed, and Ig HC was precipitated with protein A-Sepharose beads. (D) HeLa cells transiently transfected with or without SV5 HN protein and hamster BiP were pulse-labeled with [³⁵S]methionine-cysteine, and HN was immunoprecipitated with polyclonal anti-HN antiserum (lanes 1 to 2). After IP with anti-HN antisera, the supernatant of lane 2 was immunoprecipitated with polyclonal anti-BiP antiserum (lane 3). In lane 2, the position of T37G mutant BiP is indicated by an arrow and the position of SV5 HN protein is indicated by an arrowhead.

FIG. 3.

ER stress-induced BiP release is specific to ATF6 and IRE1. HeLa cells were transiently transfected with ATF6, IRE1α, Ig HC, and VSVG-T7 (tsO45) constructs as indicated to the right of each pair of panels. The S1P mutant variant of ATF6 was used to preclude any possibility of S1P digestion causing apparent dissociation of BiP. The cells were treated with 5 mM DTT for the times indicated in minutes at the top before the cells were lysed and immunoprecipitated with the indicated antibodies (or protein A-Sepharose for Ig HC). The immunocomplexes were immunoblotted for association of BiP and expression of the indicated expressed proteins.

FIG. 4.

ATF6 contains responsive sequences required for ER stress-induced BiP dissociation. (A) Diagram of constructs encoding LZIP-ATF6 chimeras. These proteins were N terminally tagged with the 3× FLAG epitope. (B to F) The above constructs were transiently transfected into HeLa cells, and the cells were treated with 5 mM DTT for the times indicated in minutes at the top. The chimeras were immunoprecipitated with anti-FLAG antibodies, and the immunocomplexes were immunoblotted with either anti-FLAG (bottom) or anti-BiP (top) antibodies. Panels B to F correspond to constructs 1 to 5 in panel A. TM, transmembrane.

FIG. 5.

(A) Diagram of constructs encoding wt ATF6 and the ATF6 LD. (B) Full-length ATF6 or the ATF6 LD was transiently expressed in HeLa cells and extracted by sonication. Cell lysates were centrifuged at 13,000 × g for 15 min before the supernatant and pellet were collected for immunoblotting analysis. (C) Localization of ATF6 LD. The ATF6 LD was transiently expressed in HeLa cells, and the cells were treated with or without 5 mM DTT as indicated. The cells were immunostained with anti-FLAG antibodies as previously described (34). (D) BiP association with the ATF6 LD. The ATF6 LD was immunoprecipitated with anti-FLAG antibodies, and BiP association was detected by immunoblotting with anti-BiP antibodies. The transfected HeLa cells were treated with 5 mM DTT as indicated to induce ER stress. (E) The ATF6 LD was extracted from HeLa cells by either 1% Triton X-100 IP buffer or sonication as indicated. The extracts were treated with or without 0.1 mM ATP at 25°C for 30 min before IP with anti-FLAG antibodies (lanes 2 and 3) and immunoblotting with either anti-BiP or anti-FLAG antibodies. For lane 1, no antibodies were added as a control for the specificity of the IP. TM, transmembrane.

FIG. 6.

ATF6 is mobile in the ER in unstressed cells. Cos-7 cells transiently expressing either GFP-ATF6 or KDELR-GFP were analyzed by FRAP. (A) Cells were untreated or incubated with BFA or BFA and DTT for at least 30 min. The indicated boxes (4-μm strips) were photobleached and analyzed for recovery as described in Materials and Methods. (B) Representative recovery curves of FRAP experiments in panel A. The recovery intensities have been transformed for comparisons of recovery rates with the recovery asymptote designated as 100%.