Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker
- University of Cambridge, United Kingdom
- Charles Darwin, Sapienza University of Rome, Italy
- Universidad de Málaga, Spain
- St. Jude Children's Research Hospital, United States
Figures
Figure 1
ATP increases sensitivity of BiP to cleavage at its interdomain linker.
(A) Immunoblot of endogenous BiP in lysates of CHO-K1 cells, following exposure (10 min at 30°C) to the indicated concentration of a bacterial protease (SubA) that selectively cleaves the …
Figure 2 with 3 supplements
Mutations that disrupt substrate binding or alter the interdomain linker interfere with BiP oligomerization.
(A) Peptide bond absorbance traces (A230 nm) of solutions of bacterially expressed and purified wildtype (wt) BiP, or the indicated mutants (all at 50 µM), following fractionation by size-exclusion …
Figure 2—figure supplement 1
Multiangle light scattering (SEC-MALS) measurement of purified BiP eluted from a size-exclusion chromatography column.
Purified wildtype BiP (90 µM) was passed through a size-exclusion chromatography (SEC) column and the molar masses of BiP complexes in elution peaks I, II and III were measured by MALS. …
Figure 2—figure supplement 2
ATP binding rather than ATP hydrolysis induces dissociation of BiP oligomers.
(A) Colorimetric measurement of ATP hydrolysis by purified wildtype BiP and the ATPase activity-deficient mutant BiPT229A. Shown are mean ± SD of three technical replicates (n = 3). Samples of the …
Figure 2—figure supplement 3
Glutaraldehyde crosslinking confirms the nucleotide dependence of BiP oligomerization and its susceptibility to competition by substrate peptides.
(A) SDS-PAGE and Coomassie staining of purified wildtype BiP or substrate binding-deficient BiPV461F following brief crosslinking with gluteraldehyde. Protein samples (at 10 µM) were pre-incubated …
Figure 3 with 1 supplement
Cross-protomer engagement of the interdomain linker in BiP oligomers protects it against cleavage by SubA.
(A) Coomassie-stained SDS-gel of wildtype (wt) and ADDA mutant BiP (12 µM) before and after cleavage with SubA (0.25 ng/µl) for 15 min. Note that the ADDA mutation precludes cleavage by SubA. (B) …
Figure 3—figure supplement 1
Removal of BiP's lid does not abrogate cross-protomer engagement of the interdomain linker in BiP oligomers.
(A) Peptide bond absorbance traces (A230 nm) of purified BiP (50 µM) lacking its C-terminal ‘lid’ (∆554–654) without (lidless) or with a compounding ADDA mutation (ADDA-lidless) following …
Figure 4 with 1 supplement
In oligomers, only a subset of protomers expose their interdomain linker to SubA.
(A) Purified wildtype (wt) and V461F mutant BiP (both at 7.5 µM) were exposed to SubA (20 ng/µl) at 30°C for the indicated time in presence or absence of 2 mM ATP and analyzed by SDS-PAGE and …
Figure 4—figure supplement 1
BiP protomers have high affinity for each other.
(A) Immunoblot of purified BiP resolved by native-PAGE. BiP was diluted to the indicated concentrations and allowed to equilibrate for 48 hr at room temperature. To obtain similar signal intensities …
Figure 5 with 2 supplements
BiPADDA fails to form discrete oligomers in contrast to other BiP mutants that are locked in the domain-uncoupled state.
(A) Purified wildtype (wt) and mutant BiP proteins (BiPADDA, BiPT37G and BiPG226D) were incubated (all at 30 µM) for 15 min at 30°C with or without 3 mM ATP in HKM buffer and analyzed by native-PAGE …
Figure 5—figure supplement 1
Partial proteolysis to analyze the nucleotide-dependent conformational states of wildtype and mutant BiP proteins.
10 µg of purified wildtype (wt) and mutant BiP proteins (BiPADDA, BiPT37G and BiPG226D) were digested with 2 µg Proteinase K in HKM buffer for the indicated times without or with added nucleotide …
Figure 5—figure supplement 2
Characterization of the proteolytic fragments generated by digestion of BiP with Proteinase K.
Wildtype (wt) and mutant BiP proteins (BiPADDA, BiPT37G and BiPG226D) carrying N-terminal hexahistidine tags (His6-tag) were digested for 25 min with Proteinase K in presence or absence of added …
Figure 6 with 1 supplement
Site-specific photo-crosslinking to probe the trans-engagement of the interdomain linker in BiP oligomers.
(A) Residues of BiP that were individually replaced by the photoactivatable crosslinking amino acid p-benzoyl-L-phenylalanine (pBpa) are highlighted (green) on a model of BiP in the ADP-bound state …
Figure 6—figure supplement 1
Site-specific photo-crosslinking yields a specific adduct.
(A) Coomassie-stained SDS-gel of otherwise identical C-terminally hexahistidine- (His6-) tagged BiPV461F expressed from plasmids lacking (lanes 1–4) or bearing site specific amber (TAG) codons …
Figure 7 with 1 supplement
Trans-engagement of the linker by the substrate binding domain (SBD) as the basis for BiP oligomerization in vivo.
(A) Immunoblot of endogenous BiP from lysates of CHO-K1 cells resolved by native-PAGE. Where indicated the lysate was depleted of ATP by incubation with hexokinase and glucose (HK). The major …
Figure 7—figure supplement 1
Defective homo-oligomerization of ADDA mutant BiP.
(A) Coomassie-stained native gel of wildtype (wt), V461F, ADDA and compound ADDA-V461F mutant BiP (each at 50 µM) purified from E. coli and allowed to equilibrate 16 hr at room temperature before …
Figure 8 with 1 supplement
BiP oligomeric status responds to changes in ER unfolded protein load.
(A) Immunoblot of endogenous BiP from CHO-K1 cell lysates resolved by native-PAGE. Where indicated the cells were exposed to cycloheximide (CHX, 100 µg/ml), novobiocin (NB, 0.5 mM) or both and the …
Figure 8—figure supplement 1
Time-course of evolution of the cycloheximide-dependent ‘B’ form of BiP.
(A) Immunoblot of endogenous BiP from ATP-depleted lysates of CHO-K1 cells resolved by native-PAGE after the indicated time of exposure to cycloheximide (CHX, 100 µg/ml). The major species observed …
Figure 9
Rapid BiP oligomerization in response to ER calcium depletion.
(A) Immunoblot of endogenous BiP from ATP-depleted lysates of CHO-K1 cells resolved by native-PAGE. Where indicated the cells were exposed to cycloheximide (CHX, 100 µg/ml) followed by thapsigargin …
Figure 10
Purinergic receptor engagement promotes BiP oligomerization at physiological levels of ER calcium depletion.
(A) Plot of time-dependent change if the donor fluorescence lifetime of ER localized D1ER cameleon in CHO-K1 cells treated for 1.5 hr with cycloheximide (100 µg/ml) followed by exposure to …
Figure 11 with 1 supplement
Lumenal calcium depletion-mediated dissociation of BiP from its client proteins.
(A) SDS-PAGE and immunoblot of BiP and FLAG-tagged misfolded null Hong Kong variant of α1-antitrypsin (AAT-NHK-3xFLAG) in ATP-depleted lysates of transfected CHO-K1 cells (Input) or recovered in an …
Figure 11—figure supplement 1
Cholecystokinin-mediated lumenal calcium depletion promotes BiP oligomerization in AR42j cells.
Immunoblot of endogenous BiP from ATP-depleted lysates of AR42j cells resolved by native-PAGE. Where indicated the cells were treated with dexamethasone (Dex, 100 nM, to promote differentiation into …
Figure 12
Schema of the proposed relationships between the different forms of BiP.
The monomer (‘A’ form) at the center of the schema binds clients and is shifted to BiP:Substrate complexes by mounting unfolded protein load. With diminishing clients it partitions to inactive …
Additional files
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Supplementary file 1
Plasmids used.
- https://doi.org/10.7554/eLife.08961.026
