AMPylation matches BiP activity to client protein load in the endoplasmic reticulum
- University of Cambridge, United Kingdom
- MRC Mitochondrial Biology Unit, United Kingdom
Figures
Figure 1
Native gel electrophoresis tracks activity state-dependent changes in BiP’s quaternary structure.
(A) Schematic representation of BiP’s domain organization in the ATP- and ADP-bound states. BiP consists of an N-terminal nucleotide binding domain (NBD, pink) and a C-terminal substrate binding …
Figure 2 with 1 supplement
FICD deletion abolishes BiP modification in cultured cells.
(A) Schematic illustration of the hamster FICD protein. The transmembrane region (TM), the two tetratricopeptide repeats (TPR) as well as the FIC-domain (purple) with its core sub-domain (dark …
Figure 2—figure supplement 1
Time-dependent changes in BiP abundance, BiP ‘B’ form and the fraction of BiP resistant to cleavage by SubA in cells exposed to the reversible ER stress-inducing agent 2-deoxy-D-glucose (2-DG).
(A) Immunoblots of BiP from lysates of untreated cells or cells exposed to 2-deoxy-D-glucose (2-DG, 3 mM). Where indicated, 2-DG was washed out before lysis (in the presence of ATP). The samples in …
Figure 3 with 1 supplement
AMPylation of purified BiP in vitro recapitulates features of BiP modified in vivo.
(A) Coomassie (CBB)-stained native-PAGE gel (left panel) or SDS-PAGE gel (right panel) of recombinant BiP purified from bacteria (10 µM) exposed to ATP (1.5 mM) in the absence or presence of …
Figure 3—figure supplement 1
Comparison of the differential susceptibility of unmodified and AMPylated BiP to cleavage by SubA in vitro.
(A) Schema of the experimental design. ATP hydrolysis-deficient BiPT229A protein was AMPylated in presence of radioactive α-32P-ATP with catalytically active GST-FICDE234G coupled to GSH-Sepharose …
Figure 4 with 3 supplements
FICD-mediated incorporation of a single AMP molecule onto the substrate binding domain of BiP in vitro.
(A) Electrospray mass spectra of bacterially expressed hamster BiP (27-654, with a His6-tag) after reverse-phase HPLC purification. The spectra contain protein ions with between 36 and 100 …
Figure 4—figure supplement 1
Chromatographic profile and reconstructed mass spectrum of unmodified BiP and BiP modified in vitro with FICD and ATP.
(A) Total ion current chromatograms from the reverse-phase separation of unmodified and AMPylated BiP, respectively. The peak regions denoted by the red arrow were used to generate the mass spectra …
Figure 4—figure supplement 2
Modification of BiP with a single AMP molecule in vivo.
(A) Electrospray ionization mass spectrum of endogenous BiP eluted from a reverse-phase HPLC column after immunoaffinity purification from wildtype CHO-K1 lysates. The cells were treated with …
Figure 4—figure supplement 3
Evidence for the absence of modification of Ser365 or Thr366 in mono-AMPylated BiP.
(A). Amino acid sequence of Chinese hamster BiP (with the cleaved signal peptide in lower case letters) with Ser365 and Thr366 highlighted in red. The SubA cleavage site is marked by the grey arrow …
Figure 5
Mutation of threonine 518 in the substrate binding domain of BiP abolishes its AMPylation in vitro.
(A) Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of recombinant bacterially-expressed wildtype (wt) BiP and the indicated mutants exposed in vitro to active GST-FICDE234G coupled to …
Figure 6 with 2 supplements
Reciprocal loss of unmodified and gain of AMPylated BiP511-532 purified from CHO-K1 cells treated with cycloheximide.
(A) Schema of the design of the SILAC experiment to quantify relative changes in abundance unmodified and AMPylated BiP peptides from untreated and cycloheximide (CHX)-treated wildtype and FICD-/- …
Figure 6—figure supplement 1
No change detected in abundance of unmodified BiP337-367 purified from CHO-K1 cells treated with cycloheximide.
Shown are LC-MS spectra of unmodified quadruply-charged BiP337-367 peptides from a SILAC experiment in which untreated “heavy” and cycloheximide-treated “light” samples from wildtype cells or FICD-/-…
Figure 6—figure supplement 2
Fragmentation spectra of unmodified and AMPylated BiP Arg-C peptide 511-532 pinpoints AMPylation to Thr518.
(A) High energy collision dissociation (HCD) fragmentation spectra of unmodified and AMPylated BiP511-532 peptides obtained from Arg-C digests of endogenous BiP (‘1’, upper panels) immunopurified …
Figure 7 with 2 supplements
AMPylation of BiP is sensitive to its conformational state.
(A) Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of wildtype (wt) BiP and the indicated mutants exposed in vitro to active GST-FICDE234G coupled to GSH-Sepharose beads (lanes 2-5) or …
Figure 7—figure supplement 1
The isolated BiP substrate binding domain is not measurably AMPylated by FICD in vitro.
Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of wildtype BiP and a fusion of the isolated substrate binding domain (SBD) to Smt3 (Smt3-SBD) following exposure in vitro to active …
Figure 7—figure supplement 2
Loop 7,8 of the BiP substrate binding domain is destabilized in the ATP-bound conformation.
The structure of the substrate binding domain (SBD) of human BiP in the apo/ADP state (PDB 5E86) and ATP state (PDB 5E84) rendered in cartoon form with the loop encompassing Thr518 (L7,8) in stick …
Figure 8
Functional consequences of BiP AMPylation in vitro.
(A) Bar diagram of ATP hydrolysis by BiP and BiP AMPylated to completion (BiP-AMP), as reflected in phosphate release (detected colorimetrically). Samples containing either purified BiP or BiP-AMP …
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Figure 8—source data 1
Data from three independent repeats (each performed in triplicates) of the experiment presented in Figure 8E are shown.
The insert on top of each graph shows the absolute fluorescence polarization (FP) signals in arbitrary units (A.U.) of a reference sample containing only free fluorescent substrate peptide, which were used to create normalized FP traces of samples containing BiP + peptide. The initial values (after reference signal subtraction) were set to 100%. The fit to a single phase decay curve (tabulated here) was better than to a two phase model. The fit values from the three experiments were used to calculate the average values for “koff” and the half-lives. Experiment 3 is shown in Figure 8E.
- https://doi.org/10.7554/eLife.12621.020
Figure 9 with 1 supplement
Overexpression of active FICDE234G activates the UPR.
(A) Flow cytometry analysis of CHO-K1 CHOP::GFP UPR reporter cells transiently transfected with plasmids encoding wildtype FICD, the constitutively active FICDE234G or the inactive FICDE234G-H363A …
Figure 9—figure supplement 1
Overexpression of active FICDE234G induces UPR in FICD-/- cells.
Flow cytometry analysis of CHO-K1 FICD-/- CHOP::GFP UPR reporter cells transiently transfected with plasmids encoding wildtype FICD, the constitutively active FICDE234G or the inactive FICDE234G-H363…
Figure 10 with 2 supplements
Over-chaperoning in FICD-deficient cells delays UPR signaling.
(A) Schematic illustration of the rat FICD protein. Protein domains are highlighted and the mutations introduced by CRISPR-Cas9-mediated genome editing are presented as in Figure 2A. (B) Isoelectric …
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Figure 10—source data 1
Data from three independent repeats used for quantification shown in the graph in Figure 10E.
- https://doi.org/10.7554/eLife.12621.024
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Figure 10—source data 2
Source file of the flow cytometry data used to generate the plot in Figure 10—figure supplement 2D.
- https://doi.org/10.7554/eLife.12621.025
Figure 10—figure supplement 1
Undetectable FICD protein in AR42j FICD-/- cell lysates.
(A) Immunoblot analysis of the sensitivity of anti-FICD antibodies. Indicated amounts of purified bacterially-expressed mouse FICD104-458 were applied to SDS-PAGE gels followed by immunoblotting …
Figure 10—figure supplement 2
Absence of FICD does not measurably affect the induction kinetics of the transcriptional response to unfolded protein stress.
(A) Schematic illustration of the hamster FICD protein. Protein domains are highlighted and the mutations introduced by CRISPR-Cas9-mediated genome editing into CHO-K1 CHOP::GFP UPR reporter cell …
Figure 11
Schema depicting the hypothesized relationship between AMPylation and the BiP chaperone cycle.
FICD-mediated AMPylation on Thr518 allosterically traps BiP in a low substrate-affinity ATP-like state that is refractory to J protein-mediated stimulation of its ATPase activity. Removal of the …
Additional files
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Supplementary File 1
List of plasmids used.
- https://doi.org/10.7554/eLife.12621.029
