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FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

Abstract

Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoan-specific FIC-domain-containing ER-localized enzyme (FICD) rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here we show that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD−/− hamster cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation-defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.

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Figure 1: Introduction of wild-type FICD into FICD−/− cells fails to restore BiP AMPylation.
Figure 2: FICD de-AMPylates BiP in vitro.
Figure 3: FICD-mediated de-AMPylation releases AMP and restores BiP to its pre-AMPylation state.
Figure 4: Enzymatic properties of the FICD de-AMPylase.
Figure 5: Engagement of E234 in the active site switches FICD from AMPylation to de-AMPylation.
Figure 6: FICD counteracts BiP AMPylation in cells.
Figure 7: A hypothetical model depicting regulation of the FICD-mediated BiP AMPylation and de-AMPylation cycle by the disposition of E234.

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Acknowledgements

We thank R. Antrobus (CIMR mass spectrometry), R. Schulte and the CIMR flow cytometry team for assistance; H.P. Harding, N. Amin-Wetzel and J. Chambers (CIMR) for advice and comments on the manuscript; and C. Flandoli (Cambridge, UK) for the cartoon. Supported by Wellcome Trust Principal Research Fellowship to D.R. (Wellcome 200848/Z/16/Z), a UK Medical Research Council PhD studentship to L.A.P. and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140).

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S.P. conceived, designed and led the project; conducted in vitro experiments, analysis and interpretation of data; and drafted and revised the article. C.R. designed, conducted and interpreted the in vivo experiments and contributed to drafting and revising the article. L.A.P. contributed to protein purification and fluorescence polarization experiments and revised the manuscript. V.S. provided valuable insights and discussions and revised the manuscript. D.R. oversaw the project conception and design, construction of plasmid DNA, analysis and interpretation of data and drafting and revising of the article.

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Correspondence to Steffen Preissler or David Ron.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 AMPylation with ATP-FAM generates BiP specifically labeled on its substrate-binding domain.

(a) SDS-PAGE gel of AMPylated BiP incubated without or with SubA protease. BiP was AMPylated in vitro with FICDE234G in presence of a fluorescently labeled ATP derivative (ATP-FAM) and the resulting AMPylated BiP, with a fluorescent AMP attached (BiPT518-AMP-FAM) was re-purified (as in Fig. 2d). After prolonged treatment without or with SubA (4 hours at 30°C) the samples were denatured and applied to SDS-PAGE. The fluorescence signals of the fluorophore in the gel were detected (excitation: 488 nm, emission: 526 nm; upper panel) and the proteins were visualized by staining with Coomassie (CBB; lower panel). Uncleaved full-length BiPT518-AMP-FAM (FL), the nucleotide binding domain (NBD), the substrate binding domain (SBD), FICD, and SubA are indicated.

(b) Time-dependent plot of fluorescence polarization (FP) of BiP AMPylated with FAM-labeled AMP (BiPT518-AMP-FAM, from the sample shown in “a” above) after incubation without (black line) or with wildtype FICD protein (red line). The decrease in the FP signal reflects release of the fluorophore from BiP.

Uncropped gel images are shown in Supplementary Data Set 1.

Supplementary Figure 2 Absorbance spectra of ion pair chromatography elution profiles.

(a) 3D absorbance plots of the nucleotide standard (upper panel) and the ‘BiP-AMP + FICD’ de-AMPylation sample (lower panel) shown in Fig. 3c (grey and red traces therein). The elution profiles between 6 and 13 minutes are shown. Note that the absorbance characteristics of the AMP standard and the de-AMPylation product (both eluting at ~11.1 minutes) are qualitatively indistinguishable with an absorbance maximum at ~260 nm (arrows).

(b) Direct comparison of the absorbance spectra at 11.14 minutes of the profiles shown in “a”.

Supplementary Figure 3 Analysis of FICD overexpression in cells.

(a) FICD immunoblot of FICD-deficient (-/-) CHO-K1 cells transfected with plasmids encoding the indicated FICD derivatives. The eIF2α below serves as a loading control. Note the higher protein levels of the weaker AMPylation active/de-AMPylation defective FICDE234V/L/Q/K mutants compared to the AMPylation hyperactive FICDE234G.

(b) Flow cytometry source data from a representative experiment (one of three) used to generate the plot in Fig. 6b.

Uncropped blot images are shown in Supplementary Data Set 1.

Supplementary Figure 4 Analysis of FICD overexpression by flow cytometry and native PAGE.

(a) Flow cytometry source data from one of three independent repeats plotted in Fig. 6c.

(b) Immunoblot of endogenous BiP from wildtype and FICD-deficient (-/-) CHO-K1 cells resolved by native-PAGE. The cells were co-transfected with the indicated pairs of plasmids as in Fig. 6c and allowed to grow for 36 hours. The AMPylated ‘B’ form of BiP is indicated (as are the other major species, see Fig. 1 legend). Immunoblots of the same samples resolved by SDS-PAGE report on FICD, total BiP and total eIF2α (which also serves as a loading control). Data representative of two independent experiments are shown.

Note the absence of AMPylated BiP (‘B’ form) in cells co-transfected with wildtype FICD and the de-AMPylation defective/AMPylation active FICDE234G.

Uncropped blot images are shown in Supplementary Data Set 1.

Supplementary Figure 5 Wild-type and FICD–/– cells respond indistinguishably to unfolded protein stress in the ER.

(a) Flow cytometry analysis of wildtype and FICD-deficient (-/-) CHO-K1 CHOP::GFP UPR reporter cells treated with the UPR-inducing compounds, tunicamycin (2.5 μg/ml) or thapsigargin (0.5 μM), for 16 hours before analysis. Note the equal accumulation of CHOP::GFP-positive cells in tunicamycin- or thapsigargin-treated wildtype and FICD-/- cells.

(b) Plot of the median values ± SD of the GFP fluorescent signal of the samples described in “a” from three independent experiments (fold change relative to untreated wildtype cells).

(c) Flow cytometry analysis of wildtype and FICD-deficient (-/-) CHO-K1 CHOP::GFP UPR reporter cells transiently transfected with plasmids encoding the Cas9 nuclease and single guide RNAs targeting hamster BiP. Note the similar levels of UPR signaling in wildtype and FICD-/- cells.

(d) Plot of the median values ± SD of the CHOP::GFP fluorescent signal in the transfected subpopulation of the cells shown in “c” from three independent experiments. Transfected cells were identified by co-expression of a mCherry marker (not shown) carried by the Cas9 plasmid (fold change relative to wildtype cells transfected with plasmid DNA encoding Cas9 and mCherry only).

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Supplementary Table 1

Description of plasmids used for the study. (XLSX 44 kb)

Supplementary Data Set 1

Uncropped blot, autoradiograph and gel images. (PDF 15392 kb)

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Preissler, S., Rato, C., Perera, L. et al. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP. Nat Struct Mol Biol 24, 23–29 (2017). https://doi.org/10.1038/nsmb.3337

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