title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
abstract: The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr(518). AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr(518) AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Steffen
authors.identifier: http://orcid.org/0000-0001-7936-9836
authors.initials: S
authors.lastname: Preissler
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Rato
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Ruming
authors.initials: R
authors.lastname: Chen
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Robin
authors.initials: R
authors.lastname: Antrobus
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, United Kingdom.
authors.forename: Shujing
authors.initials: S
authors.lastname: Ding
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, United Kingdom.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Fearnley
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: David
authors.identifier: http://orcid.org/0000-0002-3014-5636
authors.initials: D
authors.lastname: Ron
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D017353:Gene Deletion; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases; D011499:Protein Processing, Post-Translational
chemical_list: D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; C541422:FICD protein, human; D009713:Nucleotidyltransferases
keywords: AMPylation; BiP/GRP78; FICD/HYPE; Hsp70; biochemistry; cell biology; chaperones; endoplasmic reticulum; none