Significance statementSome 25 years ago it was discovered that the activity of the ER chaperone BiP is regulated by covalent modification, the nature of which, AMPylation (not ADPribosylation, as had long been thought) and the enzyme responsible, FICD, have only recently been identified. Genetic inactivation of FICD and in vitro studies of the purified enzyme and substrate have done much to clarify the biochemical consequences of the modification and its underlying logic: As ER stress wanes, FICD uses ATP to AMPylate Thr518 of BiP locking BiP in a relatively inactive conformation. As ER stress levels re-mount the cells draw on this pool of inactive chaperone, which is de-AMPylated and restored to its fully active state.Here we report on the identity of the de-AMPylating enzyme - and with it on the surprising finding that both AMPylation and de-AMPylation of BiP are carried out by the same polypeptide (FICD) using the same active site, both in vivo and in vitro. Analysis of the reaction products reveals that de-AMPylation does not involve trivial concentration-dependent micro-reversibility of an enzymatic reaction, but rather a switch in the active site of FICD that facilitates two antagonistic thermodynamically favored reactions.Surprisingly BiP de-AMPylation (not AMPylation) is the default activity of FICD. The side-chain of a single regulatory residue, E234, toggles the enzyme between de-AMPylation and AMPylation in vitro. Our studies thereby uncover an active mechanism that must exist in the ER for coupling waning levels of unfolded protein stress to the conversion of FICD from its default de-AMPylation mode to BiP AMPylation. Whilst the details of this active switch remain to be discovered, we are able to suggest a plausible mechanism by which it may come about.Identification of the enzyme that de-modifies BiP to reactivate it will be of interest to cell biologists, whereas the novel features of FICD as a dualfunctioning enzyme with a single bi-functional active site will be of broad interest to enzymologists and molecular biologists.AbstractProtein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response (UPR) that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoanspecific FIC-domain containing ER-localized enzyme, FICD/HYPE, rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here it is shown 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−/− 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.
AMPylation matches BiP activity to client protein load in the endoplasmic reticulum
