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, Thr518. AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr518 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.DOI: http://dx.doi.org/10.7554/eLife.12621.001

A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities

“…We observed changes in the conformational dynamics for Kar2 alleles that behave like oxidation mimetics and confer a protective activity during oxidative stress (Figures 6, 7). Intriguingly, the efficiency of AMPylation and the propensity to form oligomers both appear to be altered based on the conformation adopted by BiP [15, 17]. Together these data suggest the possibility that BiP cysteine oxidation could impact and/or excluded AMPylation, and/or modulate the pool of BiP oligomers, providing additional layers of regulation for BiP activities.…”

“…Several post-translational modifications have been suggested to regulate BiP activities, including AMPylation [14–16] and oligomerization [17]. In addition, it has been proposed that BiP is ADP-ribosylated [18, 19] and phosphorylated [19], although it has been put forward that these two postulated modifications may reflect misattributed AMPylation events [15]. …”

Chemical stresses fail to mimic the unfolded protein response resulting from luminal load with unfolded polypeptides

“…Also, substantially obscure remains the relevance of post-translational cellular responses to intra-compartmental load with misfolded proteins. These poorly characterized events consists in regulated covalent modification that modulate chaperone activities (e.g., ampylation of BiP (60,61) or calnexin palmitoylation (62)), regulated formation/disassembly of functional complexes or regulated turnover of specific ER-resident factors that control, for example, degradation of misfolded proteins (ERAD tuning) (57,63,64). As a final note on this subject, although comparison of transcriptomics and proteomics nicely revealed the transcriptional nature of the UPR, our LFQ-MS analyses additionally identified a number of proteins, whose level is enhanced on ER load with unfolded BACE457, BACE457∆ and CD3δQQQ in the absence of transcriptional induction of the corresponding genes.…”

Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response

“…To test this, we expressed and purified recombinant C. elegans HSP-1 (cytosolic Hsp70 ortholog), HSP-3 (BiP ortholog), and DAF-21 (cytosolic Hsp90), as well as human Hsp40 and Hsp70 and performed in vitro AMPylation assays. Because recombinant wild-type AMPylases (FIC-1, HYPE) function only poorly in vitro (9,11,14,17,23), we performed all experiments using only the constitutively active versions (FIC-1 E274G, HYPE E234G). FIC-1 (E274G) efficiently AMPylated HSP-1, HSP-3, and DAF-21 (Fig.…”

“…Recent work on BiP's function in ER homeostasis identified a major role for a particular posttranslational modification, AMPylation, in the regulation of BiP's ATPase and chaperone activity (9)(10)(11). Protein AMPylation involves the transfer of AMP from ATP to a Ser or Thr side chain and is carried out by enzymes that contain a Fic (filamentation-induced by cyclic AMP) domain (Fic proteins), an evolutionarily conserved protein family present in both bacteria and metazoans, but lacking in fungi and plants (12,13).…”