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.
Highlights
Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by signal transduction pathways that match the complement of chaperones and enzymes to the burden of unfolded protein within the compartment
Changes in the disposition of BiP induced by manipulation of conditions in the ER are conveniently tracked by native gel electrophoresis and immunoblotting (Freiden et al, 1992) coupled with site-directed proteolysis (Preissler et al, 2015) (Figure 1A)
Inhibition of protein synthesis led to accumulation of a prominent high mobility species tentatively named ‘B’ form, whereas depletion of ER calcium progressively drew on this pool to promote the assembly of BiP oligomers (Preissler et al, 2015) (Figure 1B and C)
Summary
Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by signal transduction pathways that match the complement of chaperones and enzymes to the burden of unfolded protein within the compartment. BiP is subject to activity-dependent post-translational modification(s) This is reflected in transfer of metabolic label from intracellular pools of tritiated adenosine and 32P phosphate onto BiP, covalently modifying the protein (Carlsson and Lazarides, 1983; Hendershot et al, 1988) and imparting upon it a lower isoelectric point (pI) (Carlsson and Lazarides, 1983; Laitusis et al, 1999). It has been reported that BiP is subject to a different modification, AMPylation, which results in the formation of an phosphodiester bond between the alpha phosphate of ATP and a hydroxyl amino acid side chain (with release of pyrophosphate) This modification of BiP is effected by a broadly conserved, ER-localized enzyme, FICD ( known as HYPE) (Ham et al, 2014; Sanyal et al, 2015). Our findings fit best a parsimonious model whereby AMPylation of BiP on a single residue, Thr518, is the only quantitatively significant modification of the chaperone and provide clues to its functional significance
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