Uncovering Reversible AMPylation of BiP Mediated by dFic During ER Homeostasis

Abstract

AMPylation is a posttranslational modification involving a covalent attachment of an AMP moiety from ATP to hydroxyl side chains of target substrates. Fic domain which mediates AMPylation is highly conserved across species, including higher eukaryotes, implicating an essential role of this modification in cellular function. Despite the recent discoveries and characterization of a number of bacterial AMPylators and their targets during pathogenesis, the knowledge of AMPylation in eukaryotic system is still elusive. Therefore, the goal of my thesis is to determine the eukaryotic function of AMPylation and identifying the endogenous substrates of this novel modification. In an attempt to understand the physiological function of AMPylation in eukaryotes, we used Drosophila melanogaster as our genetic model organism and created mutant flies lacking functional Drosophila Fic (dFic). We found that the flies without enzymatic function of dFic exhibit blind phenotype due to impaired synaptic transmission. dFic enzymatic activity is required in glial cells for the normal visual neurotransmission. This suggests that a target of dFic may be a component of the visual signaling pathway. dFic was observed in the cell surface of the glial cells particularly enriched in capitate projections. However, dFic is localized to the ER in a number of fly tissues and also in the S2 cells, indicating that there may be another target of dFic in the ER that plays a more general role in the cellular function. In this study, we identified an ER molecular chaperone BiP/GRP78 as a novel substrate for dFic-mediated AMPylation. BiP was predominantly labeled with AMP by dFic in S2 cell lysate. AMPylation of BiP decreases during ER stress but increases upon the reduction of unfolded proteins. Both dFic and BiP are transcriptionally activated upon ER stress induction, implicating a role for dFic in the UPR. We identified a conserved threonine residue, Thr366, as the AMPylation site, which is in close proximity to the ATP binding site of BiP's ATPase domain. Our study presents the first substrate of AMPylation by a eukaryotic protein and proposes a new mode of posttranslational regulation of BiP, which is likely to serve a crucial role in maintaining ER protein homeostasis.