BIP orchestrates bidirectional ER protein trafficking via co-chaperone complexes

Background: Interorganellar protein redistribution is an emerging but underexplored aspect of proteostasis and cellular adaptation. Beyond canonical transcriptional and translational regulation, cells dynamically reprogram the spatial distribution of proteins to rapidly respond to environmental stress. This spatial plasticity enables single gene products to acquire novel, context-dependent functions on the basis of subcellular localization. Such relocalization is particularly pronounced in pathological conditions, such as cancer and viral infections, where proteome remodeling enhances cellular survival and adaptability. We previously defined endoplasmic reticulum (ER)-to-cytosol signaling (ERCYS) as a stress-responsive mechanism that alleviates ER burden by redistributing proteins into the cytosol. Despite growing interest, the molecular mechanisms driving ERCYS and related forms of spatial proteome remodeling remain poorly defined. Methods: To investigate these mechanisms, we employed siRNA- and CRISPR-based depletion of BIP, SGTA, and DNAJB12/14, coupled with subcellular fractionation and immunoblotting to assess protein localization under stress. Co-immunoprecipitation was used to examine protein–protein interactions, and unfolded protein response (UPR) activation was quantified via quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results: Our results reveal a previously unrecognized role for the UPR in mediating ER protein reflux. Specifically, we show that ATF6 and IRE1, but not PERK, are essential for initiating ERCYS. Notably, IRE1 simultaneously promotes ERCYS while suppressing BAX/BAK-mediated ER membrane permeabilization. Furthermore, we uncover a noncanonical, signaling-independent function of the ER-resident chaperone BIP in protein reflux. BIP forms a complex with membrane-bound DNAJB12/14 and cytosolic SGTA, facilitating chaperone-guided export of proteins from the ER lumen. This process depends on an intact DNAJB12 J-domain and requires BIP to originate within the ER, supporting a directional, regulated export mechanism. These findings challenge the classical view of BIP as solely mediating inward translocation and reveal a bidirectional role in protein trafficking. Our work uncovers a novel layer of UPR-regulated spatial proteome remodeling with potential relevance in cancer biology.