DNAJB12/14 redox switching directs chaperone- and Bax/Bak-dependent ER protein reflux

ABSTRACT Maintenance of endoplasmic reticulum (ER) proteostasis is essential for cellular homeostasis and survival during stress. Beyond canonical quality control pathways, ER-to-cytosol signaling (ERCYS) enables the reflux of ER-resident proteins into the cytosol, where they can acquire noncanonical functions that promote cell survival. However, the mechanisms governing ERCYS and its relationship to ER stress remain poorly understood. Here, we show that ER protein reflux is restricted to a defined stress window and is governed by the ER redox environment. Mild ER stress maximizes protein reflux, whereas severe or reductive stress markedly suppresses this process. Mechanistically, we identify the ER-resident cochaperones DNAJB12 and DNAJB14 as redox-sensitive regulators of ERCYS. Under mild stress, intramolecular disulfide bonds stabilize DNAJB12 and DNAJB14, thereby supporting efficient protein reflux. In contrast, severe or reductive stress increases intracellular glutathione, reducing these disulfide bonds and promoting degradation of DNAJB12 and DNAJB14, resulting in the loss of chaperone-mediated reflux. We further show that protein reflux requires cysteine-dependent interactions between refluxed substrates and the cytosolic cochaperone SGTA, revealing a previously unrecognized redox-sensitive step in the ERCYS pathway. When ERCYS is impaired during severe ER stress, cells instead engage an alternative apoptosis-associated pathway mediated by BAX/BAK-dependent ER membrane permeabilization. This transition is driven by enhanced recruitment of BAX and BAK to the ER by the BH3-only protein BIK, amplifying apoptotic signaling. Together, these findings establish redox regulation as a molecular switch that determines whether cells mount an adaptive ER protein reflux response or commit to BAX/BAK-dependent ER membrane permeabilization and apoptosis.