Chaperone-mediated reflux of secretory proteins to the cytosol during endoplasmic reticulum stress

Abstract

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such “ER stress,” we employed an ER-targeted, redox-responsive, green fluorescent protein—eroGFP—that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to “reflux” back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.

Size‐dependent secretory protein reflux into the cytosol in association with acute endoplasmic reticulum stress

“…Together, these experiments argue that ER‐sfGFP enters the ER, undergoes SS cleavage and correct folding, and then is retrotranslocated by a mechanism that appears to be distinct from ERAD‐L. Furthermore, these data are in agreement with Igbaria et al that showed deletion of HRD1 , DOA10 (which is required for ERAD‐M for membrane proteins), as well as the resulting double mutant do not impair movement into the cytoplasm of ER‐localized GFP reporters during stress 14 . Thus, the phenomenon appears to be distinct from previously described forms of ERAD.…”

“…Instead, we uncovered a novel pathway, distinct from ERAD, for the movement of correctly folded soluble ER proteins back to the cytoplasm. A similar concurrent study by the Papa group using the eroGFP reporter drew similar conclusions 14 . To distinguish this novel ER stress induced mechanism from canonical ERAD pathways, we have termed this phenomenon “ER reflux.”…”

“…Cells treated grown at 40°C and then stressed with Tm exhibited substantial protection against Tm‐induced ER reflux (Figure S1A) 38 . The heat shock response upregulates expression of 165 genes including hlj1, sse1, 39 which Igbaria et al have implicated in ER reflux 14 . Interestingly, activation of the heat shock response can alleviate ER stress, 38,40 suggesting that preventive heat exposure can adapt and enhance the ER folding environment, eliminating a stimulus for reflux.…”

“…Thus, the phenomenon appears to be distinct from previously described forms of ERAD. To highlight this difference, we and the lab of Feroz Papa have termed the ERAD‐independent phenomenon “ER reflux.” 14 …”

A guide to assessing endoplasmic reticulum homeostasis and stress in mammalian systems

“…First, as targets of ERO1‐PDI, rxYFP/roGFPs may also directly report on their activity independently of GSH . Second, the probe may wrongly report on ER redox modulation if mistargeted or refluxed from the ER to the cytosol upon stress . A solution is to tether the probe to the ER membrane .…”