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 agents 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 S. cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and co-chaperones 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. Igbaria et al 2019 SIGNIFICANCEApproximately one third of eukaryotic proteins are synthesized on ribosomes attached to the endoplasmic reticulum (ER) membrane. Many of these polypeptides co-or posttranslationally translocate into the ER, wherein they fold and mature. An ER qualitycontrol system proofreads these proteins by facilitating their folding and modification, while eliminating misfolded proteins through ER-associated degradation (ERAD). Yet, the fate of many secretory proteins during ER stress is not completely understood. Here, we uncovered an ER-stress induced "protein reflux" system that delivers intact, folded ER luminal proteins back to the cytosol without degrading them. We found that ER protein reflux works in parallel to ERAD and requires distinct ER-resident and cytosolic chaperones and co-chaperones. \body

Sequestration of TDP-43216-414aggregates by cytoplasmic expression of the proSAAS chaperone

“…Following ER stress, such as thapsigargin treatment, the secretory chaperone clusterin translocates to the cytoplasm and binds TDP-43 (Gregory et al, 2017;Nizard et al, 2007). ER reflux to the cytoplasm is a phenomenon also recognized in yeast (Igbaria et al, 2019). In our hands, expression of TDP-43 216-414 together with clusterin in the absence of stressors was itself sufficient to induce a large change in the distribution of both clusterin and TDP-43 ; and following the addition of ER stressors, we confirmed clusterin retrotranslocation (as indicated by co-localization with TDP-43 ).…”

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 [47,48]. Second, the probe may wrongly report on ER redox modulation if mistargeted or refluxed from the ER to the cytosol upon stress [49]. A solution is to tether the probe to the ER membrane [40].…”

Reflux of Endoplasmic Reticulum proteins to the cytosol yields inactivation of tumor suppressors

“…We next sought to identify factors regulating ER-to-cytosol protein reflux. Recently, we reported that ER luminal proteins were refluxed to the cytosol upon ER stress in the yeast S. cerevisiae (Igbaria et al 2019;Lajoie and Snapp 2020) in a chaperone-mediated process (Igbaria et al 2019). We tested whether ER stress/UPR activation -two factors that showed a correlation with protein reflux in S. cerevisiae -would also cause ER protein reflux in mammalian cells.…”

“…Recently, we showed that protein folding stress causes ER resident proteins to be refluxed to the cytosol in the yeast Saccharomyces Cerevisiae (Igbaria et al 2019). This mechanism requires ER and cytosolic chaperones and co-chaperones but is independent of the ERAD machinery and protein degradation (Igbaria et al 2019). Here we found that ER stress mediated protein reflux is conserved in mammalian cells and in isolated cancer cells from human and murine tumors and aims at debulking the ER upon stress.…”