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Chaperone-Mediated Reflux of Secretory Proteins to the Cytosol During Endoplasmic Reticulum Stress

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Chaperone-Mediated Reflux of Secretory Proteins to the Cytosol During Endoplasmic Reticulum Stress Chaperone-mediated reflux of secretory proteins to the cytosol during endoplasmic reticulum stress Aeid Igbariaa,b,c,1, Philip I. Merksamera,b,c,d,1, Ala Trusinae, Firehiwot Tilahuna,b,c, Jeffrey R. Johnsond,f, Onn Brandmanc,f,g, Nevan J. Krogand,f, Jonathan S. Weissmanc,f,g, and Feroz R. Papaa,b,c,2 aDepartment of Medicine, University of California, San Francisco, CA 94143; bDiabetes Center, University of California, San Francisco, CA 94143; cQuantitative Biosciences Institute, University of California, San Francisco, CA 94143; dGladstone Institute of Virology and Immunology, San Francisco, CA 94158; eCenter for Models of Life, Niels Bohr Institute, University of Copenhagen, DK 2100 Copenhagen, Denmark; fDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143; and gHoward Hughes Medical Institute, University of California, San Francisco, CA 94143 Edited by Randy Schekman, University of California, Berkeley, CA, and approved April 5, 2019 (received for review March 18, 2019) Diverse perturbations to endoplasmic reticulum (ER) functions com- UPR activation). We previously showed that differential, real- promise the proper folding and structural maturation of secretory time, quantitative eroGFP changes occurred dynamically upon proteins. To study secretory pathway physiology during such “ER general loss of ER protein-folding homeostasis in wild-type stress,” we employed an ER-targeted, redox-responsive, green cells and in a small, select group of yeast mutants (6). Here, fluorescent protein—eroGFP—that reports on ambient changes using high-throughput flow cytometry, we have extended this in oxidizing potential. Here we find that diverse ER stress regimes analysis to the entire yeast genome to query nearly all non- cause properly folded, ER-resident eroGFP (and other ER luminal essential and essential genes. Through this screen, we have proteins) to “reflux” back to the reducing environment of the cy- identified and characterized a process by which eroGFP, and a tosol as intact, folded proteins. By utilizing eroGFP in a compre- number of ER-resident luminal proteins, are “refluxed” back to hensive genetic screen in Saccharomyces cerevisiae, we show that the cytosol as intact folded proteins during ER stress. The ER protein reflux during ER stress requires specific chaperones and protein reflux process occurs independent of Hrd1 and Doa10 cochaperones residing in both the ER and the cytosol. Chaperone- E3 ligases and does not require polyubiquitinylation. Instead, mediated ER protein reflux does not require E3 ligase activity, and ER protein reflux requires specific chaperones and cochaper- proceeds even more vigorously when these ER-associated degra- ones both in the ER and cytosol, and is reminiscent of a mo- CELL BIOLOGY dation (ERAD) factors are crippled, suggesting that reflux may lecular ratchet that promotes translocation, but proceeding work in parallel with ERAD. In summary, chaperone-mediated ER vectorially in the opposite direction (7, 8). protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during Results ER protein-folding stress. ER-to-Cytosol Reflux of ER-Targeted eroGFP. Designed to be a re- porter of ambient redox potential, eroGFP has an engineered re- reflux | UPR | ERAD | endoplasmic reticulum stress versible disulfide bond that alters fluorescence excitability from its two maxima of 490 and 400 nm, such that reduction of the disulfide n eukaryotic cells, secretory and membrane proteins begin increases fluorescence from 490 nm excitation, at the expense of Itranslation in the cytoplasm and are then either co- or post- that from 400 nm (6, 9). Thus, eroGFP is ratiometric by excitation, translationally translocated through the Sec61 translocon chan- nel into the endoplasmic reticulum (ER) (1). The ER is crowded Significance with molecular chaperones and protein-modifying enzymes that promote folding and structural maturation of these nascent, Approximately one-third of eukaryotic proteins are synthe- maturing secretory pathway client proteins as they traverse the sized on ribosomes attached to the endoplasmic reticulum (ER) early secretory pathway (2). To ensure stringent quality control membrane. Many of these polypeptides co- or posttransla- over these secretory cargoes, those proteins that fail to correctly tionally translocate into the ER, wherein they fold and mature. fold and mature are retrieved from the ER, ubiquitylated, and An ER quality control system proofreads these proteins by fa- degraded by the 26S proteasome in the cytosol in a process cilitating their folding and modification, while eliminating termed ER-associated degradation (ERAD) (3). misfolded proteins through ER-associated degradation (ERAD). Diverse environmental perturbations or genetic mutations can Yet the fate of many secretory proteins during ER stress is not elevate misfolding of maturing proteins in the ER. During such completely understood. Here, we uncovered an ER stress- “ER stress,” cells trigger an intracellular signaling pathway called induced “protein reflux” system that delivers intact, folded the unfolded protein response (UPR) that augments protein- ER luminal proteins back to the cytosol without degrading folding reactions through transcriptional up-regulation of genes them. We found that ER protein reflux works in parallel with encoding ER chaperones, oxidoreductases, lipid biosynthetic ERAD and requires distinct ER-resident and cytosolic chaper- enzymes, and ERAD components (4). If these adaptive UPR ones and cochaperones. outputs prove successful in reducing the concentration of un- Author contributions: A.I., P.I.M., and F.R.P. designed research; A.I., P.I.M., F.T., J.R.J., and folded proteins in the ER, cells become restored to a homeo- O.B. performed research; A.T., O.B., N.J.K., and J.S.W. contributed new reagents/analytic static state (5). tools; A.I., P.I.M., A.T., J.R.J., and F.R.P. analyzed data; and A.I., P.I.M., and F.R.P. wrote However, because the UPR’s activating inputs—(i.e., unfolded the paper. proteins)—are unfeasible to monitor in vivo, it is often unclear if The authors declare no conflict of interest. and when the UPR has successfully restored homeostasis. To This article is a PNAS Direct Submission. address this problem orthogonally, we previously developed an Published under the PNAS license. ER-targeted redox-sensitive green fluorescent protein (GFP)— 1A.I. and P.I.M. contributed equally to this work. called eroGFP—to follow oxidative protein folding in the ER, 2To whom correspondence should be addressed. Email: [email protected]. reasoning that this essential ER physiological function may de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. viate during ER stress and thereby provide an independent 1073/pnas.1904516116/-/DCSupplemental. measure of ER health (i.e., that is distinct from solely measuring www.pnas.org/cgi/doi/10.1073/pnas.1904516116 PNAS Latest Articles | 1of8 Downloaded by guest on September 26, 2021 which facilitates internally controlled measurement of its oxi- tein glycosylation in the ER, also led to partial reduction of the dation state. Through flow cytometry, the eroGFP ratio—defined eroGFP reporter, but with slower dynamics compared with as fluorescence from excitation at 488 versus 405 nm in log2 treatment with DTT [as previously shown (6)] (SI Appendix, space—can be measured in single yeast cells growing in pop- Fig. S1 A–C). Indeed, the general utility of the eroGFP tool is ulations (Fig. 1A). Targeted to the oxidizing environment of the that it deflects differentially (by reduction) in response to di- ER through an N-terminal Kar2 signal peptide sequence (and verse ER stress agents (including forced expression of unfolded retained in the organelle through a C-terminal HDEL sequence), secretory proteins) (6). eroGFP (which has a redox midpoint potential of −282 mV) is Since our original study, which was based on single-cell in- nearly completely oxidized at baseline. Treatment with hydrogen terrogations using real-time flow cytometry, it was also reported that partial cytoplasmic localization of eroGFP occurs during peroxide (H2O2) only slightly further decreases the eroGFP ratio (6). However, a wide dynamic range exists for reduction, since expression of a mutant of the UPR master regulator, Ire1, that titration with increasing amounts of the reductant DTT—an ER cannot deactivate the UPR (10); this result implied that cyto- stress agent—dose dependently increases the eroGFP ratio until plasmic localization of the eroGFP reporter may occur due to a the reporter becomes fully reduced (Fig. 1B). As previously translocation defect under unresolved ER stress signaling. But in shown, acute treatment of cells with (saturating) DTT causes rapid our original study, through using a yeast strain expressing a GAL1/ elevation of the eroGFP ratio to its new steady-state level due to 10 promoter-driven eroGFP construct gene that no longer ex- in situ and complete reduction of the reporter (6) (SI Appendix, presses new eroGFP after glucose shutoff, we had established that eroGFP reduction due to Tm provision occurred after the glucose Fig. S1 A–C). Tunicamycin (Tm), which impairs N-linked pro- shutoff (figure S7 in ref. 6). Thus, we had reasonably concluded that ER stress induced by Tm caused reduction of preexisting eroGFP that was already residing in the ER lumen. We revisited these experimental systems by showing again that S S SH ABLasers Cell Path Bandpass Detects: SH HDEL HDEL 1 H O provision of Tm to wild-type yeast cells dynamically caused Filters (nm) 2 2 gol (mM) 2 0 ∼ S ore S eroGFP eroGFP reporter reduction (to 50% of basal levels) (detectable Blue 4( 0.25 510/20 0.5 490nm FG ′ ′ (488nm) HDEL 88 DTT in a time course of 4-acetamido-4 -maleimidylstilbene-2,2 - P 1 e x counts 2 r (mM) 4/ a 4 disulphonic acid (AMS) modification, resolution on nonreducing S 0 8 S it 5 Violet eroGFP o 12 e 515/20 x 16 SDS/PAGE, and then followed by immunoblotting against GFP) (405nm) HDEL 400nm ) −1 −0.50 0.5 1 1.5 2 2.5 3 eroGFP ratio – waste (Fig.
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