This is the first study of “ER stress” using real-time analysis to interrogate the stressed state of single cells within large populations. We utilized separate reporters to measure two independent variables: UPR-RFP for UPR activity, and a redox-sensitive reporter—eroGFP—to follow oxidation of cysteine sulfhydryls to disulfides. Thus eroGFP was positioned as a “proximal” reporter for the critical ER function of disulfide formation. eroGFP is internally controlled because it is ratiometric by excitation, obviating the need to consider and correct for confounding factors affecting absolute reporter levels. Although an individual eroGFP molecule is either reduced or oxidized (binary), cumulative fluorescence from many eroGFP molecules provided a continuous (analog) readout of eroGFP’s average oxidation state within single cells. Adding UPR-RFP as a “distal” reporter for UPR signaling provided complementary information that could not have been gathered using either reporter alone. Our design strategy of positioning proximal and distal reporters may be useful for studying other intracellular signaling pathways where input signals are not easily quantifiable.
We propose a model that accounts for our observations and analyses, and provides conceptual advances (). Using our systems, we found (as expected) that chemical reduction is a “primary” stress causing under-oxidation of eroGFP and other ER proteins—we refer to this as ER oxidative folding stress. Unexpectedly, other challenges to protein folding—including under-glycosylation, inositol deprivation, and increased protein secretion—“secondarily” caused varying degrees of ER oxidative folding stress. Based on these results, we propose that the ER’s protein folding, modification, and quality control systems are functionally interlinked with the oxidative folding machinery—and by extension perhaps with each other. Thus, eroGFP can measure many discrete challenges to ER function that have historically been grouped and studied under the rubric of “ER stress”.
Conceptual model of ER stress and UPR-mediated compensation
Our model highlights four extreme states of ER oxidation state and UPR activity. Consistent with previous estimates of a highly oxidizing ER redox potential obtained through disruptive methods (Hwang et al., 1992
), eroGFP was almost completely oxidized in wild-type cells without imposed stress. Unexpectedly, eroGFP remained similarly oxidized in all mutants tested in this study. This unexpected result points to a remarkable plasticity in ER oxidative folding. Only through actively perturbing ER functions are eroGFP differences between mutants revealed. Unstressed UPR mutants can still support ER oxidation, perhaps because the UPR is only required to increase expression of ER oxidoreductases during stress. ER oxidation, however, is essential, and hypomorphs in oxidative folding activities exhibit an adapted state through low-level UPR activation.
The ER-stressed (UPR-activated) state can be achieved in any cell competent for UPR signaling. Here the combination of eroGFP and UPR-RFP revealed subtle differences between mutants under distinct stresses. Both ero1-DAmP and pdi1-DAmP resisted challenges to glycosylation better than oxidation. This difference may occur because UPR activation resets ER oxidation to minimum levels for viability, but in the process augments other ER folding activities such as those supporting glycosylation. Thus these mutants come “pre-conditioned” against challenge to other functions. We predict that many such pre-conditioned states could exist, and dynamic monitoring with eroGFP should delineate these at the genomic level, allowing assignment of primary functions to uncharacterized genes. Pre-conditioning could also be achieved through chemical-genetic activation of Ire1, which revealed slight hyperoxidation in the absence of stress. Future versions of eroGFPs exhibiting more oxidizing midpoint potentials may allow more accurate quantification of these changes under both resting and stressed states.
The ER-stressed (decompensated) state of UPR mutants was revealed through larger and sustained eroGFP deflections than wild-type under stress, and could not have been described using the distal UPR-RFP reporter alone. Therefore measuring eroGFP oxidation changes provides information on drift from ER homeostasis, while measuring UPR activity provides information on compensation.
How can eroGFP become reduced during myriad ER stresses? Like any ER protein containing cysteines, eroGFP must be oxidized through dithiol-disulfide exchange reactions through specific protein-protein interactions mediated by oxidoreductases. These activities may become compromised during stress. Indeed, oxidation of Ero1p decreased when under-glycosylated. Additionally, as the vast majority of secretory proteins contain cysteines, their accumulation in unfolded form may saturate the ER’s oxidative machinery due to futile cycling attempts to form disulfide bonds (Haynes et al., 2004
). This effect may occur in some cells during overexpression of the cysteine-containing protein, CPY and its misfolded variants. Interestingly, overexpression of a cysteine-less CPY* variant still caused eroGFP deflection in some cells (although to a lesser degree than a variant containing one cysteine) indicating that a saturation mechanism cannot be the sole explanation for eroGFP changes.
Because we also observed reduction of pre-existing eroGFP, its disulfide bond must be labile and may become reduced by enzymatic activities or ER permeable reductants such as glutathione. PDI has been shown to have reductase activity, facilitating removal of unfolded proteins from the ER (Tsai et al., 2001
). More recently a mammalian ER reductase, ERdj5, was described (Ushioda et al., 2008
). Interestingly, a conserved disulfide in the ER-luminal domain of the UPR sensor ATF6 is reduced during ER stress, allowing ATF6 to traffic to the Golgi for processing and activation (Nadanaka et al., 2006
). When protein oxidation becomes generally compromised during ER stress as we have shown, ATF6’s labile disulfide bond may become reduced, raising the exciting prospect that ER homeostasis is maintained through sensing both redox state as well as unfolded proteins.
While we observed eroGFP co-localized with the ER (i.e. in reticular structures containing Sec61) during stresses we imposed, it is conceivable that dynamic remodeling of ER to terminal or salvage organelles may expose eroGFP to less oxidizing milieus, also contributing to its reduction.
Finally, eroGFP revealed unanticipated heterogeneities between individual cells in a number of our experiments. During inositol starvation, the eroGFP ratio rose in UPR-deficient mother cells, but not in daughters. We speculate that asymmetric segregation of ER tubules containing under-oxidized proteins may occur under stress. This observation adds to reports that oxidatively damaged cytosolic and mitochondrial proteins are retained in mothers (Aguilaniu et al., 2003
). The heterogeneity observed for CPY* expression—which could represent a stress to which cells can successfully adapt—also suggests that some individuals escape homeostatic control. Such differences may account for different cell fate outcomes in metazoans, which eliminate highly stressed cells through apoptosis. The metazoan UPR can alternatively transmit survival or apoptotic signals though it is unclear how the UPR separates and relegates some cells to an apoptotic fate, while allowing others to successfully adapt. The tools and concepts we have developed may be applicable to the study and understanding of such questions.