Ergosterol acts as a permissive regulator of Ire1 responsiveness during ER stress

The unfolded protein response (UPR) safeguards endoplasmic reticulum (ER) homeostasis by integrating signals arising from both protein-folding defects and membrane stress. While activation of Ire1 by unfolded proteins has been extensively characterized, the contribution of membrane lipid composition to this process remains incompletely understood. It is unclear whether ergosterol acts as a primary activating signal for Ire1 or instead modulates the activation threshold and amplitude of the UPR response. Using {beta}-mercaptoethanol (BME) as a proteotoxic perturbation, we found that this reducing agent exerts opposing effects on the two major inputs that converge on Ire1 signaling. Although BME induces proteotoxic stress, it triggers only a moderate UPR response while simultaneously causing a pronounced reduction in ergosterol biosynthesis, a behavior distinct from that generated by classical ER stressors. Pharmacological, proteomic, genetic, and lipidomic analyses revealed a causal relationship between reduced ergosterol levels and attenuated Ire1 activation. Importantly, elevated ergosterol levels alone were insufficient to activate the UPR, indicating that sterols do not directly trigger the pathway. Instead, our findings support a model in which ergosterol functions as a permissive determinant of Ire1 responsiveness, tuning the amplitude and gain of UPR signaling in response to ER proteotoxic stress. Moreover, the differential effects of endogenous and exogenous sterol accumulation on Ire1 activation raise the possibility that not only total ergosterol abundance, but also its intracellular distribution and accessibility contribute to amplify Ire1 response. Together, these results identify sterol homeostasis as a key regulator of ER stress signaling and reveal how membrane composition influences the efficiency with which luminal stress information is translated into productive Ire1 activation.