Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated, and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention.

Phospholipid homeostasis in biological membranes is essential to maintain functions of organelles such as the endoplasmic reticulum. Phospholipid perturbation has been associated to cellular stress responses. However, in most cases, the implication of membrane lipid changes to homeostatic cellular response has not been clearly defined. Previously, we reported that Saccharomyces cerevisiae adapts to lipid bilayer stress by upregulating several protein quality control pathways such as the endoplasmic reticulum-associated degradation (ERAD) pathway and the unfolded protein response (UPR). Surprisingly, we observed certain ER-resident transmembrane proteins, which form part of the UPR programme, to be destabilised under lipid bilayer stress. Among these, the protein translocon subunit Sbh1 was prematurely degraded by membrane stiffening at the ER. Moreover, our findings suggest that the Doa10 complex recognises free Sbh1 that becomes increasingly accessible during lipid bilayer stress, perhaps due to the change in ER membrane properties. Premature removal of key ER-resident transmembrane proteins might be an underlying cause of chronic ER stress as a result of lipid bilayer stress.

The unfolded protein response (UPR) is classically viewed as a stress response pathway to maintain protein homeostasis at the endoplasmic reticulum (ER). However, it has recently emerged that the UPR can be directly activated by lipid perturbation, independently of misfolded proteins. Comprising primarily phospholipids, sphingolipids and sterols, individual membranes can contain hundreds of distinct lipids. Even with such complexity, lipid distribution in a cell is tightly regulated by mechanisms that remain incompletely understood. It is therefore unsurprising that lipid dysregulation can be a key factor in disease development. Recent advances in analysis of lipids and their regulators have revealed remarkable mechanisms and connections to other cellular pathways including the UPR. In this Review, we summarize the current understanding in UPR transducers functioning as lipid sensors and the interplay between lipid metabolism and ER homeostasis in the context of metabolic diseases. We attempt to provide a framework consisting of a few key principles to integrate the different lines of evidence and explain this rather complicated mechanism.

SUMMARYMembrane integrity at the endoplasmic reticulum (ER) is tightly regulated and is implicated in metabolic diseases when compromised. Using an engineered sensor that exclusively activates the unfolded protein response (UPR) during aberrant ER membrane lipid composition, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and are conserved in C. elegans. To systematically validate yeast mutants disrupting ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensing switch in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation (ChIP) analyses pinpoint the UPR as a broad-spectrum compensatory pathway in which LBS and proteotoxic stress-induced UPR deploy divergent transcriptional programs. Together, these findings reveal the UPR program as the sum of two independent stress events and could be exploited for future therapeutic intervention.

Age-related maculopathy susceptibility 2 (ARMS2) is a small (11 kDa), primate-specific protein found in the extracellular matrix of the choroid layer in the eye. Variants in the corresponding genetic locus are highly associated with age-related macular degeneration, a leading cause of blindness in the elderly. So far, the physiological function of ARMS2 has remained enigmatic. It has been demonstrated that ARMS2 is a genuine secreted protein devoid of an N-terminal leader sequence, yet the mechanism how it exits the cells and enters the choroidal matrix is not understood. Here, we show that ARMS2 efficiently recruits lectin chaperones from the cytosol and colocalizes with calnexin-positive and protein disulfide isomerase-negative vesicle-like structures. Site-directed mutagenesis revealed critical elements for this interaction. Mutant forms proving unable to interact with the calnexin/calreticulin system failed secretion. On the other hand, blocking the endoplasmic reticulum to Golgi transport with brefeldin A had no effect on ARMS2 secretion. As we found ARMS2 colocalizing with GRASP65, a marker for unconventional protein secretion, autophagic factors are likely to be key in its export. Interleukin-1ß (IL-1ß) is the most established example of secretory autophagy. Co-expression experiments, however, suggest that the transport of ARMS2 is different from that of IL-1ß. In conclusion, in this work we show that ARMS2 is externalized via an unconventional pathway bypassing Golgi. Its intracellular separation from the classical secretion pathway suggests that the maturation of the protein requires a specific biochemical niche and/or may be needed to impede the premature formation of unwanted protein-protein interactions.