Highlights d Akita mutant proinsulin forms detergent-insoluble aggregates d Akita aggregate formation is actively prevented by the ERresident chaperone Grp170 d RTN3-dependent ER-phagy clears Akita and other prohormone aggregates d RTN-mediated clearance of Akita aggregates partially restores WT proinsulin secretion

Protein misfolding in the endoplasmic reticulum (ER) is accompanied by adaptive cellular responses to promote cell survival. We now show that activation of mitochondrial respiration is a critical component of an adaptive ER stress response, requiring the unfolded protein response (UPR) sensor Ire1, and also calcium signaling via calcineurin. In yeast and mammalian cells lacking Ire1 or calcineurin, respiratory activation is impaired in response to ER stress; accumulation of mitochondrial reactive oxygen species (ROS) triggers cell death as abrogation of ROS by antioxidants or loss of the electron transport chain (in yeast) can rescue cells from death. Significantly, cells are rescued from ER stress-induced death by mitochondrial uncoupling by CCCP to increase O consumption (and increase the efficiency of electron transfer). Remarkably, genetic and pharmacologic strategies to promote mitochondrial biogenesis and increase O consumption also alleviate ER stress-mediated ROS and death in yeast and mammalian cells. Moreover, in a yeast genetic screen, three mitochondrial proteins Mrx9, Mrm1, and Aim19 that increase mitochondrial biogenesis were identified as high copy suppressors of ER stress-mediated cell death. Our results show that enhanced mitochondrial biogenesis, linked to improved efficiency of the electron transport chain, is a powerful strategy to block ROS accumulation and promote cell survival during ER stress in eukaryotic cells.

The reticulon-3 (RTN3)-driven targeting complex promotes clearance of misfolded prohormones from the endoplasmic reticulum (ER) for lysosomal destruction by ER-phagy. Because RTN3 resides in the cytosolic leaflet of the ER bilayer, the mechanism of selecting misfolded prohormones as ER-phagy cargo on the luminal side of the ER membrane remains unknown. Here we identify the ER transmembrane protein PGRMC1 as an RTN3-binding partner. Via its luminal domain, PGRMC1 captures misfolded prohormones, targeting them for RTN3-dependent ER-phagy. PGRMC1 selects cargos that are smaller than the large size of other reported ER-phagy substrates. Cargos for PGRMC1 include mutant proinsulins that block secretion of wildtype proinsulin through dominant-negative interactions within the ER, causing insulin-deficiency. Chemical perturbation of PGRMC1 partially restores WT insulin storage by preventing ER-phagic degradation of WT and mutant proinsulin. Thus, PGRMC1 acts as a size-selective cargo receptor during RTN3-dependent ER-phagy, and is a potential therapeutic target for diabetes.

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways.Sphingolipids are critical structural molecules in cell membranes, forming membrane microdomains by associating with cholesterol and specific proteins.1 Sphingolipid metabolites are also important signaling molecules linked to multiple other metabolic pathways with kinases and phosphatases as regulatory targets.2,3 Sphingolipids have roles in numerous cell processes, including regulation of mitochondrial function, cell death and aging.2,4 Cellular sphingolipid homeostasis is maintained by control of synthesis, breakdown and interorganellar transport of sphingolipid metabolites.1 The importance of sphingolipids is underscored by several lysosomal storage disorders, including Tay Sachs, Gaucher and Nieman-Pick diseases, which are attributable to defective sphingolipid breakdown; similarly, a hereditary sensory neuropathy is caused by accumulation of abnormal sphingolipid metabolites.

Three principal ER quality-control mechanisms, namely, unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the three pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β-cells. SEL1L-HRD1 ERAD deficiency in β-cells triggers activation of autophagy, at least in part, via IRE1α [an endogenous ERAD substrate]. In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β-cells leads to diabetes in mice shortly after weaning, with premature death by ~11 weeks of age, associated with marked ER retention of proinsulin and β-cell loss. Using focus-ion beam scanning electron microscopy (FIB-SEM) powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β-cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the three ER quality-control mechanisms in the dynamic regulation of organellar architecture and β-cell function.

One major cause of endoplasmic reticulum (ER) stress is homeostatic imbalance between biosynthetic protein folding and protein folding capacity. Cells utilize mechanisms such as the unfolded protein response (UPR) to cope with ER stress. Nevertheless, when ER stress is prolonged or severe, cell death may occur, accompanied by production of mitochondrial reactive oxygen species (ROS). Using a yeast model (Saccharomyces cerevisiae), we describe an innate, adaptive response to ER stress to increase select mitochondrial proteins, O 2 consumption and cell survival. The mitochondrial response allows cells to resist additional ER stress. The ER stressinduced mitochondrial response is mediated by activation of retrograde (RTG) signaling to enhance anapleurotic reactions of the tricarboxylic acid cycle. Mitochondrial response to ER stress is accompanied by inactivation of the conserved TORC1 pathway, and activation of Snf1/AMPK, the conserved energy sensor and regulator of metabolism. Our results provide new insight into the role of respiration in cell survival in the face of ER stress, and should help in developing therapeutic strategies to limit cell death in disorders linked to ER stress. This article has an associated First Person interview with the first author of the paper.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as ER-associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, and ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition influences ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very-long-chain ceramides leads to a marked increase in the level of ubiquitinated substrates in the ER membrane and concomitantly reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized mechanism in which ER membrane lipid remodeling changes the activity of ERAD.

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