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BiP is a major endoplasmic reticulum (ER) chaperone and is suggested to act as primary sensor in the activation of the unfolded protein response (UPR). How BiP operates as a molecular chaperone and as an ER stress sensor is unknown. Here, by reconstituting components of human UPR, ER stress and BiP chaperone systems, we discover that the interaction of BiP with the luminal domains of UPR proteins IRE1 and PERK switch BiP from its chaperone cycle into an ER stress sensor cycle by preventing the binding of its co-chaperones, with loss of ATPase stimulation. Furthermore, misfolded protein-dependent dissociation of BiP from IRE1 is primed by ATP but not ADP. Our data elucidate a previously unidentified mechanistic cycle of BiP function that explains its ability to act as an Hsp70 chaperone and ER stress sensor.

The unfolded protein response (UPR) is a key signaling system that regulates protein homeostasis within the endoplasmic reticulum (ER). The primary step in UPR activation is the detection of misfolded proteins, the mechanism of which is unclear. We have previously suggested an allosteric mechanism for UPR induction (Carrara et al., 2015) based on qualitative pull-down assays. Here, we develop an in vitro Förster resonance energy transfer (FRET) UPR induction assay that quantifies IRE1 luminal domain and BiP association and dissociation upon addition of misfolded proteins. Using this technique, we reassess our previous observations and extend mechanistic insight to cover other general ER misfolded protein substrates and their folded native state. Moreover, we evaluate the key BiP substrate-binding domain mutant V461F. The new experimental approach significantly enhances the evidence suggesting an allosteric model for UPR induction upon ER stress.

The genes coding for the enzymes of oxidative degradation of nicotinic acid have recently been identified in several species of aerobic bacteria, namely, Pseudomonas putida KT2440, Bordetella bronchiseptica RB50, and Bacillus niacini. One of the enzymes involved in an early step of this pathway is a flavin-dependent monooxygenase (6-hydroxynicotinic acid 3-monooxygenase; NicC) that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP), with concomitant oxidation of NADH to NAD+. The nicC genes from B. bronchiseptica RB50 and P. putida have been cloned, and the purified enzymes have been characterized functionally and structurally. Global fits of the steady-state kinetic data show that both enzymes are efficient catalysts, with an apparent kcat/KM6-HNA of 5.0 × 104 M–1 s–1 for B. bronchiseptica NicC. The pH dependence of Vmax/[E]t fits a double-bell model showing an optimum around pH 8 with apparent pKas of 7.24 ± 0.08 and 8.64 ± 0.08, whereas the...

The unfolded protein response (UPR) is an essential cell signaling system that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER) and initiates a cellular response in order to maintain homeostasis. How cells detect the accumulation of misfolded proteins remains unclear. In this study, we identify a noncanonical interaction between the ATPase domain of the ER chaperone BiP and the luminal domains of the UPR sensors Ire1 and Perk that dissociates when authentic ER unfolded protein CH1 binds to the canonical substrate binding domain of BiP. Unlike the interaction between chaperone and substrates, we found that the interaction between BiP and UPR sensors was unaffected by nucleotides. Thus, we discover that BiP is dual functional UPR sensor, sensing unfolded proteins by canonical binding to substrates and transducing this event to noncanonical, signaling interaction to Ire1 and Perk. Our observations implicate BiP as the key component for detecting ER stress and suggest an allosteric mechanism for UPR induction. DOI: http://dx.doi.org/10.7554/eLife.03522.001, eLife digest Proteins perform many essential tasks in cells, but to be able to work they first have to correctly fold into a specific three-dimensional shape. Within the cell, many proteins are folded with the help of ‘chaperone’ proteins. If any proteins fold incorrectly, the normal workings of the cell can be disturbed, which may damage the cell. This is more likely to happen if a cell suddenly requires a large number of proteins to be made, which can overwhelm the chaperone proteins. In humans and other eukaryotic organisms, many proteins are folded in a compartment within the cell called the endoplasmic reticulum. Inside this compartment there is a system called the unfolded protein response that detects misfolded proteins and boosts the cell's capacity to re-fold them. As part of this system, two sensor proteins detect when misfolded proteins are present, but it is not clear how they do so. It has been suggested that a chaperone protein called BiP may be able to activate these sensor proteins in order to turn on the unfolded protein response. In this study, Carrara et al. studied the sensor proteins and BiP using an artificial set-up in the laboratory. The experiments show that both of the sensor proteins can bind to a section of the BiP chaperone called the ATPase domain. However, in the presence of an unfolded protein, BiP stopped interacting with the sensor proteins, which could allow the sensor proteins to activate the unfolded protein response. The experiments also show that BiP must bind to the unfolded protein to activate the unfolded protein response. Carrara et al.'s findings suggest that BiP has a dual role in cells: to sense unfolded proteins by binding to them, and then to activate the sensor proteins that trigger the unfolded protein response. Together, these results suggest a new model for how cells detect and respond to misfolded proteins within the endoplasmic reticulum, and may provide new targets for therapies to treat diseases caused by defects in protein folding. DOI: http://dx.doi.org/10.7554/eLife.03522.002