When endoplasmic reticulum (ER) homeostasis is perturbed, an adaptive mechanism is triggered and named the unfolded protein response (UPR). Thus far, three known UPR signaling branches (IRE-1, PERK, and ATF-6) mediate the reestablishment of ER functions but can also lead to apoptosis if ER stress is not alleviated. However, the understanding of the molecular mechanisms integrating the UPR to other ER functions, such as membrane traffic or endomembrane signaling, remains incomplete. We consequently sought to identify new regulators of UPR-dependent transcriptional mechanisms and focused on a family of proteins known to mediate, among other, ER-related functions: the small GTP-binding proteins of the RAS superfamily. To this end, we used transgenic UPR reporter Caenorhabditis elegans strains as a model to specifically silence small-GTPase expression. We show that the Rho subfamily member CRP-1 is an essential component of UPR-induced transcriptional events through its physical and genetic interactions with the AAA ؉ ATPase CDC-48. In addition, we describe a novel signaling module involving CRP-1 and CDC-48 which may directly link the UPR to DNA remodeling and transcription control.The endoplasmic reticulum (ER) is an organelle found in eukaryotic cells, which is mainly involved in calcium sequestration, lipid biosynthesis and translation, folding, and transport of secretory proteins (9). These functions require specialized and integrated molecular machines (7). Most of the proteins distributed in organelles of the secretory pathway, expressed at the cell surface or secreted, transit through the ER before reaching their final destination. Indeed, polypeptide chains, translated on ER membrane-bound ribosomes, are first translocated into the ER lumen via the translocon and then processed through the ER folding machineries, which include a chaperone component (e.g., BiP or GRP94), a posttranslational modification machinery (e.g., N glycosylation with the oligosaccharyl transferase complex, S-S bound formation with the protein disulfide isomerases), and a quality control component (e.g., calnexin, UDP-glucose:glycoprotein glucosyltransferase). Proteins which do not acquire a correct conformation are retained in the ER by ER-specific quality control mechanisms and are consequently granted further folding attempts. If this fails again, terminally misfolded proteins are degraded via the ER-associated degradation (ERAD) machinery (9).Under basal conditions, these integrated mechanisms maintain the ER protein load in equilibrium with ER's folding and export capacities. However, if one of those components is dysfunctional, the entire chain reaction is perturbed and ER homeostasis is disrupted. This leads to an increased amount of improperly folded proteins, which accumulate within the ER. As a mechanism for adaption to this phenomenon, cells have evolved the unfolded protein response (UPR), which aims at restoring ER homeostasis (38, 41) by (i) attenuating protein translation, (ii) increasing ER folding capacity, (iii) incre...

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IntroductIon Large-scale technologies used within the life science industry for drug discovery have recently proven useful in academic research laboratories, facilitating chemical genomics and the investigation of specific biological functions. these technologies, including homogeneous luminescence (e.g., alphascreen ® and aequorin), fluorescence intensity (fi) or polarization (fP), time-resolved fluorescence (trf), laser scanning cytometry, or microscopy-based approaches, have become enabling tools to characterize complex functions of biological targets.in the present study, we used alphascreen ® to investigate cell signaling pathways 1 and in particular those emanating from the endoplasmic reticulum (er) under stress conditions. the er ensures proper protein folding and export to later compartments of the secretory pathway. this is achieved through complex machineries, including protein synthesis, translocation/ folding, quality control, er-associated degradation (erad), and export.2 in addition to these functional attributes, the er has evolved a highly conserved adaptive signaling pathway, referred to as the unfolded protein response (uPr), whose activation occurs upon accumulation of improperly folded proteins in the er. 3 uPr signaling is mediated by 3 er resident transmembrane proteins-the PKr-like er kinase (PerK), the activating transcription factor 6 (atf6), and the inositol requiring enzyme 1 alpha (ire1α).3,4 our work has focused on ire1α, which is a transmembrane sensor of er stress. the luminal domain of ire1α contains binding sites for the chaperone BiP, whereas the cytosolic region has 2 main catalytic elements: a serine/threonine kinase and an endoribonuclease domain.4-6 under basal conditions, ire1α is thought to exist as a monomer, and in response to the accumulation of misfolded proteins in the er, ire1 proteins oligomerize, resulting in its trans-autophosphorylation and triggering of its endoribonuclease activity. 4 however, structural studies in Saccharomyces cerevisiae showed that the luminal domain of ire1 exists as a dimer/oligomer and suggested that it could potentially bind directly to unfolded peptides similar to Mhc class i molecules.7 this was confirmed by structural studies on the cytosolic domain of ire1 in S. cerevisiae, which revealed a dimer conformation that is subjected to conformational changes upon er stress, leading to activation of its kinase and rnase activities.8 recently, the cytosolic domain of ire1 was crystallized in an oligomeric form, which was promoted by the presence of the kinase inhibitor sunitinib.9,10 this highly ordered molecular structure was found assay technologies that were originally developed for high-throughput screening (hts) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (adMet) of hts. here the authors investigated and characterized the biological properties of a novel target, ire1α, a bifunctional kinase/rnase stress sensor of the endoplasmic reticulum (er). they...