Molecular Immunology 47 (2010) 1719–1727 Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Prolonged endoplasmic reticulum stress promotes mislocalization of immunoglobulins to the cytoplasm Adi Drori a , Shahram Misaghi b , Joseph Haimovich c , Martin Messerle d , Boaz Tirosh a,∗ a The Institute of Drug Research, The School of Pharmacy, The Hebrew University, Jerusalem 91120, Israel b Physiological Chemistry Department, Genentech Inc, 1 DNA Way, South San Francisco, CA 94080, USA c Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Israel d Department of Virology, Hannover Medical School, Hannover, Germany article info Article history: Received 8 February 2010 Accepted 4 March 2010 Available online 28 March 2010 Keywords: B cells Cell differentiation Antibodies Endoplasmic reticulum Unfolded protein response abstract Signal peptide-dependent insertion of newly synthesized proteins into the endoplasmic reticulum (ER) is a multi-step process, whose fidelity varies with the identity of the protein and the cell type. ER translo- cation of prions is sensitive to conditions of acute ER stress in a manner that pre-emptively prevents their aggregation and proteo-toxicity. While this has been documented for extreme ER stress conditions and for a special type of proteins, the impact of chronic ER stress on protein translocation in general has not been well characterized. The unfolded protein response (UPR) is a cytoprotective signaling pathway activated by ER stress. The transcription factor X-box-binding protein 1 (XBP-1) is a key element of the mammalian UPR, which is activated in response to ER stress. Deletion of XBP-1 generates constitutive chronic ER stress conditions. Chronic ER stress can also be produced pharmacologically, for example by prolonged treatment with proteasome inhibitors, which abrogates XBP-1 activation. We tested the impact of chronic ER stress on protein insertion into the ER with special emphasis on antibody secreting cells (ASCs), as these cells cope physiologically with prolonged stress conditions. We show that XBP-1 in plasmablasts and fibroblasts controls the ER translocation of US2, a viral-encoded protein with a priori poor insertion efficiency. Using monoclonal antibodies that preferentially recognize ER-mis-inserted  Ig chains we demonstrate that prolonged treatment of plasmablasts with proteasome inhibitors, as well as deletion of XBP-1, impaired the translocation of  chains to the ER. Our data sug- gest that ASCs under prolonged ER stress conditions endure cytoplasmic mislocalization of Ig proteins. This mislocalization may further explain the exquisite sensitivity of multiple myeloma to proteasome inhibitors. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction In mammalian cells, it is estimated that 30% of all proteins are targeted to the endoplasmic reticulum (ER), the port of entry into the secretory pathway. The influx of newly synthesized polypeptide chains into the ER is variable, and can change rapidly in response to environmental signals, infection by pathogens or differentia- Abbreviations: ASCs, antibody secreting cells; ATF6, activating transcription factor 6; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GS, glycosy- lation site; HCMV, human cytomegalovirus; IP, immunoprecipitation; IRE-1, inositol requiring enzyme 1; PERK, PKR-like ER kinase; Tg, thapsigargin; Tm, tunicamycin; UPR, unfolded protein response; XBP-1, X-box-binding protein 1. ∗ Corresponding author at: Department of Pharmacology, School of Pharmacy, Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel. Tel.: +972 2 6758730; fax: +972 2 6758741. E-mail address: boazt@ekmd.huji.ac.il (B. Tirosh). tion programs (Gass et al., 2002; He, 2006; Iwakoshi et al., 2003; Lee et al., 2005; Ozcan et al., 2004). ER stress occurs when cel- lular protein synthesis demand exceeds its folding capacity. In an effort to maintain homeostasis, a network of signaling cas- cades known as the unfolded protein response (UPR) is activated, resulting in reduced protein synthesis, enhancement of the ER fold- ing capacity, increased degradation of ER misfolded proteins and enhanced ER biogenesis (Ron and Walter, 2007; Sriburi et al., 2004, 2007). In the mammalian UPR three ER transmembrane sensors coop- erate to attenuate the ER load: activating transcription factor 6 (ATF6), inositol requiring enzyme 1 (IRE-1) and PKR-like ER kinase (PERK). The first two pathways increase the folding capacity of the cell by upregulation of ER resident chaperones, expand the ER and increase the clearance of misfolded proteins through ER-associated degradation pathways, while the PERK pathway transiently inhibits protein translation (Ron and Walter, 2007). Continued activation of 0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2010.03.006