Content Of Endoplasmic Reticulum Research Articles

Enhancing the Antibody Production Efficiency of Chinese Hamster Ovary Cells through Improvement of Disulfide Bond Folding Ability and Apoptosis Resistance.

The complex structure of monoclonal antibodies (mAbs) expressed in Chinese hamster ovary (CHO) cells may result in the accumulation of unfolded proteins, triggering endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). If the protein folding ability cannot maintain ER homeostasis, the cell will shut down protein translation and ultimately induce apoptosis. We co-overexpressed HsQSOX1b and survivin proteins in the antibody-producing cell line CHO-PAb to obtain a new cell line, CHO-PAb-QS. Compared with CHO-PAb cells, the survival time of CHO-PAb-QS cells in batch culture was extended by 2 days, and the antibody accumulation and productivity were increased by 52% and 45%, respectively. The proportion of (HC-LC)2 was approximately doubled in the CHO-PAb-QS cells, which adapted to the accelerated disulfide bond folding capacity by upregulating the UPR's strength and increasing the ER content. The results of the apoptosis assays indicated that the CHO-PAb-QS cell line exhibited more excellent resistance to apoptosis induced by ER stress. Finally, CHO-PAb-QS cells exhibited mild oxidative stress but did not significantly alter the redox status. This study demonstrated that strategies based on HsQSOX1b and survivin co-overexpression could facilitate protein disulfide bond folding and anti-apoptosis ability, enhancing antibody production efficiency in CHO cell lines.

L-type Ca2+ channel activation of STIM1–Orai1 signaling remodels the dendritic spine ER to maintain long-term structural plasticity

Learning and memory require coordinated structural and functional plasticity at neuronal glutamatergic synapses located on dendritic spines. Here, we investigated how the endoplasmic reticulum (ER) controls postsynaptic Ca2+ signaling and long-term potentiation of dendritic spine size, i.e., sLTP that accompanies functional strengthening of glutamatergic synaptic transmission. In most ER-containing (ER+) spines, high-frequency optical glutamate uncaging (HFGU) induced long-lasting sLTP that was accompanied by a persistent increase in spine ER content downstream of a signaling cascade engaged by N-methyl-D-aspartate receptors (NMDARs), L-type Ca2+ channels (LTCCs), and Orai1 channels, the latter being activated by stromal interaction molecule 1 (STIM1) in response to ER Ca2+ release. In contrast, HFGU stimulation of ER-lacking (ER-) spines expressed only transient sLTP and exhibited weaker Ca2+ signals noticeably lacking Orai1 and ER contributions. Consistent with spine ER regulating structural metaplasticity, delivery of a second stimulus to ER- spines induced ER recruitment along with persistent sLTP, whereas ER+ spines showed no additional increases in size or ER content in response to sequential stimulation. Surprisingly, the physical interaction between STIM1 and Orai1 induced by ER Ca2+ release, but not the resulting Ca2+ entry through Orai1 channels, proved necessary for the persistent increases in both spine size and ER content required for expression of long-lasting late sLTP.

Less is better: various means to reduce protein load in the endoplasmic reticulum.

The endoplasmic reticulum (ER) is an important organelle that controls the intracellular and extracellular environments. The ER is responsible for folding almost one-third of the total protein population in the eukaryotic cell. Disruption of ER-protein folding is associated with numerous human diseases, including metabolic disorders, neurodegenerative diseases, and cancer. During ER perturbations, the cells deploy various mechanisms to increase the ER-folding capacity and reduce ER-protein load by minimizing the number of substrates entering the ER to regain homeostasis. These mechanisms include signaling pathways, degradation mechanisms, and other processes that mediate the reflux of ER content to the cytosol. In this review, we will discuss the recent discoveries of five different ER quality control mechanisms, including the unfolded protein response (UPR), ER-associated-degradation (ERAD), pre-emptive quality control, ER-phagy and ER to cytosol signaling (ERCYS). We will discuss the roles of these processes in decreasing ER-protein load and inter-mechanism crosstalk.

Neuroprotective Effect of Antiapoptotic URG7 Protein on Human Neuroblastoma Cell Line SH-SY5Y.

Up-regulated Gene clone 7 (URG7) is a protein localized in the endoplasmic reticulum (ER) and overexpressed in liver cells upon hepatitis B virus (HBV) infection. Its activity has been related to the attenuation of ER stress resulting from HBV infection, promoting protein folding and ubiquitination and reducing cell apoptosis overall. While the antiapoptotic activity of URG7 in HBV-infected cells may have negative implications, this effect could be exploited positively in the field of proteinopathies, such as neurodegenerative diseases. In this work, we aimed to verify the possible contribution of URG7 as a reliever of cellular proteostasis alterations in a neuronal in vitro system. Following tunicamycin-induced ER stress, URG7 was shown to modulate different markers of the unfolded protein response (UPR) in favor of cell survival, mitigating ER stress and activating autophagy. Furthermore, URG7 promoted ubiquitination, and determined a reduction in protein aggregation, calcium release from the ER and intracellular ROS content, confirming its pro-survival activity. Therefore, in light of the results reported in this work, we hypothesize that URG7 offers activity as an ER stress reliever in a neuronal in vitro model, and we paved the way for a new approach in the treatment of neurodegenerative diseases.

Endoplasmic Reticulum Stress and Autophagy in the Pathogenesis of Non-alcoholic Fatty Liver Disease (NAFLD): Current Evidence and Perspectives.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease with rising prevalence worldwide. Herein, we provide a comprehensive overview of the current knowledge supporting the role of ER stress and autophagy processes in NAFLD pathogenesis and progression. We also highlight the interrelation between these two pathways and the impact of ER stress and autophagy modulators on NAFLD treatment. The pathophysiological mechanisms involved in NAFLD progression are currently under investigation. The endoplasmic reticulum (ER) stress and the concomitant unfolded protein response (UPR) seem to contribute to its pathogenesis mainly due to high ER content in the liver which exerts significant metabolic functions and can be dysregulated. Furthermore, disruption of autophagy processes has also been identified in NAFLD. The crucial role of these two pathways in NAFLD is underlined by the fact that they have recently emerged as promising targets of therapeutic interventions. There is a greater need for finding the natural/chemical compounds and drugs which can modulate the ER stress pathway and autophagy for the treatment of NAFLD. Clarifying the inter-relation between these two pathways and their interaction with inflammatory and apoptotic mechanisms will allow the development of additional therapeutic options which can better target and reprogram the underlying pathophysiological pathways, aiming to attenuate NAFLD progression.

ESCRT-III-driven piecemeal micro-ER-phagy remodels the ER during recovery from ER stress

The endoplasmic reticulum (ER) produces about 40% of the nucleated cell’s proteome. ER size and content in molecular chaperones increase upon physiologic and pathologic stresses on activation of unfolded protein responses (UPR). On stress resolution, the mammalian ER is remodeled to pre-stress, physiologic size and function on activation of the LC3-binding activity of the translocon component SEC62. This elicits recov-ER-phagy, i.e., the delivery of the excess ER generated during the phase of stress to endolysosomes (EL) for clearance. Here, ultrastructural and genetic analyses reveal that recov-ER-phagy entails the LC3 lipidation machinery and proceeds via piecemeal micro-ER-phagy, where RAB7/LAMP1-positive EL directly engulf excess ER in processes that rely on the Endosomal Sorting Complex Required for Transport (ESCRT)-III component CHMP4B and the accessory AAA+ ATPase VPS4A. Thus, ESCRT-III-driven micro-ER-phagy emerges as a key catabolic pathway activated to remodel the mammalian ER on recovery from ER stress.

The various shades of ER-phagy.

Endoplasmic reticulum (ER) is a large and dynamic cellular organelle. ER morphology consists of sheets, tubules, matrixes, and contact sites shared with other membranous organelles. The capacity of the ER to fulfill its numerous biological functions depends on its continuous remodeling and the quality control of its proteome. Selective turnover of the ER by autophagy, termed ER‐phagy, plays an important role in maintaining ER homeostasis. ER network integrity and turnover rely on specific ER‐phagy receptors, which influence and coordinate alterations in ER morphology and the degradation of ER contents and membranes via the lysosome, by interacting with the LC3/GABARAP family. In this commentary, we discuss general principles and identify the major players in this recently characterized form of selective autophagy, while simultaneously highlighting open questions in the field.

Single particle trajectories reveal active endoplasmic reticulum luminal flow.

The Endoplasmic Reticulum (ER), a network of membranous sheets and pipes, supports functions encompassing biogenesis of secretory proteins and delivery of functional solutes throughout the cell1,2. Molecular mobility through the ER network enables these functionalities, but diffusion alone is not sufficient to explain luminal transport across supramicron distances. Understanding the ER structure-function relationship is critical in light of mutations in ER morphology regulating proteins that give rise to neurodegenerative disorders3,4. Here, super-resolution microscopy and analysis of single particle trajectories of ER luminal proteins revealed that the topological organization of the ER correlates with distinct trafficking modes of its luminal content: with a dominant diffusive component in tubular junctions and a fast flow component in tubules. Particle trajectory orientations resolved over time revealed an alternating current of the ER contents, whilst fast ER super-resolution identified energy-dependent tubule contraction events at specific points as a plausible mechanism for generating active ER luminal flow. The discovery of active flow in the ER has implications for timely ER content distribution throughout the cell, particularly important for cells with extensive ER-containing projections such as neurons.

STIM1 Ca2+ Sensor Control of L-type Ca2+-Channel-Dependent Dendritic Spine Structural Plasticity and Nuclear Signaling

Potentiation of synaptic strength relies on postsynaptic Ca2+ signals, modification of dendritic spine structure, and changes in gene expression. One Ca2+ signaling pathway supporting these processes routes through L-type Ca2+ channels (LTCC), whose activity is subject to tuning by multiple mechanisms. Here, we show in hippocampal neurons that LTCC inhibition by the endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule 1 (STIM1), is engaged by the neurotransmitter glutamate, resulting in regulation of spine ER structure and nuclear signaling by the NFATc3 transcription factor. In this mechanism, depolarization by glutamate activates LTCC Ca2+ influx, releases Ca2+ from the ER, and consequently drives STIM1 aggregation and an inhibitory interaction with LTCCs that increases spine ER content but decreases NFATc3 nuclear translocation. These findings of negative feedback control of LTCC signaling by STIM1 reveal interplay between Ca2+ influx and release from stores that controls both postsynaptic structural plasticity and downstream nuclear signaling.

Sequestosome 1/p62 Protein Is Associated with Autophagic Removal of Excess Hepatic Endoplasmic Reticulum in Mice

Xenobiotics exposure increases endoplasmic reticulum (ER) proliferation and cytochrome P-450 (CYP) induction to sustain metabolic requirements. Whether autophagy is essential for the removal of excess ER and CYP and whether an autophagy receptor is involved in this process in mammals remains elusive. In this study, we show that autophagy is induced in mouse livers after withdrawal of the hepatic mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Although isolated autophagosomes, autolysosomes, and lysosomes from mouse livers after withdrawal of TCPOBOP contained ER proteins, those in control mouse livers did not. Liver-specific Atg5 knockout mice had higher basal hepatic ER content that was further increased and sustained after withdrawal of TCPOBOP compared with wild-type mice. In addition to regulating ER degradation, our results also suggest that autophagy plays a role in regulating the homeostasis of hepatic CYP because blocking autophagy led to increased CYP2B10 accumulation either at the basal level or following TCPOBOP withdrawal. Furthermore, we found that the autophagy receptor protein sequestosome 1 (SQSTM1)/p62 is associated with the ER. After withdrawal of TCPOBOP, p62 knockout mice had increased ER content in the liver compared with wild-type mice. These results suggest that p62 may act as an autophagy receptor for the autophagic removal of excess ER in the mouse liver. Taken together, our results indicate that autophagy is important for the removal of excess ER and hepatic CYP enzymes in mouse livers, a process associated with the autophagy receptor protein p62.

Rapid regulation of endoplasmic reticulum dynamics in dendritic spines by NMDA receptor activation

Endoplasmic reticulum (ER) is motile within dendritic spines, but the mechanisms underlying its regulation are poorly understood. To address this issue, we have simultaneously imaged morphology and ER content of dendritic spines in cultured dissociated mouse hippocampal neurons. Over a 10 min period, spines were highly dynamic, with spines both increasing and decreasing in volume. ER was present in approximately 50% of spines and was also highly dynamic, with a net increase over this period of time. Inhibition of the endogenous activation of NMDA receptors resulted in a reduction in ER growth. Conversely, augmentation of the synaptic activation of NMDA receptors, by elimination of striatal-enriched protein tyrosine phosphatase (STEP), resulted in enhanced ER growth. Therefore, NMDA receptors rapidly regulate spine ER dynamics.

Application of statistical techniques for elucidating flow cytometric data of batch and fed‐batch cultures

The objective of this work is to develop structured, segregated stochastic models for bioprocesses using time-series flow cytometric (FC) data. To this end, mammalian CHO cells were grown in both batch and fed-batch cultures, and their viable cell numbers (VCDs), monoclonal antibody (MAb), cell cycle phases, mitochondria membrane potential/mitochondria mass, Golgi apparatus, and endoplasmic reticulum (ER) were analyzed. For the fed-batch mode, soy hydrolysate was introduced at 24-H intervals. The cytometric data were analyzed for early indicators of growth and productivity by multiple linear regression analysis, which involved taking into account multicollinearity diagnostics, Durbin-Watson statistics, and Houston tests to determine and refine statistically significant correlations between categorical variables (FC parameters) and response variables (yield parameters). The results indicate that the percentage of G1 cells and ER was significantly correlated with VCD and MAb in the case of batch culture, whereas for fed-batch culture, the percentage of G2 cells and ER was correlated significantly. There was a significant difference between cells in the batch and fed-batch cultures in their ER content, suggesting that the increase in protein synthesis as reflected by the ER content and consequent increase in growth rate and MAb productivity both can be monitored at the cellular level by FC analysis of ER content.

Non-Ligand Mobilization of Intracellular Free Calcium by Nanosecond Pulsed Electric Field (NsPEF)

NsPEF makes membrane and intracellular structures in living cells permeable to ions and small molecules thus can be used to modulate cell's signaling pathways and homeostasis. We utilized Fura-2 fast ratiometric recordings to measure changes of intracellular free calcium concentration ([Ca2+]i) in single CHO cells stimulated by 10, 60 and 300 ns PEF. 10 and 300 ns PEF were used for comparison with 60 ns PEF in regard to their potency to make plasma membrane (PM) and/or endoplasmic reticulum (ER) permeable to Ca2+ ions. Single 60 ns PEF evoked transient [Ca2+]i rise due to Ca2+ influx via PM and Ca2+ efflux from ER with thresholds ∼9 and ∼19 kV/cm, respectively. The amplitude of Ca2+ rise increased with nsPEF intensity linearly by 8-10nM per 1 kV/cm (PM) or 5-6 nM per 1 kV/cm when permeabilization of ER was assessed in Ca2+ free solution. This rate of change increased drastically (to 174nM per 1 kV/cm) when amplitude of Ca2+ rise reached 100-150nM. Depleting Ca2+ content of ER showed that such increase was a result of calcium induced calcium release (CICR) activation. Specific blockers 2-APB and dantrolene proved that the activation of IP3 receptors was responsible for physiological mechanism of CICR triggered by nsPEF. 10, 60 and 300 ns PEF had different efficiencies in regard to PM and ER permeabilization for Ca2+ ions. The thresholds of PM and ER permeabilization were indistinguishable for 10 ns pulse while for 60 ns pulse the ER threshold was twofold higher. 300 ns pulse had further reduced potency towards ER permeabilization in comparison to either 10 or 60 ns pulses. These results show that nsPEF can be effectively used to manipulate [Ca2+]i and activate intracellular signaling pathways bypassing PM receptors.Supported by R01CA125482; R01GM088303

Temporal Regulation of Intracellular Organelle Homeostasis in T Lymphocytes by Autophagy

The highly conserved self-degradation pathway known as autophagy plays important roles in regulating T lymphocyte homeostasis. Recently, we found that T lymphocytes lacking the autophagy-related gene Atg5 or Atg7 have defective survival and contain expanded mitochondria and endoplasmic reticulum (ER); however, whether these defects are caused by impaired autophagy or by defects in their autophagy-independent signaling capacity of Atg5 or Atg7 in T lymphocytes remains unknown. Furthermore, the function of the microtubule-associated protein L chain 3 (LC3) conjugation system in T lymphocytes remains unclear. To address these questions, we generated conditional knockout mice with specific deletion of Atg3, a ubiquitin enzyme E2-like molecule involved in the LC3 conjugation system, in T lymphocytes. Atg3-deficient T lymphocytes displayed a phenotype similar to those of Atg7- and Atg5-deficient T cells. The survival of Atg3-deficient naive CD4(+) and CD8(+) T cells was defective. Furthermore, the mitochondria and ER were expanded in Atg3-deficient T cells. Interestingly, mitochondrial and ER content did not change instantly upon inducible deletion of Atg3 in mature T lymphocytes in vitro. Instead, it began to expand 10 d after inducible deletion of Atg3 in mature T lymphocytes, and mitochondrial content continued to increase on day 18. Cell death began to increase 24 d after inducible deletion of Atg3. These data show that the LC3 conjugation system is essential for autophagy in T lymphocytes. Our data suggest that autophagy promotes T lymphocyte survival by regulating organelle homeostasis and that the decreased survival of autophagy-deficient T cells is due to the temporal accumulation of these autophagy-related defects.

The Myosin Va Head Domain Binds to the Neurofilament-L Rod and Modulates Endoplasmic Reticulum (ER) Content and Distribution within Axons

The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon. Our data are consistent with a novel function for NFs as a scaffold in axons for maintaining the content and proper distribution of vesicular organelles, mediated in part by Myo Va. Based on observations that the Myo Va motor domain binds to intermediate filament (IF) proteins of several classes, Myo Va interactions with IFs may serve similar roles in organizing organelle topography in different cell types.

Autophagy Regulates Endoplasmic Reticulum Homeostasis and Calcium Mobilization in T Lymphocytes

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens, presenting endogenous Ags, and regulating T lymphocyte survival and proliferation. In this study, we have investigated the role of autophagy in regulating the endoplasmic reticulum (ER) compartment in T lymphocytes. We found that ER content is expanded in mature autophagy-related protein (Atg) 7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the TCR display impaired influx, but not efflux, of calcium, and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes, suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore, we found that the stimulation-induced redistribution of stromal interaction molecule-1, a critical event for the store-operated Ca(2+) release-activated Ca(2+) channel opening, is impaired in Atg7-deficient T cells. Together, these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores, and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes.

Endoplasmic reticulum dynamics in hippocampal dendritic spines induced by agonists of type I metabotropic glutamate but not by muscarinic acetylcholine receptors

Neurons in the hippocampus exhibit subpopulations of dendritic spines that contain endoplasmic reticulum (ER). ER in spines is important for synaptic activity and its associated Ca(2+) signaling. The dynamic distribution of ER to spines is regulated by diacylglycerol and partly mediated by protein kinase C, metalloproteinases and γ-secretase. In this study, we explored whether pharmacological activation of type I metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs) known to activate phospholipase C would have any effect on spine ER content. We found that DHPG (100 μM) but not carbachol (10 μM) caused a reduction in the number of spines with ER. We further found that ER Ca(2+) depletion triggered by thapsigargin (200 nM) had no effect on ER localization in spines.

The Bcl-2 Gene Polymorphism rs956572AA Increases Inositol 1,4,5-Trisphosphate Receptor–Mediated Endoplasmic Reticulum Calcium Release in Subjects with Bipolar Disorder

Bipolar disorder (BPD) is characterized by altered intracellular calcium (Ca(2+)) homeostasis. Underlying mechanisms involve dysfunctions in endoplasmic reticulum (ER) and mitochondrial Ca(2+) handling, potentially mediated by B-cell lymphoma 2 (Bcl-2), a key protein that regulates Ca(2+) signaling by interacting directly with these organelles, and which has been implicated in the pathophysiology of BPD. Here, we examined the effects of the Bcl-2 gene single nucleotide polymorphism (SNP) rs956572 on intracellular Ca(2+) dynamics in patients with BPD. Live cell fluorescence imaging and electron probe microanalysis were used to measure intracellular and intra-organelle free and total calcium in lymphoblasts from 18 subjects with BPD carrying the AA, AG, or GG variants of the rs956572 SNP. Analyses were carried out under basal conditions and in the presence of agents that affect Ca(2+) dynamics. Compared with GG homozygotes, variant AA-which expresses significantly reduced Bcl-2 messenger RNA and protein-exhibited elevated basal cytosolic Ca(2+) and larger increases in inositol 1,4,5-trisphosphate receptor-mediated cytosolic Ca(2+) elevations, the latter in parallel with enhanced depletion of the ER Ca(2+) pool. The aberrant behavior of AA cells was reversed by chronic lithium treatment and mimicked in variant GG by a Bcl-2 inhibitor. In contrast, no differences between SNP variants were found in ER or mitochondrial total Ca(2+) content or in basal store-operated Ca(2+) entry. These results demonstrate that, in patients with BPD, abnormal Bcl-2 gene expression in the AA variant contributes to dysfunctional Ca(2+) homeostasis through a specific ER inositol 1,4,5-trisphosphate receptor-dependent mechanism.

Comparative ultrastructural analysis of diestrous and anestrous canine Grade 1 cumulus-oocyte complexes

The objective of the present study was to characterize, by means of transmission electron microscopy, immature Grade 1 cumulus-oocyte complexes (COCs) obtained from ovaries collected from bitches at diestrus and anestrus, after routine ovariohysterectomy. Cumulus-oocyte complexes were recovered after slicing the ovarian cortex and Grade 1 COCs were selected and prepared for transmission electron microscopy. All oocytes were at germinal vesicle stage in two different configurations. In 29 out of 37 COCs, oocytes presented a central or eccentric located nucleus (GVc), frequently containing a reticulated nucleolus, with a predominance of profound follicular cell processes (FCP), abundant endoplasmic reticulum (ER) and a moderated number of lipid droplets. In eight out of 37 COCs, oocytes had a peripheral located nucleus (GVp) containing small compact nucleolus, a thin perivitelline space, both superficial and profound FCP, moderate ER content and abundant lipid droplets. A higher ( p < 0.05) proportion of GVp was found in oocytes collected in diestrus (36.8% – 7 out of 19) than in anestrus (5.6% – 1 out of 18). In addition, distinct ultrastructure characteristics among GVc associated with estrous phase were noted. Furthermore, a number of structures, some of which had not been reported before, were present in canine prophase I oocytes at variable frequencies. The evident variation in the presence, quantity and distribution of cell organelles in canine immature Grade 1 oocytes is discussed in relation to the biological phase of the oocyte and the reproductive stage of the donor bitch.

XBP-1-Deficient Plasmablasts Show Normal Protein Folding but Altered Glycosylation and Lipid Synthesis

The accumulation of misfolded secreted IgM in the endoplasmic reticulum (ER) of X-box binding protein 1 (XBP-1)-deficient B cells has been held responsible for the inability of such cells to yield plasma cells, through the failure to mount a proper unfolded protein response. LPS-stimulated B cells incapable of secreting IgM still activate the XBP-1 axis normally, as follows: XBP-1 is turned on by cues that trigger differentiation and not in response to accumulation of unfolded IgM, but the impact of XBP-1 deficiency on glycoprotein folding and assembly has not been explored. The lack of XBP-1 compromised neither the formation of functional hen egg lysozyme-specific IgM nor the secretion of free kappa-chains. Although XBP-1 deficiency affects the synthesis of some ER chaperones, including protein disulfide isomerase, their steady state levels do not drop below the threshold required for proper assembly and maturation of the Igalpha/Igbeta heterodimer and MHC molecules. Intracellular transport and surface display of integral membrane proteins are unaffected by XBP-1 deficiency. Given the fact that we failed to observe any defects in folding of a variety of glycoproteins, we looked for other means to explain the requirement for XBP-1 in plasma cell development. We observed significantly reduced levels of phosphatidylcholine, sphingomyelin, and phosphatidylinositol in total membranes of XBP-1-deficient B cells, and reduced ER content. Terminal N-linked glycosylation of IgM and class I MHC was altered in these cells. XBP-1 hence has important roles beyond folding proteins in the ER.