Endoplasmic Reticulum Stress Pathway Required for Immune Homeostasis Is Neurally Controlled by Arrestin-1

In response to pathogen infection, the host innate immune system activates microbial killing pathways and cellular stress pathways that need to be balanced because insufficient or excessive immune responses have deleterious consequences. Recent studies demonstrate that two G protein-coupled receptors (GPCRs) in the nervous system of Caenorhabditis elegans control immune homeostasis. To investigate further how GPCR signaling controls immune homeostasis at the organismal level, we studied arrestin-1 (ARR-1), which is the only GPCR adaptor protein in C. elegans. The results indicate that ARR-1 is required for GPCR signaling in ASH, ASI, AQR, PQR, and URX neurons, which control the unfolded protein response and a p38 mitogen-activated protein kinase signaling pathway required for innate immunity. ARR-1 activity also controlled immunity through ADF chemosensory and AFD thermosensory neurons that regulate longevity. Furthermore, we found that although ARR-1 played a key role in the control of immunity by AFD thermosensory neurons, it did not control longevity through these cells. However, ARR-1 partially controlled longevity through ADF neurons.     

Wnt Signaling Is Regulated by Endoplasmic Reticulum Retention

Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed oto gene causes loss of the anterior forebrain during mouse embryogenesis. Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts.     

African Swine Fever Virus Is Wrapped by the Endoplasmic Reticulum

African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249–278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.African swine fever (ASF) virus is a large icosahedral enveloped DNA virus that causes a lethal hemorrhagic disease in domestic pigs. The virus is endemic in areas of southern Europe and in Africa where it causes major problems for the development of pig industries. At present there are no vaccines, and the disease is controlled through the slaughter of infected animals. The economic importance of ASF virus has made the virus the focus of much research since it was first described in 1921 (32). ASF virus is unique among animal viruses, and its classification has been controversial. ASF virus shares the striking icosahedral symmetry of iridoviruses (5, 8, 13, 34), while the presence of inverted terminal repeats and covalently linked ends in the 170-kDa genome suggests similarities with poxviruses (16). The ASF virus genome encoding at least 150 proteins has been sequenced (17, 51), and the amino acid sequences of at least 11 structural proteins are known. p73 is the major structural protein (14, 28) and has sequence similarities to the capsid protein of iridoviruses (39). The ordered proteolysis of pp220 produces p150, p37/p34 and p14 (40), which together comprise 25% of the viral proteins (3). These proteins localize to the interior of the virion (3). Three proteins, J13L/p54, I1L/p17, and p22, with membrane-spanning domains localize to the viral envelope (10, 37, 41, 43). Three other structural proteins, p14.5 encoded by E120R (30), p10 encoded by K78R (35), and p5AR encoded by A104R (7), have DNA-binding properties (51) and may be involved in DNA packaging. The virus has been the subject of several detailed electron microscopy studies (2–4, 8, 9, 11, 13, 34, 47). Electron micrographs of sections taken through ASF virus assembly sites reveal fully assembled virions as 200-nm hexagons and an ordered series of assembly intermediates with one to six sides of a hexagon. Close inspection of intracellular virions identifies multiple concentric layers of differing electron densities. According to recent models, the layers represent a central electron-dense nucleocapsid core, surrounded by an inner core shell, an inner envelope, and an outer capsid layer (3). The mechanism of formation of the inner envelope of ASF virus has not been resolved.Most viruses gain a single membrane envelope by budding into intracellular membrane compartments or from the plasma membrane, as reviewed in reference 21. When viruses bud into an intracellular compartment, the domains of the membrane proteins that are initially located in the lumen of membrane compartments are exposed on the outside of the virion after release from the cell (Fig. ​(Fig.1a).1a). A second mechanism of envelopment, described recently for poxviruses and herpesviruses (18, 20, 24, 38, 42, 46, 50), is more complex and involves the wrapping of virions by membrane cisternae derived from specific membrane compartments. Wrapping provides two membrane envelopes in one step and leaves the virion free in the cytoplasm. When compared with budding, wrapping reverses the orientation of membrane proteins within the virus such that the domains of membrane proteins located in the lumen of the wrapping organelle are confined to the interior of the virus after release from the cell, whereas cytoplasmic tails are exposed on the outside of the virus (Fig. ​(Fig.1b).1b). Given these important consequences for understanding the mechanism of assembly of the virus and for determining the final orientation of membrane proteins in virions, we have set out to determine whether ASF virus acquires its membranes by the conventional budding mechanism or whether the virus is wrapped by intracellular membrane compartments before release from the cell. Open in a separate windowFIG. 1Schematic comparison of budding and wrapping mechanisms of virus envelopment. (a) Budding. Viral nucleoprotein complexes bind to the cytoplasmic domains of virally encoded integral membrane proteins (|, membrane glycoproteins). Interactions between viral proteins lead to membrane curvature, and the virion gains a single membrane by budding into the lumen of the membrane compartment. When the virion is released from the cell, oligosaccharides () are exposed on the surface of the virus, and the cytoplasmic tail of the membrane glycoprotein is buried within the virion. (b) Wrapping. Viral nucleoprotein complexes bind to the cytoplasmic domains of virally encoded integral membrane proteins. The nucleoprotein complex is then wrapped by the membrane cisternae, and the virus gains two membranes. The particle remains in the cytosol. When the virion is released from the cell by cell lysis, oligosaccharides () are buried within the two membranes of the virion while the cytoplasmic tail of the membrane glycoprotein is exposed on the surface of the virus.In this study we have taken advantage of thin cryoelectron microscopic sections to enhance the definition of viral membranes. The micrographs show two membranes within mature intracellular virions and all structural intermediates. They also show assembly intermediates in continuity with cellular membrane compartments. Consistent with our earlier study showing that p73 was enveloped by the endoplasmic reticulum (ER) (15), immunogold labelling experiments show resident proteins of the ER within membranes found at assembly sites, in virus assembly intermediates, and in mature virions. Importantly, we have identified a protein (pXP124L) encoded by ASF virus that translocates completely into the lumen of the ER and is incorporated as a structural protein of the virus. The presence of two membranes within intracellular virions and structural intermediates and the packaging of a structural protein from the lumen of the ER into the virus, strongly suggest that ASF virus is wrapped by the ER.     

Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex.     

Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

CNIH4 Interacts with Newly Synthesized GPCR and Controls Their Export from the Endoplasmic Reticulum

The molecular mechanisms regulating G protein‐coupled receptors (GPCRs) trafficking from their site of synthesis in the endoplasmic reticulum (ER) to their site of function (the cell surface) remain poorly characterized. Using a bioluminescence resonance energy transfer‐based proteomic screen, we identified a novel GPCR‐interacting protein; the human cornichon homologue 4 (CNIH4). This previously uncharacterized protein is localized in the early secretory pathway where it interacts with members of the 3 family of GPCRs. Both overexpression and knockdown expression of CNIH4 caused the intracellular retention of GPCRs, indicating that this ER‐resident protein plays an important role in GPCR export. Overexpression of CNIH4 at low levels rescued the maturation and cell surface expression of an intracellularly retained mutant form of the β2‐adrenergic receptor, further demonstrating a positive role of CNIH4 in GPCR trafficking. Taken with the co‐immunoprecipitation of CNIH4 with Sec23 and Sec24, components of the COPII coat complex responsible for ER export, these data suggest that CNIH4 acts as a cargo‐sorting receptor, recruiting GPCRs into COPII vesicles .      

PARM-1 Is an Endoplasmic Reticulum Molecule Involved in Endoplasmic Reticulum Stress-Induced Apoptosis in Rat Cardiac Myocytes

To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1). While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER). In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease.     

African Swine Fever Virus Is Enveloped by a Two-Membraned Collapsed Cisterna Derived from the Endoplasmic Reticulum

During the cytoplasmic maturation of African swine fever virus (ASFV) within the viral factories, the DNA-containing core becomes wrapped by two shells, an inner lipid envelope and an outer icosahedral capsid. We have previously shown that the inner envelope is derived from precursor membrane-like structures on which the capsid layer is progressively assembled. In the present work, we analyzed the origin of these viral membranes and the mechanism of envelopment of ASFV. Electron microscopy studies on permeabilized infected cells revealed the presence of two tightly apposed membranes within the precursor membranous structures as well as polyhedral assembling particles. Both membranes could be detached after digestion of intracellular virions with proteinase K. Importantly, membrane loop structures were observed at the ends of open intermediates, which suggests that the inner envelope is derived from a membrane cisterna. Ultraestructural and immunocytochemical analyses showed a close association and even direct continuities between the endoplasmic reticulum (ER) and assembling virus particles at the bordering areas of the viral factories. Such interactions become evident with an ASFV recombinant that inducibly expresses the major capsid protein p72. In the absence of the inducer, viral morphogenesis was arrested at a stage at which partially and fully collapsed ER cisternae enwrapped the core material. Together, these results indicate that ASFV, like the poxviruses, becomes engulfed by a two-membraned collapsed cisterna derived from the ER.     

Yet1p and Yet3p,the Yeast Homologs of BAP29 and BAP31, Interact with the Endoplasmic Reticulum Translocation Apparatus and Are Required for Inositol Prototrophy

The mammalian B-cell receptor-associated proteins of 29 and 31 kDa (BAP29 and BAP31) are conserved integral membrane proteins that have reported roles in endoplasmic reticulum (ER) quality control, ER export of secretory cargo, and programmed cell death. In this study we investigated the yeast homologs of BAP29 and BAP31, known as Yet1p and Yet3p, to gain insight on cellular function. We found that Yet1p forms a complex with Yet3p (Yet complex) and that complex assembly was important for subunit stability and proper ER localization. The Yet complex was not efficiently packaged into ER-derived COPII vesicles and therefore does not appear to act as an ER export receptor. Instead, a fraction of the Yet complex was detected in association with the ER translocation apparatus (Sec complex). Specific mutations in the Sec complex or Yet complex influenced these interactions. Moreover, associations between the Yet complex and Sec complex were increased by ER stress and diminished when protein translocation substrates were depleted. Surprisingly, yet1Δ and yet3Δ mutant strains displayed inositol starvation-related growth defects. In accord with the biochemical data, these growth defects were exacerbated by a combination of certain mutations in the Sec complex with yet1Δ or yet3Δ mutations. We propose a model for the Yet-Sec complex interaction that places Yet1p and Yet3p at the translocation pore to manage biogenesis of specific transmembrane secretory proteins.     

MAIGO5 Functions in Protein Export from Golgi-Associated Endoplasmic Reticulum Exit Sites in Arabidopsis

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A–green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.     

Nonvesicular Lipid Transfer from the Endoplasmic Reticulum

The transport of lipids from their synthesis site at the endoplasmic reticulum (ER) to different target membranes could be mediated by both vesicular and nonvesicular transport mechanisms. Nonvesicular lipid transport appears to be the major transport route of certain lipid species, and could be mediated by either spontaneous lipid transport or by lipid-transfer proteins (LTPs). Although nonvesicular lipid transport has been extensively studied for more than four decades, its underlying mechanism, advantage and regulation, have not been fully explored. In particular, the function of LTPs and their involvement in intracellular lipid movement remain largely controversial. In this article, we describe the pathways by which lipids are synthesized at the ER and delivered to different cellular membranes, and discuss the role of LTPs in lipid transport both in vitro and in intact cells.The endoplasmic reticulum (ER) is a large interconnected membrane network that plays a major role in lipid biosynthesis in eukaryotic cells (Borgese et al. 2006). Newly synthesized lipids at the ER are then delivered to different cellular membranes or organelles, each of which shows unique lipid and protein composition and executes distinct cellular function (Holthuis et al. 2003). The transport of lipids from the ER can be mediated by both vesicular and nonvesicular transport mechanisms. Vesicular transport, as opposed to nonvesicular lipid transport, requires metabolic energy, intact cytoskeleton, and connection to the vesicular transport machinery (Kaplan and Simoni 1985a; Voelker 1990; Vance et al. 1991). Although vesicular lipid transport mediates the bulk transport of many lipids, increasing lines of evidence suggest that nonvesicular lipid transport is the major transport route for certain lipid types (Lev 2010). Nonvesicular lipid transport between membranes could be mediated by spontaneous lipid transport, in which a lipid monomer is diffused through the cytosol from a donor to the acceptor membrane. Given that most cellular lipids are highly hydrophobic, their diffusion through an aqueous phase is very slow and insufficient to support substantial transport of most lipids (Jones and Thompson 1989; Mesmin and Maxfield 2009). Nevertheless, spontaneous lipid transport can be greatly facilitated at membrane contact sites (MCSs) (Levine 2004; Holthuis and Levine 2005) and/or by lipid-transfer proteins (LTPs) (Lev 2010). MCSs are defined as small cytosolic gaps of 10–20 nm between the ER membranes and virtually all cellular organelles (Levine 2004; Lebiedzinska et al. 2009), whereas LTPs are intracellular proteins that can carry a lipid monomer in a hydrophobic pocket and transfer it between membranes through an aqueous phase. LTPs were initially discovered as soluble factors that accelerate the exchange or net transfer of different lipid species between membranes in vitro (Wirtz and Zilversmit 1968). Subsequently, many LTPs have been isolated, cloned, and crystallized. LTPs have been identified in all eukaryotes, in plants, and in bacteria, and according to their sequence and structure similarity have been subdivided into different protein families including SEC14, PITP (phosphatidylinositol-transfer protein), START (StAR-related lipid transfer), GLTP (glycolipid transfer protein), SCP-2 (nonspecific LTPs), and OSBP (oxysterol-binding protein)/ORP (OSBP-related proteins) (D’Angelo et al. 2008). In general, LTPs show specificity for one or more lipid types, and may contain only a single lipid-transfer domain (LTD), or additional structural domains with varying functions (Lev 2010). Over the past 40 years, LTPs have been extensively studied and the major principles of their action mode have been established from both biophysical measurements in vitro and structural data (Lev 2010). Nevertheless, the precise function of LTPs in intact cells remains controversial and a subject of an active field of research. In this article, we briefly describe how lipids are synthesized in the ER and delivered to different target membranes, and discuss how LTPs influence lipid transport in vitro and in intact cells.     

Endoplasmic Reticulum Stress Is Chronically Activated in Chronic Pancreatitis

The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T^−/−), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T^−/− mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies.     

Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

Proteins to be secreted are transported from the endoplasmic reticulum (ER) to the Golgi apparatus. The transport of these proteins requires the localization and activity of proteins that create ER exit sites, coat proteins to collect cargo and to reshape the membrane into a transport container, and address labels—SNARE proteins—to target the vesicles specifically to the Golgi apparatus. In addition some proteins may need export chaperones or export receptors to enable their exit into transport vesicles. ER export factors, SNAREs, and misfolded Golgi-resident proteins must all be retrieved from the Golgi to the ER again. This retrieval is also part of the organellar homeostasis pathway essential to maintaining the identity of the ER and of the Golgi apparatus. In this review, I will discuss the different processes in retrograde transport from the Golgi to the ER and highlight the mechanistic insights we have obtained in the last couple of years.Proteins that are exposed at the plasma membrane or populate a membrane-bounded organelle are synthesized into the endoplasmic reticulum (ER). In the ER, the folding of these proteins takes place and posttranslational modifications such as N-glycosylation and disulfide bridge formation occur. Upon adopting a suitable, often correct, conformation, proteins destined to locations beyond the ER are concentrated at so-called ER exit sites (ERES) and incorporated into nascent COPII-coated vesicles. These COPII vesicles eventually bud off the ER membrane and are transported to the Golgi (in yeast, Drosophila, and C. elegans) or the ER-Golgi intermediate compartment (in mammalian cells) (Schweizer et al. 1990; Kondylis and Rabouille 2003; Spang 2009; Witte et al. 2011).It is assumed that the vesicle coat is at least partially destabilized through the hydrolysis of GTP by the small GTPase Sar1 (Oka and Nakano 1994; Springer et al. 1999). However, some of the destabilized coat components have to stay on the vesicle until it has reached the Golgi apparatus because coat components participate in the recognition and the tethering process (Barlowe 1997; Cai et al. 2007; Lord et al. 2011; Zong et al. 2012). Subsequently, SNARE proteins on the vesicles (v-SNAREs) zipper up with cognate SNAREs on the Golgi (target SNAREs, t-SNAREs) to drive membrane fusion (Hay et al. 1998; Cao and Barlowe 2000; Parlati et al. 2002). The content of the ER-derived COPII vesicles is thereby released into the lumen of the cis-cisterna of the Golgi apparatus. Most proteins will continue their journey through the Golgi apparatus and encounter further modifications such as extension of the glycosylation tree or lipidation. However, some proteins, especially those involved in the fusion process, i.e., the v-SNAREs or proteins that act as export factors of the ER, such as Vma21, which is essential for export of the correctly folded and assembled V0 sector of the V-ATPase, need to be recycled back to the ER for another round of transport (Ballensiefen et al. 1998; Malkus et al. 2004). Moreover, cis-Golgi proteins are returned to the ER for quality/functional control (Todorow et al. 2000; Sato et al. 2004; Valkova et al. 2011). Finally, some ER-resident proteins, such as the ER Hsp70 chaperone BiP/Kar2, can escape the ER, but are captured at the cis-Golgi by the H/KDEL receptor Erd2 and returned to the ER (Lewis et al. 1990; Semenza et al. 1990; Aoe et al. 1997).Unfortunately, the retrograde transport route is also hijacked by toxins. For example, endocytosed cholera toxin subunit A contains a KDEL sequence and can thereby exploit the system to access the ER (Majoul et al. 1996, 1998). From there, it is retro-translocated into the cytoplasm where it can exert its detrimental function.     

Neuronal Apoptosis Induced by Endoplasmic Reticulum Stress

Apoptosis is a conserved active cellular mechanism occurring under a range of physiological and pathological conditions. In the nervous system, apoptosis plays crucial roles in normal development and neuronal degenerating diseases. Various deleterious conditions, including accumulation of the mutant proteins in the endoplasmic reticulum (ER) and inhibition of ER to Golgi transport of proteins, may result in apoptosis. In this study, we examined the downstream events of apoptosis in differentiated PC 12 cells under ER stress induced by brefeldin A, an inhibitor of ER to Golgi protein transport. Activation of NF-B and degradation of I-B were observed within 2 hours, followed by up-regulation of GRP78 protein level in treated cells. Caspase-12 only appeared around 24 hours after brefeldin A treatment, coincident with cell nuclei fragmentation. These results suggest that neuronal apoptosis may be induced by ER stress through a NF-B and caspase related pathway.     

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro²⁴⁷, in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.     

内质网相关细胞器互作的研究进展

真核细胞中内质网是由片状和管状两种不同形态组成的连续的生物膜结构,参与细胞内蛋白质和脂质的合成以及钙离子稳态的调控等。内质网通过蛋白-蛋白及蛋白-脂质的相互作用与多种膜性细胞结构建立膜接触位点,进行物质的交换、信号转导、膜动态性调控等生理活动。内质网与膜性细胞结构互作的缺陷也会引发许多人类重大疾病。该文介绍了内质网与一系列膜性细胞结构接触位点形成的分子机制及其潜在功能。