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.     

Lipid Transport between the Endoplasmic Reticulum and Mitochondria

Mitochondria are partially autonomous organelles that depend on the import of certain proteins and lipids to maintain cell survival and membrane formation. Although phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine are synthesized by mitochondrial enzymes, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sterols need to be imported from other organelles. The origin of most lipids imported into mitochondria is the endoplasmic reticulum, which requires interaction of these two subcellular compartments. Recently, protein complexes that are involved in membrane contact between endoplasmic reticulum and mitochondria were identified, but their role in lipid transport is still unclear. In the present review, we describe components involved in lipid translocation between the endoplasmic reticulum and mitochondria and discuss functional as well as regulatory aspects that are important for lipid homeostasis.Biological membranes are major structural components of all cell types. They protect the cell from external influences, organize the interior in distinct compartments and allow balanced flux of components. Besides their specific proteome, organelles exhibit unique lipid compositions, which influence their shape, physical properties, and function. Major lipid classes found in biological membranes are phospholipids, sterols, and sphingolipids.The major “lipid factory” within the cell is the endoplasmic reticulum (ER). It is able to synthesize the bulk of structural phospholipids, sterols, and storage lipids such as triacylglycerols and steryl esters (van Meer et al. 2008). Furthermore, initial steps of ceramide synthesis occur in the ER providing precursors for the formation of complex sphingolipids in other organelles (Futerman 2006). Besides the export of ceramides, the ER supplies a large portion of lipids to other organelles, which cannot produce their own lipids or have a limited capacity to do so. Organelle interaction and transport of lipids require specific carrier proteins, membrane contact sites, tethering complexes, and/or vesicle flux. These processes are highly important for the maintenance of cell structure and survival but are still a matter of dispute. Most prominent organelle interaction partners are the ER and mitochondria. A subfraction of the ER named mitochondria-associated membrane (MAM) (Vance 1990) was described to be involved in lipid translocation to mitochondria. MAM is part of the ER network, which was shown to be in contact or close proximity to the outer mitochondrial membrane (OMM). Contact sites between MAM and mitochondria were assumed to facilitate exchange of components between the two compartments. Interestingly, MAM harbor a number of lipid synthesizing enzymes (Gaigg et al. 1994). Recently, molecular components governing membrane contact between the two organelles were identified (Dolman et al. 2005; Csordás et al. 2006; de Brito and Scorrano 2008; Kornmann et al. 2009; Friedman et al. 2010; Lavieu et al. 2010), although the specific role of these components in lipid translocation is not yet clear.     

The Highly Conserved COPII Coat Complex Sorts Cargo from the Endoplasmic Reticulum and Targets It to the Golgi

Protein egress from the endoplasmic reticulum (ER) is driven by a conserved cytoplasmic coat complex called the COPII coat. The COPII coat complex contains an inner shell (Sec23/Sec24) that sorts cargo into ER-derived vesicles and an outer cage (Sec13/Sec31) that leads to coat polymerization. Once released from the ER, vesicles must tether to and fuse with the target membrane to deliver their protein and lipid contents. This delivery step also depends on the COPII coat, with coat proteins binding directly to tethering and regulatory factors. Recent findings have yielded new insight into how COPII-mediated vesicle traffic is regulated. Here we discuss the molecular basis of COPII-mediated ER–Golgi traffic, focusing on the surprising complexity of how ER-derived vesicles form, package diverse cargoes, and correctly target these cargoes to their destination.The port of entry into the secretory pathway is the endoplasmic reticulum (ER). Approximately one-third of the eukaryotic proteome traffics from this multifunctional organelle (Huh et al. 2003). This diverse set of cargo is translocated into the ER, folded, and modified before it travels to the Golgi, where further modifications occur. From the Golgi, cargo is sorted to other subcellular compartments to perform a variety of cellular functions. The highly conserved machinery required for these transport events was initially identified through genetic screens in the yeast Saccharomyces cerevisiae, and insights into the function of this machinery were provided through the use of in vitro transport assays. Advances in microscopy, in particular, the use of GFP fusion proteins and live cell imaging, have also played a critical role in understanding the dynamics of membrane traffic. In this article, we describe the mechanistic advances that have helped us to understand how diverse cargo correctly traffics from the ER to the Golgi complex in lower and higher eukaryotes. Even though these mechanisms are largely conserved, they are more complex at the molecular and organizational levels in metazoans.     

The Human Alpha Defensin HD5 Neutralizes JC Polyomavirus Infection by Reducing Endoplasmic Reticulum Traffic and Stabilizing the Viral Capsid

Progressive multifocal leukoencephalopathy (PML) is a fatal disease with limited treatment options, both clinically and in the research pipeline. Potential therapies would target and neutralize its etiologic agent, JC polyomavirus (JCPyV). The innate immune response to JCPyV infection has not been studied, and little is known about the initial host response to polyomavirus infection. This study examined the ability of a human alpha defensin, HD5, to neutralize JCPyV infection in human fetal glial cells. We show that HD5, by binding to the virion, blocks infection. The JCPyV-HD5 complexes bind to and enter host cells but are reduced in their ability to reach the endoplasmic reticulum (ER), where virions are normally uncoated. Furthermore, HD5 binding to the virion stabilizes the capsid and prevents genome release. Our results show that HD5 neutralizes JCPyV infection at an early postentry step in the viral life cycle by stabilizing the viral capsid and disrupting JCPyV trafficking. This study provides a naturally occurring platform for developing antivirals to treat PML and also expands on the known capabilities of human defensins.     

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.     

冠状病毒感染与内质网应激研究进展

内质网（Endoplasmic reticulum,ER）是真核细胞细胞器的重要组成部分,主要负责蛋白质合成和翻译后修饰等过程,还参与调控了钙离子储存和脂类合成,具有重要生理功能。冠状病毒感染细胞后,在复制其遗传信息的同时也在合成大量病毒蛋白,造成未折叠/错误折叠蛋白堆积,进而增加内质网工作负担,诱发内质网应激（Endoplasmic reticulum stress,ERS）,激活未折叠蛋白反应（Unfolded protein response,UPR）,引起一系列信号级联反应,如诱导细胞凋亡等,进而影响病毒复制。本文就冠状病毒感染与ERS及UPR信号通路的研究进展做一综述,为新型抗冠状病毒药物的研发提供新视角。     

Alteration of Endoplasmic Reticulum Lipid Rafts Contributes to Lipotoxicity in Pancreatic β-Cells

Chronic saturated fatty acid exposure causes β-cell apoptosis and, thus, contributes to type 2 diabetes. Although endoplasmic reticulum (ER) stress and reduced ER-to-Golgi protein trafficking have been implicated, the exact mechanisms whereby saturated fatty acids trigger β-cell death remain elusive. Using mass spectroscopic lipidomics and subcellular fractionation, we demonstrate that palmitate pretreatment of MIN6 β-cells promoted ER remodeling of both phospholipids and sphingolipids, but only the latter was causally linked to lipotoxic ER stress. Thus, overexpression of glucosylceramide synthase, previously shown to protect against defective protein trafficking and ER stress, partially reversed lipotoxic reductions in ER sphingomyelin (SM) content and aggregation of ER lipid rafts, as visualized using Erlin1-GFP. Using both lipidomics and a sterol response element reporter assay, we confirmed that free cholesterol in the ER was also reciprocally modulated by chronic palmitate and glucosylceramide synthase overexpression. This is consistent with the known coregulation and association of SM and free cholesterol in lipid rafts. Inhibition of SM hydrolysis partially protected against ATF4/C/EBP homology protein induction because of palmitate. Our results suggest that loss of SM in the ER is a key event for initiating β-cell lipotoxicity, which leads to disruption of ER lipid rafts, perturbation of protein trafficking, and initiation of ER stress.     

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.     

Endoplasmic Reticulum Stress Impairs Insulin Receptor Signaling in the Brains of Obese Rats

The incidence of obesity is increasing worldwide. It was reported that endoplasmic reticulum stress (ERS) could inhibit insulin receptor signaling by activating c-Jun N-terminal kinase (JNK) in the liver. However, the relationship between ERS and insulin receptor signaling in the brain during obesity remains unclear. The aim of the current study was to assess whether ERS alters insulin receptor signaling through the hyper-activation of JNK in the hippocampus and frontal cortex in the brains of obese rats. Obesity was induced using a high fat diet (HFD). The Morris water maze test was then performed to evaluate decreases in cognitive function, and western blot was used to verify whether abnormal insulin receptor signaling was induced by ERS in HFD rats exhibiting cognitive decline. In addition, to determine whether ERS activated JNK and consequently impaired insulin receptor signaling, SH-SY5Y cells were treated with the JNK inhibitor, SP600125, followed by tunicamycin or thapsigargin, and primary rat hippocampal and cortical neurons were transfected with siRNA against IRE1α and JNK. We found that the expression of phosphorylation of PKR-like kinase (PERK), phosphorylation of α subunit of translation initiation factor 2 (eIF2α), and phosphorylation of inositol-requiring kinase-1α (IRE-1α) were increased in the brains of rats with HFD when compared with control rats. The level of serine phosphorylation of insulin receptor substrate-1 (IRS-1) was also increased, while protein kinase B (PKB/Akt) was reduced. ERS was also found to inhibit insulin receptor signaling via the activation of JNK in SH-SY5Y cells, primary rat hippocampal, and cortical neurons. These results indicate that ERS was increased, thereby resulting in impaired insulin receptor signaling in the hippocampus and frontal cortex of obese rats.     

Murine Intracisternal A Type Particles Fail to Separate from the Membrane of the Endoplasmic Reticulum

Analysis of serial sections of murine cells containing intracisternal A particles revealed that over 99% of all A particles remain in a budding configuration. This indicates that these particles fail to detach from the membrane of the endoplasmic reticulum. This observation explains how, despite their intracellular abundance in certain murine tumors, no extracellular A-type particles can be found.     

The Spike Protein of Murine Coronavirus Regulates Viral Genome Transport from the Cell Surface to the Endoplasmic Reticulum during Infection

We observed that the nonfusogenic mouse hepatitis virus (MHV) strain MHV-2 reached a titer of ∼2 log₁₀ higher than that of the fusogenic strain A59 in astrocytoma DBT cells. To determine whether the spike protein is responsible for the difference, a recombinant virus, Penn-98-1, that contains the A59 genome with a spike from MHV-2 was used to infect DBT cells. Results showed that Penn-98-1 behaved like MHV-2, thus establishing a role for the spike protein in viral growth. The inverse correlation between viral fusogenicity and growth was further established in four different cell types and with a fusogenic mutant, the S757R mutant, derived from isogenic Penn-98-1. While both A59 and Penn-98-1 entered cells at similar levels, viral RNA and protein syntheses were significantly delayed for A59. Interestingly, when the genomic RNAs were delivered directly into the cells via transfection, the levels of gene expression for these viruses were similar. Furthermore, cell fractionation experiments revealed that significantly more genomic RNAs for the nonfusogenic MHVs were detected in the endoplasmic reticulum (ER) within the first 2 h after infection than for the fusogenic MHVs. Pretreatment of Penn-98-1 with trypsin reversed its properties in syncytium formation, virus production, and genome transport to the ER. These findings identified a novel role for the spike protein in regulating the uncoating and delivery of the viral genome to the ER after internalization.Murine coronavirus mouse hepatitis virus (MHV) is a member of the family Coronaviridae. It is an enveloped, positive-strand-RNA virus. The viral envelope contains three or four structural proteins, depending on the virus strain (21). The spike (S) protein is a glycoprotein with a molecular mass of approximately 180 kDa. For some MHV strains, such as JHM and A59, the S protein is cleaved by a furin-like proteinase into two subunits, the amino-terminal S1 and the carboxyl-terminal S2. The S1 subunit is thought to form the globular head of the spike and is responsible for the initial attachment of the virus to the receptor on the cell surface. The S2 subunit, which forms the stalk portion of the spike and which anchors the S protein to the viral envelope, facilitates the fusion between the viral envelope and the cell membrane and cell-cell fusion (4, 7, 20, 25, 39). In contrast, the S protein of some other MHV strains, such as MHV-2, does not undergo cleavage and usually does not cause cell-cell fusion (15, 34). It appears that the cleavability of the MHV S protein is associated usually, though not always, with its fusogenicity (10, 36). It has been suggested that the fusogenicity of the S protein may determine the route of virus entry, i.e., via direct fusion with plasma membranes or following endocytosis (11, 34), although the mechanism for virus-induced cell-cell fusion may differ from that for virus-cell fusion during entry (8). The S protein also elicits the induction of neutralizing antibodies and cell-mediated immunity in infected hosts (3). It is therefore an important determinant for viral infectivity, pathogenicity, and virulence (2, 5, 31, 38). The hemagglutinin-esterase (HE) protein is present only in certain MHV strains (22, 42) and may play a role in viral pathogenesis (44, 45). The small envelope (E) protein and the membrane (M) protein play a key role in virus assembly (40). The nucleocapsid (N) protein is a phosphoprotein of approximately 50 kDa and is associated with the RNA genome to form the nucleocapsid inside the envelope (21, 37).Infection of host cells by MHV is mediated through the interaction between the S protein and the cellular receptors that are members of the carcinoembryonic antigen (CEA) family of the immunoglobulin superfamily (9). This interaction then triggers fusion between the viral envelope and the plasma membrane or the endosomal membrane, the latter of which follows receptor-mediated endocytosis, thus allowing the nucleocapsid to deliver into the cytoplasm. Direct entry from the plasma membrane appears to be the predominant route for most MHV strains (19, 28), although entry by some mutant MHVs, such as OBLV60 and MHV-2, is low pH dependent, i.e., via endocytosis (11, 34). However, nothing is known about how the genomic RNA is transported to the rough endoplasmic reticulum (ER) for translation. Once on the ER, the viral genomic RNA is translated into a polymerase polyprotein from the 5′-end two open reading frames (two-thirds of the genome) via ribosomal frameshifting. The polymerase polyproteins in turn synthesize genomic and multiple species of subgenomic mRNAs. These mRNAs are then translated into nonstructural and structural proteins, the latter of which are essential for generation of progeny viruses.MHV can infect rodents, causing hepatitis, enteritis, nephritis, and central nervous system diseases. In the mouse central nervous system, some MHV strains, such as JHM and A59, are neurovirulent, causing acute encephalitis and chronic demyelination (1, 13), while others, such as MHV-2, exhibit extremely low neurovirulence, causing only meningitis without apparent encephalitis and demyelination (6, 16, 41). Extensive mutagenesis studies in combination with targeted RNA recombination have identified that the S protein is the major determinant of MHV pathogenicity in animals, although other viral genes also appear to modulate viral pathogenicity (17, 32). For example, the recombinant MHV Penn-98-1, which contains the S protein of MHV-2 in an A59 genome background, causes acute meningoencephalitis similar to that caused by A59 but does not cause demyelination similar to that observed for MHV-2 (6). It has also been shown that the amounts of antigen staining and necrosis in the liver correlate with the viral titer, which is determined largely by the S protein (29). However, how the S protein affects viral titer in cell culture and in animals is not known.In the present study, we initially observed that the levels of production of infectious viruses in an astrocytoma DBT cell line were markedly different among three MHV strains. Using the recombinant MHV Penn-98-1 and its isogenic S757R mutant, we further established that the S protein is responsible for the observed difference. The difference in virus production between A59 and Penn-98-1 was detected as early as 4 to 6 h postinfection (p.i.) and likely occurred during the early stages of the virus life cycle but after virus internalization. Interestingly, when the genomic RNAs were delivered directly into the cells via transfection, the levels of gene expression for these viruses were similar. Furthermore, cell fractionation experiments revealed that significantly more genomic RNAs for nonfusogenic MHVs were delivered to the ER within the first 2 h after infection than for fusogenic MHVs. These results demonstrate that the spike protein of MHV can regulate the intracellular transport of the viral genome to the ER following internalization. To our knowledge, this is the first study identifying a role for a coronavirus S protein in genome delivery in addition to its well-established role in receptor binding and virus-cell and cell-cell fusions during infection.     

Calcium-sensing Receptor Biosynthesis Includes a Cotranslational Conformational Checkpoint and Endoplasmic Reticulum Retention

Metabolic labeling with [³⁵S]cysteine was used to characterize early events in CaSR biosynthesis. [³⁵S]CaSR is relatively stable (half-life ∼8 h), but maturation to the final glycosylated form is slow and incomplete. Incorporation of [³⁵S]cysteine is linear over 60 min, and the rate of [³⁵S]CaSR biosynthesis is significantly increased by the membrane-permeant allosteric agonist NPS R-568, which acts as a cotranslational pharmacochaperone. The [³⁵S]CaSR biosynthetic rate also varies as a function of conformational bias induced by loss- or gain-of-function mutations. In contrast, [³⁵S]CaSR maturation to the plasma membrane was not significantly altered by exposure to the pharmacochaperone NPS R-568, the allosteric agonist neomycin, or the orthosteric agonist Ca²⁺ (0.5 or 5 mm), suggesting that CaSR does not control its own release from the endoplasmic reticulum. A CaSR chimera containing the mGluR1α carboxyl terminus matures completely (half-time of ∼8 h) and without a lag period, as does the truncation mutant CaSRΔ868 (half-time of ∼16 h). CaSRΔ898 exhibits maturation comparable with full-length CaSR, suggesting that the CaSR carboxyl terminus between residues Thr⁸⁶⁸ and Arg⁸⁹⁸ limits maturation. Overall, these results suggest that CaSR is subject to cotranslational quality control, which includes a pharmacochaperone-sensitive conformational checkpoint. The CaSR carboxyl terminus is the chief determinant of intracellular retention of a significant fraction of total CaSR. Intracellular CaSR may reflect a rapidly mobilizable “storage form” of CaSR and/or may subserve distinct intracellular signaling roles that are sensitive to signaling-dependent changes in endoplasmic reticulum Ca²⁺ and/or glutathione.     

内质网压力响应相关的蛋白质降解

内质网相关蛋白质降解途径(ERAD),即蛋白质分泌过程中错误折叠或未折叠的蛋白质在内质网中被识别并逆向运输到细胞质经聚泛素化后由蛋白酶体降解的过程.自从发现该途径后对其机制的阐明一直处于不断探索的阶段.近年来,对ERAD底物识别、逆向运输和泛素化新组分的发现以及新技术的应用,使得该途径的具体分子机制更加清晰.本文全面梳理并综述了内质网应激响应、ERAD降解过程与机理的最新进展,并对模式蛋白底物和最新研究方法进行了总结,以期展示该领域的研究概况.     

Endoplasmic Reticulum Stress: A New Pathway to Induce Autophagy

Autophagy is a response to the stress of nutrient limitation in yeast, whereby cytosolic long-lived proteins and organelles are non-selectively degraded, and the resulting macromolecules are recycled to allow new protein synthesis that is essential for survival. We recently revealed that endoplasmic reticulum (ER) stress induces autophagy. When misfolded proteins accumulate in the ER the resulting stress activates the unfolded protein response (UPR) to induce the expression of chaperones and proteins involved in the recovery process. Under conditions of ER stress, the pre-autophagosomal structure is assembled, and transport of autophagosomes to the vacuole is stimulated in an Atg protein-dependent manner. Interestingly, Atg1 has high kinase activity during ER stress-induced autophagy similar to the situation in starvation-induced autophagy.

Addendum to:

Endoplasmic Reticulum Stress Triggers Autophagy

T. Yorimitsu, U. Nair, Z. Yang and D.J. Klionsky

J Biol Chem 2006; 281:30299-304     

From Endoplasmic Reticulum to Mitochondria: Absence of the Arabidopsis ATP Antiporter Endoplasmic Reticulum Adenylate Transporter1 Perturbs Photorespiration

The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO₂ concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.     

PKR-Like Endoplasmic Reticulum Kinase Is Necessary for Lipogenic Activation during HCMV Infection

PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-associated stress sensor protein which phosphorylates eukaryotic initiation factor 2α (eIF2α) to induce translation attenuation in response to ER stress. PERK is also a regulator of lipogenesis during adipocyte differentiation through activation of the cleavage of sterol regulatory element binding protein 1 (SREBP1), resulting in the upregulation of lipogenic enzymes. Our recent studies have shown that human cytomegalovirus (HCMV) infection in human fibroblasts (HF) induces adipocyte-like lipogenesis through the activation of SREBP1. Here, we report that PERK expression is highly increased in HCMV-infected cells and is necessary for HCMV growth. Depletion of PERK, using short hairpin RNA (shRNA), resulted in attenuation of HCMV growth, inhibition of lipid synthesis and reduction of lipogenic gene expression. Examination of the cleavage of SREBP proteins showed PERK depletion inhibited the cleavage of SREBP1, but not SREBP2, in HCMV-infected cells, suggesting different cleavage regulatory mechanisms for SREBP1 and 2. Further studies showed that the depletion of SREBP1, but not SREBP2, reduced lipid synthesis in HCMV infection, suggesting that activation of SREBP1 is sufficient to induce lipogenesis in HCMV infection. The reduction of lipid synthesis by PERK depletion can be partially restored by expressing a Flag-tagged nuclear form of SREBP1a. Our studies also suggest that the induction of PERK in HCMV-infected cells stimulates SREBP1 cleavage by reducing levels of Insig1 (Insulin inducible gene 1) protein; this occurs independent of the phosphorylation of eIF2α. Introduction of an exogenous Insig1-Myc into HCMV infected cells significantly reduced HCMV growth and lipid synthesis. Our data demonstrate that the induction of PERK during HCMV infection is necessary for full activation of lipogenesis; this effect appears to be mediated by limiting the levels of Insig1 thus freeing SREBP1-SCAP complexes for SREBP1 processing.