A Cytosolic Chaperone Complexes with Dynamic Membrane J-Proteins and Mobilizes a Nonenveloped Virus out of the Endoplasmic Reticulum

Nonenveloped viruses undergo conformational changes that enable them to bind to, disrupt, and penetrate a biological membrane leading to successful infection. We assessed whether cytosolic factors play any role in the endoplasmic reticulum (ER) membrane penetration of the nonenveloped SV40. We find the cytosolic SGTA-Hsc70 complex interacts with the ER transmembrane J-proteins DnaJB14 (B14) and DnaJB12 (B12), two cellular factors previously implicated in SV40 infection. SGTA binds directly to SV40 and completes ER membrane penetration. During ER-to-cytosol transport of SV40, SGTA disengages from B14 and B12. Concomitant with this, SV40 triggers B14 and B12 to reorganize into discrete foci within the ER membrane. B14 must retain its ability to form foci and interact with SGTA-Hsc70 to promote SV40 infection. Our results identify a novel role for a cytosolic chaperone in the membrane penetration of a nonenveloped virus and raise the possibility that the SV40-induced foci represent cytosol entry sites.     

Protein Translocation Across the Membrane of the Endoplasmic Reticulum

Saccharomyces cerevisiae and mammals concerning the mechanisms of the translocation step and discuss the roles of the proteins implicated in this process. Received: 5 June 1996/Revised: 20 September 1996     

Interaction of Newly Synthesized Apolipoprotein B with Calnexin and Calreticulin Requires Glucose Trimming in the Endoplasmic Reticulum

Apolipoprotein B (ApoB) is the only protein component of the low density lipoproteins (LDL) in plasma. It is a glycoprotein with a molecular mass of about 550 kDa (4536 amino acids) containing 16 N-glycans. We have studied the interaction of ApoB with two lectin-like chaperones of the Endoplasmic Reticulum (ER)—Calnexin (CN) and Calreticulin (CR). Using a co-immunoprecipitation approach we observed that newly synthesized ApoB associates with CN and CR. The interaction was transient; within 30–60 min after synthesis bulk of newly formed ApoB dissociated. Using McA Rh7777 cells expressing an N-terminal fragment of ApoB we found that inhibition of glucosidases in the ER prevented the association of CN and CR to newly synthesized ApoB. The results showed that like for association with other glycoprotein substrates, trimming of glucose residues was essential for ApoB binding to CN and CR.     

Identification of a Novel Stage of Ribosome/Nascent Chain Association with the Endoplasmic Reticulum Membrane

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.     

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.     

Environmental Transition of Signal-Anchor Sequences during Membrane Insertion via the Endoplasmic Reticulum Translocon

In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence, the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence, the signal-anchor became inaccessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.     

Endoplasmic Reticulum Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway

Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their carboxyl terminus. Many of these tail-anchored (TA) proteins are posttranslationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the guided-entry of TA protein insertion (GET) pathway. In recent years, most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway-targeting factor that uses nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pretargeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.The mechanism of membrane protein insertion into the endoplasmic reticulum (ER) has been extensively studied for many years (Shao and Hegde 2011). From this work, the signal recognition particle (SRP)/Sec61 pathway has emerged as a textbook example of a cotranslational membrane insertion mechanism (Grudnik et al. 2009). The SRP binds a hydrophobic segment (either a cleavable amino-terminal signal sequence or a transmembrane domain) immediately after it emerges from the ribosomal exit tunnel. This results in a translational pause that persists until SRP engages its receptor in the ER and delivers the ribosome-nascent chain complex to the Sec61 channel. Last, the Sec61 channel enables protein translocation into the ER lumen along with partitioning of hydrophobic transmembrane domains into the lipid bilayer through the Sec61 lateral gate (Rapoport 2007).Approximately 5% of all eukaryotic membrane proteins have an ER targeting signal in a single carboxy-terminal transmembrane domain that emerges from the ribosome exit tunnel following completion of protein synthesis and is not recognized by the SRP (Stefanovic and Hegde 2007). Nonetheless, because hydrophobic peptides in the cytoplasm are prone to aggregation and subject to degradation by quality control systems (Hessa et al. 2011), these tail-anchored (TA) proteins still have to be specifically recognized, shielded from the aqueous environment, and guided to the ER membrane for insertion. In the past five years, the guided-entry of TA proteins (GET) pathway has come to prominence as the major machinery for performing these tasks and the enabler of many key cellular processes mediated by TA proteins including vesicle fusion, membrane protein insertion, and apoptosis. This research has rapidly yielded biochemical and structural insights (​and2)2) into many of the GET pathway components (Hegde and Keenan 2011; Chartron et al. 2012a; Denic 2012). In particular, Get3 is an ATPase that uses metabolic energy to bridge recognition of TA proteins by upstream pathway components with TA protein recruitment to the ER for membrane insertion. However, the precise mechanisms of nucleotide-dependent TA protein binding to Get3 and how the GET pathway inserts tail anchors into the membrane are still poorly understood. Here, we provide an overview of the budding yeast GET pathway with emphasis on mechanistic insights that have come from structural studies of its membrane-associated steps and make a speculative juxtaposition with the ABC transporter mechanism.

Table 1.

A catalog of GET pathway component structures

The Endoplasmic Reticulum Provides the Membrane Platform for Biogenesis of the Flavivirus Replication Complex

The cytoplasmic replication of positive-sense RNA viruses is associated with a dramatic rearrangement of host cellular membranes. These virus-induced changes result in the induction of vesicular structures that envelop the virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile virus strain Kunjin virus (WNV_KUN) to show that the virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV_KUN replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV_KUN replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.West Nile virus (WNV) belongs to the Flaviviridae, which is a large family of enveloped, positive-strand RNA viral pathogens that are responsible for causing severe disease and mortality in humans and animals each year. The Australian WNV strain Kunjin virus (WNV_KUN) is a relatively low-pathogenic virus that is closely related to the pathogenic WNV strain New York 99 (WNV_NY99), the causative agent of the 1999 epidemic of encephalitis in New York City (11).It has become increasingly known that the replication of most, if not all, positive-sense RNA viruses, whether they infect plants, insects, or humans, is associated with dramatic membrane alterations resulting in the formation of membranous microenvironments that facilitate efficient virus replication. In most cases the induced membrane structures house the actively replicating viral RNA and comprise 70- to 100-nm membrane “vesicles” (sometimes referred to as spherules). Although this distinct morphology is shared across virus families, the cellular origins of these membranes is diverse: the endoplasmic reticulum (ER), mitochondria, peroxisomes, and trans-Golgi membranes have been implicated in different viral systems (1, 8, 13, 23, 31, 38, 41, 45). This diversity implies that the processes involved in inducing the membrane vesicles/spherules are shared, rather than the composition of the membrane itself, although the exact purpose for utilizing membranes derived from different cellular compartments is still not completely resolved or understood.The replication of the flavivirus WNV_KUN is associated with the induction of morphologically distinct membrane structures that have defined roles during the WNV_KUN replication cycle. Three well-defined structures can be seen as large convoluted membranes (CM), paracrystalline arrays (PC), or membrane sacs containing small vesicles, termed vesicle packets (VP) (18, 20, 48). Based on localization studies with viral proteins of specific functions, we observed that components of the virus protease complex (namely, nonstructural protein 3 [NS3] with cofactor NS2B) localize specifically to the CM/PC, whereas viral double-stranded RNA (dsRNA) and the viral RNA-dependent RNA polymerase (RdRp) NS5 localized primarily to VP (20-22, 47, 48). Additionally, we observed that the CM and PC originate from and are modified membranes of the intermediate compartment (IC) and rough endoplasmic reticulum (RER), whereas the VP appear to be derived from trans-Golgi network (TGN) membranes (19). Recently, we have found that the WNV_KUN NS4A protein by itself has the intrinsic capacity to induce the CM and PC structures (35), a property also subsequently shown for Dengue virus (DENV) NS4A (29). Additionally, we have shown that upon WNV infection cellular cholesterol and cholesterol-synthesizing proteins are redistributed to the virus-induced membranes and that this redistribution severely disrupted the formation of cholesterol-rich microdomains (23). Furthermore, we have shown that the membranous structures induced during WNV replication provide partial protection of the WNV replication components from the interferon (IFN)-induced antiviral MxA protein, suggesting that the distinct compartmentalization of viral replication and components of the cellular antiviral response may be an evolutionary mechanism by which flaviviruses can protect themselves from host surveillance (6).In this study we focused on three-dimensional (3D) modeling to give insight into the 3D structure of the VP and provide evidence of how these complexes are organized and formed within the RER membrane. These results add valuable information to our understanding of how the WNV replication complex (RC) functions.     

Protein N-Myristoylation Plays a Critical Role in the Endoplasmic Reticulum Morphological Change Induced by Overexpression of Protein Lunapark,an Integral Membrane Protein of the Endoplasmic Reticulum

N-myristoylation of eukaryotic cellular proteins has been recognized as a modification that occurs mainly on cytoplasmic proteins. In this study, we examined the membrane localization, membrane integration, and intracellular localization of four recently identified human N-myristoylated proteins with predicted transmembrane domains. As a result, it was found that protein Lunapark, the human ortholog of yeast protein Lnp1p that has recently been found to be involved in network formation of the endoplasmic reticulum (ER), is an N-myristoylated polytopic integral membrane protein. Analysis of tumor necrosis factor-fusion proteins with each of the two putative transmembrane domains and their flanking regions of protein Lunapark revealed that transmembrane domain 1 and 2 functioned as type II signal anchor sequence and stop transfer sequence, respectively, and together generated a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. Immunofluorescence staining of HEK293T cells transfected with a cDNA encoding protein Lunapark tagged with FLAG-tag at its C-terminus revealed that overexpressed protein Lunapark localized mainly to the peripheral ER and induced the formation of large polygonal tubular structures. Morphological changes in the ER induced by overexpressed protein Lunapark were significantly inhibited by the inhibition of protein N-myristoylation by means of replacing Gly2 with Ala. These results indicated that protein N-myristoylation plays a critical role in the ER morphological change induced by overexpression of protein Lunapark.     

过表达apoA-I可缓解BEL-7402细胞中衣霉素诱导的内质网应激

内质网应激（endoplasmic reticulum stress,ER stress）对非酒精性脂肪性肝病（non alcoholic fatty liver disease,NAFLD）的发生发展具有十分重要的作用。本实验室前期结果证实,载脂蛋白A1（apolipoprotein A-I,apoA-I）可以通过减少肝细胞脂质堆积来减轻蛋氨酸胆碱缺乏饲料造成的非酒精性肝炎(non-alcoholic steatohepatitis,NASH),但相关机制仍不十分清楚。为探索apoA I对内质网应激的影响,本研究采用衣霉素处理人肝癌BEL-7402细胞。Western印迹结果证实,衣霉素确实可以诱导BEL-7402细胞内质网应激,并具有时间和剂量依赖性。通过将apoA-I表达载体及其对照载体转染到BEL-7402细胞,再加入衣霉素处理,结果显示,与对照组相比,过表达apoA-I的细胞内质网应激标志分子表达明显减轻,同时与脂质合成相关的固醇调节元件结合蛋白1、脂肪酸合成酶和乙酰辅酶A羧化酶1蛋白质水平明显降低。脂质检测结果表明,细胞内甘油三酯和游离胆固醇水平也明显降低（P<0.05）。上述结果表明,apoA-I能够减轻衣霉素引起的内质网应激,可能机制是通过调控固醇调节元件结合蛋白1减少肝细胞的脂质堆积。     

Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic polypeptide-conducting channel, the heterotrimeric Sec61 complex. Previous work has characterized the Sec61 complex as a potential ER Ca²⁺ leak channel in HeLa cells and identified ER lumenal molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) as limiting Ca²⁺ leakage via the open Sec61 channel by facilitating channel closing. This BiP activity involves binding of BiP to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344. Of note, the Y344H mutation destroys the BiP binding site and causes pancreatic β-cell apoptosis and diabetes in mice. Here, we systematically depleted HeLa cells of the BiP co-chaperones by siRNA-mediated gene silencing and used live cell Ca²⁺ imaging to monitor the effects on ER Ca²⁺ leakage. Depletion of either one of the ER lumenal BiP co-chaperones, ERj3 and ERj6, but not the ER membrane-resident co-chaperones (such as Sec63 protein, which assists BiP in Sec61 channel opening) led to increased Ca²⁺ leakage via Sec6 complex, thereby phenocopying the effect of BiP depletion. Thus, BiP facilitates Sec61 channel closure (i.e. limits ER Ca²⁺ leakage) via the Sec61 channel with the help of ERj3 and ERj6. Interestingly, deletion of ERj6 causes pancreatic β-cell failure and diabetes in mice and humans. We suggest that co-chaperone-controlled gating of the Sec61 channel by BiP is particularly important for cells, which are highly active in protein secretion, and that breakdown of this regulatory mechanism can cause apoptosis and disease.     

The Endoplasmic Reticulum Is the Main Membrane Source for Biogenesis of the Lytic Vacuole in Arabidopsis

Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H⁺-pyrophosphatase and the vacuolar H⁺-adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)–Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.     

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.     

Integral Membrane Proteins of the Nuclear Envelope Are Dispersed throughout the Endoplasmic Reticulum during Mitosis

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC₆ and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.     

The Saccharomyces cerevisiae Prenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.     

A Novel Membrane Protein Complex on the Endoplasmic Reticulum and Early Golgi Compartments in the Yeast Saccharomyces cerevisiae

A novel membrane protein, Yml067c in the systematic ORF name, was discovered as a component of immunoisolated vesicles of the early Golgi compartment of the yeast Saccharomyces cerevisiae (Cho et al., FEBS Lett. 469, 151-154 (2000)). Conserved sequences having sequence similarity to Yml067c were widely distributed in the eukaryotes and one of them, Yal042w, was found in the Saccharomyces genome database. In the yeast cell, Yml067c and Yal042w were found to form a heterooligomeric complex by immunoprecipitation of their tagged derivatives from the detergent-solubilized membrane. Cell fractionation and indirect immunofluorescent staining indicated that the majority of these proteins were localized on the ER membrane. Therfore, the Yml067c-Yal042w complex should shuttle between the ER and the early Golgi compartment as well as the p24-family proteins.     

Association of the Yeast RNA-binding Protein She2p with the Tubular Endoplasmic Reticulum Depends on Membrane Curvature

Localization of mRNAs contributes to the generation and maintenance of cellular asymmetry in a wide range of organisms. In Saccharomyces cerevisiae, the so-called locasome complex with its core components Myo4p, She2p, and She3p localizes more than 30 mRNAs to the yeast bud tip. A significant fraction of these mRNAs encodes membrane or secreted proteins. Their localization requires, besides the locasome, a functional segregation apparatus of the cortical endoplasmic reticulum (ER), including the machinery that is involved in the movement of ER tubules into the bud. Colocalization of RNA-containing particles with these tubules suggests a coordinated transport of localized mRNAs and the cortical ER to the bud. Association of localized mRNAs to the ER requires the presence of the locasome component She2p. Here we report that She2p is not only an RNA-binding protein but can specifically bind to ER-derived membranes in a membrane curvature-dependent manner in vitro. Although it does not contain any known curvature recognizing motifs, the protein shows a binding preference for liposomes with a diameter resembling that of yeast ER tubules. In addition, membrane binding depends on tetramerization of She2p. In an in vivo membrane-tethering assay, She2p can target a viral peptide GFP fusion protein to the cortical ER, indicating that a fraction of She2p associates with the ER in vivo. Combining RNA- and membrane-binding features makes She2p an ideal coordinator of ER tubule and mRNA cotransport.     

编码内质网膜蛋白的新基因家族RTN的研究进展

蛋白质的分泌渠道是近几年生命科学研究焦点之一.RTN（reticulon）是定位于内质网膜上的一个基因家族,现已发现RTN1、RTN2、RTN3、RTN4等多个家族成员.其中,这些基因在蛋白质分泌加工过程中的具体作用,以及RTN1（即NSP）作为神经内分泌特异性的标志或与神经元分化的关系都在进一步探讨之中.