Endoplasmic reticulum: Membrane contact sites

This paper presents a review of modern data on the functional designation of membrane contact sites (MCSs) of endoplasmic reticulum. Problems of traffic of lipids and calcium in are discussed. It is to be emphasized that the function of MCSs is not yet clear, while the mechanism providing contact of two membranes (the problem of “anchors”) remains poorly studied. Data are discussed that testify in favor of MCSs being able to participate both in selective traffic of lipids and in free diffusion of any small molecule (apparently, up to 1.5 kDa) and ions between cell compartments that are in contact.     

Endoplasmic reticulum: a metabolic compartment

Several biochemical reactions and processes of cell biology are compartmentalized in the endoplasmic reticulum (ER). The view that the ER membrane is basically a scaffold for ER proteins, which is permeable to small molecules, is inconsistent with recent findings. The luminal micro-environment is characteristically different from the cytosol; its protein and glutathione thiols are remarkably more oxidized, and it contains a separate pyridine nucleotide pool. The substrate specificity and activity of certain luminal enzymes are dependent on selective transport of possible substrates and co-factors from the cytosol. Abundant biochemical, pharmacological, clinical and genetic data indicate that the barrier function of the lipid bilayer and specific transport activities in the membrane make the ER a separate metabolic compartment.     

Endoplasmic reticulum architecture: structures in flux

The endoplasmic reticulum (ER) is a dynamic pleiomorphic organelle containing continuous but distinct subdomains. The diversity of ER structures parallels its many functions, including secretory protein biogenesis, lipid synthesis, drug metabolism and Ca2+ signaling. Recent studies are revealing how elaborate ER structures arise in response to subtle changes in protein levels, dynamics, and interactions as well as in response to alterations in cytosolic ion concentrations. Subdomain formation appears to be governed by principles of self-organization. Once formed, ER subdomains remain malleable and can be rapidly transformed into alternative structures in response to altered conditions. The mechanisms that modulate ER structure are likely to be important for the generation of the characteristic shapes of other organelles.     

Endoplasmic reticulum bodies: solving the insoluble

Plant cells produce and accumulate insoluble triglycerides, proteins, and rubber that are assembled into inert, ER-derived organelles broadly termed as ER bodies. ER bodies appear to originate from tubular ER domains that are maintained by cytoskeletal interactions and integral ER proteins. ER bodies sequestering insoluble substances usually are transferred to the vacuole but sometimes remain as cytoplasmic organelles. Some otherwise soluble ER-synthesized proteins are converted to insoluble aggregates to produce ER bodies for transfer to the vacuole. This process constitutes an alternate secretory system to assemble and traffic transport-incompetent insoluble materials.     

Endoplasmic reticulum calcium release is modulated by actin polymerization

Intracellular calcium ions regulate the structure and functions of cytoskeletal proteins. On the other hand, recent studies have shown that the cytoskeleton, and actin filaments in particular, can modulate calcium influx through plasma membrane ligand- and voltage-gated channels. We now report that calcium release from inositol trisphosphate (IP3) and ryanodine-sensitive endoplasmic reticulum (ER) stores is modulated by polymerization and depolymerization of actin filaments in cultured hippocampal neurons. Depolymerization of actin filaments with cytochalasin D attenuates calcium release induced by carbamylcholine (CCh; a muscarinic agonist for IP3 pathway), caffeine (a ryanodine receptor agonist) and thapsigargin (an inhibitor of the ER calcium- ATPase) in both the presence and absence of extracellular calcium. Conversely, the actin polymerizing agent jasplakinolide potentiates calcium release induced by CCh, caffeine and thapsigargin. Cytochalasin D attenuated, while jasplakinolide augmented, thapsigargin-induced JNK activation and neuronal cell death. Our data show that the actin cytoskeleton regulates ER calcium release, suggesting roles for actin in the various physiological and pathological processes that involve calcium release.     

Endoplasmic reticulum: anatomy of a biologic membrane

Endoplasmic reticulum (ER) is a large membranous network containing a wide variety of lipid and protein constituents which play important roles in cellular physiology. In this review, selection of experimental results are presented which have shaped our concepts of the molecular organization of ER. The morphological approach--electron microscope examination of ultra-thin sections of a variety of cells--led to the dualistic distinction between rough ER and smooth ER. Consequently, various attempts were made to separate the 2 entities and to demonstrate that they are endowed with distinct functional properties. Studies on the biogenesis of ER showed that enzymes associated with this organelle turn over independently, which was interpreted in terms of ER being biochemically organized as a mosaic. The results of isopycnic centrifugation of rat liver microsomes led us to conclude that the ER is comprised of three biochemically distinct domains and that the distribution of integral proteins in the lateral plane of the membrane (lateral topography) is primarily determined by their transmembrane disposition. Data on the transverse topography and the mode of biogenesis of ER enzymes are confronted with the predictions of this model.     

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.     

Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells

The endoplasmic reticulum (ER) is a multifaceted cellular organelle both structurally and functionally, and its cell cycle–dependent morphological changes are poorly understood. Our quantitative confocal and EM analyses show that the ER undergoes dramatic reorganization during cell division in cultured mammalian cells as mitotic ER profiles become shorter and more branched. 3D modeling by electron tomography reveals that the abundant interphase structures, sheets, are lost and subsequently transform into a branched tubular network that remains continuous. This is confirmed by observing the most prominent ER subdomain, the nuclear envelope (NE). A NE marker protein spreads to the mitotic ER tubules, although it does not show a homogenous distribution within the network. We mimicked the mitotic ER reorganization using puromycin to strip the membrane-bound ribosomes from the interphase ER corresponding to the observed loss of ribosomes normally occurring during mitosis. We propose that the structural changes in mitotic ER are linked to ribosomal action on the ER membranes.     

Endoplasmic reticulum stress-induced cell death mediated by the proteasome

Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis, such as cysteine aspartyl protease (caspase) activation, cytochrome c release, and DNA fragmentation. Here, we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stress-induced degradation of Bcl-2, release of cytochrome c, processing of effector caspase-3, and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release, we propose that the pro-apoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus, the proteasome serves as a principal mediator of ER stress-induced cell death in this system.     

Endoplasmic reticulum stress is induced and modulated by enterovirus 71

Picornavirus infection alters the endoplasmic reticulum (ER) membrane but it is unclear whether this induces ER stress. Infection of rhabdomyosarcoma cells with enterovirus 71 (EV71), a picornavirus, caused overexpression of the ER‐resident chaperone proteins, BiP and calreticulin, and phosphorylation of eIF2α, but infection with UV‐inactivated virus did not, indicating that ER stress was induced by viral replication and not by viral attachment or entry. Silencing (si)RNA knockdown demonstrated that phosphorylation of eIF2α was dependent on PKR: eIF2α phosphorylation was reduced by siPKR but not by siPERK. We provided evidence showing that PERK is upstream of PKR and is thus able to negatively regulate the PKR‐eIF2α pathway. Pulse‐chase experiments revealed that EV71 infection inhibited translation and activation of ATF6. Expression of BiP at the protein level was activated by a virus‐dependent, ATF6‐independent mechanism. EV71 upregulated XBP1 mRNA level, but neither IRE1‐mediated XBP1 splicing nor its active spliced protein was detected, and its downstream gene, EDEM, was not activated. Epigenetic BiP overexpression alleviated EV71‐induced ER stress and reduced viral protein expression and replication. Our results suggest that EV71 infection induces ER stress but modifies the outcome to assist viral replication.     

Endoplasmic reticulum glucosidase II is inhibited by its end products

The calnexin/calreticulin cycle is a quality control system responsible for promoting the folding of newly synthesized glycoproteins entering the endoplasmic reticulum (ER). The association of calnexin and calreticulin with the glycoproteins is regulated by ER glucosidase II, which hydrolyzes Glc 2Man X GlcNAc 2 glycans to Glc 1Man X GlcNAc 2 and further to Glc 0Man X GlcNAc 2 ( X represents any number between 5 and 9). To gain new insights into the reaction mechanism of glucosidase II, we developed a kinetic model that describes the interactions between glucosidase II, calnexin/calreticulin, and the glycans. Our model accurately reconstructed the hydrolysis of glycans with nine mannose residues and glycans with seven mannose residues, as measured by Totani et al. [Totani, K., Ihara, Y., Matsuo, I., and Ito, Y. (2006) J. Biol. Chem. 281, 31502-31508]. Intriguingly, our model predicted that glucosidase II was inhibited by its nonglucosylated end products, where the inhibitory effect of Glc 0Man 7GlcNAc 2 was much stronger than that of Glc 0Man 9GlcNAc 2. These predictions were confirmed experimentally. Moreover, our model suggested that glycans with a different number of mannose residues can be equivalent substrates of glucosidase II, in contrast to what had been previously thought. We discuss the possibility that nonglucosylated glycans, existing in the ER, might regulate the entry of newly synthesized glycoproteins into the calnexin/calreticulin cycle. Our model also shows that glucosidase II does not interact with monoglucosylated glycans while they are bound to calnexin or calreticulin.     

Endoplasmic reticulum localization of DHHC palmitoyltransferases mediated by lysine-based sorting signals

Intracellular palmitoylation dynamics are regulated by a family of 24 DHHC (aspartate-histidine-histidine-cysteine) palmitoyltransferases, which are localized in a compartment-specific manner. The majority of DHHC proteins localize to endoplasmic reticulum (ER) and Golgi membranes, and a small number target to post-Golgi membranes. To date, there are no reports of the fine mapping of sorting signals in mammalian DHHC proteins; thus, it is unclear how spatial distribution of the DHHC family is achieved. Here, we have identified and characterized lysine-based sorting signals that determine the restricted localization of DHHC4 and DHHC6 to ER membranes. The ER targeting signal in DHHC6 conforms to a KKXX motif, whereas the signal in DHHC4 is a distinct KXX motif. The identified dilysine signals are sufficient to specify ER localization as adding the C-terminal pentapeptide sequences from DHHC4 or DHHC6, which contain these KXX and KKXX motifs, to the C terminus of DHHC3, redistributes this palmitoyltransferase from Golgi to ER membranes. Recent work proposed that palmitoylation of newly synthesized peripheral membrane proteins occurs predominantly at the Golgi. Indeed, previous analyses of the peripheral membrane proteins, SNAP25 and cysteine string protein, are fully consistent with their initial palmitoylation being mediated by Golgi-localized DHHC proteins. Interestingly, ER-localized DHHC3 is able to palmitoylate SNAP25 and cysteine string protein to a similar level as wild-type Golgi-localized DHHC3 in co-expression studies. These results suggest that targeting of intrinsically active DHHC proteins to defined membrane compartments is an important factor contributing to spatially restricted patterns of substrate palmitoylation.     

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 nonselectively 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 preautophagosomal 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.     

Endoplasmic reticulum composition and form: Proteins in and out

The endoplasmic reticulum (ER) is the main harbor for newly synthesized proteins in eukaryotic cells. Through a continuous membrane network of sheets and tubules, the ER hosts secretory proteins, integral membrane proteins, and luminal proteins of the endomembrane system. These proteins are translated by ribosomes outside the ER and require subsequent integration into or translocation across the lipid bilayer of the ER. They are then modified post-translationally and folded in the ER. Some of these proteins are packaged into coat protein complex II–coated vesicles for export. Here, we review recent advances in understanding the mechanism of protein translocation and transmembrane domain insertion in the ER, summarize new insights into selective cargo packaging, and discuss the roles of ER morphological dynamics in these processes.     

Endoplasmic reticulum stress caused by aggregate-prone proteins containing homopolymeric amino acids

Many human proteins have homopolymeric amino acid (HPAA) tracts, but their physiological functions or cellular effects are not well understood. Previously, we expressed 20 HPAAs in mammalian cells and showed characteristic intracellular localization, in that hydrophobic HPAAs aggregated strongly and caused high cytotoxicity in proportion to their hydrophobicity. In the present study, we investigated the cytotoxicity of these aggregate-prone hydrophobic HPAAs, assuming that the ubiquitin proteasome system is impaired in the same manner as other well-known aggregate-prone polyglutamine-containing proteins. Some highly hydrophobic HPAAs caused a deficiency in the ubiquitin proteasome system and excess endoplasmic reticulum stress, leading to apoptosis. These results indicate that the property of causing excess endoplasmic reticulum stress by proteasome impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress is not a common cytotoxic effect of aggregate-prone proteins such as polyglutamine.     

鼻病毒非结构蛋白2B 诱导细胞发生内质网应激

为探讨鼻病毒非结构蛋白2B诱导内质网应激和细胞凋亡的机制,本研究构建了鼻病毒非结构蛋白2B的真核表达载体p2B‐GFP ,通过转染BHK‐21细胞检测相关标志蛋白的变化情况。结果显示,非结构蛋白2B定位表达于BHK‐21细胞内质网,诱导内质网应激标志蛋白Grp78、CHOP的表达增加,并使活化转录因子6（ATF6）的转录活性增加,还诱导BHK‐21细胞发生核浓缩而凋亡,使凋亡标志蛋白PARP发生降解而减少。结果提示,鼻病毒非结构蛋白2B可诱导细胞发生内质网应激,并经该途径诱导细胞凋亡。     

Endoplasmic reticulum and Golgi complex: Contributions to, and turnover by, autophagy

The degradation of cytoplasmic contents, especially organelles [mitochondria, peroxisomes, endoplasmic reticulum (ER), Golgi complex (GC)], cannot be accomplished solely by the cytosolic degradation machinery, of which the most prominent component is the proteasome. However, it is possible that such organelles (or portions thereof) can be degraded by the cell's autophagic machinery. In this manner, organelles can be either specifically or non-specifically targeted to the vacuole/lysosome for degradation. These processes can be triggered in response to different environmental cues. Here, we focus on two particular organelles, the ER and the GC, and their relationship with the autophagic process. Firstly, we briefly consider how these two organelles contribute to the synthesis and delivery of hydrolytic enzymes involved in autophagy as well as how they may potentially contribute to their own degradation by addressing the origin of the autophagic membrane. Secondly, we summarize the evidence for the turnover of these two organelles by autophagic processes in different organisms.     

Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation

We had previously shown that endoplasmic reticulum (ER)-associated degradation (ERAD) of glycoproteins in mammalian cells involves trimming of three to four mannose residues from the N-linked oligosaccharide Man(9)GlcNAc(2). A possible candidate for this activity, ER mannosidase I (ERManI), accelerates the degradation of ERAD substrates when overexpressed. Although in vitro, at low concentrations, ERManI removes only one specific mannose residue, at very high concentrations it can excise up to four alpha1,2-linked mannose residues. Using small interfering RNA knockdown of ERManI, we show that this enzyme is required for trimming to Man(5-6)GlcNAc(2) and for ERAD in cells in vivo, leading to the accumulation of Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2) on a model substrate. Thus, trimming by ERManI to the smaller oligosaccharides would remove the glycoprotein from reglucosylation and calnexin binding cycles. ERManI is strikingly concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC) that we had described previously. ERManI knockdown prevents substrate accumulation in the ERQC. We suggest that the ERQC provides a high local concentration of ERManI, and passage through this compartment would allow timing of ERAD, possibly through a cycling mechanism. When newly made glycoproteins cannot fold properly, transport through the ERQC leads to trimming of a critical number of mannose residues, triggering a signal for degradation.