Exiting the endoplasmic reticulum

Vesicular transport from the endoplasmic reticulum (ER) to the Golgi complex constitutes the initial step in protein secretion. COPII-coated vesicles mediate the export of newly synthesized proteins from the ER, and this transport step is coupled with COPI-mediated retrograde traffic to form a transport circuit that supports the compositional asymmetry of the ER-Golgi system. Biochemical and structural studies have advanced our understanding of the mechanisms that control vesicle formation and cargo-protein capture. Recent work has highlighted the function of transitional ER regions in specifying the location of COPII budding.     

Structure and assembly of the endoplasmic reticulum. Biosynthetic sorting of endoplasmic reticulum proteins

We have studied the post-translational processing and the biosynthetic sorting of three protein components of murine endoplasmic reticulum (ER), ERp60, ERp72, and ERp99. In pulse-labeled MOPC-315 (where MOPC-315 represents mineral oil-induced plasmacytoma cells) plasmacytoma cells, no precursor forms of these proteins were detected and only ERp99 was sensitive to endoglycosidase H. The ERp99 oligosaccharide remained endoglycosidase H sensitive during a 3-h chase, and analysis by high performance liquid chromatography showed the predominant structure to be Man8GlcNAc2. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 plasmacytoma cells in order to directly study the biosynthetic sorting of both glycosylated and nonglycosylated ERps and have found no strong evidence to suggest these proteins ever leave the endoplasmic reticulum. In spite of their common sorting pathway, these proteins differ in their membrane orientation. Both ERp60 and ERp72 are entirely protected by the endoplasmic reticulum membrane while ERp99 appears to have a large domain exposed on the cytoplasmic face of the endoplasmic reticulum.     

Construction of the endoplasmic reticulum

To study the construction of the ER, we used the microtubule-disrupting drug nocodazole to induce the complete breakdown of ER structure in living cells followed by recovery in drug-free medium, which regenerates the ER network within 15 min. Using the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide to visualize the ER, we have directly observed the network construction process in living cells. In these experiments, the ER network was constructed through an iterative process of extension, branching, and intersection of new ER tubules driven by the ER motility previously described as tubule branching. We have tested the cytoskeletal requirements of this process. We find that newly formed ER tubules are aligned with single microtubules but not actin fibers or vimentin intermediate filaments. Microtubule polymerization preceded the extension of ER tubules and, in experiments with a variety of different drugs, appeared to be a necessary condition for the ER network formation. Furthermore, perturbations of the pattern of microtubule polymerization with microtubule-specific drugs caused exactly correlated perturbations of the pattern of ER construction. Induction of abnormally short, nonintersecting microtubules with 20 microM taxol prevented the ER network formation; ER tubules only extended along the few microtubules contacting the aggregated ER membranes. This requirement for a continuous network of intersecting microtubules indicates that ER network formation takes place through the branching and movement of ER membranes along microtubules. Cytochalasin B had no apparent effect on the construction of the ER network during recovery, despite apparently complete disruption of actin fibers as stained by phalloidin. Blockage of protein synthesis and disorganization of intermediate filaments with cycloheximide pretreatment also failed to perturb ER construction.     

疱疹病毒与内质网应激

内质网是分泌型蛋白和膜蛋白折叠及翻译后修饰的主要场所.病毒感染所引起的宿主细胞内环境的改变可使细胞或病毒的未折叠和/或错误折叠蛋白在内质网中大量聚集,使内质网处于生理功能紊乱的应激状态.为了缓解这种应激压力,细胞会启动未折叠蛋白反应(UPR),并通过一系列分子的信号转导维持内质网稳态;同时病毒也会通过对UPR的精密调控...     

Subcompartments of the endoplasmic reticulum

The endoplasmic reticulum (ER) is the largest continuous endomembrane structure in the cytoplasm. It may be viewed as a series of unique subcompartments. In this review, we examine the rough ER, nuclear envelope and several smooth ER subcompartments. Consideration is given to the characteristic properties and functions of the ER and its domains, and to the formation and maintenance of subcompartments. Associations within the ER membrane bilayer, and with constituents of the cytoplasm and the ER lumen, contribute to the formation of domains and lead to the establishment of subcompartments that reflect specialized functions and vary according to the physiologic state and phenotype of the individual cell. Although the structural complexity of some ER subcompartments (such as the sarcoplasmic reticulum) is highly elaborate, the ER remains a dynamic organelle, subject to assembly and disassembly, capable of extensive remodelling and active in exchange with other organelles through mechanisms of membrane transport.     

Presynaptic endoplasmic reticulum and neurotransmission

Synaptic transmission relies on rapid calcium (Ca²⁺) influx into presynaptic terminal via voltage-gated Ca²⁺ channels. However, smooth ER is present in presynaptic terminals and accumulating evidence indicate that ER Ca²⁺ signaling may play a modulatory role in synaptic transmission. Most recent publication by Lindhout and colleagues (EMBO J, 38 (2019) e101345) suggested that the fragmentation state of the ER affects synaptic vesicle release. Here we discuss these results as well as several key publications that addressed a connection between ER Ca²⁺ signaling and synaptic transmission.     

Plant G proteins interact with endoplasmic reticulum luminal protein receptors to regulate endoplasmic reticulum retrieval

Maintaining endoplasmic reticulum (ER) homeostasis is essential for the production of biomolecules. ER retrieval, i.e., the retrograde transport of compounds from the Golgi to the ER, is one of the pathways that ensures ER homeostasis. However, the mechanisms underlying the regulation of ER retrieval in plants remain largely unknown. Plant ERD2‐like proteins (ERD2s) were recently suggested to function as ER luminal protein receptors that mediate ER retrieval. Here, we demonstrate that heterotrimeric G protein signaling is involved in ERD2‐mediated ER retrieval. We show that ERD2s interact with the heterotrimeric G protein Gα and Gγ subunits at the Golgi. Silencing of Gα, Gβ, or Gγ increased the retention of ER luminal proteins. Furthermore, overexpression of Gα, Gβ, or Gγ caused ER luminal proteins to escape from the ER, as did the co‐silencing of ERD2a and ERD2b. These results suggest that G proteins interact with ER luminal protein receptors to regulate ER retrieval.     

The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.     

Compartmentation of the rough endoplasmic reticulum

It has become evident during recent years that a wide variety of proteins are synthesized on membrane-bound polysomes, very many of which are not ultimately secreted from the cell. The majority of proteins appear to go through some form of post-translational modification before the final appearance of an 'active' product, and in some cases the polypeptide chain may be modified before the completed protein molecule is released from the ribosome. This then raises the question concerning the possibility of the organization of the rough endoplasmic reticulum into individual domains, or compartments, each of which may have the responsibility of performing definite and well defined functions. During recent years the behaviour of two subfractions of the rough endoplasmic reticulum in a variety of cell types and under a variety of conditions has been studied in order to gain insight into a possible compartmentation of this organelle. Throughout the studies disruption of cells has been performed by nitrogen cavitation. This technique was chosen in order to provide conditions of homogenization which were extremely reproducible since shearing forces, mechanical damage and the effects of local heating were eliminated. Endoplasmic reticulum (ER) membranes isolated from the post-mitochondrial supernatant have been separated into subfractions by centrifugation on discontinuous sucrose gradients. By virtue of their high density imparted by the association of ribosomes, rough ER (RER) membranes penetrate 1.4 M sucrose accumulating above either 2.0 M sucrose (light rough -LR membranes) or a cushion of 2.3 M sucrose (heavy rough -HR membranes). Smooth (S) membranes, which are virtually devoid of ribosomes, collect above 1.4 M sucrose. The HR, LR and S subfractions in MPC-11 cells differ in a number of respects: RNA/protein and RNA/phospholipid ratios, polysome profiles and marker enzymes. When cells were homogenized in buffer containing 25 mM KCl then all three ER subfractions were observed, however, when the buffer contained 100 mM KCl then only the LR and S subfractions were observed in gradients, radioactivity equivalent to that in the HR fraction was not recovered in the other two subfractions. Four times as many light chain immunoglobulin polypeptides were found associated with polysomes of HR membranes compared to LR membranes. The nuclear associated ER (NER), though very active in protein synthesis, was only 20% as active in the synthesis of light chain as the combined LR/HR fraction. Studies with MPC-11 cells showed that the relative amounts of the three ER subfractions were related to the phase of the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

ATM blocks tunicamycin-induced endoplasmic reticulum stress

Endoplasmic reticulum stress (ER-stress) is associated with ataxia telangiectasia mutated (ATM) gene. We present here conclusive data showing that ATM blocks ER-stress induced by tunicamycin or ionizing radiation (IR). X-box protein-1 (XBP-1) splicing, GRP78 expression and caspase-12 activation were increased by tunicamycin or IR in Atm-deficient AT5BIVA fibroblasts. Activation of caspase-12 and caspase-3 by tunicamycin was significantly reduced in cells transfected with wild-type Atm (AT5BIVA/wtATM). Atm knockdown by siRNA, however, noticeably elevated ER-stress and chemosensitivity to tunicamycin. In summary, we present substantial data demonstrating that ATM blocks the ER stress signaling associated with cancer cell proliferation.     

Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum

Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.     

Rat liver endoplasmic reticulum protein kinases

• 1.1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography.
• 2.2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase and casein kinases.
• 3.3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis.
• 4.4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca²⁺ /calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

     

The endoplasmic reticulum of mung-bean cotyledons

Germination and seedling growth of mungbean (Vigna radiata (L.) Wilczek) are accompanied by the incorporation of radioactive amino acids, glycerol, galactose, and glucosamine in an organelle fraction of the cotyledons which co-equilibrates with NADH-cytochrome-c-reductase activity at 1.13 g·cm^–3 on isopycnic gradients containing 1 mM EDTA. Up to 20% of the newly synthesized proteins accumulate in this organelle fraction. The organelle fraction has been identified as rough endoplasmic reticulum (ER) on the basis of its increased density (1.16 g·cm^–3) when 3 mM MgCl₂ is included in all media. Seedling growth is also accompanied by a marked rise (more than 5-fold) in ER-associated NADH- and NADPH-cytochrome-c-reductase activity, and by the incorporation of⁵⁹Fe into ER-associated heme. Other manifestations of the reorganization of the ER in the cotyledons include a relative increase in membrane-associated RNA (from 12% of total RNA after 12 h of imbibition to 23% after 6 d of growth), and a change in the pattern of polypeptides associated with the ER. These results provide further evidence for the extensive reorganization of the ER of the cotyledons which accompanies seedling growth. The reorganization includes the simultaneous breakdown of the pre-existing tubular ER and the biosynthesis of new ER components.This is the fourth paper in a series on the endoplasmic reticulum of mung-bean cotyledons. The first three papers are referenced as Gilkes and Chrispeels (in press); Harris and Chrispeels 1980; Van der Wilden et al. (in press)     

The endoplasmic reticulum and calcium storage

Calcium storage is one of the functions commonly attributed to the endoplasmic reticulum (ER) in nonmuscle cells. Several recent studies have added support to this concept. Analysis of reticuloplasm, the luminal ER content, has shown that it contains several proteins (reticuloplasmins) which are prospective calcium storage proteins. One of these, calreticulin, is also present in the sarcoplasmic reticulum (SR). In sea urchin eggs, a calsequestrin-like protein has been clearly localised to the ER. The recent demonstration that the IP3 receptor, which has similarities with the calcium release channel in the SR is also localised in the ER membrane suggests that calcium stored in the ER is important for intracellular signalling. The alternative view, that the physiologically important calcium store is a specialised organelle, the calciosome, is not supported by these observations. Recent evidence also suggests that ER calcium might be important in ER structure and in the retention of the luminal ER proteins.