Characterization of the rough endoplasmic reticulum ribosome-binding activity.

The rough endoplasmic reticulum membranes of mammalian cells contain specific ribosome-binding sites. A purification to apparent homogeneity of a negatively charged protein (ERp180) of relative molecular mass 180,000 (180 K) was reported which was proposed to function as a rough endoplasmic reticulum ribosome receptor. We report here that ribosome-binding site activity quantitatively solubilized from rough endoplasmic reticulum membranes does not cofractionate with ERp180. By contrast, ribosome-binding site activity fractionates as a much smaller, positively charged protein.     

A protein of the endoplasmic reticulum involved early in polypeptide translocation.

To identify components of the mammalian endoplasmic reticulum involved in the translocation of secretory proteins, crosslinking and reconstitution methods were combined. A multispanning abundant membrane glycoprotein was found which is in proximity to nascent chains early in translocation. In reconstituted proteoliposomes, this protein is stimulatory or required for the translocation of secretory proteins.     

Quality control in the endoplasmic reticulum protein factory

The endoplasmic reticulum (ER) is a factory where secretory proteins are manufactured, and where stringent quality-control systems ensure that only correctly folded proteins are sent to their final destinations. The changing needs of the ER factory are monitored by integrated signalling pathways that constantly adjust the levels of folding assistants. ER chaperones and signalling molecules are emerging as drug targets in amyloidoses and other protein-conformational diseases.     

Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum.

Shiga toxin and some other protein toxins that act on targets in the cytosol have previously been shown to enter the trans-Golgi network. Transport by this route may be necessary for translocation of the toxin to the cytosol and for intoxication, but it is not known whether the enzymatically active part of the toxins actually enters the cytosol from the trans-Golgi network. It has been suggested that such toxins are transported in a retrograde manner to the endoplasmic reticulum and that translocation occurs in this organelle, but retrograde transport of endocytosed material beyond the trans-Golgi network has never been demonstrated. Here we show that in butyric acid-treated A431 cells endocytosed Shiga toxin is not only transported to the trans-Golgi network, but also to all Golgi stacks, to the endoplasmic reticulum and to the nuclear envelope. Furthermore, butyric acid sensitizes the cells to Shiga toxin, which is consistent with the possibility that retrograde transport is required for translocation of the toxin to the cytosol.     

Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum.

Being topologically equivalent to the extracellular space, the lumen of the endoplasmic reticulum (ER) provides a unique folding environment for newly synthesized proteins. Unlike other compartments in the cell where folding occurs, the ER is oxidizing and therefore can promote the formation of disulphide bonds. The reducing agent dithiothreitol, when added to living cells, inhibits disulphide formation with profound effects on folding. Taking advantage of this effect, we demonstrate here that folding of influenza haemagglutinin is energy dependent. Metabolic energy is required to support the correct folding and disulphide bond formation in this well characterized viral glycoprotein, to rescue misfolded proteins from disulphide-linked aggregates, and to maintain the oxidized protein in its folded and oligomerization-competent state.     

A signal sequence receptor in the endoplasmic reticulum membrane

Protein translocation across the endoplasmic reticulum (ER) membrane is triggered at several stages by information contained in the signal sequence. Initially, the signal sequence of a nascent secretory protein upon emergence from the ribosome is recognized by a polypeptide of relative molecular mass 54,000 (Mr54K) which is part of the signal recognition particle (SRP). Binding of SRP may induce a site-specific elongation arrest of translation in vitro. Attachment of the arrested translation complex to the ER membrane is mediated by the SRP-receptor (docking protein) and is accompanied by displacement of the SRP from both the ribosome and the signal sequence. We have investigated the fate of the signal sequence following the disengagement of SRP and its receptor by a crosslinking approach. We report here that the signal sequence of nascent preprolactin, after its release from the SRP, interacts with a newly discovered component, a signal sequence receptor (SSR), which is an integral, glycosylated protein of the rough ER membrane (Mr approximately 35K).     

Identification of a ribosome receptor in the rough endoplasmic reticulum

Attachment of ribosomes to the membrane of the endoplasmic reticulum is one of the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells. The process is mediated by an integral membrane protein of relative molecular mass 180,000 (Mr 180K), having a large (at least 160K) cytosolic domain that, when proteolytically detached from the membrane, can competitively inhibit the binding of ribosomes to intact membranes. Isolation of this domain has led to the identification, purification and characterization of the intact ribosome receptor, as well as its functional reconstitution into lipid vesicles.     

Recognition of transmembrane helices by the endoplasmic reticulum translocon

Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.     

An endoplasmic reticulum retention signal in the CD3 epsilon chain of the T-cell receptor.

Isolated polypeptide chains of the T-cell antigen receptor complex are degraded or retained in the endoplasmic reticulum (ER). Assembly of the multisubunit complex allows the individual chains to escape retention in the ER and to be expressed on the cell surface. We engineered a series of deletions in the CD3 epsilon subunit of the human T-cell receptor in order to find the sequences responsible for its retention in the ER. Deletion of amino acids 171 to 180 in the cytosolic tail resulted in the cell-surface expression of the isolated chain. This sequence also promotes retention when it is appended to CD4, a plasma membrane protein. Mutagenesis of the 10-amino-acid CD3 epsilon sequence established that the tyrosine and serine residues are important for ER retention. This and other ER retention signals must be hidden when a complete T-cell receptor complex is assembled in order to allow its expression on the cell surface.     

Independent phosphatidylinositol synthesis in pituitary plasma membrane and endoplasmic reticulum

Phosphatidylinositol (PtdIns), the most abundant phosphoinositide, is the precursor of phosphatidylinositol 4-monophosphate which is converted to phosphatidylinositol 4,5-bisphosphate, the lipid hydrolysed as an early step in signal transduction by many stimuli. It is generally thought that a single enzyme in the endoplasmic reticulum, PtdIns synthase (CDP-diglyceride:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11), is responsible for PtdIns synthesis and that newly synthesized PtdIns is transported to the plasma membrane by exchange proteins. Several investigators have proposed that there are two functionally distinct pools of PtdIns, one responsive to stimulation and the other not, and that the stimulus-responsive pool may be synthesized at a different site within the cell, perhaps within the plasma membrane. Indeed, it was suggested that there is PtdIns synthase activity in plasma membrane isolated from rat liver. GH3 rat pituitary tumour cells are an excellent model system to study stimulation of phosphoinositide metabolism by thyrotropin-releasing hormone (TRH). Conversion of PtdIns to polyphosphoinositides and TRH (and GTP)-activated phosphoinositide hydrolysis are known to occur in plasma membrane isolated from GH3 cells. Here we report that PtdIns synthase activity in the plasma membrane of GH3 cells is distinct from that present in the endoplasmic reticulum. The plasma membrane PtdIns synthase may be responsible for a portion of PtdIns re-synthesis that occurs during cell stimulation.     

A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum

The segregation of secretory and membrane proteins to the mammalian endoplasmic reticulum is mediated by remarkably diverse signal sequences that have little or no homology with each other. Despite such sequence diversity, these signals are all recognized and interpreted by a highly conserved protein-conducting channel composed of the Sec61 complex. Signal recognition by Sec61 is essential for productive insertion of the nascent polypeptide into the translocation site, channel gating and initiation of transport. Although subtle differences in these steps can be detected between different substrates, it is not known whether they can be exploited to modulate protein translocation selectively. Here we describe cotransin, a small molecule that inhibits protein translocation into the endoplasmic reticulum. Cotransin acts in a signal-sequence-discriminatory manner to prevent the stable insertion of select nascent chains into the Sec61 translocation channel. Thus, the range of substrates accommodated by the channel can be specifically and reversibly modulated by a cell-permeable small molecule that alters the interaction between signal sequences and the Sec61 complex.     

Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes

A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.     

Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.     

内质网应激对小鼠脂肪组织的影响

为了探究内质网应激对小鼠白色脂肪组织的影响,通过腹腔注射衣霉素(tunicamycin, TM)诱导小鼠体内内质网应激,构建内质网应激小鼠模型,然后获取小鼠附睾处白色脂肪组织,并采用RT-PCR和Western blot检测白色脂肪组织中内质网应激相关因子的mRNA水平及蛋白表达情况,通过荧光显微镜观察白色脂肪组织细胞的变化,测定甘油三酯、游离脂肪酸含量及脂肪细胞因子和炎症因子分泌情况.结果表明：1.0 mg/kg TM处理72 h诱导小鼠内质网应激效果最佳;内质网应激可以导致白色脂肪组织比例减少,脂肪细胞直径减小,脂解反应增多,脂肪细胞因子分泌减少及炎症反应增多等.该研究结果说明内质网应激可以诱导小鼠脂肪组织中发生脂解,进而介导脂代谢紊乱.     

Topology of signal recognition particle receptor in endoplasmic reticulum membrane

The signal recognition particle (SRP) receptor is an integral membrane protein of the endoplasmic reticulum which, in conjunction with SRP, ensures the correct targeting of nascent secretory proteins to this membrane system. From the complementary DNA sequence we have deduced the complete primary structure of the SRP receptor and established that its amino-terminal region is anchored in the membrane. The anchor fragment and the cytoplasmic fragment contribute jointly to a functionally important region which is highly charged and may function in nucleic acid binding.     

AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.     

ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum

The ability of killer T cells carrying the CD8 antigen to detect tumours or intracellular pathogens requires an extensive display of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of potential target cells. These peptides are derived from almost all intracellular proteins and reveal the presence of foreign pathogens and mutations. How cells produce thousands of distinct peptides cleaved to the precise lengths required for binding different MHC class I molecules remains unknown. The peptides are cleaved from endogenously synthesized proteins by the proteasome in the cytoplasm and then trimmed by an unknown aminopeptidase in the endoplasmic reticulum (ER). Here we identify ERAAP, the aminopeptidase associated with antigen processing in the ER. ERAAP has a broad substrate specificity, and its expression is strongly upregulated by interferon-gamma. Reducing the expression of ERAAP through RNA interference prevents the trimming of peptides for MHC class I molecules in the ER and greatly reduces the expression of MHC class I molecules on the cell surface. Thus, ERAAP is the missing link between the products of cytosolic processing and the final peptides presented by MHC class I molecules on the cell surface.     

Calcium content of mitochondria and endoplasmic reticulum in liver frozen rapidly in vivo

The recognition that the endoplasmic reticulum (ER), rather than the mitochondria, is the main organelle regulating the cytoplasmic Ca2+ concentration in non-muscle cells supports the notion that an alternative physiological role of mitochondrial Ca transport is the modulation of Ca-sensitive mitochondrial enzymes through small (micromolar) fluctuations in the concentration of mitochondrial matrix Ca2+. The latter mechanism could operate only if the mitochondrial Ca concentration were low, as it is in muscle and retinal rods, below the levels saturating the regulated enzymes. In contrast, if the ER serves as an intracellular Ca store, its Ca content would be expected to be high. In view of the major metabolic function of the liver, the question of whether hepatic mitochondrial matrix Ca2+ regulates metabolism is particularly important, but the range of Ca concentrations reported for isolated liver mitochondria is too wide to provide a conclusive answer. Therefore, we have used electron probe X-ray microanalysis (EPMA) to measure the subcellular distribution of Ca in liver snap-frozen in vivo, and report here that the endoplasmic reticulum is a major intracellular store of Ca, while the concentration of Ca in mitochondria is low and compatible with the regulation of mitochondrial enzymes.