Internalized Pseudomonas exotoxin A can exploit multiple pathways to reach the endoplasmic reticulum

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the intoxication of mammalian cells by Pseudomonas exotoxin A (PEx). The toxin binds the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Here, we show that in HeLa cells, PEx recruits a proportion of this receptor to detergent-resistant microdomains (DRMs). Uptake of receptor-bound PEx involves transport steps both directly from early endosomes to the trans-Golgi network (TGN) independently of Rab9 function and from late endosomes to the TGN in a Rab9-dependent manner. Furthermore, treatments that simultaneously perturb both Arf1-dependent and Rab6-dependent retrograde pathways show that PEx can use multiple routes to reach the ER. The Rab6-dependent route has only been described previously for cargo with lipid-sorting signals. These findings suggest that partial localization of PEx within DRM permits a choice of trafficking routes consistent with a model that DRM-associated toxins reach the ER on a lipid-dependent sorting pathway whilst non-DRM-associated PEx exploits the previously characterized KDEL receptor-mediated uptake pathway. Thus, unexpectedly, an ER-directed toxin with a proteinaceous receptor shows promiscuity in its intracellular trafficking pathways, exploiting routes controlled by both lipid- and protein-sorting signals.     

Retro-translocation of proteins from the endoplasmic reticulum into the cytosol

Proteins that are misfolded in the endoplasmic reticulum are transported back into the cytosol for destruction by the proteasome. This retro-translocation pathway has been co-opted by certain viruses, and by plant and bacterial toxins. The mechanism of retro-translocation is still mysterious, but several aspects of this process are now being unravelled.     

Insertion and topology of a plant viral movement protein in the endoplasmic reticulum membrane

Virus-encoded movement proteins (MPs) mediate cell-to-cell spread of viral RNA through plant membranous intercellular connections, the plasmodesmata. The molecular pathway by which MPs interact with viral genomes and target plasmodesmata channels is largely unknown. The 9-kDa MP from carnation mottle carmovirus (CarMV) contains two potential transmembrane domains. To explore the possibility that this protein is in fact an intrinsic membrane protein, we have investigated its insertion into the endoplasmic reticulum membrane. By using in vitro translation in the presence of dog pancreas microsomes, we demonstrate that CarMV p9 inserts into the endoplasmic reticulum without the aid of any additional viral or plant host components. We further show that the membrane topology of CarMV p9 is N(cyt)-C(cyt) (N and C termini of the protein facing the cytoplasm) by in vitro translation of a series of truncated and full-length constructs with engineered glycosylation sites. Based on these results, we propose a topological model in which CarMV p9 is anchored in the membrane with its N- and C-terminal tail segments interacting with its soluble, RNA-bound partner CarMV p7, to accomplish the viral cell-to-cell movement function.     

Palmitylation of viral membrane glycoproteins takes place after exit from the endoplasmic reticulum

Palmitylation of vesicular stomatitis virus G and Sindbis virus E1 glycoproteins has been studied in relation to the transport from the endoplasmic reticulum (ER) to the Golgi complex. Incubation of infected cells at 15 degrees C prevents the transport of newly synthesized membrane proteins from the ER to the Golgi (Saraste, J., and Kuismanen, E. (1984) Cell 38, 535-549). In these conditions, also palmitylation of G protein and of E1 glycoprotein is blocked. When the transport is restored by increasing the temperature, palmitylation occurs quickly and is followed by the complete trimming of peripheral mannose residues due to mannosidase I (a putative cis-Golgi function). Immunofluorescence analysis showed that the G glycoprotein accumulated at 15 degrees C in structures distinct from both ER and Golgi. These studies suggest that transport from the ER to the cis-Golgi involves intermediate compartments.     

EDEM is involved in retrotranslocation of ricin from the endoplasmic reticulum to the cytosol

The plant toxin ricin is transported retrogradely from the cell surface to the endoplasmic reticulum (ER) from where the enzymatically active part is retrotranslocated to the cytosol, presumably by the same mechanism as used by misfolded proteins. The ER degradation enhancing alpha-mannosidase I-like protein, EDEM, is responsible for directing aberrant proteins for ER-associated protein degradation. In this study, we have investigated whether EDEM is involved in ricin retrotranslocation. Overexpression of EDEM strongly protects against ricin. However, when the interaction between EDEM and misfolded proteins is inhibited by kifunensin, EDEM promotes retrotranslocation of ricin from the ER to the cytosol. Furthermore, puromycin, which inhibits synthesis and thereby transport of proteins into the ER, counteracted the protection seen in EDEM-transfected cells. Coimmunoprecipitation studies revealed that ricin can interact with EDEM and with Sec61alpha, and both kifunensin and puromycin increase these interactions. Importantly, vector-based RNA interference against EDEM, which leads to reduction of the cellular level of EDEM, decreased retrotranslocation of ricin A-chain to the cytosol. In conclusion, our results indicate that EDEM is involved in retrotranslocation of ricin from the ER to the cytosol.     

Protein folding includes oligomerization - examples from the endoplasmic reticulum and cytosol

A correct three-dimensional structure is a prerequisite for protein functionality, and therefore for life. Thus, it is not surprising that our cells are packed with proteins that assist protein folding, the process in which the native three-dimensional structure is formed. In general, plasma membrane and secreted proteins, as well as those residing in compartments along the endocytic and exocytic pathways, fold and oligomerize in the endoplasmic reticulum. The proteins residing in the endoplasmic reticulum are specialized in the folding of this subset of proteins, which renders this compartment a protein-folding factory. This review focuses on protein folding in the endoplasmic reticulum, and discusses the challenge of oligomer formation in the endoplasmic reticulum as well as the cytosol.     

Protein transport across the endoplasmic reticulum membrane

A decisive step in the biosynthesis of many eukaryotic proteins is their partial or complete translocation across the endoplasmic reticulum membrane. A similar process occurs in prokaryotes, except that proteins are transported across or are integrated into the plasma membrane. In both cases, translocation occurs through a protein-conducting channel that is formed from a conserved, heterotrimeric membrane protein complex, the Sec61 or SecY complex. Structural and biochemical data suggest mechanisms that enable the channel to function with different partners, to open across the membrane and to release laterally hydrophobic segments of membrane proteins into lipid.     

Regulation of calcium homeostasis and flux between the endoplasmic reticulum and the cytosol

The concentration of Ca²⁺ in the endoplasmic reticulum (ER) is critically important for maintaining its oxidizing environment as well as for maintaining luminal ATP levels required for chaperone activity. Therefore, local luminal Ca²⁺ concentrations and the dynamic Ca²⁺ flux between the different subcellular compartments are tightly controlled. Influx of Ca²⁺ into the ER is enabled by a reductive shift, which opens the sarcoendoplasmic reticulum calcium transport ATPase pump, building the Ca²⁺ gradient across the ER membrane required for ATP import. Meanwhile, Ca²⁺ leakage from the ER has been reported to occur via the Sec61 translocon following protein translocation. In this review, we provide an overview of the complex regulation of Ca²⁺ homeostasis, Ca²⁺ flux between subcellular compartments, and the cellular stress response (the unfolded protein response) induced upon dysregulated luminal Ca²⁺ metabolism. We also provide insight into the structure and gating mechanism at the Sec61 translocon and examine the role of ER-resident cochaperones in assisting the central ER-resident chaperone BiP in the control of luminal Ca²⁺ concentrations.     

A class of membrane proteins shaping the tubular endoplasmic reticulum

How is the characteristic shape of a membrane bound organelle achieved? We have used an in vitro system to address the mechanism by which the tubular network of the endoplasmic reticulum (ER) is generated and maintained. Based on the inhibitory effect of sulfhydryl reagents and antibodies, network formation in vitro requires the integral membrane protein Rtn4a/NogoA, a member of the ubiquitous reticulon family. Both in yeast and mammalian cells, the reticulons are largely restricted to the tubular ER and are excluded from the continuous sheets of the nuclear envelope and peripheral ER. Upon overexpression, the reticulons form tubular membrane structures. The reticulons interact with DP1/Yop1p, a conserved integral membrane protein that also localizes to the tubular ER. These proteins share an unusual hairpin topology in the membrane. The simultaneous absence of the reticulons and Yop1p in S. cerevisiae results in disrupted tubular ER. We propose that these "morphogenic" proteins partition into and stabilize highly curved ER membrane tubules.     

Inefficient targeting to the endoplasmic reticulum by the signal recognition particle elicits selective defects in post-ER membrane trafficking

The signal recognition particle (SRP) is required for protein translocation into the endoplasmic reticulum (ER). With RNA interference we reduced its level about ten-fold in mammalian cells to study its cellular functions. Such low levels proved insufficient for efficient ER-targeting, since the accumulation of several proteins in the secretory pathway was specifically diminished. Although the cells looked unaffected, they displayed noticeable and selective defects in post-ER membrane trafficking. Specifically, the anterograde transport of VSV-G and the retrograde transport of the Shiga toxin B-subunit were stalled at the level of the Golgi whereas the endocytosed transferrin receptor failed to recycle to the plasma membrane. Endocytic membrane trafficking from the plasma membrane to lysosomes or Golgi was undisturbed and major morphological changes in the ER and the Golgi were undetectable at low resolution. Selective membrane trafficking defects were specifically suppressed under conditions when low levels of SRP became sufficient for efficient ER-targeting and are therefore a direct consequence of the lower targeting capacity of cells with reduced SRP levels. Selective post-ER membrane trafficking defects occur at SRP levels sufficient for survival suggesting that changes in SRP levels and their effects on post-ER membrane trafficking might serve as a mechanism to alter temporarily the localization of selected proteins.     

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.     

Phospholipase A2 antagonists inhibit constitutive retrograde membrane traffic to the endoplasmic reticulum

Eukaryotic cells contain a variety of cytoplasmic Ca²⁺-dependent and Ca²⁺-independent phospholipase A₂s (PLA₂s; EC 2.3.1.2.3). However, the physiological roles for many of these ubiquitously-expressed enzymes is unclear or not known. Recently, pharmacological studies have suggested a role for Ca²⁺-independent PLA₂ (iPLA₂) enzymes in governing intracellular membrane trafficking events in general and regulating brefeldin A (BFA)-stimulated membrane tubulation and Golgi-to-endoplasmic reticulum (ER) retrograde membrane trafficking, in particular. Here, we extend these studies to show that membrane-permeant iPLA₂ antagonists potently inhibit the normal, constitutive retrograde membrane trafficking from the trans -Golgi network (TGN), Golgi complex, and the ERGIC-53-positive ER-Golgi-intermediate compartment (ERGIC), which occurs in the absence of BFA. Taken together, these results suggest that iPLA₂ enzymes play a general role in regulating, or directly mediating, multiple mammalian membrane trafficking events.     

Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol

Assays for two distinct phosphatidate phosphohydrolase activities were established based upon a differential inhibition by N-ethylmaleimide (NEM). The activity that is insensitive to this reagent in rat liver is predominantly in the plasma membrane fraction, whereas the NEM-sensitive activity is in the cytosolic and microsomal fractions. The NEM-insensitive activity is further distinguished from the NEM-sensitive phosphohydrolase by: (a) being relatively stable to heat; (b) not being inhibited by phenylglyoxal, butane-2,3-dione, cyclohexane-1,2-dione, 2,4-dinitrofluorobenzene, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and diethyl pyrocarbonate; (c) being inhibited by NaF and phosphatidylcholine; and (d) not being stimulated by Mg2+. The NEM-insensitive activity was specific for phosphatidate. Both phosphohydrolase activities could be inhibited by chlorpromazine, propranolol, sphingosine, and spermine. The NEM-sensitive phosphatidate phosphohydrolase activity was increased by incubating hepatocytes for 12 h with glucagon and dexamethasone, and this effect was antagonized by insulin. The NEM-sensitive phosphohydrolase is concluded to be involved in glycerolipid synthesis. The activity of the NEM-insensitive phosphohydrolase was not altered by preincubation of rat hepatocytes in the short or long term with vasopressin, glucagon, insulin, triiodothyronine, or dexamethasone, but it might be modulated indirectly by sphingosine. The NEM-insensitive enzyme of the plasma membranes could be involved in signal transduction via the agonist-stimulated degradation of phosphatidylcholine through the phospholipase D pathway.     

The endoplasmic reticulum membrane is permeable to small molecules

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of approximately 5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER.     

Transport of cholesterol from the endoplasmic reticulum to the plasma membrane

We have studied the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane in Chinese hamster ovary cells using a cell fractionation assay. We found that transport is dependent on metabolic energy, but that the maintenance of the high differential concentration of cholesterol in the plasma membrane is not an energy-requiring process. We have tested a variety of inhibitors for their effect on cholesterol transport and found that cytochalasin B, colchicine, monensin, cycloheximide, and NH4Cl did not have any effect. The cholesterol transport process shows a sharp temperature dependence; it ceases at 15 degrees C, whereas cholesterol synthesis continues. When synthesis occurs at 15 degrees C, the newly synthesized cholesterol accumulates in the endoplasmic reticulum and in a low density, lipid-rich vesicle fraction. These results suggest that cholesterol is transported via a vesicular system.     

Folding of viral envelope glycoproteins in the endoplasmic reticulum

Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality control system of the endoplasmic reticulum. A special characteristic that distinguishes viral fusion proteins from most cellular proteins is the extensive conformational change they undergo during fusion of the viral and cellular membrane. Many viral proteins fold in conjunction with and dependent on a viral partner protein, sometimes even synthesized from the same mRNA. Relevant for folding is that viral glycoproteins from the same or related virus families may consist of overlapping sets of domain modules. The consequences of these features for viral protein folding are at the heart of this review.     

Protein folding and translocation across the endoplasmic reticulum membrane

Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen.