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Schematic representation of Shiga toxin B-fragment constructs. Panel A, the recombinant protein B-Glyc-KDEL is composed of amino-terminally located Shiga toxin B-fragment, an additional Tyr residue for efficient iodination, an N-glycosylation site, and the carboxyl-terminally located KDEL peptide. The sequence of B-Glyc- KDELGL differs from that of B-Glyc-KDEL by the addition of Gly and Leu at its carboxyl terminus. Panel B, B-Glyc-Sulf-KDEL carries a sulfation site in addition to the other elements.
Source publication
To investigate retrograde transport along the biosynthetic/secretory pathway, we have constructed a recombinant Shiga toxin B-fragment carrying an N-glycosylation site and a KDEL retrieval motif at its carboxyl terminus (B-Glyc-KDEL). After incubation with HeLa cells, B-Glyc-KDEL was progressively glycosylated in the endoplasmic reticulum (ER) and...
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Retrograde transport (RT) allows cells the retrieval of receptors and other cellular cargoes to the Golgi contributing to the maintenance of cellular homeostasis. This transport route is also commonly used by several bacterial toxins to exert their deleterious actions on eukaryotic cells. While the retrograde transport process has been well charact...
The pH within individual organelles of the secretory pathway is believed to be an important determinant of their biosynthetic activity. However, little is known about the determinants and regulation of the pH in the secretory organelles, which cannot be readily accessed by [H+]-sensitive probes. We devised a procedure for the dynamic, noninvasive m...
Citations
... Sulfation is not the latest developed approach to assay cell surface/endosome-to-TGN traffic. Already decades ago, several groups have independently designed tools based on sulfation site sequences that can be either expressed as tag part of a recombinant cargo or chemically coupled to protein (Sandvig and van Deurs, 1994;Johannes et al., 1997;Rapak et al., 1997;Mallard et al., 1998;Amessou et al., 2006). Particularly, recombinant fragments of ricin and Shiga toxin subunits were modified with tyrosine sulfation (TS) consensus sequences (Johannes et al., 1997;Rapak et al., 1997;Mallard and Johannes, 2003;Utskarpen et al., 2006;Shiba et al., 2010). ...
... Already decades ago, several groups have independently designed tools based on sulfation site sequences that can be either expressed as tag part of a recombinant cargo or chemically coupled to protein (Sandvig and van Deurs, 1994;Johannes et al., 1997;Rapak et al., 1997;Mallard et al., 1998;Amessou et al., 2006). Particularly, recombinant fragments of ricin and Shiga toxin subunits were modified with tyrosine sulfation (TS) consensus sequences (Johannes et al., 1997;Rapak et al., 1997;Mallard and Johannes, 2003;Utskarpen et al., 2006;Shiba et al., 2010). Using these recombinant toxin subunits, it could be shown that these proteins were sulfated, revealing their passage through the TGN en route to the ER. ...
Retrograde transport from endosomes to the trans-Golgi network is essential for recycling of protein and lipid cargoes to counterbalance anterograde membrane traffic. Protein cargo subjected to retrograde traffic include lysosomal acid-hydrolase receptors, SNARE proteins, processing enzymes, nutrient transporters, a variety of other transmembrane proteins, and some extracellular non-host proteins such as viral, plant, and bacterial toxins. Efficient delivery of these protein cargo molecules depends on sorting machineries selectively recognizing and concentrating them for their directed retrograde transport from endosomal compartments. In this review, we outline the different retrograde transport pathways governed by various sorting machineries involved in endosome-to-TGN transport. In addition, we discuss how this transport route can be analyzed experimentally.
... Some toxins contain an ER retrieval sequence (ER retrieval sequence KDEL: amino acids Lys-Asp-Glu-Leu), like Pseudomonas exotoxin or cholera toxin [27][28][29][30]. Other toxins, like Shiga toxin or ricin, do not have a KDEL signal [30][31][32][33]. Transport to the ER can depend on the KDEL receptor or it can be independent from it. ...
Numerous toxins translocate to the cytosol in order to fulfil their function. This demonstrates the existence of routes for proteins from the extracellular space to the cytosol. Understanding these routes is relevant to multiple aspects related to therapeutic applications. These include the development of anti-toxin treatments, the potential use of toxins as shuttles for delivering macromo-lecular cargo to the cytosol or the use of drugs based on toxins. Compared with other strategies for delivery, such as chemicals as carriers for macromolecular delivery or physical methods like electroporation, toxin routes present paths into the cell that potentially cause less damage and can be specifically targeted. The efficiency of delivery via toxin routes is limited. However, low-delivery efficiencies can be entirely sufficient, if delivered cargoes possess an amplification effect or if very few molecules are sufficient for inducing the desired effects. This is known for example from RNA-based vaccines that have been developed during the coronavirus disease 2019 pandemic as well as for other approved RNA-based drugs, which elicited the desired effect despite their typically low delivery efficiencies. The different mechanisms by which toxins enter cells may have implications for their technological utility. We review the mechanistic principles of the translocation pathway of toxins from the extracellular space to the cytosol, the delivery efficiencies, and therapeutic strategies or applications that exploit toxin routes for intracellular delivery.
... This seven transmembrane domain containing protein localizes mainly to the cis-cisternae of the Golgi and is predicted to behave like a G-protein coupled receptor (GPCR) [38]. KDELR recognizes the KDEL (Lys-Asp-Glu-Leu) motif found on ER resident proteins and retrieves them back to the ER in a pH-dependent manner [39,40]. It was recently proposed that three amino acids of the signal binding pocket of KDELR (Y158, E127 and H12) could play an essential role in KDELR activation and cargo locking. ...
Lysosomes are key regulators of many fundamental cellular processes such as metabolism, autophagy, immune response, cell signalling and plasma membrane repair. These highly dynamic organelles are composed of various membrane and soluble proteins, which are essential for their proper functioning. The soluble proteins include numerous proteases, glycosidases and other hydrolases, along with activators, required for catabolism. The correct sorting of soluble lysosomal proteins is crucial to ensure the proper functioning of lysosomes, and is achieved through the coordinated effort of many sorting receptors, resident ER and Golgi proteins, and several cytosolic components. Mutations in a number of proteins involved in sorting soluble proteins to lysosomes result in human disease. These can range from rare diseases such as lysosome storage disorders, to more prevalent ones, such as Alzheimer’s disease, Parkinson’s disease and others, including rare neurodegenerative diseases that affect children. In this review, we discuss the mechanisms that regulate the sorting of soluble proteins to lysosomes, and highlight the effects of mutations in this pathway that cause human disease. More precisely, we will review the route taken by soluble lysosomal proteins from their translation into the ER, their maturation along the Golgi apparatus, and sorting at the trans-Golgi network. We will also highlight the effects of mutations in this pathway that cause human disease.
... STx, CTx and other AB 5 -toxins have since provided robust tools to study the mechanisms and components responsible for endosomal sorting and membrane trafficking in the retrograde pathway (summarized before in [11,77,152]). Toxin trafficking was measured by immune or direct labeling of the toxins with fluorophores or nano-gold particles or biochemically by tagging the toxins with N-glycosylation motifs that became glycosylated when the toxins entered the ER lumen [33,156]. These studies were highly informative, but none were able to measure retrograde trafficking in real time or quantitatively. ...
Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.
... Thus, searching for compounds that protect against protein toxins not only can be of benefit in connection with disease or intoxication by these toxins but can help us to clarify transport pathways in the cells. The construction of genetically modified toxin molecules with sulfation sites, which are modified in the trans-Golgi, and glycosylation sites that can be modified in the ER [13,14] has been of great help in revealing which step is affected. Thus, toxins have contributed to our knowledge about transport from the cell surface to endosomes, to the Golgi apparatus, and to the ER and nuclear envelope. ...
... Thus, one can study the effects of different molecules or drugs on cell intoxication to learn about interference with this transport step. Importantly, chemical modifications of genetically modified ricin or Shiga B-chain with sulfate in the trans Golgi or by glycosylation of ricin in the ER [13,14] have facilitated our studies of this pathway. Moreover, electron and fluorescence microscopy have contributed to a large amount of the knowledge we have today about this transport. ...
Protein toxins secreted by bacteria and found in plants can be threats to human health. However, their extreme toxicity can also be exploited in different ways, e.g., to produce hybrid toxins directed against cancer cells and to study transport mechanisms in cells. Investigations during the last decades have shown how powerful these molecules are as tools in cell biological research. Here, we first present a partly historical overview, with emphasis on Shiga toxin and ricin, of how such toxins have been used to characterize processes and proteins of importance for their trafficking. In the second half of the article, we describe how one can now use toxins to investigate the role of lipid classes for intracellular transport. In recent years, it has become possible to quantify hundreds of lipid species using mass spectrometry analysis. Thus, it is also now possible to explore the importance of lipid species in intracellular transport. The detailed analyses of changes in lipids seen under conditions of inhibited toxin transport reveal previously unknown connections between syntheses of lipid classes and demonstrate the ability of cells to compensate under given conditions.
... In an attempt to gain insight into the mechanism of PC transport, we next examined whether interorganelle lipid transport was affected by vesicle trafficking using nocodazole (Noc), which interferes with retrograde vesicle transport between the ER and Golgi through microtubule disruption and thereby increases Golgi-lysosome contacts 23,24 , or brefeldin A (BFA), which inhibits Golgi-mediated anterograde membrane traffic 25 . After labeling, cells were treated with Noc or BFA for 4 h and subsequently analyzed by CLSM (Extended Data Fig. 8a). ...
Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.
... The B-subunit is a receptorbinding protein. Binding to either the globotriose (Gb3)-lipopolysaccharide (LPS) or the globotetraose (Gb4)-LPS, glycolipids on the cell membrane mediate holotoxin entry into host cells through endocytosis (both clatherin dependent and independent) and ultimate retro-transport from the ER to the cytosol [16][17][18]. While all Stxs conform to the basic AB5 structure and act by inhibiting translation, STEC pathogenesis is highly dependent on the type(s) of Stx expressed by the organism [19,20]. ...
Shiga toxin (Stx) is the major virulence factor of Shiga toxin-producing Escherichia coli (STEC). Stx evolves rapidly and, as such, new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies. A new subtype, Stx2k, was recently identified in E. coli isolated from a wide range of sources including diarrheal patients, animals, and raw meats, and was poorly detected by existing immunoassays. In this study, the structure of Stx2kE167Q was determined at 2.29 Å resolution and the conservation of structure with Stx2a was revealed. A novel polyclonal antibody capable of neutralizing Stx2k and an immunoassay, with a 10-fold increase in sensitivity compared to assays using extant antibodies, were developed. Stx2k is less toxic than Stx2a in Vero cell assays but is similar to Stx2a in receptor-binding preference, thermostability, and acid tolerance. Although Stx2k does not appear to be as potent as Stx2a to Vero cells, the wide distribution and blended virulence profiles of the Stx2k-producing strains suggest that horizontal gene transfer through Stx2k-converting phages could result in the emergence of new and highly virulent pathogens. This study provides useful information and tools for early detection and control of Stx2k-producing E. coli, which could reduce public risk of infection by less-known STECs.
... The retrograde pathways, however, have been difficult to measure with precision, sensitivity, and in real time. Biochemical assays for toxin transport into the Golgi and ER using protein sulfation and N-glycosylation to mark entry into TGN and ER respectively have proved highly informative, as has the use of ultrastructural electron and confocal light microscopy (Guimaraes et al., 2011;Johannes et al., 1997;Saslowsky et al., 2010;Torgersen et al., 2001). ...
... Retrograde transport into the Golgi was also blocked by BFA (Fig. 2c), as observed before using other measures of retrograde transport (Fujinaga et al., 2003). Also as expected (Johannes et al., 1997), we found that treatment with nocodazole to depolymerize microtubules resulted in inhibition of retrograde ER trafficking in both cell lines. ...
Retrograde membrane trafficking from plasma membrane (PM) to Golgi and endoplasmic reticulum (ER) typifies one of the key sorting steps emerging from the early endosome that affects cell surface and intracellular protein dynamics underlying cell function. While some cell surface proteins and lipids are known to sort retrograde, there are few effective methods to quantitatively measure the extent or kinetics of these events. Here we took advantage of the well-known retrograde trafficking of cholera toxin and newly defined split fluorescent protein technology to develop a quantitative, sensitive, and effectively real-time single-cell flow cytometry assay for retrograde membrane transport. The approach can be applied in high throughput to elucidate the underlying biology of membrane traffic, and how endosomes adapt to the physiologic needs of different cell types and cell states.
... Similarly, ricin and Shiga toxin subunits had previously been modified with sulfation sites to demonstrate a transport route through the TGN (e.g., refs. [56][57][58]. TS peptides had also been chemically coupled to antibodies to assay retrograde transport of GFP-CIMPR and endogenous TGN46 to the TGN (19). The VHH-2xTS nanobodies have the advantage of easy and reproducible production and of a 1:1 stoichiometry with the target protein. ...
Significance
Retrograde transport from the cell surface to intracellular compartments is essential for homeostasis and cell physiology. We developed derivatized nanobodies to follow proteins from the cell surface to endosomes and the trans -Golgi network. Nanobodies are an emerging class of protein binders with many advantages over conventional antibodies: they are small and noncross-linking, and can be produced in bacteria. Using a nanobody against GFP, our tool is widely applicable to any GFP-tagged protein of interest. It allows the quantitative analysis of endocytic and retrograde transport biochemically, by fixed- and live-cell imaging and by electron microscopy. As proof-of-principle, we applied them to determine the contribution of adaptor protein-1/clathrin in retrograde transport of the mannose-6-phosphate receptors to the trans -Golgi.
... In general, two different endosomal escape mechanisms have been observed for A-B toxins. Long-trip A-B (5) -toxins, such as Shiga toxin, pertussis toxin or cholera toxin, are routed through the trans-Golgi network and are released from the endoplasmic reticulum into the cytosol (Sandvig and van Deurs, 1996;Johannes et al., 1997;Majoul et al., 1998;Dixit et al., 2008;Capitani and Sallese, 2009;Sandvig et al., 2010;Wernick et al., 2010). Short-trip toxins, such as clostridial toxins, anthrax or diphtheria toxin translocate already across endosomal membranes (Barth et al., 2000;Puhar et al., 2004;Liu et al., 2014). ...
Effector proteins secreted by the type 3 secretion system (T3SS) of pathogenic bacteria have been shown to precisely modulate important signaling cascades of the host for the benefit of the pathogens. Among others, the non-LEE encoded T3SS effector protein NleC of enteropathogenic Escherichia coli (EPEC) is a Zn-dependent metalloprotease and suppresses innate immune responses by directly targeting the NF-κB signaling pathway. Many pathogenic bacteria release potent bacterial toxins of the A-B type, which—in contrast to the direct cytoplasmic injection of T3SS effector proteins—are released first into the environment. In this study, we found that NleC displays characteristics of bacterial A-B toxins, when applied to eukaryotic cells as a recombinant protein. Although lacking a B subunit, that typically mediates the uptake of toxins, recombinant NleC (rNleC) induces endocytosis via lipid rafts and follows the endosomal-lysosomal pathway. The conformation of rNleC is altered by low pH to facilitate its escape from acidified endosomes. This is reminiscent of the homologous A-B toxin AIP56 of the fish pathogen Photobacterium damselae piscicida (Phdp). The recombinant protease NleC is functional inside eukaryotic cells and cleaves p65 of the NF-κB pathway. Here, we describe the endocytic uptake mechanism of rNleC, characterize its intracellular trafficking and demonstrate that its specific activity of cleaving p65 requires activation of host cells e.g., by IL1β. Further, we propose an evolutionary link between some T3SS effector proteins and bacterial toxins from apparently unrelated bacteria. In summary, these properties might suggest rNleC as an interesting candidate for future applications as a potential therapeutic against immune disorders.
