Targeting of Shiga Toxin B-Subunit to Retrograde Transport Route in Association with Detergent-resistant Membranes

2001 ◽  
Vol 12 (8) ◽  
pp. 2453-2468 ◽  
Author(s):  
Thomas Falguières ◽  
Frédéric Mallard ◽  
Carole Baron ◽  
Daniel Hanau ◽  
Clifford Lingwood ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Shiga Toxin ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Toxin B ◽  
B Subunit ◽  
Early Endosomes ◽  
Golgi Network ◽  
Triton X

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to thetrans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more

2002 ◽  
Vol 283 (1) ◽  
pp. G1-G7 ◽  
Author(s):  
Ludger Johannes
Keyword(s):  
Endoplasmic Reticulum ◽  
Shiga Toxin ◽  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
Physiological Function ◽  
Retrograde Transport ◽  
Toxin B ◽  
B Subunit ◽  
New Methods ◽  
Therapeutic Compounds

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

scholarly journals Retrograde Transport from Early Endosomes to thetrans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions

2016 ◽  
Vol 90 (19) ◽  
pp. 8891-8905 ◽  
Author(s):  
Gilad Sivan ◽  
Andrea S. Weisberg ◽  
Jeffrey L. Americo ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Actin Polymerization ◽  
Secretory Pathway ◽  
Retrograde Transport ◽  
Double Membrane ◽  
Retrograde Trafficking ◽  
Early Endosomes ◽  
Trafficking Inhibitor ◽  
Golgi Network ◽  
Cell Spread

ABSTRACTThe anterograde pathway, from the endoplasmic reticulum through thetrans-Golgi network to the cell surface, is utilized bytrans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in thetrans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to thetrans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane.IMPORTANCEEfficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization. Interference with wrapping or subsequent steps results in severe attenuation of the virus. Some previous studies had suggested that the wrapping membrane arises from thetrans-Golgi network, whereas others suggested an origin from early endosomes. Some nonenveloped viruses use retrograde trafficking for entry into the cell. In contrast, we provided evidence that retrograde transport from early endosomes to thetrans-Golgi network is required for the membrane-wrapping step in morphogenesis of vaccinia virus and egress from the cell. The potentin vitroinhibition of this step by the drug Retro-2 suggests that derivatives with enhanced pharmacological properties might serve as useful antipoxviral agents.

scholarly journals Blood group P1 antigen–bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors

2020 ◽  
Vol 295 (28) ◽  
pp. 9490-9501 ◽  
Author(s):  
Kanta Morimoto ◽  
Noriko Suzuki ◽  
Isei Tanida ◽  
Soichiro Kakuta ◽  
Yoko Furuta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Blood Group ◽  
Shiga Toxin ◽  
Retrograde Transport ◽  
Host Cells ◽  
Mammalian Host ◽  
B Subunit ◽  
Early Endosomes ◽  
Hydrophobic Portion

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

scholarly journals TSSC1 is novel component of the endosomal retrieval machinery

2016 ◽  
Vol 27 (18) ◽  
pp. 2867-2878 ◽  
Author(s):  
David C. Gershlick ◽  
Christina Schindler ◽  
Yu Chen ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Affinity Purification ◽  
Critical Role ◽  
Gel Filtration ◽  
Retrograde Transport ◽  
Recycling Endosomes ◽  
B Subunit ◽  
Multiple Protein ◽  
Early Endosomes ◽  
Golgi Network

Endosomes function as a hub for multiple protein-sorting events, including retrograde transport to the trans-Golgi network (TGN) and recycling to the plasma membrane. These processes are mediated by tubular-vesicular carriers that bud from early endosomes and fuse with a corresponding acceptor compartment. Two tethering complexes named GARP (composed of ANG2, VPS52, VPS53, and VPS54 subunits) and EARP (composed of ANG2, VPS52, VPS53, and Syndetin subunits) were previously shown to participate in SNARE-dependent fusion of endosome-derived carriers with the TGN and recycling endosomes, respectively. Little is known, however, about other proteins that function with GARP and EARP in these processes. Here we identify a protein named TSSC1 as a specific interactor of both GARP and EARP and as a novel component of the endosomal retrieval machinery. TSSC1 is a predicted WD40/β-propeller protein that coisolates with both GARP and EARP in affinity purification, immunoprecipitation, and gel filtration analyses. Confocal fluorescence microscopy shows colocalization of TSSC1 with both GARP and EARP. Silencing of TSSC1 impairs transport of internalized Shiga toxin B subunit to the TGN, as well as recycling of internalized transferrin to the plasma membrane. Fluorescence recovery after photobleaching shows that TSSC1 is required for efficient recruitment of GARP to the TGN. These studies thus demonstrate that TSSC1 plays a critical role in endosomal retrieval pathways as a regulator of both GARP and EARP function.

scholarly journals Intracellular Neutralization of Shiga Toxin 2 by an A Subunit-Specific Human Monoclonal Antibody

2008 ◽  
Vol 76 (5) ◽  
pp. 1931-1939 ◽  
Author(s):  
Greice Krautz-Peterson ◽  
Susan Chapman-Bonofiglio ◽  
Karen Boisvert ◽  
Hanping Feng ◽  
Ira M. Herman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Shiga Toxin ◽  
Human Monoclonal Antibody ◽  
Retrograde Transport ◽  
Systemic Complications ◽  
B Subunit ◽  
Early Endosomes ◽  
Shiga Toxin 2 ◽  
A Subunit

ABSTRACT Infection of children with Shiga toxin (Stx)-producing Escherichia coli (STEC) is the leading cause of hemolytic-uremic syndrome (HUS). Stx2, one of two toxins liberated by the bacteria, is directly linked with HUS. We have previously shown that Stx2-specific human monoclonal antibodies (HuMAbs) protect mice and piglets from fatal systemic complications of Stx2. The present study investigates the mechanisms by which our most efficacious A- and B-subunit-specific HuMAbs neutralize the cytotoxic effects of Stx2 in vitro. Whereas the B-subunit-specific HuMAb 5H8 blocked binding of Stx2 to its receptor on the cell surface, the A-subunit-specific HuMAb 5C12 did not interfere with the toxin-receptor binding. Further investigations revealed that 5C12 did not block endocytosis of Stx2 by HeLa cells as both Stx2 and 5C12 colocalized with early endosomes. However, 5C12 blocked the retrograde transport of the toxin into the Golgi and the endoplasmic reticulum, preventing the toxin from entering the cytosol where the toxin exerts its cytotoxic effect. The endocytosed 5C12/Stx2 complexes appear to be rapidly transported to the plasma membrane and/or to the slow recycling perinuclear compartments, followed by their slow recycling to the plasma membrane, and release into the extracellular environment.

scholarly journals Rab6 Coordinates a Novel Golgi to ER Retrograde Transport Pathway in Live Cells

1999 ◽  
Vol 147 (4) ◽  
pp. 743-760 ◽  
Author(s):  
Jamie White ◽  
Ludger Johannes ◽  
Frédéric Mallard ◽  
Andreas Girod ◽  
Stephan Grill ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Live Cells ◽  
Cell Periphery ◽  
Wild Type ◽  
Transport Pathway ◽  
Toxin B ◽  
Kdel Receptor

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.

Shiga Toxin B-Subunit as a Tool to Study Retrograde Transport

E. coli ◽  
2003 ◽  
pp. 209-220 ◽  
Author(s):  
Frédéric Mallard ◽  
Ludger Johannes
Keyword(s):  
Shiga Toxin ◽  
Retrograde Transport ◽  
Toxin B ◽  
B Subunit

scholarly journals Passage through the Golgi is necessary for Shiga toxin B subunit to reach the endoplasmic reticulum

FEBS Journal ◽  
2009 ◽  
Vol 276 (6) ◽  
pp. 1581-1595 ◽  
Author(s):  
Jenna McKenzie ◽  
Ludger Johannes ◽  
Tomohiko Taguchi ◽  
David Sheff
Keyword(s):  
Endoplasmic Reticulum ◽  
Shiga Toxin ◽  
Toxin B ◽  
B Subunit

scholarly journals The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans-Golgi Network

2007 ◽  
Vol 18 (12) ◽  
pp. 4979-4991 ◽  
Author(s):  
Zi Zhao Lieu ◽  
Merran C. Derby ◽  
Rohan D. Teasdale ◽  
Charles Hart ◽  
Priscilla Gunn ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Transport Pathways ◽  
Recycling Endosomes ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Cargo Proteins ◽  
Mannose 6 Phosphate ◽  
Golgi Network

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane–TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.

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