Cytosolic entry of Shiga-like toxin a chain from the yeast endoplasmic reticulum requires catalytically active Hrd1p.

BACKGROUND: Escherichia coli Shiga-like toxin 1 normally traffics to the endoplasmic reticulum (ER) in sensitive mammalian cells from where the catalytic A chain (SLTxA1) dislocates to the cytosol to inactivate ribosomes. Currently, no molecular details of the dislocation process are available. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae. METHODOLOGY AND PRINCIPAL FINDINGS: Using a combination of growth studies and biochemical tracking in yeast knock-out strains we show that SLTxA1 follows an ER-associated degradation (ERAD) pathway to enter the cytosol in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex. ER-to-cytosol dislocation of the bulk population of SLTxA1 requires Cdc48p and its ubiquitin-handling co-factor Npl4p, and this population of toxin is terminally dispatched by proteasomal degradation. A small sub-population of SLTxA1 uncouples from this classical ERAD pathway and recovers catalytic activity in the cytosol. The pathway that leads to toxicity is also Hrd1p-dependent but, unlike that for the related ricin A chain toxin, SLTxA1 dislocation does require the catalytic cysteine of Hrd1p. However it does not depend on canonical ubiquitylation since toxin variants lacking endogenous lysyl residues also utilize this pathway, and furthermore there is no requirement for a number of Cdc48p co-factors. CONCLUSIONS AND SIGNIFICANCE: The fraction of SLTxA1 that disengages from the ERAD pathway thus does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation. Mechanistically therefore, the dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus.

Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways.

BACKGROUND: The small molecule Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and subsequent degradation of ERAD (ER associated protein degradation) substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx) are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilise ERAD-like mechanisms to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation. METHODOLOGY AND PRINCIPAL FINDINGS: Using cytotoxicity measurements, we found no role for ES(I) as an inhibitor of toxin dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin delivery to the ER. Microscopy of the trafficking of labelled SLTx and its B chain (lacking the toxic A chain) showed a delay in its accumulation at a peri-nuclear location, confirmed to be the Golgi by examination of SLTx B chain metabolically labelled in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking. CONCLUSIONS AND SIGNIFICANCE: ESI acts on one or more components that function during vesicular transport, whilst at least one retrograde trafficking pathway, that of ricin, remains unperturbed.

Folding-competent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum.

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTA(Delta)), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER-Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTA(Delta), although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.

Ricin B chain targeted to the endoplasmic reticulum of tobacco protoplasts is degraded by a CDC48- and vacuole-independent mechanism.

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway.

The role of CDC48 in the retro-translocation of non-ubiquitinated toxin substrates in plant cells.

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterize the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that although RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesized RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated.

Dynamic interplay between the neutral glycosphingolipid CD77/Gb3 and the therapeutic antibody target CD20 within the lipid bilayer of model B lymphoma cells.

The centroblast-specific differentiation marker CD77 (Gb(3)), is the receptor for Shiga-like toxin (SLT). The dynamic relationship between Gb(3)/CD77 and key B-cell membrane proteins was studied in Burkitt's lymphoma cells with a focus on CD20. Engagement of Gb(3)/CD77 with SLT-B reduced the amount of CD20 and CXCR4 available, but levels of BCR, MHC Class II, CD21, CD27 and CD54 remained unchanged. Cholesterol depletion promoted a decrease in the number of sites accessed by CD20, CXCR4 and Gb(3)/CD77 antibodies. Constitutive localisation of Gb(3)/CD77 to lipid rafts was unperturbed by either SLT-B binding or cholesterol depletion, whereas the opposite was true for CD20. The effects were specific to SLT-B, highlighted by the inability of cholera toxin B-subunit to alter CD20 availability. Thus, the binding of Gb(3)/CD77 by its cognate ligand transmits information within the lipid bilayer of model lymphoma cells to impact the behaviour of selective proteins, most notably CD20, via a mechanism influenced by the level of cholesterol within the membrane.

Quality control: linking retrotranslocation and degradation.

Misfolded proteins in the ER require the p97 AAA ATPase for dislocation across the membrane prior to degradation by the cytosolic proteasome. The mechanism by which dislocated proteins are delivered to the proteasome from p97 is unclear, but recent studies suggest an important regulatory role for the protein ataxin-3.

The N-terminal ricin propeptide influences the fate of ricin A-chain in tobacco protoplasts.

The plant toxin ricin is synthesized in castor bean seeds as an endoplasmic reticulum (ER)-targeted precursor. Removal of the signal peptide generates proricin in which the mature A- and B-chains are joined by an intervening propeptide and a 9-residue propeptide persists at the N terminus. The two propeptides are ultimately removed in protein storage vacuoles, where ricin accumulates. Here we have demonstrated that the N-terminal propeptide of proricin acts as a nonspecific spacer to ensure efficient ER import and glycosylation. Indeed, when absent from the N terminus of ricin A-chain, the non-imported material remained tethered to the cytosolic face of the ER membrane, presumably by the signal peptide. This species appeared toxic to ribosomes. The propeptide does not, however, influence catalytic activity per se or the vacuolar targeting of proricin or the rate of retrotranslocation/degradation of A-chain in the cytosol. The likely implications of these findings to the survival of the toxin-producing tissue are discussed.

Retrograde transport pathways utilised by viruses and protein toxins.

A model has been presented for retrograde transport of certain toxins and viruses from the cell surface to the ER that suggests an obligatory interaction with a glycolipid receptor at the cell surface. Here we review studies on the ER trafficking cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A and ricin, and compare the retrograde routes followed by these protein toxins to those of the ER trafficking SV40 and polyoma viruses. We conclude that there is in fact no obligatory requirement for a glycolipid receptor, nor even with a protein receptor in a lipid-rich environment. Emerging data suggests instead that there is no common pathway utilised for retrograde transport by all of these pathogens, the choice of route being determined by the particular receptor utilised.

The association of Shiga-like toxin with detergent-resistant membranes is modulated by glucosylceramide and is an essential requirement in the endoplasmic reticulum for a cytotoxic effect.

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb(3)) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.

Quality control: another player joins the ERAD cast.

The quality control system known as ERAD removes misfolded proteins from the ER to the cytosol for degradation. The AAA ATPase Cdc48p and ubiquitin ligases play crucial roles; their relationship has been unclear, but recent work has shown that the membrane protein Ubx2p links their functions in yeast.

Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells.

Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER-associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL-like C-terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome-inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome-inactivating protein that can enter cells by receptor-mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi-independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin-based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi-mediated retrograde transport to reach their retrotranslocation site.

Endoplasmic reticulum-associated degradation of ricin A chain has unique and plant-specific features.

Proteins that fail to fold in the endoplasmic reticulum (ER) or cannot find a pattern for assembly are often disposed of by a process named ER-associated degradation (ERAD), which involves transport of the substrate protein across the ER membrane (dislocation) followed by rapid proteasome-mediated proteolysis. Different ERAD substrates have been shown to be ubiquitinated during or soon after dislocation, and an active ubiquitination machinery has been found to be required for the dislocation of certain defective proteins. We have previously shown that, when expressed in tobacco (Nicotiana tabacum) protoplasts, the A chain of the heterodimeric toxin ricin is degraded by a pathway that closely resembles ERAD but is characterized by an unusual uncoupling between the dislocation and the degradation steps. Since lysine (Lys) residues are a major target for ubiquitination, we have investigated the effects of changing the Lys content on the retrotranslocation and degradation of ricin A chain in tobacco protoplasts. Here we show that modulating the number of Lys residues does not affect recognition events within the ER lumen nor the transport of the protein from this compartment to the cytosol. Rather, the introduced modifications have a clear impact on the degradation of the dislocated protein. While the substitution of the two Lys residues present in ricin A chain with arginine slowed down degradation, the introduction of four extra lysyl residues had an opposite effect and converted the ricin A chain to a standard ERAD substrate that is disposed via a process in which dislocation and degradation steps are tightly coupled.

Genetics of ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) are a heterogeneous group of enzymes found mainly in plants and a few bacteria that possess N-glycosidase activity on ribosomes and a related polynucleotide adenosine glycosidase activity on naked nucleic acids. They encompass single enzymatic chains, heterodimeric toxic lectins and related agglutinins. Plants commonly produce several RIP isoforms encoded by multi-gene families. The toxic lectins possess adaptations related to their cytotoxic role.

Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation.

In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8(+) T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.

The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol.

Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells by endocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmic reticulum (ER) membrane. To achieve this export, such toxins exploit the ER-associated protein degradation (ERAD) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates. Toxins that translocate from the ER have an unusually low lysine content. Since lysyl residues are potential ubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitination and subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residues within RTA were changed to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitin attachment. However, when four additional lysines were introduced into RTA in a way that did not compromise the activity, structure, or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasome inhibitor clasto-lactacystin beta-lactone increased the cytotoxicity of the lysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysyl residues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased the cytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomal inhibition. Our data support the hypothesis that the evolution of a low lysine content is a degradation-avoidance strategy for toxins that retrotranslocate from the ER.

ER dislocation: Cdc48p/p97 gets into the AAAct.

Misfolded or unassembled proteins present in the lumen of the endoplasmic reticulum are exported to the cytosol and degraded. Recent studies have implicated a complex containing the AAA ATPase Cdc48p/p97 in the export process.

Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer.

Ricin is a heterodimeric protein toxin in which a catalytic polypeptide (the A-chain or RTA) is linked by a disulfide bond to a cell-binding polypeptide (the B-chain or RTB). During cell entry, ricin undergoes retrograde vesicular transport to reach the endoplasmic reticulum (ER) lumen, from where RTA translocates into the cytosol, probably by masquerading as a substrate for the ER-associated protein degradation (ERAD) pathway. In partitioning studies in Triton X-114 solution, RTA is predominantly found in the detergent phase, whereas ricin holotoxin, native RTB, and several single-chain ribosome-inactivating proteins (RIPs) are in the aqueous phase. Fluorescence spectroscopy and far-UV circular dichroism (CD) demonstrated significant structural changes in RTA as a result of its interaction with liposomes containing negatively charged phospholipid (POPG). These lipid-induced structural changes markedly increased the trypsin sensitivity of RTA and, on the basis of the protein fluorescence determinations, abolished its ability to bind to adenine, the product resulting from RTA-catalyzed depurination of 28S ribosomal RNA. RTA also released trapped calcein from POPG vesicles, indicating that it destabilized the lipid bilayer. We speculate that membrane-induced partial unfolding of RTA during cell entry may facilitate its recognition as an ERAD substrate.

Proteolytic cleavage of the A subunit is essential for maximal cytotoxicity of Escherichia coli O157:H7 Shiga-like toxin-1.

Members of the bacterial Shiga toxin family consist of a single A subunit that is non-covalently associated with a pentamer of B subunits. These toxins bind to receptors on susceptible mammalian cells and enter the cells by endocytic uptake. During cell entry, the 32 kDa A subunit is cleaved by the membrane-anchored protease furin to generate a catalytically active, 27.5 kDa A1 fragment and a 4.5 kDa A2 fragment. Previous studies have shown that mutating the furin site to prevent cleavage did not significantly affect toxin potency, suggesting that cleavage is not required for toxin activity. Here it is confirmed that preventing cleavage at the usual processing site does not prevent proteolytic processing of the Escherichia coli Shiga-like toxin-1 A subunit. However, simultaneous mutation of both the primary furin-recognition site and a nearby putative furin cleavage site did prevent intracellular processing of the A subunit. Comparison of the cytotoxicities of purified recombinant toxins to cultured mammalian cells demonstrated that even on prolonged incubation with toxin, the unprocessed mutant was 60-fold less toxic than the wild-type protein or other mutants still capable of being proteolytically processed during cell entry.