The intracellular voyage of cholera toxin: going retro

Cholera toxin (CT) and related AB₅-subunit toxins move from the plasma membrane through the trans-Golgi and endoplasmic reticulum (ER) to the cytosol of host cells. The toxins exploit a specific glycolipid pathway rather than a protein pathway. They bind glycolipids that associate with lipid rafts at the cell surface, which carry the toxins retrograde to the Golgi and ER. In the ER, the A1-chain of the CT unfolds and enters the cytosol by hijacking the cellular machinery that enables misfolded proteins to cross the membrane for degradation by the proteasome, a process termed retro-translocation. Upon entering the cytosol, the A1-chain rapidly refolds, avoids the proteasome and induces toxicity.     

Retrotranslocation of a viral A/B toxin from the yeast endoplasmic reticulum is independent of ubiquitination and ERAD

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca(2+) homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where beta is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.     

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.     

Role of HERP and a HERP-related Protein in HRD1-dependent Protein Degradation at the Endoplasmic Reticulum

Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the proteasome via a process termed ER-associated degradation (ERAD). The precise mechanism of retrotranslocation is unclear. Here, we use several lumenal ERAD substrates targeted for degradation by the ubiquitin ligase HRD1 including SHH (sonic hedgehog) and NHK (null Hong Kong α1-antitrypsin) to study the geometry, organization, and regulation of the HRD1-containing ERAD machinery. We report a new HRD1-associated membrane protein named HERP2, which is homologous to the previously identified HRD1 partner HERP1. Despite sequence homology, HERP2 is constitutively expressed in cells, whereas HERP1 is highly induced by ER stress. We find that these proteins are required for efficient degradation of both glycosylated and nonglycosylated SHH proteins as well as NHK. In cells depleted of HERPs, SHH proteins are largely trapped inside the ER with a fraction of the stabilized SHH protein bound to the HRD1-SEL1L ligase complex. Ubiquitination of SHH is significantly attenuated in the absence of HERPs, suggesting a defect in retrotranslocation. Both HERP proteins interact with HRD1 through a region located in the cytosol. However, unlike its homolog in Saccharomyces cerevisiae, HERPs do not regulate HRD1 stability or oligomerization status. Instead, they help recruit DERL2 to the HRD1-SEL1L complex. Additionally, the UBL domain of HERP1 also seems to have a function independent of DERL2 recruitment in ERAD. Our studies have revealed a critical scaffolding function for mammalian HERP proteins that is required for forming an active retrotranslocation complex containing HRD1, SEL1L, and DERL2.     

Ufd1-Npl4 is a negative regulator of cholera toxin retrotranslocation

The A1 chain of the cholera toxin (CT) undergoes retrotranslocation to the cytosol across the endoplasmic reticulum (ER) membrane by hijacking ER-associated degradation (ERAD). In the cytosol the CT A1 chain stimulates adenylyl cyclase. The VCP(Ufd1-Npl4) complex mediates retrotranslocation of emerging ER proteins. While one group reported that VCP is required for CT retrotranslocation, another group concluded the opposite. We show that VCP is dispensable for CT retrotranslocation, however RNAi of either Ufd1 or Npl4 induces an increase in adenylyl cyclase activity induced by CT. RNAi of VCP, Ufd1 or Npl4 did not affect adenylyl cyclase activity induced by forskolin. These findings are coherent with our previous report showing that depletion of Ufd1-Npl4 accelerates ERAD of reporter substrates. To integrate contradictory results we propose a new model, where Ufd1-Npl4 is a negative regulator of retrotranslocation, delaying the retrotranslocation of ERAD substrates independently of its association with VCP.     

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.     

Inhibition of p97-dependent protein degradation by Eeyarestatin I

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-associated degradation involves substrate retrotranslocation from the ER lumen into the cytosol for degradation by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extracted by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chemical inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can associate with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to negatively influence a deubiquitinating process that is mediated by p97-associated deubiquitinating enzymes. We further show that ataxin-3, a p97-associated deubiquitinating enzyme previously implicated in ER-associated degradation, is among those affected. Interestingly, p97-associated deubiquitination is also involved in degradation of a soluble substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.     

A role for the protease-sensitive loop region of Shiga-like toxin 1 in the retrotranslocation of its A1 domain from the endoplasmic reticulum lumen

Shiga-like toxin I (Slt-I) is a ribosome-inactivating protein that undergoes retrograde transport to the endoplasmic reticulum to exert its cytotoxic effect on eukaryotic cells. Its catalytically active A(1) domain subsequently migrates from the endoplasmic reticulum (ER) lumen to the cytoplasm. To study this final retrotranslocation event, a suicide assay was developed based on the cytoplasmic expression and ER-targeting of the cytotoxic Slt-I A(1) fragment in Saccharomyces cerevisiae. Expression of the Slt-I A(1) domain (residues 1-251) with and without an ER-targeting sequence was lethal to the host and demonstrated that this domain can efficiently migrate from the ER compartment to the cytosol. Deletion analyses revealed that residues 1-239 represent the minimal A(1) segment displaying full enzymatic activity. This fragment, however, accumulates in the ER lumen when directed to this compartment. The addition of residues 240-251 restores the translocation property of the A(1) chain in yeast. However, single mutations within this region do not significantly alter this function in the context of the 251-residue long A(1) domain or affect the toxicity of the resulting Slt-I variants toward Vero cells in the context of the holotoxin. Since this mechanism of retrotranslocation is common to other protein toxins lacking a peptide motif similar in sequence to residues 240-251, the present results suggest that the ER export mechanism may involve the recognition of a more universal structural element, such as a misfolded or altered peptide domain localized at the C terminus of the A(1) chain (residues 240-251) rather than a unique ER export signal sequence.     

The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover

Recent studies have revealed a role for the ubiquitin/proteasome system in the regulation and turnover of outer mitochondrial membrane (OMM)-associated proteins. Although several molecular components required for this process have been identified, the mechanism of proteasome-dependent degradation of OMM-associated proteins is currently unclear. We show that an AAA-ATPase, p97, is required for the proteasomal degradation of Mcl1 and Mfn1, two unrelated OMM proteins with short half-lives. A number of biochemical assays, as well as imaging of changes in localization of photoactivable GFP-fused Mcl1, revealed that p97 regulates the retrotranslocation of Mcl1 from mitochondria to the cytosol, prior to, or concurrent with, proteasomal degradation. Mcl1 retrotranslocation from the OMM depends on the activity of the ATPase domain of p97. Furthermore, p97-mediated retrotranslocation of Mcl1 can be recapitulated in vitro, confirming a direct mitochondrial role for p97. Our results establish p97 as a novel and essential component of the OMM-associated protein degradation pathway.     

Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events.

Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, mu and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of mu and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all mu chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of mu chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates.     

Role of p97 AAA-ATPase in the retrotranslocation of the cholera toxin A1 chain, a non-ubiquitinated substrate

The enzymatic A1 chain of cholera toxin retrotranslocates across the endoplasmic reticulum membrane into the cytosol, where it induces toxicity. Almost all other retrotranslocation substrates are modified by the attachment of polyubiquitin chains and moved into the cytosol by the ubiquitin-interacting p97 ATPase complex. The cholera toxin A1 chain, however, can induce toxicity in the absence of ubiquitination, and the motive force that drives retrotranslocation is not known. Here, we use adenovirus expressing dominant-negative mutants of p97 to test whether p97 is required for toxin action. We find that cholera toxin still functions with only a small decrease in potency in cells that cannot retrotranslocate other substrates at all. These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function.     

The p97 ATPase Dislocates MHC Class I Heavy Chain in US2-expressing Cells via a Ufd1-Npl4-independent Mechanism

The human cytomegalovirus (HCMV) protein US2 hijacks the endoplasmic reticulum (ER)-associated degradation machinery to dispose of MHC class I heavy chain (HC) at the ER. This process requires retrotranslocation of newly synthesized HC molecules from the ER membrane into the cytosol, but the mechanism underlying the dislocation reaction has been elusive. Here we establish an in vitro permeabilized cell assay that recapitulates the retrotranslocation of MHC HC in US2-expressing cells. Using this assay, we demonstrate that the dislocation process requires ATP and ubiquitin, as expected. The retrotranslocation also involves the p97 ATPase. However, the mechanism by which p97 dislocates MHC class I HC in US2 cells is distinct from that in US11 cells: the dislocation reaction in US2 cells is independent of the p97 cofactor Ufd1-Npl4. Our results suggest that different retrotranslocation mechanisms can employ distinct p97 ATPase complexes to dislocate substrates.     

The pertussis toxin S1 subunit is a thermally unstable protein susceptible to degradation by the 20S proteasome

Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before the A chain passes into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain.     

The viral E3 ubiquitin ligase mK3 uses the Derlin/p97 endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class I proteins

Ubiquitin E3 ligases are important cellular components for endoplasmic reticulum (ER)-associated degradation due to their role in substrate-specific ubiquitination, which is required for retrotranslocation (dislocation) of most unwanted proteins from the ER to the cytosol for proteasome degradation. However, our understanding of the molecular mechanisms of how E3 ligases confer substrate-specific recognition, and their role in substrate retrotranslocation is limited especially in mammalian cells. mK3 is a type III ER membrane protein encoded by murine gamma herpesvirus 68. As conferred by its N-terminal RING-CH domain, mK3 has E3 ubiquitin ligase activity. In its role as an immune evasion protein, mK3 specifically targets nascent major histocompatibility complex class I heavy chains (HC) for rapid degradation. The mechanism by which mK3 extracts HC from the ER membrane into the cytosol for proteasome-mediated degradation is unknown. Evidence is presented here that HC down-regulation by mK3 is dependent on the p97 AAA-ATPase. By contrast, the kK5 protein of Kaposi's sarcoma-associated herpesvirus is p97-independent despite the fact that it is highly homologous to mK3. mK3 protein was also found in physical association with Derlin1, an ER protein recently implicated in the retrotranslocation of HC by immune evasion protein US11, but not US2, of human cytomegalovirus. The mechanistic implications of these findings are discussed.     

The nucleotide exchange factors Grp170 and Sil1 induce cholera toxin release from BiP to enable retrotranslocation

Cholera toxin (CT) intoxicates cells by trafficking from the cell surface to the endoplasmic reticulum (ER), where the catalytic CTA1 subunit hijacks components of the ER-associated degradation (ERAD) machinery to retrotranslocate to the cytosol and induce toxicity. In the ER, CT targets to the ERAD machinery composed of the E3 ubiquitin ligase Hrd1-Sel1L complex, in part via the activity of the Sel1L-binding partner ERdj5. This J protein stimulates BiP''s ATPase activity, allowing BiP to capture the toxin. Presumably, toxin release from BiP must occur before retrotranslocation. Here, using loss-and gain-of-function approaches coupled with binding studies, we demonstrate that the ER-resident nucleotide exchange factors (NEFs) Grp170 and Sil1 induce CT release from BiP in order to promote toxin retrotranslocation. In addition, we find that after NEF-dependent release from BiP, the toxin is transferred to protein disulfide isomerase; this ER redox chaperone is known to unfold CTA1, which allows the toxin to cross the Hrd1-Sel1L complex. Our data thus identify two NEFs that trigger toxin release from BiP to enable successful retrotranslocation and clarify the fate of the toxin after it disengages from BiP.     

Selective targeting of proteins within secretory pathway for endoplasmic reticulum-associated degradation

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity.     

The Grp170 nucleotide exchange factor executes a key role during ERAD of cellular misfolded clients

When a protein misfolds in the endoplasmic reticulum (ER), it retrotranslocates to the cytosol and is degraded by the proteasome via a pathway called ER-associated degradation (ERAD). To initiate ERAD, ADP-BiP is often recruited to the misfolded client, rendering it soluble and translocation competent. How the misfolded client is subsequently released from BiP so that it undergoes retrotranslocation, however, remains enigmatic. Here we demonstrate that the ER-resident nucleotide exchange factor (NEF) Grp170 plays an important role during ERAD of the misfolded glycosylated client null Hong Kong (NHK). As a NEF, Grp170 triggers nucleotide exchange of BiP to generate ATP-BiP. ATP-BiP disengages from NHK, enabling it to retrotranslocate to the cytosol. We demonstrate that Grp170 binds to Sel1L, an adapter of the transmembrane Hrd1 E3 ubiquitin ligase postulated to be the retrotranslocon, and links this interaction to Grp170’s function during ERAD. More broadly, Grp170 also promotes degradation of the nonglycosylated transthyretin (TTR) D18G misfolded client. Our findings thus establish a general function of Grp170 during ERAD and suggest that positioning this client-release factor at the retrotranslocation site may afford a mechanism to couple client release from BiP and retrotranslocation.     

Protein disulfide isomerase-like proteins play opposing roles during retrotranslocation

Misfolded proteins in the endoplasmic reticulum (ER) are retained in the organelle or retrotranslocated to the cytosol for proteasomal degradation. ER chaperones that guide these opposing processes are largely unknown. We developed a semipermeabilized cell system to study the retrotranslocation of cholera toxin (CT), a toxic agent that crosses the ER membrane to reach the cytosol during intoxication. We found that protein disulfide isomerase (PDI) facilitates CT retrotranslocation, whereas ERp72, a PDI-like protein, mediates its ER retention. In vitro analysis revealed that PDI and ERp72 alter CT's conformation in a manner consistent with their roles in retrotranslocation and ER retention. Moreover, we found that PDI's and ERp72's opposing functions operate on endogenous ER misfolded proteins. Thus, our data identify PDI family proteins that play opposing roles in ER quality control and establish an assay to further delineate the mechanism of CT retrotranslocation.     

A binding motif for Hsp90 in the A chains of ADP‐ribosylating toxins that move from the endoplasmic reticulum to the cytosol

Cholera toxin (Ctx) is an AB‐type protein toxin that acts as an adenosine diphosphate (ADP)‐ribosyltransferase to disrupt intracellular signalling in the target cell. It moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic CtxA1 subunit then dissociates from the rest of the toxin, unfolds, and activates the ER‐associated degradation system for export to the cytosol. Translocation occurs through an unusual ratchet mechanism in which the cytosolic chaperone Hsp90 couples CtxA1 refolding with CtxA1 extraction from the ER. Here, we report that Hsp90 recognises two peptide sequences from CtxA1: an N‐terminal RPPDEI sequence (residues 11–16) and an LDIAPA sequence in the C‐terminal region (residues 153–158) of the 192 amino acid protein. Peptides containing either sequence effectively blocked Hsp90 binding to full‐length CtxA1. Both sequences were necessary for the ER‐to‐cytosol export of CtxA1. Mutagenesis studies further demonstrated that the RPP residues in the RPPDEI motif are required for CtxA1 translocation to the cytosol. The LDIAPA sequence is unique to CtxA1, but we identified an RPPDEI‐like motif at the N‐ or C‐termini of the A chains from four other ER‐translocating toxins that act as ADP‐ribosyltransferases: pertussis toxin, Escherichia coli heat‐labile toxin, Pseudomonas aeruginosa exotoxin A, and Salmonella enterica serovar Typhimurium ADP‐ribosylating toxin. Hsp90 plays a functional role in the intoxication process for most, if not all, of these toxins. Our work has established a defined RPPDEI binding motif for Hsp90 that is required for the ER‐to‐cytosol export of CtxA1 and possibly other toxin A chains as well.     

Role of ubiquitination in retro-translocation of cholera toxin and escape of cytosolic degradation

Cholera toxin travels from the cell surface of affected mammalian cells to the endoplasmic reticulum (ER), where the A1 chain is released and retro-translocated across the ER membrane into the cytosol. We have tested whether, as in other cases, retro-translocation requires poly-ubiquitination. We show that an A1 chain mutant that lacks lysines and has a blocked N-terminus, and therefore cannot be ubiquitinated, remains active in vivo. The A1 chain is not degraded in the cytosol, as demonstrated by the fact that proteasome inhibitors do not stimulate its activity. When additional lysines are introduced into the A1 chain, moderate degradation by the proteasome is observed. The unfolded A1 chain rapidly refolds in vitro. These results show that poly-ubiquitination is not required for retro-translocation of all proteins across the ER membrane and indicate that the reason why the toxin escapes degradation in the cytosol may be both its paucity of lysines and its rapid refolding.