Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum

Spooner, Robert A.; Watson, Peter; Marsden, Catherine J.; Smith, Daniel C.; Moore, Katherine A. H.; Cook, Jonathan P.; Lord, J. Michael; Roberts, Lynne M.

doi:10.1042/bj20040742

Cited by 137 publications

(133 citation statements)

References 45 publications

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“…It is possible that Derlin-1-YFP failed to interact with other unidentified ER components mediating toxin retro-translocation or, as our finding indicated, affected the function of endogenous Derlin-1. Not all toxins that interact with PDI in the ER rely on Derlin-1 for retro-translocation, because retro-translocation of ricin, a plant toxin that interacts with PDI (Spooner et al, 2004), was not affected by dominant-negative Derlin-1 (Slominska- Wojewodzka et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Derlin-1 Facilitates the Retro-Translocation of Cholera Toxin

Bernardi

Forster

Lencer

et al. 2008

MBoC

102

134

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Cholera toxin (CT) intoxicates cells by using its receptor-binding B subunit (CTB) to traffic from the plasma membrane to the endoplasmic reticulum (ER). In this compartment, the catalytic A1 subunit (CTA1) is unfolded by protein disulfide isomerase (PDI) and retro-translocated to the cytosol where it triggers a signaling cascade, leading to secretory diarrhea. How CT is targeted to the site of retro-translocation in the ER membrane to initiate translocation is unclear. Using a semipermeabilized-cell retro-translocation assay, we demonstrate that a dominant-negative Derlin-1-YFP fusion protein attenuates the ER-to-cytosol transport of CTA1. Derlin-1 interacts with CTB and the ER chaperone PDI as assessed by coimmunoprecipitation experiments. An in vitro membrane-binding assay showed that CTB stimulated the unfolded CTA1 chain to bind to the ER membrane. Moreover, intoxication of intact cells with CTB stabilized the degradation of a Derlin-1-dependent substrate, suggesting that CT uses the Derlin-1 pathway. These findings indicate that Derlin-1 facilitates the retro-translocation of CT. CTB may play a role in this process by targeting the holotoxin to Derlin-1, enabling the Derlin-1-bound PDI to unfold the A1 subunit and prepare it for transport.

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Section: Discussionmentioning

confidence: 99%

Derlin-1 Facilitates the Retro-Translocation of Cholera Toxin

Bernardi

Forster

Lencer

et al. 2008

MBoC

102

134

View full text Add to dashboard Cite

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“…HeLa cell responses to 4-h challenges with graded doses of ricin or diphtheria toxin (DTx) were measured as previously described (3). For pharmacological studies, cells were treated coevally with graded doses of toxin in medium containing carrier or solvent vehicle (control) and with toxin dilutions in medium containing both carrier/vehicle and pharmacological agent.…”

Section: Methodsmentioning

confidence: 99%

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

Spooner

Hart

Cook

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states.Hsc70 ͉ Hsp90 ͉ ricin E ndoplasmic-reticulum (ER) associated protein degradation (ERAD) comprises coordinated disposal systems that recognize and remove misfolded and unassembled proteins in the ER, dislocating them across the ER membrane to the cytosol for proteasomal destruction. Degradation is normally facilitated by polyubiquitylation, usually on internal lysyl residues of the target protein. Both membrane-bound and soluble ER proteins can be disposed of by ERAD (1, 2).The plant cytotoxin ricin traffics to the ER lumen of mammalian cells where it is reduced to its RTA and RTB subunits before RTA dislocation (3). RTA does not penetrate the ER membrane directly; instead it exploits pre-existing proteinconducting channels as a nonnative species, mimicking ER proteins dispatched via ERAD (4, 5). Thus, it enters the cytosol in a form susceptible to proteolysis or aggregation. A proportion must evade these fates to gain a catalytic conformation that depurinates target ribosomes, stopping protein synthesis. The paucity of lysine residues in RTA may facilitate uncoupling from ERAD by reducing the potential for polyubiquitylation, thereby hampering proteasomal degradation (6). This, in turn, may provide opportunities for spontaneous or chaperone-assisted folding not normally sanctioned for ERAD substrates.We show an interaction of RTA with the cytosolic heat shock (cognate) protein Hsc70. From the chaperone-bound state, nonnative cytosolic RTA can achieve a catalytic conformation or can be inactivated. Its ultimate fate depends on the activities of ...

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“…Thus, overexpression of RTB at the site of reduction should protect cells from ricin by acting as a dead-end receptor for newly-released RTA. Expression of RTB in the mammalian ER has precisely this effect (Spooner et al 2004). The ERexpressed RTB is retained for some time in the ER by a thiol anchor constituted by a mixed disulphide between RTB and PDI.…”

Section: Reductive Separation Of the Holotoxin Subunitsmentioning

confidence: 99%

“…However, when RTB is expressed exogenously in the mammalian ER, it is trapped for a while by a thiol anchor, and then disappears from the ER by two mechanisms: approximately half is secreted, whilst the remainder becomes an ERAD substrate that can be stabilised by proteasomal inhibition (Spooner et al 2004). The fates of STxB/A2 and CTxB/A2 are currently unmapped.…”

Section: Dislocation Across the Er Membranementioning

confidence: 99%