Dependence of Ricin Toxicity on Translocation of the Toxin A-chain from the Endoplasmic Reticulum to the Cytosol

Abstract

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.

Membrane cytosolic translocation of verotoxin A1 subunit in target cells

“…However, about 30 % of VTA 1 remains susceptible to proteasomal degradation, as demonstrated by the increased cytotoxicity and cytosolic translocation of VT1 (our results) and the threefold increase in ricin potency and cytosolic translocation (Deeks et al, 2002;Wesche et al, 1999), in the presence of lactacystin. As translocated VTA 1 (our results) and ricin A chain (Deeks et al, 2002) do not display increased molecular mass, indicative of ubiquitin ligation, they are likely targeted to the proteasome by ubiquitinindependent mechanisms (Kalejta & Shenk, 2003;Lee et al, 2004).…”

“…This was performed three times. 2002; Rodighiero et al, 2002;Wesche et al, 1999), in which misfolded proteins are translocated for proteosomal degradation (Werner et al, 1996). To assess the possible involvement of the ERAD pathway, the effect of lactacystin proteasomal inhibition on VTA 1 translocation was tested.…”

How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum

“…little is known beyond the implication of Sec61 as part of a dislocation channel (Simpson et al 1999;Slominska-Wojewodzka et al 2006;Wesche et al 1999) and the lack of obvious function of derlins (Slominska-Wojewodzka et al 2006). …”

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

“…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.…”