Abstract
The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated to the cytosol in a chaperone-dependent process, which is not fully explored. Recently two separate siRNA screens have demonstrated that ER chaperones have implications for ricin toxicity. ER associated degradation (ERAD) involves translocation of misfolded proteins from ER to cytosol and it is conceivable that protein toxins exploit this pathway. The ER chaperone BiP is an important ER regulator and has been implicated in toxicity mediated by cholera and Shiga toxin. In this study, we have investigated the role of BiP in ricin translocation to the cytosol. We first show that overexpression of BiP inhibited ricin translocation and protected cells against the toxin. Furthermore, shRNA-mediated depletion of BiP enhanced toxin translocation resulting in increased cytotoxicity. BiP-dependent inhibition of ricin toxicity was independent of ER stress. Our findings suggest that in contrast to what was shown with the Shiga toxin, the presence of BiP does not facilitate, but rather inhibits the entry of ricin into the cytosol.
Highlights
Folding and assembly of secretory and transmembrane proteins occur in the endoplasmic reticulum (ER) and are assisted by numerous molecular chaperones
In order to investigate the role of BiP in ricin toxicity, Human Embryonic Kidney 293 (HEK293) cells were first transiently transfected with myc-tagged wild type BiP (Figure 1A and [23])
The ER chaperone BiP has since its discovery been ascribed several functions and is today accepted as a master regulator of ER function [4]
Summary
Folding and assembly of secretory and transmembrane proteins occur in the endoplasmic reticulum (ER) and are assisted by numerous molecular chaperones. BiP has been described as the master regulator of ER function, being involved in folding and assembly of newly synthesized proteins, and in maintaining the permeability barrier of the ER during protein translocation, targeting misfolded proteins for retrograde translocation and sensing conditions of ER-stress, to activate the mammalian unfolded protein response (reviewed in [4]). Ricin is an AB protein toxin found in the seeds of the plant Ricinus communis It consists of two subunits, A and B chain, held together by a disulfide bridge. Cytosolic chaperones Hsc and Hsc were found to be essential for refolding of ricin A into a functional toxic enzyme [9]. We have investigated the role of BiP in ricin toxicity, and based on our results, we propose that BiP, in contrast to its importance for retro-translocation of other toxins studied so far, acts as a negative regulator of ricin toxicity
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