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 α-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 Sec61α, 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

BiP Negatively Affects Ricin Transport

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

Conserved amino acid sites in typical MHC I molecules.

<p>WebLogo presentation of important selected structural and functional sites for subset of MHC I sequences from Atlantic cod. Letter size indicates the probability of the particular amino acids at the given site. Coloring scheme follows standard presentation in MEGA 5.05, reflecting amino acid properties. Numbering is based on consensus sequence, starting at the α1 domain (exon 2).</p

Synonomus (<i>dS</i>) and non-synonomus (<i>dN</i>) mutations in functional sites of MHC I molecules.

<p>Identification of antigen presenting sites (APS and non-APS) follows Kaufmann et. al. (1994), with 37 and 148 sites pr. sequence, respectively. Significant values in bold.</p

Phylogeny of MHC I diversity in Atlantic cod.

<p>A) Unrooted polar cladogram of all unique cDNA sequences of MHC Ia and Ib in Atlantic cod, based on amino acid sequence alignment. Elongated branches illustrate sequences originating from at least two independent PCR reactions. B) Subset of sequences highlighted in a), rooted with additional teleost Ia and Ib sequences from Ensembl. Maximum likelihood (ML) and Bayesian posterior probabilities are shown for the basal branches. Scale bar represents number of amino acid substitutions pr site.</p