title: Adapting yeast as model to study ricin toxin a uptake and trafficking.
abstract: The plant A/B toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a toxin (RTA(HDEL)) showed a similar cytotoxic effect on yeast as a corresponding RTA(KDEL) variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B toxin transport from the plasma membrane through the endosomal compartment to the ER.
authors: Molecular and Cell Biology, Department of Biosciences (FR 8.3), Saarland University, D-66041Saarbrücken, Germany. bjoern_becker2@gmx.de
authors: Björn
authors: B
authors: Becker
authors: Manfred J
authors: MJ
authors: Schmitt
mesh_terms: D001681:Biological Assay; D015153:Blotting, Western; D004705:Endocytosis / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport; D012339:RNA, Ribosomal, 28S / Q000187:drug effects* / Q000235:genetics; D012276:Ricin / Q000493:pharmacokinetics* / Q000633:toxicity*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014118:Toxins, Biological / Q000493:pharmacokinetics / Q000633:toxicity
chemical_list: D012339:RNA, Ribosomal, 28S; D014118:Toxins, Biological; D012276:Ricin