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Adapting Yeast as Model to Study Ricin Toxin A Uptake and Trafficking†
Molecular and Cell Biology, Department of Biosciences (FR 8.3), Saarland University, D-66041 Saarbrücken, Germany
† This work is dedicated to Professor Dr. Ferdinand Radler on the occasion of his 82nd birthday.
* Author to whom correspondence should be addressed.
Received: 7 June 2011; in revised form: 7 June 2011 / Accepted: 28 June 2011 / Published: 5 July 2011
(This article belongs to the Special Issue Ricin Toxin
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 (RTAHDEL) showed a similar cytotoxic effect on yeast as a corresponding RTAKDEL 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.
Keywords: ricin toxin A chain; ER retention signal; yeast spheroplasts; toxin endocytosis and transport; oyxgen-sensor microtiter plate; H/KDEL
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MDPI and ACS Style
Becker, B.; Schmitt, M.J. Adapting Yeast as Model to Study Ricin Toxin A Uptake and Trafficking. Toxins 2011, 3, 834-847.
Becker B, Schmitt MJ. Adapting Yeast as Model to Study Ricin Toxin A Uptake and Trafficking. Toxins. 2011; 3(7):834-847.
Becker, Björn; Schmitt, Manfred J. 2011. "Adapting Yeast as Model to Study Ricin Toxin A Uptake and Trafficking." Toxins 3, no. 7: 834-847.