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Toxins (Basel). 2011 Jul;3(7):834-47. doi: 10.3390/toxins3070834. Epub 2011 Jul 5.

Adapting yeast as model to study ricin toxin a uptake and trafficking.

Author information

1
Molecular and Cell Biology, Department of Biosciences (FR 8.3), Saarland University, D-66041Saarbrücken, Germany. bjoern_becker2@gmx.de

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.

KEYWORDS:

ER retention signal; H/KDEL; oyxgen-sensor microtiter plate; ricin toxin A chain; toxin endocytosis and transport; yeast spheroplasts

PMID:
22069743
PMCID:
PMC3202858
DOI:
10.3390/toxins3070834
[Indexed for MEDLINE]
Free PMC Article

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