Molecular and Cell Biology, Department of Biosciences (FR 8.3), Saarland University, D-66041Saarbrücken, Germany.
Toxins (Basel). 2011 Jul;3(7):834-47. doi: 10.3390/toxins3070834. Epub 2011 Jul 5.
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.
植物 A/B 毒素蓖麻毒素是一种异二聚体糖蛋白，属于核糖体失活蛋白（RIPs）家族。它对真核细胞的毒性是由于蓖麻毒素 A 链（RTA）的 N-糖苷酶活性导致 28S rRNA 的脱嘌呤化。由于 RTA 通过哺乳动物特异性内质网（ER）保留信号（KDEL）的延伸，大大增加了 RTA 对哺乳动物细胞的体内毒性，因此我们在此分析了携带酵母特异性 ER 保留基序 HDEL 的 RTA 的表型效应。有趣的是，这样的毒素（RTA(HDEL)）在酵母上表现出与相应的 RTA(KDEL)变体在 HeLa 细胞上相似的细胞毒性作用。此外，我们建立了一种强大的酵母体内 RTA 摄取和转运的生物测定法，该方法基于测量毒素处理的酿酒酵母原生质球培养物中的溶解氧。我们表明酵母原生质球对外部施加的 RTA 高度敏感，并进一步证明通过用 HDEL 替换 C 末端 KDEL 基序可以大大增强其毒性。基于在早期内吞作用步骤（∆end3）和/或在 28S rRNA 上 RTA 脱嘌呤活性缺陷的酵母敲除突变体中观察到的 RTA 抗性表型（∆rpl12B），我们认为本文所述的基于酵母的生物测定法是一种强大的工具，可从质膜通过内体区室到 ER 来剖析细胞内 A/B 毒素的运输。
