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Toxins 2016, 8(12), 366; doi:10.3390/toxins8120366

Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking

Molecular and Cell Biology, Department of Biosciences and Center of Human and Molecular Biology (ZHMB), Saarland University, Saarbrücken D-66123, Germany
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Academic Editor: Tomas Girbes
Received: 11 October 2016 / Revised: 22 November 2016 / Accepted: 30 November 2016 / Published: 6 December 2016
(This article belongs to the Special Issue Ribosome Inactivating Toxins)
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Abstract

RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast. View Full-Text
Keywords: S. cerevisiae; ricin toxin A chain (RTA); ribosome inactivating protein (RIP); retrograde protein transport; trans-Golgi network (TGN); endoplasmic reticulum (ER) S. cerevisiae; ricin toxin A chain (RTA); ribosome inactivating protein (RIP); retrograde protein transport; trans-Golgi network (TGN); endoplasmic reticulum (ER)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Becker, B.; Schnöder, T.; Schmitt, M.J. Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking. Toxins 2016, 8, 366.

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