title: Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking.
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.
authors.affiliation: Molecular and Cell Biology, Department of Biosciences and Center of Human and Molecular Biology (ZHMB), Saarland University, Saarbrücken D-66123, Germany. b.becker@microbiol.uni-sb.de.
authors.forename: Björn
authors.initials: B
authors.lastname: Becker
authors.affiliation: Molecular and Cell Biology, Department of Biosciences and Center of Human and Molecular Biology (ZHMB), Saarland University, Saarbrücken D-66123, Germany. Tina.Schnoeder@med.uni-jena.de.
authors.forename: Tina
authors.initials: T
authors.lastname: Schnöder
authors.affiliation: Molecular and Cell Biology, Department of Biosciences and Center of Human and Molecular Biology (ZHMB), Saarland University, Saarbrücken D-66123, Germany. mjs@microbiol.uni-sb.de.
authors.forename: Manfred J
authors.initials: MJ
authors.lastname: Schmitt
mesh_terms: D001681:Biological Assay; D049452:Green Fluorescent Proteins / Q000235:genetics; D021381:Protein Transport; D012276:Ricin / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins; D012276:Ricin
keywords: S. cerevisiae; endoplasmic reticulum (ER); retrograde protein transport; ribosome inactivating protein (RIP); ricin toxin A chain (RTA); trans-Golgi network (TGN)