VAAM-Jahrestagung 2012 18.–21. März in Tübingen

80FUP008Asc1p’s role in MAP-kinase and cAMP-PKA signalingK. Schmitt*, N. Rachfall, S. Sanders, G.H. Braus, O. ValeriusGeorg-August University Göttingen, Mol. Microbiol. & Genetics,Göttingen, GermanyThe eukaryotic ribosomal protein Asc1p/RACK1 is required fordevelopmental processes in lower eukaryotes (S. cerevisiae) as well as inhigher eukaryotes (plants and mammals). However, there is poorknowledge about the protein’s exact mode of action and its own posttranscriptionalregulation. We could show that S. cerevisiae Asc1p controlsthe abundance of transcription factors in yeast, namely of Ste12p, Phd1p,Tec1p, Rap1p, and Flo8p. This seems to be at least partially due to anAsc1p-dependent translational regulation of the transcription factormRNAs. We dissect Asc1p’s influence on the translation rates of theencoding mRNAs from its putative influence on the stability of thementioned transcription factors. Tec1p-stability is regulated by the matingresponse pathway that targets Tec1p for degradation upon phosphorylationthrough the Fus3p-MAP-kinase. Indeed, the pheromone response pathwayis up-regulated in the asc1 strain. However, pathway inactivation bydeletion of the FUS3 gene did not restore Tec1p levels in the asc1 strain.Thus, Ascp1 affects Tec1p-abundance via a pheromone-independentmechanism. Shut-off experiments for Tec1p indicate that deletion of ASC1has no effect on its stability suggesting an Asc1p-dependent regulation ofTEC1-mRNA translation. We also analyze whether Asc1p itself is posttranslationallymodified (e.g. phosphorylated) through MAPkinase/cAMP-PKApathways. Modifications of Asc1p could regulate itsinteraction with other ribosomal proteins or the formation of Asc1phomodimers[1]. Four phospho-sites of Asc1p are known from highthroughputstudies [2,3,4]. Using mass spectrometry we could confirm twoof these sites (S166 and T168) and furthermore determined one previouslyunknown site (T72).Yatime et al. (2011). Structure of the RACK1 dimer from Saccharomyces cerevisiae. J Mol Biol411, 486-498.Chi et al. (2001). Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclindependentkinase. Genes Dev 15, 1078-1092.Smolka et al. (2007). Proteome-wide identification of in vivo targets of DNA damage checkpointkinases. Proc Natl Acad Sci USA 104, 10364-10369.Albuquerque et al. (2008). A multidimensional chromatography technology for in-depthphosphoproteome analysis. Mol Cell Proteomics 7, 1389-1396.FUP009Mode of action of a cell cycle arresting yeast killer toxinT. Hoffmann*, J. Reiter, M.J. SchmittUniversität des Saarlandes, Molekular- und Zellbiologie, Saarbrücken,GermanyK28 is a heterodimeric A/B toxin secreted by virally infected killer strainsof the yeast Saccharomyces cerevisiae. After binding to the cell wall ofsensitive yeasts the / toxin enters cells via receptor-mediatedendocytosis and is retrogradely transported to the cytosol where itdissociates into its subunit components. While is polyubiquitinated andproteasomaly degraded, the -subunit enters the nucleus and causes anirreversible cell cycle arrest at the transition from G1 to S phase. K28-treated cells typically arrest with a medium-sized bud, a single nucleus inthe mother cell and show a pre-replicative DNA content (1n).Since other cell cycle arresting killer toxins like zymocin fromKluyveromyces lactis or Pichia acaciae toxin PaT cause a similar“terminal phenotype”, we tested the effect of K28 on S. cerevisiae mutantsthat are resistant against those toxins. Agar diffusion assays showed thatdeletion of TRM9 or ELP3 did not lead to toxin resistance, indicating thatthe arrest caused by K28 differs from zymocin or PaT induced cell cyclearrest. Interestingly, RNA polymerase II deletion mutants (rpb4, rpb9)show complete resistance against K28.To gain deeper insight into the mechanism(s) of how K28 arrests the cellcycle, we further studied the influence of the toxin on transcription of cellcycle and G1-specific genes. Northern blot analyses showed that G1-specific CLN1 and CLN2 mRNA levels rapidly decrease after toxintreatment, though it is unclear if this decline is due to a direct effect.Potential toxin targets were identified in a yeast two hybrid screen andverified biochemically by coIP and GST pulldown assays. To confirm thatthe nucleus represents the compartment where in vivo toxicity occurs, weconstructed protein fusions between K28 and mRFP and analysed theirintracellular localisation.FUP010Benzene oxygenation by Agrocybe aegerita aromaticperoxygenase (AaeAPO)A. Karich*, R. Ullrich, M. Kluge, M. HofrichterInternationales Hochschulinstitut (IHI) Zittau, Department Bio- undUmweltwissenschaften, Zittau, GermanyAgrocybe aegerita aromatic peroxygenase (AaeAPO) is an extracellularenzyme secreted by the agaric basidiomycete Agrocybe aegerita. AaeAPOhydroxylates the aromatic ring of benzene using hydrogen peroxide as cosubstrate.The optimum pH for the reaction is around 7. The reactionproceeds via the primary product benzene oxide which rapidly undergoesaromatization and rearranges to phenol in aqueous solution. Existence ofbenzene oxide was proved by chemical preparation of this compound andGC/MS and LC-MS analysis. Further oxidation lead to hydroquinone;catechol; p-benzoquinone; o-benzoquinone as well as 1,2,4-trihydroxybenzene and hydroxy-p-benzoquinone. Using H 2 18 O 2 as cosubstratethe origin of the oxygen transferred into benzene and phenol wasproved to be the peroxide. The use of ascorbic acid as radical scavengerprevented dihydroxy benzenes from exchanging oxygen with water (viaquinones) in this investigation. The apparent k cat and the approximated K M-value for benzene hydroxylation were estimated to 7.9 s -1 and 3.6 mMrespectively. Benzene oxygenation is first described herein for a hemeperoxidase.FUP011ER exit of a yeast viral A/B toxin SECrets of K28N. Mueller*, M. SchmittSaarland University, Molecular and Cellbiology, Saarbrücken, GermanyK28 is a virus encoded A/B protein toxin secreted by the yeastSaccharomyces cerevisiae that enters susceptible target cells by receptormediatedendocytosis. After retrograde transport from early endosomesthrough the secretory pathway, the / heterodimeric toxin reaches thecytosol where the cytotoxic -subunit dissociates from , subsequentlyenters the nucleus and causes cell death by blocking DNA synthesis andarresting cells at the G1/S boundary of the cell cycle [1].Interestingly, K28 retrotranslocation from the ER into the cytosol isindependent of ubiquitination and does not require cellular components ofthe ER-associated protein degradation machinery (ERAD). In contrast, ERexit of a cytotoxic -variant expressed in the ER lumen depends onubiquitination, proteasomes and ERAD components, indicating (i) that amost likely masks itself as ERAD substrate and (ii) that ERretrotranslocation mechanistically differs under both scenarios [2]. Toelucidate the molecular mechanism(s) of ER-to-cytosol toxin transport inyeast as well as in mammalian cells, the major focus of the present study isto identify cellular components (including the nature of the ERtranslocation channel) involved in this process. The requirement ofproteasomal activity and ubiquitination to drive ER export, and theidentification of cellular K28 interaction partners of both, the / toxin aswell as K28 are being analysedin vitroby using isolated microsomes andIP experiments.Kindly supported by a grant from the Deutsche Forschungsgemeinschaft(GRK 845).[1] Carroll et al. (2009). Dev. Cell .....[2] Heiligenstein et al. (2006). EMBO J. .....FUP012Adapting yeast as a model to study ricin toxin A uptake andtraffickingB. Becker*, M. SchmittFR. 8.3 Biosciences, Molecular and Cell Biology, Saarbrücken, GermanyThe plant A/B toxin ricin represents a heterodimeric glycoproteinbelonging to the family of ribosome inactivating proteins, RIPs. Its toxicitytowards eukaryotic cells results from the depurination of 28S rRNA due tothe N-glycosidic activity of ricin toxin A chain, RTA. Since extention ofRTA by a mammalian-specific endoplasmic reticulum (ER) retentionsignal (KDEL) significantly increases RTA in vivo toxicity againstmammalian cells, we analyzed the phenotypic effect of RTA carrying theyeast-specific ER retention motif HDEL. Interestingly, such a toxin(RTA HDEL ) showed a similar cytotoxic effect on yeast as a correspondingRTA KDEL variant on HeLa cells. Furthermore, we established a powerfulyeast bioassay for RTA in vivo uptake and trafficking which is based onthe measurement of dissolved oxygen in toxin-treated spheroplast culturesof S. cerevisiae. We show that yeast spheroplasts are highly sensitiveagainst external applied RTA and further demonstrate that its toxicity isgreatly enhanced by replacing the C-terminal KDEL motif by HDEL.Based on the RTA resistant phenotype seen in yeast knock-out mutantsdefective in early steps of endocytosis (end3) and/or in RTA depurinationactivity on 28S rRNA (rpl12B) we feel that the yeast-based bioassaydescribed in this study is a powerful tool to dissect intracellular A/B toxintransport from the plasma membrane through the endosomal compartmentto the ER.Furthermore, we established a simple and sensitive fluorescence assaybased on thein vivotranslation of a GFP reporter to investigate intracellularRTA trafficking from the endosome to the yeast ER. Our results indicatethat both, the mammalian Rab6a homologue Ypt6p, and the yeast syntaxin5 homologue Sft2p are involved in toxin transport from the endosome tothe TGN. In addition, the GARP complex is also important for thistrafficking step, whereas defects in the retromer complex did not influenceRTA toxicity. Since our results uncovered striking similarities of toxintrafficking between yeast and mammalian cells, we feel that our screeningBIOspektrum | Tagungsband 2012