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

238YEV2-FGMechanistic insight into receptor endocytosis and endosomalA/B toxin trafficking in yeastE. Gießelmann, J. Dausend, B. Becker, M.J. Schmitt*Saarland University, Department of Biosciences (FR 8.3), Molecular &Cell Biology, Saarbrücken, GermanyYeast strains infected with the M28 dsRNA killer virus secrete aheterodimeric killer toxin (K28) belonging to the family of microbial A/Btoxins. After receptor-mediated cell entry, the toxin reaches the cytosol ofa target cell by travelling the secretion pathway in reverse [1]. The alphatoxin inhibits DNA synthesis in the nucleus and causes apoptotic cell death[1]. Key components in the intoxification process are the -C-terminalHDEL motiv of the toxin and its interaction with the HDEL receptorErd2p of the target cell. Most recent CSLM data in conjunction withErd2p-based reporter assays indicated that Erd2p colocalizes to the plasmamembrane where it functions as membrane receptor for toxin endocytosis.Sequence analysis of Erd2p revealed N- and C-terminal endocytic motifsrelevant for receptor internalization. Physical Erd2p/Rsp5p interactionidentified via BiFC analysis indicated that receptor (mono)ubiquitiationtriggers the internalization of receptor-bound toxin. Additional studiesverified the importance of early mediators of endocytosis, including thecoat building complex and the actin machinery for toxin uptake as well asAP2-complex components, which so far have only been described to beinvolved in the endocytosis of mammalian cells [2]. To further dissecttoxin trafficking, biologically active K28/mCherry fusions were expressedin Pichia pastoris and used to track the toxin's transit through theendocytic pathway, including TIRF microscopy for quantitative analysesof Erd2-GFP mobility in wild-type yeast and selected endocytic mutants.Our studies were extended by investigating uptake and endocytic transportof the plant A/B toxin ricin. This heteromeric glycoprotein belongs to thefamily of ribosome inactivating proteins (RIPs) whose in vivo toxicityresults from the depurination of 28S rRNA catalyzed by the A-chain ofricin, RTA. Since extention of RTA by a mammalian-specific ER retentionsignal (KDEL) significantly increases RTA toxicity against mammaliancells, we analyzed the phenotypic effect of RTA carrying the yeast-specificER retention motif HDEL. Interestingly RTA HDEL showed a similarcytotoxic effect on yeast as a corresponding RTA KDEL variant on HeLacells. Furthermore, we established a powerful yeast bioassay for RTA invivo uptake and trafficking. The assay verified the RTA resistantphenotype seen in yeast mutants defective in early steps ofendocytosis(end3) and/or in RTA depurination activity(rpl12B) [3].Thus K28 and RTA represent powerful tools and substrates for generalstudies of endocytosis and endosomal trafficking in eukaryotic cells.Kindly supported by grants from the Deutsche Forschungsgemeinschaft.1. M.J. Schmitt and F. Breinig (2006). Nat. Rev. Microbiol.4:, 212.2. S.Y. Carroll et al. (2009). Dev. Cell17: 552.3. B. Becker and M.J. Schmitt (2011). Toxins7: 834.YEV3-FGThe conjunction of mRNA export and translationA. Hackmann, T. Gross, C. Baierlein, H. Krebber*University of Göttingen, Institute for Microbiology and Genetics,Department Molecular Genetics, Göttingen, GermanyIn recent years it has been shown that some mRNA export factors are alsoinvolved in translation. Here we report on the identification of a noveltransport function of the yeast mRNA export factor Npl3 in the export oflarge ribosomal subunits from the nucleus to the cytoplasm. Interestingly,while mRNAs are exported via the RNA-binding protein Npl3 and itsinteracting export receptor Mex67, the export of large ribosomal subunitsalso requires Mex67, however, in this case Mex67 directly binds to the 5SrRNA and does not require the Npl3-adapter. We discovered a novelfunction of Npl3 in mediating pre-60S ribosomal subunit exportindependent of Mex67. Npl3 interacts with the 25S rRNA, ribosomal andribosome associated proteins and with the NPC. Mutations in NPL3 lead toexport defects of the large subunit and genetic interactions with other pre-60S export factors.YEV4-FGLocalization of mRNAs and endoplasmic reticulum in buddingyeastJ. Fundakowski 1 , M. Schmid 2 , C. Genz 1 , S. Lange 2 , R.-P. Jansen* 11 Eberhard-Karls-Universität, Interfaculty Institute for Biochemistry, Tübingen,Germany2 Ludwig-Maximilians-Universität München, GeneCenter, Munich, GermanyLocalization of mRNAs contributes to generation and maintenance ofcellular asymmetry, embryonic development and neuronal function [1]. Itis a widely distributed process in single-celled and multicellular eukaryotesbut has also been described for prokaryotes. In the budding yeastSaccharomyces cerevisiae, a minimal protein complex comprised of themyosin motor Myo4p, the RNA binding protein She2p, and the adapterand RNA binding protein She3p localizes >30 mRNAs to the bud tip [2].This set of mRNAs includes 13 mRNAs encoding membrane or secretedproteins. It has been observed that ribonucleoprotein (RNP) particlescontaining one of these mRNAs can co-localize with tubular ER structures.Such ER tubules form the initial elements for segregation of cortical ER(cER) [3]. Co-localization has therefore been suggested to illustrate acoordination of mRNA localization and cER distribution [4]. Byinvestigating mRNA localization in yeast mutants defective in cERsegregation, we demonstrate that proper cER segregation is required forlocalization of a subset of mRNAs. These mRNAs are expressed at thetime of tubular ER movement into the bud. Localization of ASH1 mRNAthat is expressed after tubular movement has ceased is not affected in anyof these mutants. Co-localization of RNPs and tubular ER depends on theRNA-binding protein She2p and requires its tetramerization. She2p canbind to artificial, protein-free liposomes in a membrane curvaturedependentmanner with a preference for small liposomes with a diameterresembling yeast ER tubules. In support of this finding, loss of proteinsrequired for tubule formation result in defective mRNA localization invivo. Our results demonstrate that She2p is not only an RNA- but alsolipid-binding protein that recognizes membrane curvature, which makes itan ideal coordinator of ER tubule and mRNA co-transport1. K. Martin and A. Ephrussi, Cell136(2009), p. 719.2. G. Gonsalez, C.R. Urbinati, and R.M. Long, Biol. Cell97 (2005), p. 75.3. Y. Du, S. Ferro-Novick, and P. Novick, J. Cell Sci.117(2004), p. 2871.4. M. Schmid, A. Jaedicke, T.-G. Du, and R.-P. Jansen, Curr. Biol16(2006), p. 1538.YEV5-FGEukaryotic Ribosome Biogenesis: Analysis of the NucleolarEssential Yeast Nep1 Protein and Mutations Causing theHuman Bowen-Conradi SyndromeK.-D. Entian*, B. MeyerJohann Wolfgang Goethe University, Cluster of Excellence: MacromolecularComplexes and Institute for Molecular Biosciences, Frankfurt a.M., GermanyIn eukaryotes, ribosome biogenesis needs the coordinated interaction ofrRNAs and proteins. We identified the Nep1 (Emg1) protein family as anessential protein involved in ribosome biogenesis. The yeast and thehuman Nep1 proteins are localized in the nucleolus and the humanHsNep1 can complement the Nep1 function in a yeast nep1 mutant.A mutation which abolished the yeast Nep1 RNA binding was responsiblefor the human Bown-Conradi-Syndrome (BCS) which causes early childdeath. Analysis of yeast and human mutations showed that the mutatedproteins lost their nucleolar location and their RNA-binding activity.Structure analysis of the Nep1 protein suggested its function as a methyltransferase and, recently, we could confirm that Nep1 methylated 1191 inthe decoding center of the 18S rRNA. Additionally, the Nep1 protein has adual function in ribosome biogenesis and supports Rps19 assembly to thepre-ribosome.Buchhaupt et al. (2006) Mol. Genet. Genomics. 276: 273; Buchhaupt et al. (2007) FEMS Yeast Res.7: 771,. Eschrich et al. (2002) Curr. Genet. 40: 326; Taylor et al. (2007) NAR 36: 1542; Armisteadet al. (2009) Am. J. Hum. Genet. 84, 728;Wurm et al. (2010) NAR 38: 2387, Meyer et al.(2011)NAR39: 1524.YEV6-FGHigh-level production of tetraacetyl phytosphingosine (TAPS)by combined genetic engineering of sphingoid basebiosynthesis and L-serine availability in the non-conventionalyeast Pichia ciferriiC. Schorsch, E. Boles*Johann Wolfgang Goethe University, Institute of Molecular Biosciences,Frankfurt a.M., GermanyThe non-conventional yeast Pichia ciferrii (formerly known as Hansenulaciferri) is the only known organism that is specialized in producing andsecreting large quantities of tetraacetyl phytosphingosine (TAPS), a fullyacetylated form of the sphingolipid intermediate phytosphingosine.Because of its unique feature this yeast is an attractive microorganism forthe industrial production of TAPS. Sphingolipids are important ingredientsin cosmetic applications and formulations. They play important roles inhuman stratum corneum as they are involved in skin permeability andantimicrobial barrier homeostatic functions.Our work aimed to improve TAPS production by genetic engineering of P.ciferrii. In a first step, we could increase TAPS production by improvingprecursor availability. This was achieved by blocking degradation of L-serine which - in the first committed step of sphingolipid biosynthesis - iscondensed with palmitoyl-CoA by serine palmitoyltransferase. Moreover,genetic engineering of the sphingolipid pathway further increasedsecretion of TAPS considerably. The final recombinant P. ciferrii strainproduced up to 199 mg (TAPS) * g -1 (cdw) with a maximal production rate of8.42 mg * OD 600nm -1 * h -1 and a titer of about 2 g * L -1 , and should beapplicable for industrial TAPS production.We would like to thank Evonik Industries and the German FederalMinistry of Education and Research (Bundesministerium für Bildung undForschung, BMBF; Bioindustrie 2021, “FerDi”) for support.BIOspektrum | Tagungsband 2012