VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

localization of cell end markers [1; 2]. Although the importance of SRDs isgetting clearer, the roles and formation mechanism of SRDs remain almostunknown. To analyze the functional roles of SRDs, we investigate themechanism of SRD (or raft cluster) formation and maintenance. There arenumerous studies on raft formation in different organisms and somecomponents are known. Flotillin/reggie proteins for instance werediscovered in neurons and are known to form plasma membrane domains.The flotillin/reggie protein and a related microdomain scaffolding protein,stomatin, are conserved in filamentous fungi but have not yet beencharacterized. We have started the investigation of their functions by genedeletion and GFP-tagging. It was revealed that the flotillin/reggie proteinFloA-GFP accumulated at hyphal tips. The deletion of floA showed smallercolony than that of wild-type strain and often exhibited irregular thickness ofhyphae. Moreover, the stomatin related protein StoA-GFP localized only atyoung branch tips and subapical cortex in mature hyphal tips. The deletionof stoA also showed smaller colony than that of wild-type strain andexhibited irregular hyphae and increased branching. The localization ofSRDs, cell end markers, and actin etc. are analyzed in the mutants.[1] Takeshita, N., Higashitsuji, Y., Konzack, S. & Fischer, R. (2008) Mol. Biol. Cell, 19(1):339-351.[2] Fischer, R., Zekert, N. & Takeshita, N. (2008) Mol. Microbiol., 68(4):813-826.CBP004Mode of action of a cell cycle arresting yeast killer toxinT.M. Hoffmann*, M.J. SchmittDepartment of Molecular and Cell Biology, Saarland University,Saarbrücken, GermanyK28 is a heterodimeric A/B toxin secreted by virally infected killer strains ofthe yeast Saccharomyces cerevisiae. After binding to the cell wall ofsensitive yeasts the a/b toxin enters cells via receptor-mediated endocytosisand is retrogradely transported to the cytosol where it dissociates into itssubunit components. While β is polyubiquitinated and proteasomalydegraded, the α-subunit enters the nucleus and causes an irreversible cellcycle arrest at the transition from G1 to S phase. K28-treated cells typicallyarrest with a medium-sized bud, a single nucleus in the mother cell andshow 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 „terminalphenotype”, we tested the effect of K28 on S. cerevisiae mutants that areresistant against those toxins. Agar diffusion assays showed that deletion ofTRM9 or ELP3 did not lead to toxin resistance, indicating that the arrestcaused by K28 differs from zymocin or PaT induced cell cycle arrest.Interestingly, RNA polymerase II deletion mutants (rpb4, rpb9) showcomplete resistance against K28.To gain deeper insight into the mechanism(s) of how K28α 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-specificCLN1 and CLN2 mRNA levels rapidly decrease after toxin treatment,though it is unclear if this decline is due to a direct effect. Potential toxintargets were found using the yeast two hybrid system and were verifiedbiochemically by coIP and GST pulldown assays. To confirm that thenucleus represents the compartment where in vivo toxicity occurs weconstructed protein fusions between K28α and mRFP and analysed theirintracellular localisation.[1] Schmitt et al (1996): Cell cycle studies on the mode of action of yeast K28 killer toxin.Microbiology 142: 2655-2662.[2] Reiter et al (2005): Viral killer toxins induce caspase-mediated apoptosis in yeast. J Cell Biol. 168:353-358.CBP005Reverse SECretion or ERADication?N. Müller*, M.J. SchmittDepartment of Molecular and Cell Biology, Saarland University,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 α/β heterodimeric toxin reaches thecytosol where the cytotoxic α-subunit dissociates from β, 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 α-variant expressed in the ER lumen depends onubiquitination and ERAD, indicating (i) that α masks itself as ERADsubstrate for proteasomal degradation and (ii) that ER retrotranslocationmechanistically differs under both scenarios [2]. To elucidate the molecularmechanism(s) of ER-to-cytosol toxin transport in yeast as well as inmammalian cells, the major focus of the present study is to identify cellularcomponents (including the nature of the ER translocation channel) involvedin this process. The requirement of proteasomal activity and ubiquitinationto drive ER export, and the identification of cellular K28 interaction partnersof both, the α/β toxin as well as K28α are being analysed in vitro on isolatedmicrosomes and IP experiments.[1] Carroll et al (2009): Dev. Cell 17 (4), 552-60.[2] Heiligenstein et al (2006): EMBO J. 25 (20) 4717-27.CBP006Follow the light: Visualization of K28 cell entry and itsreceptor’s mobilityE. Gießelmann*, M.J. SchmittDepartment of Molecular and Cell Biology, Saarland University,Saarbrücken, GermanyK28 toxin, secreted by virus-infected killer strains of the yeastSaccharomyces cerevisiae, is a α/β heterodimeric protein of the A/B toxinfamily. After initial toxin binding to the surface of sensitive target cells, K28is taken up by receptor-mediated endocytosis and subsequently delivered toan early endosomal compartment from where it is transported backwardsthrough the Golgi and the endoplasmic reticulum (ER) to the cytosol. Withinthe cytosol, the toxin′s β-subunit is polyubiquitinated and targeted forproteasomal degradation, while α enters the nucleus and causes a G1/S cellcycle arrest and cell death.Both, toxin uptake and intracellular transport crucially depend on thecellular HDEL receptor Erd2p which ensures that the toxin is targeted fromthe plasma membrane to the secretory pathway of intoxicated cells. ThusK28 represents a powerful tool and substrate for general studies ofendocytosis and endosomal trafficking in eukaryotic cells. To elucidate thetrafficking route of the toxin, biologically active K28/mCherry fusionproteins as well as inactive controls were expressed in Pichia pastoris andused to track the toxin′s in vivo binding to the yeast cell and transit throughthe endocytic pathway. Another approach includes the investigation of theGFP-tagged toxin receptor Erd2p with the help of TIRF microscopy. Erd2pmobility in wild-type and endocytic mutants was compared quantitatively.CBP007A bacterial dynamin-like protein promotes magnesiumassisted membrane fusionF. Buermann, N. Ebert, S. van Baarle, M. Bramkamp*Institute of Biochemistry, University of Cologne, Cologne, GermanyMembrane dynamics are of fundamental importance for all cells.Dysfunction of membrane remodeling in mitochondria plays a role at theonset of virtually all neurodegenerative diseases and hence detailedmolecular understanding of membrane dynamics are of great importance.Mitochondria are dynamic organelles that undergo constant fusion andfission events which require membrane remodeling events catalyzed by agroup of large GTPase, dynamin-related proteins (DRPs). However, theexact biochemical details as to how DRPs catalyze membrane remodelingremain largely elusive. The inner membrane of mitochondria is homologousto the cytoplasmic membrane of heterotrophic bacteria. Not surprisinglymany homologous proteins involved in vital mitochondrial processes arealso found in bacterial membranes. Strikingly, the dynamin superfamily isnot restricted to eukaryotes, but has bacterial origin with many speciescontaining an operon coding for two genes of the mitofusin class ofdynamins. Our lab uses the bacterium Bacillus subtilis as a model system tostudy membrane dynamics. In this organism we identified a bacterial DRP,DynA that is homologous to the mitofusin branch of the DRPs. DynA ofBacillus subtilis is remarkable in that it arose from a gene fusion. Usingpurified, recombinant protein we were able to study dynamin-relatedfunctions such as membrane association and lipid-binding. We found thatDynA exhibits cooperative GTP hydrolysis and that self-interaction ismodulated by both dynamin subunits, which in turn only allow homotypiccontacts. DynA is able to tether adjacent membranes via one of its dynaminsubunits. Strikingly, DynA catalyzes fusion of synthetic vesicles in vitro,spektrum | Tagungsband 2011