BZH_Report_U1_RZ.indd 3 Biochemie-Zentrum der Universität Heidelberg RESEARCH AND EDUCATION IN MOLECULAR LIFE SCIENCES REPORT 2008–2010 RESEARCH AND EDUCATIONRESEARCH AND SCIENCES LIFE MOLECULAR IN HEIDELBERG UNIVERSITY BIOCHEMISTRY UNIVERSITY CENTER HEIDELBERG REPORT 2008–2010 01.03.2011 10:26:40 Uhr Biochemie-Zentrum der Universität Heidelberg (BZH) Im Neuenheimer Feld 328 D-69120 Heidelberg Germany

Phone: +49 (0)6221 54 4154 Fax: +49 (0)6221 54 5356 www.bzh.uni-heidelberg.de

Director: Prof. Dr. Michael Brunner

Editors: Prof. Dr. Irmgard Sinning Dipl.-Kfr. Catarina Vill-Härtlein

Layout: Dipl.-Kfr. Catarina Vill-Härtlein Cover: Dipl.-Grafik-Designerin Anke Heinzelmann

For a copy of this report please contact: Barbara Bohne (BZH-Administration) e-mail: [email protected] Introduction 4

Research Groups

Michael Brunner 6

Elisabeth Davioud-Charvet 10

Tamás Fischer 12

Ed Hurt 14

Wilhelm Just 18

Martin Koš 20

Luise Krauth-Siegel 22

Johannes Lechner 24

Dimitris Liakopoulos 26

Walter Nickel 28

Heiner Schirmer 30

Irmgard Sinning 32

Thomas Söllner 36

Frank Weber 38

Felix Wieland / Britta Brügger 40

Teaching at the BZH 44

Facilities 46

Funding 50

Theses 54

Publications 56

Staff 64

Scientific Advisory Board 68

How to get to the BZH 71 Welcome to the BZH!

Virtually all cellular functions are maintained by biological machines, which consist of macromolecular proteinaceous assemblies and nucleic acid/protein particles. The biogenesis and structure of such mo- lecular machines, as well as their function, regulation and interaction are in the focus of research at the Heidelberg University Biochemistry Center) (BZH). Biological processes studied at the BZH include the biogenesis of ribosomes, molecular mechanisms of protein translocation into the endoplasmic reticulum and the biogenesis of membrane proteins, the analysis of the machinery for vesicular transport and un- conventional secretion of proteins, as well as the spatial and temporal dynamics of molecular components of the circadian clock, which controls the day-night rhythm of cells. In addition, research groups are con- cerned with the biochemical characterization of plasmodia and trypanosomes.

The BZH is a central institution for research and teaching. It was founded 1997 and today accommodates 14 research groups. Amongst these are 4 junior groups and a further junior group is presently being re- cruited. Altogether the BZH hosts about 200 scientific and non-scientific coworkers. More than 70% of the scientists are funded from external sources.

Since its establishment the BZH has developed into a leading research establishment in the area of mo- lecular life sciences. A modern department structure with a flat hierarchy and complementary interests of research creates a lively atmosphere. The group leaders at the BZH pursue their individual research interests. These topics are, however, embedded into a general main topic, to develop synergies and to allow for a meaningful use of resources.

Such synergies become evident in the research cooperatives that are hosted by the BZH. Felix Wieland is the coordinator of a large collaborative research center (SFB 638) and Thomas Söllner, who has a chair at the BZH since 2005, is the coordinator of an SFB/Transregio (TRR83) with participation of 17 research groups in Bonn, Dresden and Heidelberg.

Research in molecular life sciences is subject to rapid technological progress and requires elaborate and usually extremely expensive machinery. The BZH has an excellent infrastructure, offering state of the art equipment in a wide range of leading-edge technologies. The center accommodates under the supervision of Britta Brügger a mass spectrometry facility for the qualitative and quantitative analysis of lipids, and Johannes Lechner leads a mass spectrometry facility for protein analysis. The BZH hosts under the direction of Irmi Sinning an automated facility for protein crystallization, which was supported by the CellNetworks cluster of excellence. Research groups within and outside of the BZH use this unit that is operated by Dr. Jürgen Kopp. Furthermore, facilities at the BZH offer confocal light-microscopy and fluorescence activated cell sorting (FACS). Finally, we are presently establishing a facility for electron microscopy.

4 Introduction The BZH is responsible for teaching Biochemistry in Medicine, Biology and Chemistry, and in particular en- gages in excellent training of the next generation of molecular life scientists with emphasis in Biochemistry. Annually, about 1000 students in the fields mentioned are being trained, and approximately 70 graduate students are working at the BZH on the average. For all graduate students participation is mandatory in an extensive BZH internal graduating program, which is coordinated with HBIGS, an international graduate school supported by the German Excellence Initiative. Within the framework of this program the gradu- ate students have the possibility of learning latest techniques and methods. Starting from 2012, the BZH will host a Bachelor and a consecutive Master program in Biochemistry, which is jointly offered by the Faculties for Chemistry and for Life Sciences.

Due to its internal structure and imbedding into the research landscape in Heidelberg, the BZH is well set up to ensure also for the future excellent training of the next generations of scientists and to conduct research at an internationally competitive level.

I hope this brochure captures your attention and inspires your view on our activities in research and teaching at the BZH.

Prof. Dr. Michael Brunner

Director, BZH Heidelberg, 30.01.2011

Introduction 5 1988 Ph.D. - University of Heidelberg, Germany 1989 - 1991 PostDoc - Princeton University and Rockefeller Research Laboratory, New York, USA (Prof. James E. Rothman) 1992 - 1998 Group Leader and habilitation - Ludwig-Maximilians- Universität München, Germany (Prof. Walter Neupert) 1998 - 2000 Professor - Ludwig-Maximilians-Universität München, Germany since 2000 Full Professor - BZH since 2010 Director - BZH

Michael Brunner

The Molecular Clock of Neurospora crassa

Goal of clock-controlled genes (ccgs). Amongst the Circadian clocks are timekeeping devices genes directly controlled by the WCC are the that measure time on a molecular level and clock genes frequency (frq) and vivid (vvd). FRQ coordinate the temporal organization of glob- and VVD are circadian repressors that inhibit al gene expression. The endogenous cell-au- their own synthesis in negative feedback loops tonomous pacemakers are synchronized via by regulating the activity and abundance of the various signal transduction pathways with WCC in a rhythmic fashion. FRQ is in complex the exogenous geophysical 24 h day/night with the RNA helicase FRH and casein kinase 1a cycle. The molecular mechanisms underlying (CK-1a) and it inactivates the WCC by facilitating these phenomena are in the focus of our re- its phosphorylation by CK-1a and CK-2. FRQ search. also support accumulation of high levels of the WCC. FRQ is phosphorylated at more than 100 Background sites, which regulates turnover and function of the Circadian clocks are cell-autonomous oscillatory clock protein in complex manner. Several kinases systems that modulate rhythmic expression of and phosphatases - e.g. CK-1a, CK-2, PP1 and a large number of genes. In eukaryotes these PP4 – have been implicated in the control of its clocks are based on networks of interconnected phosphorylation status. transcriptional, translational and posttranslational feedback loops. Circadian clocks are synchronized The WCC is composed of the subunits WC-1 and with the exogenous day by environmental cues WC-2. WC-1 is a blue-light photoreceptor that such as light and temperature. In the absence of contains a flavin-binding LOV domain. Hence, the entraining cues clock-specific oscillations persist WCC can be activated by light. Light-activation with an intriguingly precise period that creates is required for synchronization of the clock with an endogenous robust self-sustained subjective external light/dark cycles. The activation of light- day-night rhythm of approximately 24 h. induced gene expression is a transient process. After an initial burst of transcription the levels of In the core of the Neurospora clock is the light-induced RNA decrease despite the presence transcription factor White Collar Complex (WCC), of continuous light and reach a steady state after which directly and indirectly activates transcription 1-2 h. The photoadaptation of light-induced gene

6 Michael Brunner expression is facilitated by Vivid (VVD), which hypophosphorylated FRQ rapidly shuttles is also a LOV domain containing blue-light between the cytosol and nuclei and equilibrates photoreceptor. between both compartments. In the course of the day FRQ is progressively hyperphosphorylated, Research Highlights which slows down its import kinetics but does not Circadian abundance and activity of FRQ and affect nuclear export. Accordingly FRQ gradually WCC accumulates in the cytosol and it is subsequently Recent fi ndings of our lab revealed the mechanism degraded in the course of the (subjective) night. underlying the circadian activity rhythm of the The circadian abundance rhythm of FRQ and WCC, which is the basis of timekeeping on a its progressive accumulation in the cytosol molecular level. We found that the subcellular facilitate rhythmic modulation of the localization distribution of FRQ and the WCC is highly and the activity of the WCC. FRAP analysis of dynamic and regulated by phosphorylation. GFP labelled WCC revealed that the circadian The nuclo-cytoplasmic distribution of FRQ transcription factor is also shuttling in the range changes in the course of a circadian day. FRQ of minutes between cytosol and nuclei. In the late synthesis starts in the early (subjective) morning subjective night, when FRQ levels are at their of a circadian period and newly synthesized, trough, the WCC is hypophosphorylated and rapid

Fig. 1: Interdependent life- and nucleo-cytoplasmic shuttling cycles of FRQ and WCC. Hyperphosphorylated, inactive WCC is activated by PP2A- (and very likely PP4) dependent dephosphorylation in the cytosol and then imported into the nucleus. Active WCC binds to frq and other target genes and activates their expression. DNA binding destabilizes WCC and leads to rapid degra- dation of the complex. Newly synthesized, hypophosphorylated FRQ forms a stable complex with CK-1a and FRH, accumulates in the nucleus and inactivates WCC by supporting its phosphorylation by CK-1a and CK2. Priming phosphorylation of the WCC by PKA is required for subsequent casein kinase mediated phosphorylation. Inactivated, hyperphosphorylated WCC is exported to the cytosol, dephosphorylated and rapidly re-imported. Thus, the steady state concentration of active, nuclear WCC is high. The inhibitory FRQ/FRH/CK-1a complex (FFCC) also shuttles rapidly between cytosol and nucleus in a phosphorylation dependent manner. Over the course of a circadian day FRQ is progressively phosphorylated and import of the FFCC is throttled. Therefore, late in the circadian cycle FRQ accumulates in high levels in the cytosol: Cytoplasmic PP2A- and PP4-dependent dephosphoryla- tion of the WCC is antagonized by FRQ-dependent phosphorylation via CK-1a and CK2, resulting in a delay in WCC reactivation and nuclear import. Progressive, sequential and clustered phosphorylation eventually leads to the degradation of FRQ, and levels of hypophosphorylated WCC rise again. Hence, phosphorylation-dependent abundance cycles of FRQ and shuttling kinetics con- trol the steady state levels of active nuclear WCC in a circadian manner.

Michael Brunner 7 nuclear import dominates kinetically over export. regulator Vivid (VVD). In the dark, the WCC is a Hypophosphorylated WCC is active and supports protomer consisting of a WC-1 and a WC-2 subunit. transcription of frq and other ccgs. Binding of We have shown that light triggers dimerization of the WCC to its target promoters triggers rapid the WCC protomers via the activated LOV-domain degradation of the transcription factor. Hence, of their WC-1 subunits. The activated WCC binds at this circadian time the WCC is predominantly to light responsive elements (LREs) and induces nuclear; it is active and unstable. When FRQ expression of the corresponding target genes. levels rise in the subjective morning it promotes One of theses genes is vvd. When light activated phosphorylation of the nuclear WCC by CK-1a. VVD accumulates it disrupts and inactivates the Phosphorylation interferes with DNA binding of WCC homo-dimers by the competitive formation the WCC. The inactivated phosphorylated WCC of WCC-VVD hetero-dimers (Fig 2A). This leads is stable and exported into the cytosol. Cytosolic to photoadaptation of light-activated transcription WCC is then rapidly dephosphorylated by (Fig 2A, box). During the day, the expression PP2a, which reactivates the transcription factor, levels of VVD correlate with light intensity, which and re-imported into the nucleus. Since the phosphatase is exclusively cytosolic, passage of the WCC through the cytosol in obligatory for its reactivation. Later in the circadian day, when high levels of FRQ have accumulated in the cytosol, the WCC is also phosphorylated in the cytosol, a reaction competing with dephosphorylation of the WCC by PP2A. This slows down the reactivation and import of the WCC into the nucleus. Thus, hyperphosphorylated, inactive and stable WCC tends to accumulate in the cytoplasm. Finally, as FRQ is degraded in the course of the night, the kinetics of phosphorylation of the WCC decreases and dephosphorylation of the WCC by cytosolic PP2A dominates and the re-activated WCC is imported into the nucleus. In summary, our data suggest that rapid nucleo- cytoplasmic shuttling cycles of the WCC are coupled to phosphorylation-dephosphorylation cycles that regulate WCC activity. The kinetics of theses cycles is modulated by FRQ on a circadian time scale (Fig 1), which thereby generates a daily Fig. 2: Molecular mechanism of light-dependent activation of the WCC and photoadaptation. (A) Light treatment of mo- activity rhythm of WCC. nomeric dark state WCC (red hexagons indicate dark state LOV domain) results in formation of the photoadduct (orange hexagons) and dimerization. vvd transcription is activated and VVD protein accumulates. VVD forms a photoadduct and can either homodimerize or heterodimerize with WCC, there- Light entrainment of the clock by competing with the assembly of the WCC light complex. Box: WCC-dependent gene expression (black line) rapidly in- Light responses and photoadaptation of creases upon light induction. Transcription adapts to steady state levels after an initial peak. Increase of light intensity Neurospora are dependent on the photosensory (red line) results in a second peak of transcription followed by adaptation of gene expression on slightly elevated levels. (B) light-oxygen-voltage (LOV) domains of the VVD provides a memory of the preceding daylight intensity, thereby protecting the circadian clock from getting reset by circadian transcription factor WCC and its negative disturbing light cues during the night, e.g. by the moon.

8 Michael Brunner Fig. 3: Genome-wide identification of WCC-controlled genes. Chromatin immunoprecipitation followed by deep sequencing re- vealed more than 400 binding sites of the WCC in the Neurospora genome. Genes near WCC binding sites fall into diverse func- tional categories.

allows photoadaptation over several orders of WCC, the key transcription factor of the circadian magnitude. At night, the previously synthesized clock. VVD serves as a molecular memory of the brightness of the preceding day and suppresses Selected Publications 2008 - 2010 responses to light cues of lower intensity (Fig 2B). Tataroğlu, O. and Schafmeier, T. (2010) Of switches and hourglasses: regulation of subcellular traffi c in circadian clocks We found that VVD is essential to discriminate by phosphorylation. EMBO Rep 11, 927-935. between day and night, in particular in naturally Malzahn, E., Ciprianidis S., Kaldi, K., Schafmeier, T. and Brunner, M. (2010) Photoadaptation in Neurospora by ambiguous photoperiods with moonlight. competitive interaction of activating and inhibitory LOV domains. Cell 142, 762-772. Smith, K.M., Sancar, G., Dekhang, R., Sullivan, C.M., Li, S., Gemome-wide analysis of WCC-controlled Tag, A.G., Sancar, C., Bredeweg, E.L., Priest, H.D., Mccormick, R.F., Thomas, T.L., Carrington J.C., Stajich, J.E., Bell-Pedersen, genes D., Brunner, M. and Freitag, M. (2010) Transcription factors in light and circadian clock signaling networks revealed by Light signaling has profound effects on the genome-wide mapping of direct targets for neurospora white collar complex. Eukaryot Cell 9, 1549-1556. development and behavior of Neurospora. We Diernfellner, A., Querfurth, C., Salazar, C., Höfer, T., Brunner, M. (2009) Phosphorylation modulates rapid nucleo-cytoplasmic used ChIP-sequencing to uncover direct targets of shuttling and cytoplasmic accumulation of Neurospora clock protein FRQ on a circadian time scale. Genes Dev 23, 2192- the WCC. We found that the light-activated WCC 2200. binds to hundreds of regions, including promoters Sancar, G, Sancar, C, Brunner M and Schafmeier, T (2009) Activity of the circadian transcription factor White Collar of known clock and light-regulated genes. Complex is modulated by phosphorylation of SP-motifs. FEBS Lett 583, 1833-1840. Amongst the genes activated by the WCC are 28 Schafmeier, T, Diernfellner, A, Schäfer, A, Dintsis, O, Neiss, transcription factor genes (Fig 3). Transcription of A, and Brunner, M (2008) Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC most, but not all, WCC target genes is induced associated with rapid nucleo-cytoplasmic shuttling. Genes Dev 22, 3397-3402. by light. Our fi ndings provide links between WC-2 Neiss, A, Schafmeier, T and Brunner, M (2008) Transcriptional regulation and function of the Neurospora clock gene white and effectors in downstream regulatory pathways collar-2 and its isoforms. EMBO Rep 9, 788-794. for light-induced behavior. Our data suggest a "fl at" hierarchical network in which 20% of all Michael Brunner Phone: +49 (0)6221-54 4207 annotated Neurospora transcription factors are E-mail: [email protected] regulated during the early light response by the

Michael Brunner 9 1985 - 2001 Ph.D. in Pharmaceutical and Chemical Sciences - Paris 11 University, France and Senior Scientist - Centre National de la Recherche Scientifi que (CNRS), France 2001 - 2002 Visiting Scientist - University of Michigan, USA

since 2001 Research Director - CNRS, France

since 2002 Group Leader - BZH

Elisabeth Davioud-Charvet

Drug Development against Disulfide Reductases from Parasites and Cancer Cells

Goal act as “Trojan horses” drugs consisting of a short Design, synthesis and mechanism of disul- chloroquine analogue – active against malaria per fide reductase inhibitors and redox-cyclers se – linked to a GR inhibitor. The strategy was also that affect the redox equilibrium of parasites validated in the current malaria project with new and cancer cells. functionalized low-weight 1,4-naphthoquinones derivatives belonging to the 3-benzylmenadione Background series. The redox-active compounds revealed The aim of our interdisciplinary research is to sub- potent antimalarial effects against chloroquine- stantiate NADPH-dependent disulfide reductase sensitive and -resistant strains of Plasmodium inhibitors as antiparasitic and cytostatic agents. falciparum in vitro and in mouse malaria models Such compounds are active per se but, in addition, (EP patent). A cascade of redox reactions for anti- they can reverse thiol-based resistance against malarial drug bioactivation involving both heme- other drugs in parasites and tumour cells. Our strategy is based on the synthesis of subversive red blood cell substrates or catalytic inhibitors, fluorine-based Plasmodium falciparum suicide-substrates, uncompetitive inhibitors, pho- toreactive inhibitors (as tools for photoaffinity la- GR beling studies) of the selected targets, namely the glutathione reductases (GR) of the malarial para- site Plasmodium falciparum and man, the thiore- doxin reductases (TrxR) of P. falciparum and man, the trypanothione reductase (TR) from Trypano- soma cruzi, and the thioredoxin-glutathione re- ductase (TGR) of Schistosoma mansoni.

Research Highlights Fig. 1: “Drowning Plasmodium in Redox” was achieved by 3-benzylmenadione derivatives with potent antimalarial Our strategy for inhibitor optimization is based on effects both in vitro and in vivo. Compounds with GR redox- cycling activity displaying the ability to reduce ironIII to ironII the design and the synthesis of dual drugs that from haemoglobin and heme species.

10 Elisabeth Davioud-Charvet NADPH, H+ NADP+ GR

O O OH [O] RedOx Cycle R R O O O OH O R benzylNQ benzoylNQ = reduced benzoylNQ biometabolite I = biometabolite II

2+ 3+ inhibition of Hb(Fe ) metHb(Fe ) hemozoin hemozoin formation PFIX(Fe2+) PFIX(Fe3+) formation and and trophozoite growth of development arrest the parasite

Fig. 2: Proposed cascade of redox reactions for bioactivation of antimalarial 3-benzyl menadione derivatives (benzylNQ). The re- action cascade in the Plasmodium-infected erythrocyte involves heme-catalyzed oxidations and glutathione reductase-catalyzed reduction leading to inhibition of P. falciparum trophozoite development and of hemozoin formation.

catalyzed oxidation reactions and the glutathione Wenzel, N. I., Chavain, N., Wang, Y., Friebolin, W., Maes, L., Pradines, B., Lanzer, M., Yardley, V., Brun, R., Herold- reductases from the Plasmodium-infected eryth- Mende, C., Biot, C., Katalin Tóth, Davioud-Charvet, E. (2010) Antimalarial versus Cytotoxic Properties of Dual Drugs rocyte was proposed to be involved in the action Derived From 4-Aminoquinolines and Mannich Bases: Interaction with DNA. J. Med. Chem. 53, 3214–3226. mechanism of the 3-benzylmenadione series. Chavain, N., Davioud-Charvet, E., Trivelli, X., Mbeki, The biometabolites were shown to act, in oxidized L., Rottmann, M., Brun, R., Biot, C. (2009) Antimalarial Antimalarial activities of ferroquine conjugates with either glutathione reductase inhibitors or glutathione depletors via form, as the most efficient subversive substrates a hydrolyzable amide linker. Bioorg. Med. Chem. 17, 8048– of both glutathione reductases of Plasmodium- 8059. Wenzel, I. N., Wong, P. E., Maes, L., Müller, T. J. J., Krauth- infected erythrocytes described so far, and, in Siegel, L. R., Barrett, M., Davioud-Charvet, E. (2009) Antitrypanosomal unsaturated Mannich bases active against reduced form, to redox-cycle methemoglobin to multidrug-resistant T. brucei brucei strains. ChemMedChem. 4,339-351. hemoglobin. Ultimately, the antimalarial naph- Davioud-Charvet E., Müller, T., Bauer, H., Schirmer, H. thoquinones are suggested to affect the redox 1,4-Naphthoquinones as Inhibitors of Glutathione Reductases and Antimalarial Agents. European Patent EP 08290278.4 equilibrium of target cells resulting in trophozoite (March 26, 2008). PCT/EP2009/053483 (25-03-2009). WO/2009/118327 (01.10.2009). development arrest, by drowning the parasite in Viry, E., Battaglia, E., Deborde, V., Müller, T., Réau, R., Davioud-Charvet, E., Bagrel, D. (2008) A sugar-modified its own metabolic products. For leishmania and phosphole gold complex with antiproliferative properties acting as a thioredoxin reductase inhibitor in MCF-7 cells. trypanosomes various unsaturated ketones de- ChemMedChem. 3, 1667-1670. rivatives acting as trypanothione-reactive agents Müller, T., Müller, T.J.J., Davioud-Charvet, E. (2008) Synthesis of photo-reactive naphthoquinones for photoaffinity labeling were produced and revealed potent trypanocidal of glutathione reductases. In Flavins and Flavoproteins 2008, 16, 443–452. Frago, S., Gómez-Moreno, C., Medina, M., Eds. effects against pentamidine-sensitive and -resist- Prensas Universitarias de Zaragoza, Spain, 2008. ant strains of Trypanosoma and Leishmania spe- Morin, C., Besset, T., Moutet, J.-C., Fayolle, M., Brückner M, Limosin, D., Becker, K., Davioud-Charvet, E. (2008) The Aza- cies in vitro. Current efforts include the chemis- Analogues of 1,4-Naphthoquinones are potent Substrates and Inhibitors of Disulfide Reductases. Org. Biomol. Chem. try of prodrugs of 1,4-naphthoquinones and bis 6, 2731-2742. (Michael acceptors) derivatives as antiparasitic drug-candidates (against malaria, trypanosomia- Elisabeth Davioud-Charvet* sis, and schistosomiasis), biochemical and en- *delegate of CNRS, in the frame of a German French cooperation with the University of Heidelberg, zymic studies on the mechanism and the regula- Germany tion of disulfide reductase in vivo. Phone: +49 (0)6221-54 4293 E-mail: [email protected]

Selected Publications 2008 - 2010 Our work is supported by:

Davioud-Charvet, E., Lanfranchi, D. A. Subversive sub- strates of glutathione reductases from P. falciparum-infected red blood cells as antimalarial agents. In K. Becker and P. Selzer (Eds.) Drug Discovery against Apicomplexan para- sites – Molecular approaches to targeted drug development, in the series “Drug Discovery in Infectious Diseases”. Wiley, 2010. In press.

Elisabeth Davioud-Charvet 11 2005 Ph.D. - University of Heidelberg, Germany

2005 - 2006 PostDoc - BZH (Prof. Ed Hurt)

2006 - 2010 PostDoc -National Cancer Institute, NIH, MD, USA (Dr. Shiv Grewal)

Since 2010 Junior Group Leader - BZH

Tamás Fischer

Epigenetics and Genomic Stability

Goal The main focus of the research in our laboratory is: To understand how epigenetic mechanisms • to understand the role of chromatin and its contribute to genome organization, mainte- modifying activities in genomic indexing; nance of genomic stability and chromosome • to understand the link between cryptic transcript segregation. accumulation and genomic instability and how it contributes to cancer development. Background A large portion of the eukaryotic transcriptome Research Highlights consists of non-protein-coding RNA transcripts Recently developed techniques give detailed and (ncRNAs or cryptic transcripts), but the function sensitive genome-wide maps of transcription ac- and significance of this widespread ncRNA tran- tivity, including the intergenic and antisense por- scription is not understood. The majority of these tion of the genome. We are using high resolution cryptic transcripts are recognized and quickly de- tiling arrays in combination with high-throughput graded by the RNA surveillance machinery. De- sequencing technology to obtain an unbiased pic- fects in the recognition and degradation of cryptic ture of eukaryotic transcriptional activity through- transcripts or increased transcriptional activity out the genome. With the help of these techniques outside of transcription units can lead to toxic ac- we have identified mutations in the fission yeast S. cumulation of these transcripts. But how do ge- pombe that lead to cryptic transcript accumulation. nomic indexing mechanisms define transcription Currently we are focusing on these mutations and units and their transcripts? The answer to this trying to understand the molecular mechanisms question lies in chromatin structure and its modi- behind the observed phenotypes. (Figure) fying activities. Combinations of epigenetic marks We have found that accumulation of ncRNAs is provide a complex indexing mechanism for defin- associated with genomic instability and sensitiv- ing transcription units. Defects in such epigenetic ity to DNA damage. Furthermore, we discovered indexing could lead to cryptic transcript accumu- that RNase H, an enzyme that degrades DNA- lation and to genomic instability. RNA hybrids, is important for the maintenance

12 Tamás Fischer Fig. 1: Genomic view of ncRNA-accumulating S. pombe mutants. Yellow bars represent increased RNA levels compared to wild type expression levels, while blue bars represent a decrease.

of genomic stability, and essential in the isolated Selected Publications 2008 - 2010

ncRNA-accumulating mutants. These results Zofall, M.*,T. Fischer*, K. Zhang, M. Zhou, B. Cui, T.D. Veenstra, S.I. Grewal. (2009). “Histone H2A.Z cooperates suggest that ncRNAs form DNA-RNA hybrids with RNAi and heterochromatin factors to eliminate anti- sense RNAs.” Nature 461(7262):419-22 (*These authors with their DNA template, which can lead to rep- contributed equally). lication fork collapses and consequently to DNA Fischer, T., B. Cui, J. Dhakshnamoorthy, M. Zhou, C. Rubin, M. Zofall, T.D. Veenstra, S.I. Grewal. (2009). “Diverse roles lesions and genomic instability. We would like to of HP1 proteins in heterochromatin assembly and functions in fission yeast.” PNAS 106(22):8998-9003. further study the molecular mechanisms leading Roguev, A., S. Bandyopadhyay, M. Zofall, K. Zhang K, T. Fischer, S.R. Collins, H. Qu, M. Shales, H.O. Park, J. Hayles, to genomic instability, and the in vivo function of K.L. Hoe, D.U. Kim, T. Ideker, S.I. Grewal, J.S. Weissman, N.J. Krogan. (2008). “Conservation and Rewiring of Functional RNase H in eukaryotic genome organization. Modules Revealed by an Epistasis Map in Fission Yeast.” Science 322(5900):405-410.

RNA may play a more significant role in nuclear processes than previously imagined. These stud- Tamás Fischer Phone: +49 (0)6221-54 4728 ies will increase our general understanding of ge- E-mail: [email protected] nomic organization, transcriptional regulation and the biological significance of ncRNAs in the eu- karyotic cell. Mutations leading to genomic insta- bility are a major cause of cancer development, which highlights the importance of studying the molecular mechanisms behind this process.

Tamás Fischer 13 1983 Ph.D. - University of Regensburg, Germany 1984 - 1986 PostDoc - Biocenter, Basel, Switzerland (Prof. G. Schatz) 1986 - 1994 Group Leader - European Molecular Biology Laboratory (EMBL) Heidelberg, Germany, Cell Biology 11.07.1990 Habilitation in Biochemistry - University of Regensburg, Germany since 1995 Full Professor - BZH 2003 - 2005 Director - BZH

Ed Hurt

The nuclear pore complex and its link to mRNP and ribosome biogenesis

Goal and metazoans, are believed to be the building Our group performs research with the goal blocks of the NPC. Currently, the reconstitution of to elucidate the structure and function of the these modules is a major challenge in this field. nuclear pore complex and the mechanism of Nuclear mRNA export depends on the formation how mRNPs and ribosomal subunits form of transport-competent mRNPs that leave the in the nucleus and are exported to the cyto- nucleus through nuclear pore complexes (NPCs). plasm. Transcription-export complexes (TREXs) com- posed of factors, which fucntion in transcription Background and mRNA export, were discovered in the past. Nuclear pore complexes (NPCs) are the sole me- These findings indicated that mRNA export fac- diator of transport between the nucleus and the cy- tors can be loaded onto the nascent mRNA dur- toplasm. Embedded into the double nuclear mem- ing transcription. In addition, it has been shown brane, this huge assembly exhibits an eight-fold that a gene locus can be tethered to the nuclear rotational symmetry with distinct substructures, envelope to either promote transcription or couple including the spoke-ring complex, cytoplasmic transcription with mRNA processing and export. pore filaments and the nuclear basket. The core The identification of Sus1 that is assembled into structure also contains a central channel through two complexes, a transcription complex (SAGA) which nucleocytoplasmic transport occurs. The and an NPC-associated export complex termed NPC consists of multiple (8, 16 or 32) copies of ~ TREX-2, suggested a physical coupling of acti- 30 different proteins named nucleoporins. While vated genes to the nuclear side of the NPC. a few of them are located asymmetrically on ei- Eukaryotic ribosome formation in the nucleus is a ther the nucleoplasmic or cytoplasmic side, most highly dynamic process, which involves the tran- of the nucleoporins are distributed symmetrically sient interaction of more than 200 non-ribosomal within the core structure of the NPC. The major- factors with the evolving pre-ribosomal particle in ity of nucleoporins are part of discrete and stable the nucleus. Biogenesis and export of ribosomal subcomplexes, which arrange in a still unknown subunits has been analyzed in the past with the way within the NPC scaffold. These conserved help of functional GFP-tagged ribosomal proteins, complexes, which have been described for yeast which served as reporters to perform genetic

14 Ed Hurt screens for ribosomal export mutants. However, it in collaboration with Peer Bork’s group (EMBL these studies not only yielded ribosome export Heidelberg). Comparison of the thermophile pro- factors, but also a number of biogenesis factors, teome with several proteomes of closely related which act ‘upstream’ of ribosome export. Moreover, mesophilic filamentous fungi gave insight into isolation of pre-ribosomal particles along the path eukaryotic protein adaptation towards thermoph- from the nucleolus to the cytoplasm yielded bio- ily. Subsequently, we used a model protein, Arx1 chemical “snapshots” of the dynamic nascent 60S from C. thermophilum (ctArx1) and its mesophilic and 40S subunits. Subsequently, a few of these counterpart C. globosum (cgArx1) to demonstrate pre-ribosomal particles were analyzed by EM. In their different thermostabilities, corresponding to addition, in vitro assays were developed, which the optimal growth temperatures of these organ- allowed to monitor pre-ribosome maturation both isms. The crystal structure of ctArx1 revealed the by structural and biochemical means. The chal- position of residues possibly contributing to ther- lenge in this field remains to assign roles to these mo-adaptation (Figure 1). Subsequently, several ca. 200 ribosome biogenesis factors. other thermophilic proteins were expressed (also in collaboration with other labs interested in ther- Research Highlights mophilic orthologs), which in many cases allowed Despite numerous efforts to elucidate the archi- to perform successful biochemical and structural tecture and function of the NPC, the principles studies, including in vitro reconstitution, electron that govern the assembly of the nucleoporins microscopy and X-ray crystallography. into the NPC remain poorly understood. The ma- In addition, we exploited the thermophilic Nups for jor obstacle is the purification of nucleoporins in vitro reconstitution. For these studies, we used in sufficient amounts to perform reconstitution information from a comprehensive yeast 2-hybrid studies. We have chosen the thermophilic eu- analysis performed with yeast Nups, which not karyote Chaetomium thermophilum, a filamen- only recapitulated some of the known interac- tous ascomycete with a growth optimum at 55°C, tions, but also revealed novel Nup connections. to gain access to the entire set of thermostable Notably, nucleoporins derived from the thermo- nucleoporins, which may have superior biochemi- philic eukaryote revealed excellent properties in cal and structural properties when compared to binding studies, since ctNups could be purified the mesophilic orthologs. Hence, we sequenced in significantly higher amounts and exhibited the genome of C. thermophilum and annotated increased thermosolubility when compared to

Fig. 1: Thermostability of Arx1 from C. thermophilum and C. globosum. a, Purifi ed Arx1 proteins were incubated for one hour at the indicated temperatures, before centrifugation into supernatant (S) and pellet (P). b, Crystal structure of ctArx1 with indicated residues possibly involved in thermostability.

Ed Hurt 15 their yeast counterparts. Using several structural In our projects related to transcription-coupled ctNups, whose yeast orthologs were difficult to mRNA export, we could provide structural in- handle, we could isolate large amounts and per- sights into the machineries, which operate at form successful binding and EM studies (Figure the interface between transcription and mRNA 2). Moreover, we could reconstitute a long-sought export. In collaboration with the Stewart group after NPC subcomplex with these thermophilic (MRC, Cambridge) we reconstituted and solved Nups that was not possible to achieve with the the crystal structure of a subcomplex of TREX-2, mesophilic Nups from yeast. which contained Sus1, Cdc31 and Sac3-CID (CID, Cdc31 Interacting Domain) and could serve as a scaffold to coordinate the interactions between transcription and mRNA export machineries at the NPC. In collaboration with the Zheng lab (Seattle, USA) we gained structural insight into another Sus1-containing complex, the Sus1-Sgf11-Ubp8- Sgf73 module, which is part of SAGA and acts as a histone H2B de-ubiquitination (DUB) complex Fig. 2: EM and 3D reconstruction of ctNup170 (Figures 3). Altogether, these findings highlight In future studies, we will include further thermo- the versatile nature of the small Sus1 molecule to philic Nups in our assembly tests to eventually act as a clamp, either as a co-factor of the histone reconstitute the entire NPC. These investigations DUB module or as a targeting device to tether could also foster the development of this thermo- TREX-2 to the NPC. philic eukaryote as a model organism for the re- During our studies to investigate the mechanisms constitution and structural determination of large of ribosome biogenesis, we obtained insight into eukaryotic supramolecular assemblies, which the function of a mechanoenzyme, the Rea1 AAA- are otherwise difficult to purify from mesophilic type ATPase, which is involved in ATP-hydrolysis organisms. dependent removal of factors from the pre-60S

Fig. 3: . Crystal Structure and model of the Ubp8-Sgf73-Sgf1-Sus1 (DUB) module.

16 Ed Hurt Thorsten Schäfer, David Tollervey and Ed Hurt: RNA helicase Prp43 and its co-factor Pfa1 promote 20S to 18S rRNA processing catalyzed by the en- donuclease Nob1. J. Biol. Chem. 284, 35079-35091 (2009). Cornelia Ulbrich, Meikel Diepholz, Jochen Baßler, Dieter Kressler, Brigitte Pertschy, Kiki Galani, Bettina Böttcher and Ed Hurt: Mechanochemical Removal of Ribosome Biogenesis Factors from Nascent 60S Ribosomal Subunits. Cell 138, 911-922 (2009). Christoph Klöckner, Maren Schneider, Sheila Lutz, Dieter Kressler, Divyang Jani, Murray Stewart, Ed Hurt and Alwin Köhler: Mutational Uncoupling of Sus1’s role in NPC-targeting of an Fig. 4: . Model of Rea1 function during 60S ribosome biogenesis. mRNA Export Complex and Histone H2B deubiquitination. J. Biol. Chem. 284, 12049-12056 (2009). ribosome. Rea1 consists of an AAA-ATPase Divyang Jani, Sheila Lutz, Neil J. Marshall, Tamas Fischer, head and a long flexible tail, both of which can Alwin Köhler, Andrew M. Ellisdon, Ed Hurt and Murray Stewart: Sus1, Cdc31 and the Sac3 CID region form a con- dock to the pre-ribosomal particle. Subsequently served interaction platform that promotes nuclear pore asso- ciation and mRNA export. Mol. Cell 33, 727-737 (2009). the molecular motor uses ATP to build up a ten- Michal Skruzny, Claudia Schneider, Attila Rácz, Julan Weng, sile force. This force can be compared to a spiral David Tollervey and Ed Hurt: An endoribonuclease function- ally linked to perinuclear mRNP quality control associates with the nuclear pore complexes. spring and is transmitted to the ribosome precur- PLoS Biology 7, e8 (2009). sor via the tail (Figure 4). This force could release Dirk Flemming, Philipp Sarges, Philipp Stelter, Andrea Hellwig, Bettina Boettcher and Ed Hurt: Two structurally dis- late biogenesis factors (such as Rsa4 or the Rix1- tinct domains of the nucleoporin Nup170 cooperate to tether a subset of nucleoporins to nuclear pores. J. Cell Biol. 185, subcomplex) from the pre-ribosomal particles in 387-395 (2009). the nucleus, which makes the pre-ribosome com- Stefanie Grund, Tamas Fischer, Ghislain G. Cabal, Oreto Antúnez, José E. Pérez-Ortín and Ed Hurt: The inner nuclear petent for export to the cytoplasm. membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression. J. Cell Biol. 182, Recent work revealed that this same mecha- 897-910 (2008). noenzyme Rea1 is used twice in the ribosome Dieter Kressler, Daniela Roser, Brigitte Pertschy and Ed Hurt: The AAA-ATPase Rix7 powers progression of ribosome bio- genesis by stripping Nsa1 from pre-60S particles. J. Cell Biol. biogenesis pathway, acting also in the nucleolus 181, 835-844 (2008). to pull off other biogenesis factors from an earlier Alwin Köhler, Maren Schneider, Ghislain Cabal, Ulf Nehrbass and Ed Hurt: An integrative role of Sgf73 in multiple steps of pre-60S particle. Overall, these studies revealed SAGA-dependent gene gating. Nat. Cell Biol. 10, 707-15 (2008). mechanistic insight into the complex pathway of Nils Schrader, Philipp Stelter, Dirk Flemming, Ruth Kunze, Ed ribosome biogenesis and clarified the function of Hurt* and Ingrid Vetter* (*corresponding authors): Structural basis of the Nic96 subcomplex organization in the nuclear some of the participating factors. pore channel. Mol. Cell 29, 46-55 (2008). Wei Yao, Malik Lutzmann and Ed Hurt: A versatile interac- tion platform on the Mex67-Mtr2 receptor creates an over- Selected Publications 2008 - 2010 lap between mRNA and ribosome export. EMBO J. 27, 6–16 (2008). Jochen Baßler, Martina Kallas, Matthias Thoms, Cornelia Ulbrich, Brigitte Pertschy and Ed Hurt: The AAA-ATPase Rea1 drives removal of biogenesis factors during multiple stages of 60S ribosome assembly. Mol. Cell 38, 712-721 Awards and Honors (2010). 2007 Feldberg Prize Alwin Köhler, Eric Zimmermann, Maren Schneider, Ed Hurt and Ning Zheng: Structural basis for assembly and activation of the heterotetrameric SAGA histone H2B deubiquitinase 2001 Gottfried Wilhelm Leibniz Prize module. Cell 141, 606-617 (2010). Since 2010 Editorial Board of EMBO Journal Dirk Flemming, Karsten Thierbach, Philipp Stelter, Bettina Boettcher and Ed Hurt: Precise mapping of subunits in multi- Since 2007 Member of ACADEMIA EUROPAEA protein complexes by a versatile EM-label Nat. Struct. Mol. Biol.17,775-778 (2010). Since 2005 Member of LEOPOLDINA Julien Batisse, Claire Batisse, Aidan Budd, Bettina Böttcher Since 1994 Member of EMBO and Ed Hurt: Purification of poly(A)-binding protein Nab2 re- veals association with the yeast transcriptome and a mes- senger ribonucleoprotein (mRNP) core structure. J. Biol. Chem. 284, 34911-34917 (2009). Ed Hurt Phone: +49 (0)6221-54 4173 Brigitte Pertschy, Claudia Schneider, Maren Gnädig, E-mail: [email protected]

Ed Hurt 17 1970 - 1978 Group Leader - Max-Planck-Institute of Brain Research, Frankfurt/M., Germany since 1978 Group Leader - Institute of Biochemistry I, University of Heidelberg, Germany / BZH 1987 Habilitation in Biochemistry - University of Heidelberg 1994 - 2008 Professor - BZH

Wilhelm Just

Functions and Biogenesis of Peroxisomes

Goal The work of the group focused on two main topics (i) mechanisms involved in peroxisome proliferation and (ii) the physiological role of ether lipids (ELs) particularly plasmalogens (PLs).

Background New peroxisomes are formed by both budding Fig. 1: Peroxisomal constrictions at an early (A) and later (B) stage of tubulated peroxisomes. from the ER and autonomous division. Investigat- ing the latter process, we studied peroxisome- potentially implicated in peroxisome division cytoskeleton interactions and searched for com- include various Arf isotypes and distinct phos- ponents involved by proteomics, biochemical and phoinositide kinases and phosphatases (Fig. 2) immunofluorescence analyses. Furthermore, we (Grunau et al 2010). used the EL-deficient mouse recently generated We suggest that RhoA-GDP dissociates from per- in our laboratory as a model to study in vivo the oxisomes enabling microtubule-based peroxisom- functions of ELs in CNS and testis, two tissues al movements whereas RhoA-GTP targets to per- exhibiting severe phenotypic changes. oxisomes favoring ROCKII recruitment. ROCKII

Research Highlights Ultrastructural analysis of proliferating rat liver peroxisomes revealed formation of multiple con- strictions in tubular peroxisomes resembling a pre-segregational state (Fig. 1).

We identified actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 as Fig. 2: The model describes molecules implicated in new components associated with peroxisomes vesicular transport between peroxisomes and the ER (steps 1and 2) and autonomous peroxisome division (Schollenberger et al, 2010). Other components (steps 3 and 4).

18 Wilhelm Just activates the acto-myosin complex that supports biogenetic functions balancing peroxisome size, shape, number, and clustering (Schollenberger et al, 2010).

We continued to investigate the effects of PL de- ficiency in CNS and testis (Teigler et al, 2009; Komljenovic et al 2009). In CNS we found: (i) de- fects in foliation patterning and delay in precursor granule cell migration, (ii) defects in myelination and a concomitant reduction in the level of myelin basic protein, (iii) disturbances in paranode orga- nization by extending the Caspr distribution and disrupting axo-glial septate-like junctions, (iv) im- paired innervation of PCs by both parallel fibers and climbing fibers (CFs) (Fig. 3)

Fig. 4: Varicosities (A, B) in PC axons of P45 EL-deficient mice comprised of IP3R-containing smooth ER struc- tures (C, D) indicating axon degeneration. CB, calbindin staining.

ficient sealing of the intermediate compartment. These results demonstrate that ELs are essential for correct myelination, PC innervation and brain functioning as well as cyclic BTB dynamics ensur- ing the sluice mechanism for leptotene transloca- tion into the adluminal compartment (Komljenovic et al 2009).

Selected Publications 2008 - 2010 Grunau S, D. Lay, S. Mindthoff, H. W. Platta, W. Girzalsky, W.W. Just, and R. Erdmann. 2010. The Phosphoinositide-3- Kinase Vps34p is required for pexophagy in Saccharomyces cerevisiae. Biochem J. in press. Fig. 3: In mutants, CF synapses (green) reside on PC (red) somata (curved arrows) and proximal dendritic Schollenberger L, T. Gronemeyer, C. M. Huber, D. Lay, S. trunk (straight arrows) and occupy a severely restricted Wiese, H. E. Meyer, B. Warscheid, R. Saffrich, J. Peranen, area of PC innervation. K. Gorgas, W.W. Just. 2010. RhoA Regulates Peroxisome Association to Microtubules and the Actin Cytoskeleton. PLoS One, 5, e13886. and (v) formation of axon swellings by the accu- Teigler A, D. Komljenovic, A. Draguhn, K. Gorgas, W.W.Just. mulation of inositol-tris-phosphate receptor 1-con- 2009. Defects in myelination, paranode organization and Purkinje cell innervation in the ether lipid-deficient mouse taining smooth ER-like tubuli (Fig. 4) (Teigler et cerebellum. Hum Mol Genet. 18, 1897-1908. al, 2009). Komljenovic D, R. Sandhoff, A. Teigler, H. Heid, W.W. Just and K. Gorgas. 2009. Disruption of blood-testis barrier dy- namics in ether-lipid-deficient mice. Cell Tissue Res. 337, 281-299. In testis, EL deficiency blocks blood testis barrier (BTB) remodeling. This block is associated with Wilhelm Just down-regulation and mis-targeting of claudin-3 Phone: +49 (0)6221-54 4286 E-mail: [email protected] and impaired BTB disassembly resulting in de-

Wilhelm Just 19 1998 - 2002 Ph.D. - European Molecular Biology Laboratory (EMBL) Heidelberg, Germany and Charles University in Prague, Czech Republic

2002 - 2008 PostDoc - Wellcome Trust Centre for Cell Biology, University of Edinburgh, Great Britain

since 2008 Junior Group Leader - Excellence Cluster “CellNetworks”, BZH

Martin Koš

Ribosome biogenesis

Goal Research Highlights Our aim is to understand how ribosomal Mature rRNAs have a very complex structure RNAs are processed, correctly folded and that appears to be incompatible with assembly. assembled with proteins to form functional Furthermore, rRNAs are extensively modified ribosomes. by methylation and pseudouridylation at approxi- mately 100 sites in a process that requires base- Background pairing of small nucleolar RNAs (snoRNAs) to Ribosome biogenesis is a major energy consum- rRNAs. Several snoRNAs are also essential for ing process in all organisms that has to be tightly specific cleavages of pre-rRNA. The extent to regulated with regard to cell growth. This highly which snoRNAs influence/participate in folding of conserved process begins with transcription of pre-rRNAs and the order of modifications is un- a large ribosomal RNA (rRNA) precursor that is clear. Thus the need for RNA unwinding during subsequently covalently modified and processed ribosome synthesis seems obvious. Among the into mature 18S, 5.8S and 25S rRNAs (Figure 1). myriad factors required for ribosome synthesis Pre-rRNA processing takes place in very large are 18 putative ATP-dependent RNA helicases. particles (>2MDa) called pre-ribosomes. These The lab is investigating the early assembly steps molecular machines ensure that the rRNA is in ribosome synthesis, with specific focus on the properly processed, folded and assembled with mechanisms by which RNA helicases modulate ribosomal proteins. At least 180 non-ribosomal the structure of large RNA-protein complexes proteins and 70 small nucleolar RNAs (snoRNAs) such as ribosome. In addition, we are examining have been implicated in ribosome biogenesis in the order of snoRNAs mediated modifications, yeast. The process of ribosome maturation is their role and mechanisms involved. The insight very complex and highly dynamic; it takes only gained will shed light on their potential functions 6 minutes to make a functional mature ribosome. or RNA helicases and small RNAs in other RNA How cells achieve this efficiency and the precise related processes. function of most of the factors remains unclear. As mentioned earlier, ribosome biogenesis is a The goal of the lab is to extend our understanding large energy consumer in the cell and as such it of the molecular mechanism underlying ribosome is tightly regulated. Although the nucleolus is tra- biogenesis and its regulation. ditionally regarded as a “ribosome factory” a large

20 Martin Koš Fig. 1. Ribosome biogenesis in yeast.

amount of data now provides substantial evidence Selected Publications that the nucleolus plays also a central role in the Ross,D.A., Barrass, J.D., Dichtl,B., Koš,M., Obtulowicz,T., Robert,M.-C., Koper,M., Karkusiewicz,I., Mariconti,L., cellular stress response and cell growth regula- Tollervey,D., Dichtl,B., Kufel,J., Bertrand,E. and Beggs,J.D. (2010). RiboSys, a high-resolution, quantitative approach to tion. We are characterizing the link between ribo- measure the in vivo kinetics of pre-mRNA splicing and 3’- end processing in Saccharomyces cerevisiae. RNA 16, 2570- some biogenesis and stress response. 2580. Boon,K.-L. and Koš,M. (2010). Deletion of Swm2p selectively impairs trimethylation of snRNAs by Trimethylguanosine syn- thase (Tgs1p). FEBS Lett. 584, 3299-3304. Koš,M. and Tollervey,D. (2010). Yeast Pre-rRNA Processing and Modification Occur Cotranscriptionally. Mol. Cell 37, 809-820. Bohnsack, M.T., Koš,M. and Tollervey,D. (2008). Quantitative analysis of snoRNA association with pre-ribosomes and re- lease of snR30 by Rok1 helicase. EMBO Rep. 9, 1230-1236. Koš,M. and Tollervey,D. (2005). The Putative RNA Helicase Dbp4p Is Required for Release of the U14 snoRNA from Preribosomes in Saccharomyces cerevisiae. Mol. Cell 20, 53-64.

Martin Koš Phone: +49 (0)6221-54 4151 E-mail: [email protected]

Fig. 2. Identification of Rcm1 as a methylase of the cyto- sine 2278 in 25S rRNA in yeast. Sequencing of the bisulfite treated rRNA shows that the highly conserved methylation of C2278 is abolished in Rcm1Δ strain. The histograms represent preserved cytosines in the 25S rRNA that were not converted to uracil. (in collaboration with Frank Lykko, DKFZ).

Martin Koš 21 1982 Ph.D. - University of Heidelberg, Germany (Max Planck Institute for Medical Research, Heidelberg) 1982 - 1995 Staff Member - Institute of Biochemistry II, University of Heidelberg 1989 Habilitation in Biochemistry 1995 - 2003 Apl. Professor for Biochemistry - BZH 2002 Call for a professorship for Pharmaceutical Chemistry (Marburg), declined since 2003 Professor for Biochemistry - BZH

Luise Krauth-Siegel

The parasite-specific trypanothione redox metabolism

Goal 2). Unsaturated Mannich bases are trypanocidal Aim of our work is to analyze the unique and irreversibly inactivate TR (Wenzel et al. 2009). trypanothione thiol redox metabolism In an attempt to create compounds that address of trypanosomatids in atomic detail and two potential binding sites within the unusually to contribute to the development of new wide active site of the enzyme, conjugates of antiparasitic drugs on the basis of specific known TR inhibitors were synthesized by the enzyme inhibitors. group of François Diederich (ETH Zürich). Indeed, conjugates between arylsulfides and mepacrine Background inhibit TR with Ki-values of <1μM and exhibit high Trypanosomes and leishmania are the causative selectivity over human GR (Eberle et al. 2009). agents of African sleeping sickness (Trypanosoma Detoxification of hydroperoxides brucei), South American Chagas' disease (T. cruzi) African trypanosomes possess 2-Cys- and other tropical diseases. All these parasitic peroxiredoxins and glutathione peroxidase- protozoa lack glutathione reductase (GR) and type enzymes (Px). Both types of enzymes thioredoxin reductases. The main non-protein are essential and act as tryparedoxin (Tpx)- thiol is the bis(glutathionyl)spermidine-conjugate dependent peroxidases (Fig. 2). In collaboration with Claudia Muhle-Goll (EMBL), Ivo Tews and trypanothione T(SH)2 which is involved in a wide variety of metabolic pathways Fig. 1 (Fig. 1; Krauth-Siegel and Comini 2008). The trypanothione metabolism

Trypanothione disulfide Research Highlights Protein biosynthesis Trypanothione Trypanothione reductase as a Thioredoxin-S Ascorbate reductase 2 potential drug target molecule homeostasis Thioredoxin-(SH)2 Trypanothione reductase (TR) which Trypanothione Glutaredoxin-S2 catalyzes the NADPH-dependent NDP Ribonucleotide Glutaredoxin-(SH)2 reduction of trypanothione disulfide reductase dNDP (TS2) to the dithiol T(SH)2 is essential Glyoxalase system and represents a key enzyme for the ROH Export/ Reduction of Conjugation of ROOH parasite antioxidant defense (Figs. 1 and Sequestration Metals and drugs hydroperoxides

22 Luise Krauth-Siegel Fig. 2: Detoxification of hydroperoxides (ROOH) by the trypanothione cas- cade.

Irmi Sinning (BZH), the 3-dimensional structure of T. brucei PxIII has been solved (Fig. 3). Unexpectedly, the reduced and oxidized forms have essentially identical structures (Melchers et al. 2008). Subjecting the peroxidase system to a high-throughput screening approach with 80,000 compounds identified potential lead inhibitors which are currently further analyzed (Florian Füller in collaboration with Joe Lewis EMBL). A detailed kinetic analysis showed that the Px- type enzymes are responsible for protecting the parasite specifically against lipid peroxidation (Michael Diechtierow, unpublished results). Fig. 3: Superposition of the structures of oxidized Px The role of parasite glutaredoxins III obtained by X-ray diffraction (gray) and NMR (black) analysis and the NMR-structure of reduced Px III (light Despite the lack of a GR, the genome of African blue). L1, L2, and L3 represent the most extended loops. Cys47 and Cys95 are involved in catalysis. trypanosomes encodes several genes for mono- and dithiol glutaredoxins (Grx). The 1-Cys-Grx1 Selected Publications 2008 - 2010 proved to be an essential iron-sulfur cluster protein in the single mitochondrion of these parasites Ceylan, S., Seidel, V., Ziebart, N., Berndt, C., Dirdjaja, N. and Krauth-Siegel, R. L. (2010) The dithiol glutaredoxins of African trypanosomes have distinct roles and are closely linked to the (Comini et al. 2008). The cytosolic 2-Cys-Grx1 unique trypanothione metabolism, J. Biol. Chem. 285, 35224- also forms an iron sulfur cluster although the 35237. Wenzel, N. I., Wong, P. E., Maes, L., Müller, T. J. J., Krauth- protein contains the canonical CPYC active site Siegel, R. L., Barrett, M. P. and Davioud-Charvet, E. (2009) Unsaturated Mannich bases active against multidrug-resistant motif previously claimed not to allow complex Trypanosoma strains. Chem. Med. Chem. 4, 339-351. formation. 2-Cys-Grx2 is an essential protein in Comini, M. A., Dirdjaja, N., Kaschel, M. and Krauth-Siegel, R. L. (2009) Preparative enzymatic synthesis of trypanothione the mitochondrial intermembrane space (Ceylan and trypanothione analogues. Intl J. Parasitol. 39, 1059-1062. et al. 2010). The oxidized form of both 2-Cys-Grxs Eberle, C., Burkhard, J. A., Stump, B., Kaiser, M., Brun, R., Krauth-Siegel, R. L. and Diederich, F. (2009) Synthesis, containing an intramolecular disulfide is reduced inhibition potency, binding mode, and antiprotozoal activities of fl uorescent inhibitors of trypanothione reductase based on mepacrine-conjugated diaryl sulfi de scaffolds. Chem. Med. by T(SH)2 at rate constants that are three orders Chem. 4, 2034-2044. of magnitude higher than those with GSH which Krauth-Siegel, R. L. and Comini, M. A. (2008) Redox control in trypanosomatids, parasitic protozoa with trypanothione-based underlines the close link between the Grx and thiol metabolism. Biochim. Biophys. Acta, 1780, 1236-1248.

T(SH)2 metabolism. Comini, M., Rettig, J., Dirdjaja, N., Hanschmann, E. M., Berndt, C. and Krauth-Siegel, R. L. (2008) Monothiol glutaredoxin-1 is Large scale synthesis of trypanothione an essential, iron sulfur protein in the mitochondrion of African trypanosomes. J. Biol. Chem. 283, 27785-27798. A prerequisite for the analysis of the parasite Melchers, J., Diechtierow, M., Fehér, C., Sinning, I., Tews, thiol redox metabolism is the availability of I., Krauth-Siegel, R. L. and Muhle-Goll, C. (2008) Structural basis for a distinct catalytic mechanism in Trypanosoma brucei trypanothione. For this purpose we developed tryparedoxin peroxidase. J. Biol. Chem. 283, 30401-30411. an enzymatic system that allows the large scale production of both reduced and oxidized Luise Krauth-Siegel Phone: +49 (0)6221 54 4187 trypanothione (Comini et al. 2009). E-mail: [email protected]

Luise Krauth-Siegel 23 1985 Ph.D. - University of Regensburg, Germany 1985 - 1987 PostDoc - University of Regensburg 1987 - 1990 PostDoc - University of California, Santa Barbara, USA 1994 Habilitation - University of Regensburg 1994 - 1999 Group Leader - University of Regensburg Since 1999 Group Leader - BZH

Johannes Lechner Kinetochore and Mitosis

Goal attached kinetochores and, when active, halts the To understand kinetochore structure and progress through mitosis at metaphase. An active function. SAC prolongs the time available for kinetochore- spindle attachment but does not actively enhance Background this step. Consequently, unattached kinetochores are likely to direct further schemes designed to Organized by centrosomes (or spindle pole bod- increase their chance of being captured by a mi- ies in yeast) the mitotic spindle executes chromo- crotubule. some segregation. It is composed of interpolar microtubules that overlap in the spindle midzone and by kinetochore microtubules that attach to Research Highlights chromosomes via the kinetochore. The S. cer- The CLASP homolog Stu1 regulates evisiae kinetochore displays considerable simi- kinetochore capturing and spindle stability larities to its counterpart in higher eukaryotes. It inversely harbors many ortholog proteins organized in sub- We observed that certain mutations in the COMA complexes that assemble in a hierarchical man- complex resulted in the permanent accumulation ner. Basically, it consists of centromeric chroma- of the S. cerevisiae CLASP homolog, Stu1, at the tin that includes a histone H3 variant (Cse4), a ki- compromised kinetochores and thus interfered netochore-microtubule interface (MIND, Spc105, with microtubule localization of Stu1. Since the Ndc80 and DASH complex) and a linker layer latter is essential to stabilize overlapping interpo- including the COMA complex that connects the lar microtubules this resulted in severely defec- two. Chromosome segregation is executed with tive metaphase and anaphase spindles. Accord- extremely high fidelity. A failure to do so is a hall- ingly, when Stu1 localization to the compromised mark of cancer cells. One particular critical step kinetochore was abolished the spindle defect was in this respect is the attachment of kinetochores rescued. Analyzing Stu1 localization in wild type to microtubule plus ends after nuclear breakdown cells revealed that Stu1 localizes specifically to ki- in higher eukaryotes or after DNA replication and netochores that are not attached to microtubules kinetochore assembly during the closed mitosis and that kinetochore localized Stu1 facilitates ki- of yeast. This step is closely supervised by the netochore capture by microtubules (Fig. 1). Un- spindle assembly checkpoint (SAC), a signaling attached kinetochores apparently not only attract pathway that is elicited by unattached or falsely Stu1 binding but also cause Stu1 oligomerization.

24 Johannes Lechner SPB

kinetochore Stu1

DASH complex microtubule

Fig. 1: Model illustrating the mutually exclusive roles of Stu1 in kinetochore capturing and spindle stabilization.

As a consequence, even a single unattached Mimicking an Ndc80 phosphorylation pattern as kinetochore is able to sequester the majority of deduced from in-vitro phosphorylation with Mps1 nuclear Stu1 and thus prevents the association and in-vivo overexpression of Mps1 results in a of Stu1 with microtubules. Therefore, while facili- constitutive activation of the SAC. SAC is normal- tating capturing of unattached kinetochores, the ly activated by unattached kinetochores or kineto- oligomerization of Stu1 at detached kinetochores chores that lack tension because they are not in also prevents the formation of a stable spindle. a bipolar attachment. Mimicking Ndc80 phospho- This leaves the spindle poles in close proximity rylation however initiates SAC signaling although and thus facilitates bipolar attachment of kineto- kinetochores are under tension and although the chores. The majority of Stu1 dissociates from a mutant Ndc80 still binds to microtubules in vitro. kinetochore after it is captured by a microtubule Consequently cells die and, importantly, can be and is then able to bind to microtubules and to re- rescued by disrupting SAC signaling. We there- locate (in anaphase) to the spindle midzone. This fore speculate that Ndc80 phosphorylation is process requires an intact DASH complex. Since an inherent step in SAC signaling. In the future the DASH complex stabilizes the interaction of it will be of interest to understand how mimick- a kinetochore with a microtubule plus end, kine- ing Ndc80 phosphorylation elicits SAC activation. tochores most likely release Stu1 in the context One approach is to identify proteins that interact of microtubule plus end binding. In the future it with mutant and wild type Ndc80 in a differential will be of interest to understand the mechanism manner. and regulation of the kinetochore-induced Stu1 oligomerization as well as its role in kinetochore Selected Publications capturing. Furthermore, it will be interesting to Ortiz, J., Funk, C., Schäfer, A., and Lechner, J. 2009. Stu1 inversely regulates kinetochore capture and spindle stability. learn whether there is a cross talk between Stu1 Genes Dev 23(23): 2778-2791. oligomerization and the SAC, since both are con- Kemmler, S., Stach, M., Knapp, M., Ortiz, J., Pfannstiel, trolled by kinetochore—microtubule interaction. J., Ruppert, T., and Lechner, J. 2009. Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint the centromeric chromatin. signalling. EMBO J 28(8): 1099-1110.

Maekawa, H., Priest, C., Lechner, J., Pereira, G., and Schiebel, Mps1, Ndc80 and SAC regulation E. 2007. The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal. J The kinase Mps1 is a component of the SAC sig- Cell Biol 179(3): 423-436. naling pathway. Ndc80 is a subunit of the Ndc80 complex that not only is directly involved in kine- Johannes Lechner tochore-microtubule interaction but also is es- Phone +49 (0)6221-54 4371 E-mail: [email protected] sential for kinetochores to execute SAC function.

Johannes Lechner 25 1988 - 1993 Chemistry Studies, University of Athens, Greece 1994 - 1998 Ph.D. - Zentrum für Molekulare Biologie Heidelberg (ZMBH), Germany (Prof. Stefan Jentsch) 2000 - 2005 PostDoc - Institute of Biochemistry, ETH Zürich, Switzerland (Prof. Yves Barral) since 10/2005 Junior Group Leader - BZH

Dimitris Liakopoulos

Spindle positioning in yeast

Goal polarized material, resulting in unequal segrega- To study how the interactions of astral spin- tion of the polarized factors. At the same time, the dle microtubules with the cortex bring the cytokinetic actomyosin ring cleaves the cell mid- spindle to its correct position during asym- way through the mitotic spindle to ensure equal metric cell divisions. segregation of chromosomes between daughters. Coordination of cell cleavage with chromosome Background segregation depend on interactions of astral Polarized cells have two options when they divide: spindle microtubules (aMTs) with the cortical ac- they can either divide symmetrically, or asym- tin cytoskeleton. A complex network of proteins metrically. Asymmetric divisions are encountered involving non-motor microtubule (+)-end tracking whenever the goal is generation of cellular diver- proteins (+TIPs), kinesins, dynein and actin-inter- sity, for example during embryonic divisions or the acting proteins mediate these interactions. divisions of stem cells. Factors that determine cell fate are asymmetrically segregated in one of the Our lab studies the mechanisms and regulation two daughters, that consequently differentiates. of astral spindle microtubules with the cortical cy- toskeleton using one of the simplest asymmetri- In an asymmetric cell division, the cytokinetic ma- cally dividing organisms, the yeast S. cerevisiae chinery must cleave the cell perpendicular to the (Fig.1).

Fig.1: Spindle positioning in yeast. Thick grey bar: metaphase spindle, red, green spots: old and new microtubule organizing centers (SPBs), thin grey lines: aMTs. Left: The protein Kar9 forms a bridge between aMTs and actin cables through its interaction with Bim1 and Myo2. Myo2 pulls aMTs from the old SPB and the metaphase spindle towards the bud. Right: Dynein is transported by the kinesin Kip2 to the (+)-ends of aMTs and binds to Num1 at the cortex. Num1-immobilized dynein pulls aMTs and orients the spindle, because its motor activity is directed towards the (-)-ends of aMTs that are anchored at the SPB. The blue-gray ring is the actomyosin-based cytokinetic apparatus and the future site of cytokinesis. Only metaphase spindles aligned with the mother-bud axis are able to elongate and partition half of the chromosomes into the bud in anaphase, so that cytokinesis can occur later midway through segregated chromosomes.

26 Dimitris Liakopoulos Fig. 2: A) During early spindle positioning repeated cycles of aMT guidance to the cleavage apparatus (upper cell), followed by detachment of aMTs from actin cables when aMTs reach the bud neck (bottom cell) bring the spindle close to the bud neck. Detachment of aMTs from actin cables occurs due to Kar9 degradation and disassembly of Kar9 complexes at the bud neck. In every cycle, only a small proportion of aMT-bound Kar9 is degraded, since only Kar9 that assembles into active aMT-guiding com- plexes reaches the bud neck. B) Green-to-red photoconversion and in vivo chase of Kar9-AA-EosFP that cannot be ubiquitylated at the bud neck during the cell cycle of a yeast cell. Note that Kar9 accumulates at the bud neck most of the time, and enters the bud app. 10 min before anaphase. The spindle pole in Kar9-AA remains associated with the bud neck resulting in spindle elongation and mispositioning in anaphase (arrow). Chromosome segregation is rescued later in this cell, because the spindle positioning checkpoint prevents spindle disassembly until the spindle manages to elongate into the bud.

Research Highlights A third project concerns the mechanics of nuclear We found that the protein Kar9, the yeast function- migration during closed mitosis. We have shown al equivalent of the Adenomatous Polyposis Coli that nuclear migration through the bud neck is fa- (APC) tumor suppressor, that links astral microtu- cilitated by nuclear membrane expansion and are bules with actin, is regulated by phosphorylation, currently investigating the molecular mechanisms sumoylation and ubiquitylation. Phosphorylation that link these two processes. of Kar 9 by the Cdc28/Clb4 kinase complex (Cdc28 is the yeast Cdk1 kinase) is required for Kar9 ubiq- Finally, we demonstrated that mitotic spindles of uitylation and degradation. Ubiquitylation of Kar9 asymmetrically dividing cells display morphologi- is spatially confined and regulates interactions of cal and functional differences. We now explore astral microtubules with the yeast cleavage appa- how spindle asymmetry feeds back to correctly ratus. When phosphorylation or ubiquitylation are position the spindle relative to cell polarity. prohibited, interactions of astral microtubules with the bud neck persist and cause mispositioning of Selected Publications 2008 - 2010 the mitotic spindle (Fig. 2). Kammerer,D., Stevermann,L. and Liakopoulos,D. (2010) Ubiquitylation regulates interactions of astral microtubules Our aim in the future is to elucidate how sumoyla- with the cleavage apparatus. Curr. Biol. 27, 1233-43. tion regulates function of Kar9 and interactions of Leisner,C., Kammerer,D., Denoth,A., Barral,Y. and aMTs with cortical actin filaments. Liakopoulos,D. (2008) Regulation of Mitotic-Spindle Asymmetry by SUMO and the Spindle-Assembly Checkpoint in Yeast, Curr. Biol. 16, 1249-55. We also showed that budding yeast GSK-3 phos- Barral, Y. and Liakopoulos, D. Role of spindle asymmetry in cellular dynamics (2009) Int. Rev. Cell Mol Biol. 278, 149- phorylates the kinesin Kip2 and reduces its af- 213. finity for aMTs and transport of dynein and Kar9 on aMTs. Interestingly, Kip2 is able to stabilize Dimitris Liakopoulos Phone: +49 (0)6221-54 4181 aMTs. Very little is known on the mechanisms of E-mail: [email protected] MT-stabilizing kinesins and their regulation. In the future, we will investigate the mechanism through which Kip2 stabilizes aMTs in vitro, in collabora- tion with with J. Howard (Dresden).

Dimitris Liakopoulos 27 1994 Ph.D. - University of Göttingen, Germany

1994 - 1997 PostDoc - Institute for Biochemistry I, University of Heidelberg, Germany (Prof. Felix T. Wieland)

1997 - 2000 PostDoc - Memorial Sloan-Kettering Cancer Center, New York, USA (Prof. James E. Rothman)

2001 Group Leader - BZH

2004 Professor of Biochemistry - BZH

Walter Nickel

Unconventional Protein Secretion

Goal Research Highlights To reveal the molecular components and As illustrated in Fig. 1, three critical components mechanisms involved in unconventional se- of the unconventional secretory machinery of cretion of fibroblast growth factor 2 (FGF2), a FGF2 have been identified all of them being as- potent mitogen mediating tumor-induced an- sociated with plasma membranes. In addition to giogenesis. our earlier findings demonstrating an essential role of heparan sulfate proteoglycans that pro- Background vide membrane-proximal FGF2 bindings sites on The vast majority of extracellular proteins are se- the extracellular side of the plasma membrane creted by the classical ER/Golgi-dependent se- (Zehe et al. 2006, Proc. Natl. Acad Sci. U.S.A. cretory pathway, however, numerous exceptions 103:15479-15484), we have identified a mem-

have been identified. As opposed to proteins that brane lipid, the phosphoinositide PI(4,5)P2, that is are transported along the classical route, uncon- required for the recruitment of FGF2 at the inner ventional secretory proteins lack a signal pep- leaflet of plasma membranes (Temmerman et al. tide and their export from cells is not affected by 2008). Based on FGF2 variant forms that fail to brefeldin A, an inhibitor of ER to Golgi traffick- bind to PI(4,5)P2 and RNAi-mediated inhibition of ing. Several kinds of unusual secretory pathways PI(4,5)P2 biosynthesis, we demonstrated an es-

have been described some of which involve in- sential role of PI(4,5)P2 in FGF2 secretion. PI(4,5)

tracellular vesicles such as secretory lysosomes P2-dependent recruitment of FGF2 at the inner or multi-vesicular bodies. By contrast, unconven- leaflet of plasma membranes does not only direct tional secretion of FGF2 has been shown to occur FGF2 into the cell periphery but also induces its by direct translocation across plasma membranes oligomerization and membrane insertion (Fig. 1). resulting in its association with heparan sulfate In a cellular context this process is likely to be fa- proteoglycans on cell surfaces. Using genome- cilitated by integral membrane proteins that may wide RNAi screening approaches as well as bio- form a microenvironment for example enriched

chemical reconstitution experiments, our labora- in PI(4,5)P2 and other membrane lipids favoring tory functionally dissects molecular components membrane curvature. Depending on such local and mechanisms involved in unconventional se- properties of the plasma membrane, multivalent cretion of FGF2. FGF2 oligomers may be able to penetrate and

28 Walter Nickel RNAi screening including integral plasma membrane proteins, a major goal of our future work will be to reconstitute FGF2 membrane translocation in vitro using chemically defined components. In this way we aim at defining the core machinery as well as regulatory components involved in unconventional secretion of FGF2 from cells.

Fig. 1: Molecular components and mechanisms involved in FGF2 translocation across plasma membranes (Nickel, Curr Opin Biotechnol, 2010; 21(5):621-6.).

break the permeabilitry barrier of the plasma Selected Publications 2008 - 2010 membrane resulting in membrane insertion. This Nickel W, Pathways of Unconventional Protein Secretion. Curr. Opin. Biotechnol., 2010; 21(5):621-6. model is also consistent with our finding that Ebert AD, Laussmann M, Wegehingel S, Kaderali L, Erfle H, FGF2 translocates across plasma membranes in Reichert J, Lechner J, Beer HD, Pepperkok R, Nickel W. Tec- kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion. Traffic. 2010; a fully folded conformation (Cespon-Torrado et al. 11(6):813-26. 2009, J. Cell Sci.). To complete membrane trans- Torrado LC, Temmerman K, Müller HM, Mayer MP, Seelenmeyer C, Backhaus R, Nickel W. An intrinsic quality- location, on the extracellular side of the plasma control mechanism ensures unconventional secretion of fi- broblast growth factor 2 in a folded conformation. membrane, heparan sulfate proteoglycans are J Cell Sci. 2009; 122(Pt 18):3322-9. required to extract FGF2 from the membrane re- Nickel W, Rabouille C. Mechanisms of regulated uncon- ventional protein secretion. Nat Rev Mol Cell Biol. 2009; sulting in its storage on cell surfaces. 10(2):148-55. Using a genome-wide RNAi screening approach, Temmerman K and Nickel W, A novel flow cytometric assay to quantify interactions between proteins and membrane lip- a third component of the FGF2 secretion ma- ids. J. Lipid Res. 2009; 50:1245-1254. Tournaviti S, Pietro ES, Terjung S, Schafmeier T, Wegehingel chinery was revealed to be Tec kinase (Ebert et S, Ritzerfeld J, Schulz J, Smith DF, Pepperkok R, Nickel W. Reversible phosphorylation as a molecular switch to regulate al., 2010), an enzyme that contains a PH domain plasma membrane targeting of acylated SH4 domain pro- teins. Traffic. 2009; 10(8):1047-60. and, alike FGF2, is recruited to the inner leaflet Nickel W, Seedorf M. Unconventional mechanisms of protein by phosphoinositides (Fig. 1; Nickel, 2010). FGF2 transport to the cell surface of eukaryotic cells. Annu Rev Cell Dev Biol. 2008; 24:287-308. has been demonstrated a target of Tec kinase re- Temmerman K, Ebert AD, Müller HM, Sinning I, Tews I, Nickel sulting in its phosphorylation at tyrosine 82. This W. A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2. Traffic. modification is essential for FGF2 secretion and 2008; 9(7):1204-17.

may play a role in PI(4,5)P2-induced FGF2 oli- gomerization and membrane insertion. Based on the components illustrated in Fig. 1 Walter Nickel Phone: +49 (0)6221-54 5425 as well as additional factors identified through E-mail: [email protected]

Walter Nickel 29 1964 - 1966 MD – Max Planck Institute for Medical Research, Heidelberg (Prof. J.C. Rüegg)

1966 - 1970 PostDoc - Dartmouth Medical School, Hanover NH, USA (Prof. L.H. Noda); Internship and residency at hospitals in Heidelberg, Stuttgart, and Karlsruhe

1970 - 1980 Group Leader in Biophysics - Max Planck Institute for Medical Research, Heidelberg

1975 Habilitation in Biochemistry

1980 - 2007 Professor - Institute of Biochemistry II, University of Heidelberg / BZH; now professor emeritus

Heiner Schirmer

Drugs und transmission blockers against pediatric malaria

Goal Research Highlights To develop affordable and accessible medi- When reducing MB, the disulfide reductases cines for malaria utilize the flavin cofactor and not the active site cysteine pair (Fig.1) for electron transfer (Buch- Background holz et al 2008). Falciparum malaria is a disease which in its dan- Pyocyanin, a social signal and respiratory pig- gerous form mainly affects preschool children, ment from Pseudomonas aeruginosa is a natu- pregnant women and tourists. ral counterpart of the synthetic drug methylene Our work focuses on redox milieu-targeting drug blue (see legends of Figs 1 and 2; Schirmer et combinations against pediatric malaria. al 2008). The biochemical networks that maintain the cyto- Antimalarial MB-combination therapies like MB- solic redox potential at values below –250 mV are artemisinine and MB-amodiaquine are currently based in many organisms on a dual system, the studied by Olaf Müller, Peter Meissner and Bou- glutathione system and the thioredoxin system bacar Coulibaly in clinical trials at the Centre de (Buchholz et al 2010). We study these systems Recherche en Santé de Nouna (CRSN) in Burki- primarily in the protozoal parasite Plasmodium fal- na Faso. Coulibaly did his thesis work at the BZH ciparum, its insect vector Anopheles gambiae and and, in 2004, was the first Burkinabé to obtain a the human host. Differences in the proteins of the PhD in biochemistry. MB is not only active against redox networks are exploited for the development Plasmodium schizonts but also against Plasmo- of species-specific and stage-specific therapeutic dium gametocytes (Coulibaly et al 2009) which agents. We focus as targets on the disulfide re- means that MB can block transmission of the ductases glutathione reductase and thioredoxin disease from patient to patient via the mosquito. reductase, as well as dihydrolipoamide dehydro- Thus MB-containing antimalarial drug combina- genase. The phenothiazine methylene blue (MB), tions may become important for malaria elimina- a subversive substrate and inhibitor of disulfide tion programs. reductases, is currently tested as a partner in an- Up to June 2010, the combination of MB and timalarial drug combinations (Bountogo et al 2010, amodiaquine was considered an ethical drug; this Müller et al 2009, Schirmer et al 2008). drug combination is effective, safe, affordable, ac-

30 Heiner Schirmer Fig. 1: Cytosolic hu- man glutathione re- ductase homodimer with bound pyocya- nin (PYO). Pyocyanin (blue) and FAD (yel- low) are represented as ball and stick models. Additionally, the surfaces of the catalytic cysteines Cys58/Cys63 and Cys58’/Cys63’ (green) and of PYO (blue) are shown. Azure B (monodemethyl MB), the major metabolite of MB, binds to the same site as pyocyanin. The MB structure itself is too large to be accom- modated here (Karin Fritz-Wolf, personal communication).

cessible and available in sufficient dosages. The of Figs. 1 and 2) may indeed be the active form rumours, however, that cationic MB might inter- of MB in a number of therapeutic indications, with fere with the growth of phospho-tau filaments and MB serving as a pro-drug of azure B. thus delay the onset of Alzheimer disease have recently contributed to a shortage of and a price Selected Publications 2008 - 2010 explosion for MB as a cGMP-grade raw material Bountogo M, Zoungrana A, Coulibaly B, Klose C, Mansmann U, Mockenhaupt FP, Burhenne J, Mikus G, Walter-Sack I, from less than € 150 to € 30000 (http://www.alzfo- Schirmer RH, Sié A, Meissner P, Müller O (2010) Effi cacy of methylene blue monotherapy in semi-immune adults with rum.org/new/Schirmer.asp). If this situation does uncomplicated falciparum malaria: a controlled trial in Burkina Faso Trop Med Int Health 15, 713-717 not change MB has no future as a drug for malaria Buchholz K, Putrianti ED, Rahlfs S, Schirmer RH, Becker K, as a disease of the poor. As a consequence, we Matuschewski K (2010) Molecular genetics evidence for the in vivo roles of the two major NADPH-dependent disulfi de reductases in the malaria parasite. J Biol Chem 285: 37388- study the cell biochemistry of azure B, the major 37395 metabolite of MB in man. Azure B (see legends Buchholz K, Schirmer RH, Eubel JK, Akoachère MB, Dandekar T, Becker K, Gromer S (2008) Interactions of methylene blue with human disulfi de reductases and their orthologues from Plasmodium falciparum. Antimicrob Agents Chemother 52, 183-191 Coulibaly B, Zoungrana A, Mockenhaupt FP, Schirmer RH, Klose C, Mansmann U, Meissner P, Müller O (2009) Strong gametocytocidal effect of methylene blue-based combination therapy against falciparum malaria: a randomised controlled trial. PloS ONE 4, e5318 Müller O, Sié A, Meissner P, Schirmer RH, Kouyaté B (2009) Artemisinin resistance on the Thai-Cambodian border. The Lancet 374, 1418-1419 Schirmer RH, Adler H, Zappe HA, Gromer S, Becker K, Coulibaly B, Meissner P (2008) Disulfi de reductases as drug targets: Methylene blue combination therapies for falciparum malaria in African children. Flavins and Flavoproteins 16, 481- 486

Awards and Honors 1976 Appointment as a Bicentennial Lecturer in Philadelphia and Boston 2002-2009 Dream Action Award of the Dutch chemical company DSM Fig. 2 Methylene blue as an H2O2-producing subversi- ve redox-cycler. The enzyme glutathione reductase and other disulfi de reductases of the malaria parasite catalyze the reduction of methylene blue to leucomethylene blue. Leucomethylene blue auto-oxidizes instantaneously regen- Heiner Schirmer Phone: +49 (0)6221-54 4165 erating MB and producing parasitocidal H2O2. Pyocyanin and azure B can undergo the same redox-cycling as MB. E-mail: [email protected]

Heiner Schirmer 31 1989 Ph.D. - Ludwig-Maximilians-Universität München, Germany (Max Planck Institute of Biochemistry, Martinsried) 1989 - 1991 PostDoc - Max Planck Institute for Biophysics, Frankfurt, Germany (Prof. Hartmut Michel) 1991 - 1993 Scientist - Biomedical Centre, Uppsala, Sweden (Prof. Alwyn Jones) 1994 - 1999 Group Leader - European Molecular Biology Laboratory (EMBL), Heidelberg, Structural Biology Programme since 2000 Full Professor - BZH 2006 - 2009 Director - BZH

Irmgard Sinning

Molecular Machines in protein targeting and membrane protein biogenesis

Goal otes and to the plasma membrane in bacteria. We To understand the structure and function study the molecular mechanisms of how SRP and of molecular machines in co- and post- SR participate in protein targeting by a combina- translational protein targeting. tion of biochemical techniques and X-ray crystal- lography as our key method. Our data provide Background structural snapshots of SRP and SR in distinct Membrane proteins comprise more than 25% of functional states that are combined into a movie the cellular proteome and their function depends of SRP driven membrane protein biogenesis. In on insertion into the correct target membrane. particular, we are interested in the role of mem- Membrane proteins utilize predominantly the uni- brane lipids in the regulation of SR activity and in versally conserved co-translational delivery path- the molecular mechanisms of SRP GTPases. way of the signal recognition particle (SRP). This In contrast to the SRP system, post-translational pathway elegantly couples protein synthesis at targeting delivers proteins when their synthesis is the ribosome to membrane targeting and insertion, already completed. Tail-anchored (TA) membrane and avoids exposure of hydrophobic transmem- proteins contain a single transmembrane domain brane domains. Although the composition of the at their C-terminus which excludes them from the SRP system differs in the three kingdoms of life, co-translational pathway (Fig. 1). They play im- the central SRP core consisting of SRP54 and its portant roles in membrane insertion, membrane binding site on the SRP RNA are conserved. SRP fusion and apoptosis. Recently, the so-called Get recognizes signal sequences at the N-terminus (guided-entry of tail-anchored membrane pro- of newly synthesized polypeptides in the context teins) pathway has been discovered that delivers of a translating ribosome (Fig. 1). Subsequent in- TA proteins to the ER. Like other post-translational teraction of SRP with the membrane bound SRP targeting pathways, the Get pathway depends on receptor (SR) involves the formation of a symmet- ATP. We started a detailed comparative analysis ric hetero dimer of the two GTPases present in of the SRP and Get pathways in order to unravel SRP and SR, which directs the ribosome nascent mechanistic details and common principles of chain (RNC)/SRP complex to the ER in eukary- regulation.

32 Irmgard Sinning Although the SRP system is conserved in evolu- tion, it can be adapted for specific requirements. The post-translational function of SRP in chlo- roplasts is particularly interesting as it guides nuclear encoded light-harvesting chlorophyll a,b binding proteins (LHCPs) to the thylakoid mem- brane. LHCPs serve as antenna complexes in photosynthesis and are the most abundant mem- brane proteins on our planet. They contain three hydrophobic transmembrane helices and have to kept in a conformation competent for membrane insertion. We study the structure and function of Fig. 1: Recognition of targeting signals by SRP and Get pathways. cpSRP43, a novel component of cpSRP, in order to understand its role in LHCP biogenesis. transit complex that enables LHCP delivery to and insertion into the thylakoids. cpSRP43 is there- Research Highlights fore more than an adaptor that allows to highjack cpSRP43 is characterized by a unique arrange- the conserved SRP system for post-translational ment of chromodomains and ankyrin repeats. Our protein targeting. It recognizes its membrane pro- crystal structure of cpSRP43 revealed that it re- tein cargo in a most SRP-unlike manner – with sembles the SRP RNA (Fig. 2). While chromodo- high sequence specificity. We could show that mains are almost exclusively known for their key cpSRP43 acts as a chaperone for membrane pro- role in the regulation of gene expression, read- teins and localize the primary chaperone function ing the so-called histone code, ankyrin repeats to the ankyrin repeats. In contrast, most chaper- are well established as versatile protein interac- ones are large proteins or assemblies that require tion modules. In cpSRP43 the ankyrin repeats ATP hydrolysis for their function. We discovered provide the binding site for an internal signal se- that cpSRP43 can even act as a disaggregase quence present in LHCPs, the L18 region (Fig. 2). that is able to dissolve LHCP aggregates with- Moreover, we could show that a ‘DPLG’ motif with- out ATP hydrolysis. We also clarified the role of in L18 is required to recruit LHCPs into a soluble the two C-terminal chromodomains of cpSRP43.

Fig. 2: cpSRP43 serves as a specific membrane protein chaperone. cpSRP43 consists of ankyrin repeats and chromodo- mains (left). Ank1-4 specifically bind a ‘DPLG’ motif within LHCPs (right) and keep it in a conformation competent for carotenoid (lutein) attachment upon membrane insertion.

Irmgard Sinning 33 we could now show that the interaction with anionic phospholipids triggers a conformational switch of the MTS. This switch allows for subsequent activation of the FtsY GTPase which is crucial for SRP mediated protein targeting. The central component of TA membrane protein biosynthesis, the ATPase Get3, is also a member of the SIMIBI class of NTP binding proteins. Structure determination of Get3 in different nucleotide loaded states (Fig. 3) together with membrane insertion assays (with B. Dobberstein, ZMBH) allowed us to propose a model for how the ATPase cycle of Get3 is linked to TA protein binding and release. HX-MS was used to localize the TA protein binding site in Get3 to a hydrophobic subdomain formed by two insertions

Fig. 3: Structure of Get3. The ATPase forms a dimer in the ATPase fold. Interestingly, the TA protein clamped together by a Zn ion and comprises two subdomains (blue, green). binding site shares the enrichment in methionine residues with the signal sequence binding site in They are involved in the interaction with cpSRP54 the M domain of SRP54. Although the co- and and with the chloroplast member of the YidC/ posttranslational functions of the SRP and Get Oxa1/Alb3 family of the membrane insertases. pathways differ, the basic principles of cargo The C-terminal tail of Alb3 contains two motifs recognition are conserved. enriched in positive charges that are required to We continued additional research activities bind cpSRP43. Our studies suggest a model for in small teams: Gert Bange studies flagella LHCP delivery to the thylakoid membrane. In or- biosynthesis in Bacillus. Flagella are one of der to understand how Alb3 and its homologs act nature’s largest molecular machines and act as membrane insertases we continue our efforts also as virulence factors - besides their role in towards the structure determination of Alb3 and locomotion. The translocation of flagella building homologs. blocks involves a type III secretion system (TTSS) The SRP GTPases form a distinct subfamily of which comprises a number of membrane proteins. the SIMIBI (for Signal Recognition Particle, MinD, FlhA is the largest component of the TTSS and BioD) class of NTP binding proteins with only the structure of its cytosolic domain provided three members: the SRP core protein SRP54, first insights into the domain architecture (Fig. 4). the SRP receptor protein FtsY (in bacteria; SRα Together with biochemical data, we clarified the in eukaryotes) and FlhF, a protein involved in the role of chaperones in the coordinated delivery of assembly of polar flagella. We have previously late flagellar building blocks to the TTSS. Valerie identified a conserved membrane targeting Panneels optimizes a novel expression system sequence (MTS) in FtsY that is required and for membrane proteins developed previously sufficient for directing the SRP receptor to the in our lab. It exploits the naturally abundant plasma membrane. Combining amide hydrogen- membrane stacks in the photoreceptor cells deuterium exchange with mass spectrometry (HX- (PRCs) in the eyes of Drosophila melanogaster. MS), X-ray crystallography and CD spectrometry We analysed how endogeneous rhodopsin is

34 Irmgard Sinning Selected Publications 2008 - 2010 Falk, S. & Sinning I. (2010) cpSRP43 is a novel chaperone specific for light-harvesting chlorophyll a,b binding proteins, J. Biol. Chem. 285: 21655-61. Bange, G., Kümmerer, N., Engel, C., Bozkurt, G., Wild, K. & Sinning, I. (2010) FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system, PNAS 107: 11295-300. Wild, K., Bange, G., Bozkurt, G., Segnitz, B., Hendricks, A. & Sinning, I. (2010) Structural insights in RNP assembly of the human and archaeal signal recognition particle, Acta Cryst. D 66: 295-303. Falk, S., Ravaud, S., Koch, J. & Sinning, I. (2010) The C-terminus of the Alb3 membrane insertase recruits cpSRP43 to the thylakoid membrane, J. Biol. Chem. 285: 5954-62. Panneels, V. & Sinning, I. (2010) Overexpression of mem- brane proteins in fly eyes. In: Heterologous Expression of Membrane Proteins: Methods and Protocols, Series: Methods in Molecular Biology, Humana Press (I. Mus-Veteau, ed.). Methods Mol. Biol. 601:135-47. Fig. 4: Domain arrangement of the FlhA cytosolic do- main. Bozkurt, G., Stjepanovic, G., Vilardi, F., Amlacher, S., Wild, K., Bange, G., Favaloro, V., Rippe, K., Hurt, E., Dobberstein, B. & Sinning, I. (2009) Structural insights into tail-anchored protein binding and membrane insertion by Get3, PNAS 106: targeted to the rhabdomeres. We could localize 21131-6. the targeting signal of Drosophila rhodopsin in Kock, I., Bulgakova, N.A., Knust, E., Sinning, I. & Panneels, V. (2009) Targeting of Drosophila rhodopsin requires helix 8 but the distal part of helix 8 which might be a useful not the distal C-terminus, PLoSOne 4: e6101. Grudnik, P., Bange, G. & Sinning, I. (2009) Protein targeting tool to improve heterologous expression of by the signal recognition particle, Biol. Chem. 390:775-82. GPCRs and transporters. Several receptors and Cross, B.C.S., Sinning, I., Luirink, J. & High, S. (2009) Delivering proteins for export from the cytosol, Nat. Rev. Mol. transporters produced in fl y eyes have entered into Cell. Biol. 10: 255-64. crystallization trials. Ivo Tews studies the molecular Sinning, I., Wild, K. & Bange, G. (2009) Signal sequences get active. Nat Chem Biol. 5: 146-7. mechanisms of Toc GTPases in chloroplast import, Stengel, K., Holdermann, I., Cain, P., Robinson, C., Wild, K. & mycobacterial adenylylcyclases and Vitamin B6 Sinning, I. (2008) Structural basis for specific substrate rec- ognition by the chloroplast signal recognition particle protein biosynthesis. Structural studies of PLP synthase cpSRP43, Science 321: 253-6. Ravaud, S., Stjepanovic, G., Wild, K. & Sinning, I. (2008) The provided insights into the assembly mechanism crystal structure of the periplasmic domain of the Escherichia coli membrane protein insertase YidC contains a substrate of this huge molecular machine and highlighted binding cleft, J. Biol. Chem. 283: 9350-8. a number of key intermediates in PLP synthesis. Chloroplasts contain a majority of proteins that Awards and Honors are nuclear encoded, synthesized in the cytosol 2010 Member of EMBO and imported into the stroma across the outer 2010 Member of LEOPOLDINA and inner envelope. Import is regulated by the 2010 Heidelberg Molecular Life Sciences GTPases Toc33 and Toc159. Structure analyses (HMLS) Investigator Award and biochemical data clarifi ed the role of Toc33

dimerization for protein import. Klemens Wild Irmgard Sinning analyses the structure and function of amyloid Phone: +49 (0)6221-54 4781 E-mail: [email protected] precursor protein (APP) complexes. APP is the central player in Alzheimer Disease pathogenesis. Structures were determined of the APP intracellular domain (AICD) in complex with a physiologically and pathologically important phosphotyrosine- binding domain Fe65-PTB2 and of the Fe65-PTB1 domain, which constitutes a main crossroad in APP signaling and trafficking.

Irmgard Sinning 35 1991 Ph.D. - Ludwig-Maximilians-Universität München, Germany 1991 - 1993 PostDoc - Sloan-Kettering Institute, New York, USA 1994 - 1997 Assistant Laboratory Member - Sloan-Kettering Institute 1998 - 2004 Assistant Professor - Sloan-Kettering Institute 2004 - 2005 Associate Professor - Sloan-Kettering Institute since 2005 Full Professor - BZH

Thomas Söllner

Regulated Membrane Fusion: Molecular Mechanisms and Machinery

Goal regulatory proteins, protein phosphorylation and To gain insight into the function of regulatory the local lipid composition (membrane microdo- proteins and lipids that control the assembly mains) provide additional means to restrain or ac- of the membrane fusion machinery and to un- celerate individual steps and contribute to synap- derstand the molecular mechanisms of regu- tic plasticity. lated exocytosis. Research Highlights Background To decipher the role of individual components in Intracellular membrane fusion is driven by the pair- this reaction cascade, we use reconstituted mem- ing of v-SNAREs on a transport vesicle with their brane fusion assays, which allow us to precisely cognate t-SNAREs on the target membrane and define the protein and lipid composition and their subsequent assembly into a stable four-helix membrane curvature. These biochemical assays bundle. The initial contact between the transport use neuronal SNAREs reconstituted into small vesicle and its target membrane, called vesicle unilamellar vesicles (SUV) and giant unilamellar tethering, requires tethering proteins such as vesicles (GUV) to study single vesicle docking/ Rabs and their effectors, which probably control fusion in vitro. In addition, we have developed a t-SNARE complex formation (Fig.1). At this stage, cellular assay, which employs NPY-pHluorin - a trans v/t-SNARE complexes (SNAREpins) have pH-sensitive GFP - targeted into the lumen of se- not formed. Regulator proteins directly bind Rab cretory vesicles to detect single exocytosis events effectors and t-SNAREs and initiate SNAREpin in vivo (Fig.2). formation, which is ultimately controlled by Sec1/ Our recent in vitro analysis of complexin has re- Munc18 (SM) proteins. In regulated exocytosis vealed both inhibitory and stimulatory roles. We these events are called vesicle priming. Primed could show that complexin stabilizes SNAREpins vesicles contain SNAREpins, which are stabi- and that its carboxy-terminus contains a puta- lized in a partially assembled ‘ready to go’ state, tive amphipathic helix that stimulates SUV fusion. by components of the calcium-sensing machin- This stimulatory function directly correlates with ery (synaptotagmin and complexin). The binding the lipid bilayer binding properties of the amphip- of calcium to the calcium-sensor synaptotagmin athic helix and is affected by the lipid composition. triggers exocytosis by the displacement of com- These data demonstrate that local lipid interac- plexin and by local perturbations of the lipid bi- tions of the complexin carboxy-terminus can mod- layer, resulting in fusion pore opening. Further ulate membrane fusion. In the presence of synap-

36 Thomas Söllner Rab3 Synaptotagmin

Rim complexin

syntaxin 1 Munc13 SNAP-25

Munc18 VAMP2 SNAREpin-independent t-SNARE complex vesicle tethering formation

vesicle docking by SNAREpins SV SV priming priming fusion pore calcium opening SV

SV

Fig. 1: Model of the cascade of events controlling exocytosis at the neuronal synapse. Only key regulatory components are in- dicated at the individual steps and the exact molecular order still needs to be established. For example, synaptotagmin together with Munc18 and syntaxin have been implicated to provide an additional SNAREpin-independent vesicle tethering/docking event, which is not depicted in this model. Putative protein-lipid interactions are indicated by colored lipids.

totagmin, reconstituted into v-SNARE SUVs, and in1. These data suggest that Munc18 and prob- t-SNAREs reconstituted into GUVs, complexin ably its SM homologues at other transport steps, inhibits synaptotagmin-stimulated membrane fu- function as molecular shields preventing the for- sion. The addition of calcium releases the block mation of noncognate SNARE complexes. In the and synchronizes liposome fusion. presence of the cognate v-SNARE, this block is In a different liposome fusion assay, which re- released and Munc18 now accelerates cognate solves t-SNARE assembly, Munc18-1 inhibits t- SNAREpin assembly and membrane fusion. SNARE (syntaxin 1, SNAP-25) complex formation We are presently testing the roles of lipids and and membrane fusion. Remarkably, this block can several other regulatory proteins in various com- be released by liposomes containing the cognate binations. This approach will reveal the exact or- v-SNARE VAMP2, but not by liposomes contain- der of events and the principle organization of the ing VAMP8. Furthermore, Munc18-1 dramatically regulatory network. In collaborative studies, we stimulates the subsequent fusion reaction. In would like to obtain structural information about contrast to the inhibition and inhibition release, distinct reaction intermediates. the stimulation strictly depends on the binding of Munc18-1 to the aminoterminal peptide of syntax- Selected Publications 2008 - 2010 Kögel T., Rudolf, R., Hodneland E., Hellwig, A., Kutnetsov, S.A., Seiler., F., Söllner., T., Barroso., J., Gerdes, H.-H. (2010). $% Distinct roles of myosin Va in membrane remodeling and exo- cytosis of secretory Granules. Traffic, 11, 637-650. Seiler, F. Malsam, J., Krause, J.M., Söllner T.H. (2009). A role of complexin-lipid interactions in membrane fusion. FEBS Letters, 583, 2343-2348.   Malsam, J., Seiler, F. Schollmeier, Y., Rusu, P., Krause, J.M., Söllner, T.H. (2009). The carboxy-terminal domain of com- plexin I stimulates liposome fusion. Proc. Natl. Acad. Sci.  USA 106, 2001-2006. Malsam, J., Kreye, S., Söllner, T.H. (2008). Membrane fusion: SNAREs and regulation. Cell. Mol. Life. Sci. 65, 2814-2832.

Fig. 2: Single vesicle exocytosis in PC12 cells is triggered by Kreye, S., Malsam, J., Söllner, T.H. (2008). In vitro assays membrane depolarization (addition of KCl) and detected by a to measure SNARE mediated membrane fusion. Methods in transient increase of NPY-pHluorin-fluorescence. (pHluorin, Molecular Biology 440, 37-50. a pH-sensitive GFP, was fused to neuropeptide Y (NPY) and thereby targeted into the lumen of large dense core granules.) (A) PC12 cell before addition of KCl, (B) a one second snapshot after KCl addition shows single vesicle fu- Thomas Söllner sion events (red circles) detected by a custom-made MatLab Phone: +49 (0)6221-54 5342 application (Kögel et al., 2010). E-mail: [email protected]

Thomas Söllner 37 1995 Diplom (Chemie), University of Heidelberg, Germany 1995 - 1997 Ph.D. - Sloan-Kettering Cancer Center, New York, USA (Prof. Franz Ulrich Hartl) 1997 - 1999 Ph.D. - Max Planck Institute of Biochemistry, Martinsried (Prof. Franz Ulrich Hartl) 1999 - 2003 PostDoc - The Scripps Research Institute, La Jolla, USA (Prof. Steve Kay) 2003 - 2004 PostDoc - University of California, San Diego, USA (Prof. Maho Niwa) since 2004 Emmy-Noether Group Leader / Junior Group Leader, BZH

Frank Weber

Circadian Regulation and Biological Timing

Goal We investigate the assembly and regulation of To understand the molecular and neuronal the circadian clock in the model organism Droso- program that facilitates a temporal synchro- phila, which is homologous to the clock in mam- nization of physiology and behaviour by the mals. Our goal is to understand how physiology circadian clock. Specific aims: and behaviour are temporally orchestrated, and 1. How transcription factor activity can be we aim to gain insights into general mechanisms precisely controlled to specific times. of biological timing that are similarly important for 2. How cellular and circadian signalling cross- accurate cell cycle and developmental regulation. talk in order to temporally coordinate ge- nome-wide transcription and physiology. Research Highlights 3. How neuronal and cellular signalling net- 1) The timing of transcription factors works control behaviour. The core oscillating activity of the circadian clock in Drosophila and mammals is formed by the Background heterodimeric complex of transcription factors Most organisms regulate their physiological, met- CLOCK (CLK) and CYCLE (CYC). Particularly abolic, and behavioural activities in a rhythmic rhythmic regulation of CLK is crucial for circadian fashion and in synchrony with the environmental clock function. We showed that a sequential and cycles of day and night. Circadian rhythms are

controlled by a set of transcription factors that P P CLK CLK CYC CLK CYC

assemble a molecular circadian clock, which is SUMO able to maintain a self-sustained 24-hour oscilla- CBP P P CYC tion. Circadian regulation provides a vital advan- CLK CLK SUMO? tage by allowing a temporal separation and co- CBP ordination of homeostatic functions, such as an P CLK CYC P per, tim up-regulation of apoptotic and DNA-repair genes E E

prior to sunrise or of metabolic enzymes prior to P P P P CLK P CLK P CLK P food uptake. Malfunction of the circadian system P P P P P P P P is associated with diseases, such as sleeping-, cytoplasm nucleus bipolar-, and depressive-disorder, diabetes, Al- Fig.1: A post-translational interval-timer of the Drosophila circadian clock based on sequential and compartment- zheimer disease and increased tumorigenesis. specific modification of the CLK protein.

38 Frank Weber compartment-specific phos- phorylation controls the life cycle of the CLK protein, un- covering a post-translational timing mechanisms of the circadian clock. Our results indicate that every step of the CLK life cycle is precise- ly controlled by co-factor and DNA interactions, as well as by a cascade of specific post- translational modifications Fig. 2: Distinct sets of neurons assemble a network that controls circadian behaviour that include phosphorylation, (figure adopted from Helfrich-Förster C et. al. J Comp Neurol. (2007) 500:47-70.). SUMOylation and ubiquitination. The sequence structures that underlay circadian behaviour we of specific interactions and modifications allows a investigate the siesta-phenotype in flies. At low precise timing of CLK accumulation, nucleo-cyto- temperature flies like humans are highly active dur- plasmic transport, localization to PML-like nuclear ing midday, while at high temperature behavioural bodies, transcriptional activation, inhibition, and activity is shifted to morning and evening hours finally degradation (Fig. 1). We were able to iden- with a pronounced ‘siesta’ during midday. Morning tify specific phosphorylation sites and kinases and evening behavioural activity are controlled by that control individual steps in the life cycle of the distinct groups of neurons (Fig. 2). We investi- CLK protein. Unravelling the regulation of the CLK gated neuropeptide signalling between circadian protein provides important insights into molecular neurons, which we found to contribute to the reg- mechanisms that allow a precise temporal control ulation of siesta time. In addition, we showed that of transcription factors in general and of circadian the chaperone HSP90 is important for fine tuning transcription in particular. variability and stability of circadian behavioural 2) Temporal regulation of physiology phenotypes, which is particularly interesting with We investigate the cross-talk between circadian regard to the evolution of new behavioural traits. and cellular signalling pathways to better under- stand the signalling network that allows a rhyth- Selected Publications mic organisation of homeostatic functions such H-C. Hung, C. Maurer, D. Zorn, W-L. Chang and F. Weber (2009) Sequential and compartment-specifi c phosphorylation as metabolism, cell proliferation, and neuronal controls the life cycle of the circadian CLOCK protein. J. Biol. Chem. 284:23734-23742. activity. We found that cyclic-nucleotide/PKA, C. Maurer, H-C. Hung and F. Weber (2009) Cytoplasmic calcium/CaMKII and Ras/MAPK pathways con- interaction with CYCLE promotes the post-translational processing of the circadian CLOCK protein. FEBS letters tribute to the regulation of circadian transcription, 583:1561-1566. partially by direct phosphorylation of the CLK pro- H-C. Hung, S. Kay and F. Weber (2009) HSP90, a capacitor of behavioural variation. J. Biol. Rhythms. 24:183-192. tein and partially through regulation of the CREB- R. Brunsing, S.A. Omori, F. Weber, A. Bicknell, L. Friend, binding protein (CBP), which we showed to act R. Rickert, M. Niwa (2008) B- and T-cell development both involve activity of the unfolded protein response pathway. J. as a co-activator and regulatory factor of CLK/ Biol. Chem. 283:17954-17961. CYC-dependent transcription. These signalling H-C. Hung, C. Maurer, S.A. Kay and F. Weber (2007) Circadian transcription depends on limiting amounts of the transcription pathways are likely involved in the regulation of co-activator nejire/CBP. J. Biol. Chem. 282:31349-31357. circadian transcription by metabolic and behav- ioural activity. Frank Weber Phone: +49 (0)6221-54 8573 3) Neurobiology of circadian behaviour E-mail: [email protected] In order to gain insights into neuronal network

Frank Weber 39 1978 Ph.D. - Ludwig-Maximilians-Universität München, Germany (Max Planck Institute of Biochemistry, Martinsried) 1978 - 1986 PostDoc and Group Leader - University of Regensburg, Germany 1986 - 1988 Visiting Scientist - Dept. of Biochemistry, Stanford University, USA 1988 - 1997 Full Professor and Chairman of Biochemistry I - University of Heidelberg, Germany since 1991 Chairman of SFB 352 and of SFB 638 1997 - 2002 Director - BZH since 2001 Managing Editor FEBS Letters 2003 - 2005 President elect German Society of Biochemistry and Molecular Biology (GBM) 2005 - 2007 President GBM Felix Wieland

Molecular mechanisms of COPI transport

Goal the donor membrane, vesicle fission and initiation We are interested in the molecular of uncoating. In contrast to COPII and clathrin mechanisms underlying vesicular transport, coats, the heptameric large COPI coat compo- and are characterizing the components nent coatomer is recruited en bloc to the mem- and their coordinate action that allow brane, so that both the inner and outer shell of formation and fission of Golgi-derived COPI- the vesicle are formed at the same time. Recently, coated vesicles. This includes proteomics the two coatomer subunits γ-COP and ζ-COP and lipidomics of isotypic COPI vesicles, were found to exist in two isoforms. Each isoform functional in vitro assays and reconstitution is, like all other subunits, present in coatomer as of vesicle formation, fission and uncoating in one copy, resulting in four possible different hep- a chemically defined liposomal system. tameric protein complexes. We found that these coatomer isoforms localize differently within the Background Golgi of mammalian cells, suggesting different In the eukaryotic cell, vesicular transport repre- sites of budding for each of them. sents the basic mechanism for i) maintaining the homeostasis of the endomembrane system, ii) In our view, the formation of a COPI transport biosynthetic transport of newly synthesized pro- vesicle involves the following minimal set of com- teins and lipids, and iii) the uptake and intracellular ponents: donor membranes with transmembrane transport of exogenous macromolecules. Three proteins acting as coat and/or cargo receptors classes of coated vesicles are well established to (e.g. members of the p24 family), cytosolic Arf1, mediate transport of proteins and lipids in the exo- cytosolic coatomer and auxiliary enzymes that and endocytic pathway: COPII vesicles for ER ex- serve activation on the membrane of Arf1 (GBF1) port, COPI vesicles for retrograde transport from and the activation of GTP hydrolysis by Arf1 (Arf the Golgi to the ER and bidirectional intra-Golgi GAPs). transport, and clathrin-coated vesicles operat- ing in the late secretory and endocytic pathway. A schematic view of individual steps in COPI Coat components are involved in multiple tasks vesicle biogenesis, with key events during coated such as cargo selection, curvature formation at highlighted, is given in Fig. 1.

40 Felix Wieland / Britta Brügger Ph.D. - University of Frankfurt, Germany 1998

PostDoc - Memorial Sloan Kettering Cancer Center, 1998 - 2000 New York, USA (Prof. James E. Rothman)

PostDoc - BZH (Prof. Felix T. Wieland) 2000 - 2002

Research Fellow - BZH since 2002

Habilitation in Biochemistry, University of Heidelberg, 2007 Medical Faculty

Britta Brügger

Fig. 1: COPI vesicle biogenesis.

Research Highlights Molecular mechanisms of COPI vesicle bio- Arf1 mutant, which does not display the ability genesis to modulate membrane curvature in vitro or to Roles of dimeric Arf1 in vesicle formation and fis- drive formation of coated vesicles, is able to re- sion: Formation of coated vesicles requires two cruit coatomer to allow formation of COPI-coated striking manipulations of the lipid bilayer. First, buds, but does not support scission. Chemical membrane curvature is induced to drive bud for- cross-linking of this Arf1 mutant restores vesicle mation. Second, a scission reaction at the bud release. These studies show that initial curvature neck releases the vesicle. Using a reconstituted of the bud is driven primarily by coatomer, where- system for COPI vesicle formation from puri- as the membrane curvature modulating activity of fied components we find that a non-dimerizing dimeric Arf1 is required for membrane scission.

Felix Wieland / Britta Brügger 41 AB

Fig. 2: A) Molecular mechanisms of COPI vesicle biogenesis. Cryo-electron microscopy of COPI-coated vesicles generated with Arf wt (left hand panels) and COPI-coated buds generated with a non-dimerizing Arf mutant (right hand panels). B) Structure of a SM 18-binding motif. Molecular dynamics simulation of p24 TMD (blue, with the motif highlighted in red) and SM 18:0 (green, hydrocarbon chains; yellow, headgroup of SM 18:0) in a POPC bilayer.

Structures of coatomer and of the COPI coat: p24 family, p24. SM 18:0 binding favors dimeriza- Together with John Briggs’ group at the EMBL tion of p24. Dimeric p24, in turn, recruits coatomer we investigate the structure of soluble coatomer and triggers a conformational change of the com- by single particle electron microscopy, and of plex resulting in polymerization, initiating COPI the coatomer shell on coated vesicles. With the bud formation. Thus, a membrane lipid molecu- first data of a coat on a membrane, a structure lar species can serve as a cofactor in controlling emerges that is strikingly different from those of vesicle budding. the COPII and the clathrin systems as delineated from protein assemblies. Structural principles of transmembrane pro- Differential sorting of cargoes and tethers into iso- tein/membrane lipid interactions formic COPI vesicle populations: We have used A signature within the p24 transmembrane do- an in vitro reconstitution system for COPI vesicles main for recognition of a sphingolipid molecular from Golgi membranes and recombinant isofor- species: We have discovered a peptide signature mic coatomer complexes to compare cargo within within the transmembrane span of p24 for sphin- various COPI vesicle isoforms. In this system we golipid binding. When transplanted, the signature have identified several cargo proteins with a pref- confers sphingolipid binding to a non-sphingolipid erence for individual COPI isoforms. binding transmembrane domain. Results from a Regulation of COPI transport by a unique sphin- data mining approach indicate that this signature golipid/cargo-receptor complex: We have dis- represents a conserved binding site for sphingo- covered a specific binding of the sphingomyelin lipids in several transmembrane proteins. molecular species SM 18:0 exclusively to the Defining the lipid environments of membrane pro- transmembrane domain of one member of the teins: Our mass spectrometry-based approach

42 Felix Wieland / Britta Brügger to quantify membrane lipids has allowed us to Osman C, Haag M, Potting C, Rodenfels J, Dip PV, Wieland FT, Brügger B, Westermann B, Langer T. The genetic probe the boundary lipids of all subunits of the interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in membrane protein protease complex -secretase. mitochondria. J Cell Biol. 2009 Feb 23;184(4):583-96. Weimer C, Beck R, Eckert P, Reckmann I, Moelleken J, As a result we can forward a model in which Brügger B, Wieland F. Differential roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI traffi cking. J Cell Biol. 2008 Nov -secretase is organized within the membrane 17;183(4):725-35. between microdomains with its substrate binding Beck R, Sun Z, Adolf F, Rutz C, Bassler J, Wild K, Sinning I, Hurt E, Brügger B, Béthune J, Wieland F. Membrane curvature site oriented towards a liquid-ordered phase, im- induced by Arf1-GTP is essential for vesicle formation. Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11731-6. plicating liquid-ordered phase as entrance doors Trajkovic K, Hsu C, Chiantia S, Rajendran L, Wenzel D, to proteolytic degradation. Wieland F, Schwille P, Brügger B, Simons M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science. 2008 Feb 29;319(5867):1244-7. These investigations are based on a wide range of Haberkant P, Schmitt O, Contreras FX, Thiele C, Hanada K, Sprong H, Reinhard C, Wieland FT, Brügger B. Protein- sphingolipid interactions within cellular membranes. J Lipid methods, including live cell imaging (with Rainer Res. 2008 Jan;49(1):251-62. Pepperkok; EMBL), bioinformatics (with Gunnar von Heijne and Arne Elofsson, Stockholm), mo- lecular dynamics simulations (with Erik Lindahl, Awards and Honors Felix Wieland Stockholm) microinjection studies (together with 1993 Honorary Member of Charité, Medical Faculty of the Graham Warren, Vienna), in vivo and in vitro FRET Humboldt University, Berlin studies, cryo-electron microscopy (with John Since 2000 EMBO Member Briggs, EMBL), protein chemistry, molecular biol- 2001 Heinrich-Wieland Award ogy, and quantitative nano-mass spectrometry of since 2003 Member of Deutsche Akademie der lipids, as well as chemical biology approaches. Naturforscher Leopoldina 2006 Feldberg Foundation Award

Our research is supported by the German Research Council (SFB 638: Dynamics of mac- Felix Wieland romolecular complexes in biosynthetic transport, Phone: +49 (0)6221-54 4150 E-mail: [email protected] SFB/TRR83: Molecular architecture and cellular functions of protein/lipid assemblies, GRK 1188: Britta Brügger Quantitative analysis of dynamic processes in Phone: +49 (0)6221-54 5426 E-mail: [email protected] membrane transport and translocation, SPP1175: Dynamics of cellular membranes and their exploi- tation by viruses) and CellNetworks Heidelberg.

Selected Publications 2008 - 2010

Osman C, Haag M, Wieland FT, Brügger B, Langer T. A mitochondrial phosphatase required for cardiolipin biosynthesis: the PGP phosphatase Gep4. EMBO J. 2010 Jun 16;29(12):1976-87. Lavieu G, Orci L, Shi L, Geiling M, Ravazzola M, Wieland F, Cosson P, Rothman JE. Induction of cortical endoplasmic reticulum by dimerization of a coatomer-binding peptide anchored to endoplasmic reticulum membranes. Proc Natl Acad Sci U S A. 2010 Apr 13;107(15):6876-81. Rutz C, Satoh A, Ronchi P, Brügger B, Warren G, Wieland FT. Following the fate in vivo of COPI vesicles generated in vitro. Traffi c. 2009 Aug;10(8):994-1005. Beck R, Adolf F, Weimer C, Brügger B, Wieland FT. ArfGAP1 activity and COPI vesicle biogenesis. Traffi c. 2009 Mar;10(3):307-15.

Felix Wieland / Britta Brügger 43 1989 Ph.D. - ETH Zürich, Switzerland 1990 - 1992 PostDoc - Yale University School of Medicine, New Haven, USA 1992 - 1999 PostDoc - Institute of Biochemistry I, University of Heidelberg / BZH 1999 Habilitation in Biochemistry, University of Heidelberg, Medical Faculty 2000 - 2002 Scientific Director - German Cystic Fibrosis Association since 2002 Head of the teaching unit and lecturer - BZH

Cordula Harter

Teaching at the BZH

Our dedication to biochemical education metabolites. For advanced courses, a cell culture is unique. On one hand, more than 900 stu- lab, a cold room, a dark room and equipment for dents of three different faculties (Medicine, large scale preparations, like centrifuges and in- Biosciences, Chemistry) are trained in a large cubators, are available. In a computer room with variety of courses at different levels. On the 14 workstations students are taught in the use of other hand, selected master and graduate special software or online tools, like databases students work on individual projects in the for gene and protein analysis or virtual patients. research groups. In addition, we engage in Outside the BZH-course hours, the entire infra- the development of curricula and novel forms structure of the teaching unit can be used by other of teaching. groups on the campus.

The teaching unit Undergraduate Program All teaching activities are centrally organized by Approximately 800 medical students, 150 biology an independent teaching unit in cooperation with students and 70 chemistry students participate in the group leaders and the deans’ offices of the basic biochemistry courses each year. All basic faculties. Besides coordinating the large variety courses consist of lectures, seminars and prac- of courses and programs, the teaching unit pro- ticals and are individually organized for the stu- vides services and advice for students and teach- dents of the respective subject. ers, maintains the electronic platform for the stu- The medical students’ courses extend from the dents, handles examinations and is responsible second throughout the fourth semester. They for the teaching laboratories. are systematically structured from the basics of Since the initial establishment of its teaching unit biomolecules to complex metabolic pathways and in 2002, the BZH has invested a considerable cellular functions. The preclinical curriculum at portion of its resources to adapt its teaching pro- Heidelberg University is unique in Germany as all grams to state-of-the art education in biochemis- topics are taught interdisciplinary with physiology try. With the reopening in 2006 after the recon- and anatomy. Our curriculum is not only very well struction of the building, the teaching unit was accepted by the students and led to better success completely reorganized. It offers lab space for up rates in internal examinations but also allowed to to 100 students with about half of the benches improve our position in the state examinations: In equipped with basic instruments for biochemi- the last 5 years Heidelberg always ranked within cal analysis of proteins, nucleic acids, lipids and the top 6 German medical faculties (out of 34).

44 Teaching at the BZH In the biosciences, bachelor and master pro- grams were gradually introduced between 2004 and 2008 with a major revision of the bachelor program in 2009. The curricula consist of mod- ules, with basic biochemistry subdivided in theo- retical and practical courses in the second or third semester and advanced courses in the fourth or fifth semester. The BZH has restructured the ba- sic lecture in 2009 (which was transferred from the third to the second semester in the revised curriculum) and introduced a new form of inter- Practical training is at the heart our teaching activities: Here, medical school students analyse the lipid disciplinary multiple-choice examination together composition of blood samples. with cell and molecular biology. In addition, lec- In the last 3 years, more than 30% of the class turers of the BZH offer new seminars which at- registered for biochemistry (although the students tract many new students. In the major “Molecular can choose among more than 20 electives) and and Cellular Biology” of the international master many students are attracted to biochemistry. The program “Molecular Biosciences” the BZH con- elective modules provide training in state-of-the- tributes to various modules with lectures, tutori- art biochemistry and molecular biology as well as als, practical courses and offers lab rotations and a specific introduction into structural biology. master theses to selected students.

Graduate program Graduate education is of high significance at the BZH. Our 65-70 graduate students are enrolled in the internal doctoral program with the major aims to provide intense and professional super- vision and to promote discussions and scientific interactions. To this end, each student discusses the project on a regular basis with a thesis advi- sory committee and presents his or her work in a weekly seminar series. In addition, opportunity is given to discuss science issues in guest speakers’ Work in small groups in an undergraduate seminar promotes active learning and problem solving. seminars and at an annual retreat, both of which are organized by the graduate students them- With the introduction of bachelor and master pro- selves. Students are also encouraged to partici- grams in chemistry, the BZH has reorganized the pate in activities of other programs on campus, basic, obligatory module and established new, like the Hartmut Hoffmann-Berling international elective modules for chemistry students. The graduate school (HBIGS) and the DFG-funded basic, obligatory module consists of two parts: A research training group 1188, to foster inspiring lecture and a one week practical with accompa- discussions and scientific collaborations. nying seminars. To support the students in their preparation for the examinations at the end of Cordula Harter each part, tutorials are offered parallel to the lec- Phone: +49 (0)6221 / 54 6758 tures and the students are intensely supervised E-mail: [email protected] during the practical. This led to a high success rate in the examinations and a strong increase in the demand for elective courses in biochemistry.

Teaching at the BZH 45 Facilities

Protein Mass Spectrometry

We provide the following analytical service: labelling with amino acids in cell culture; Fig.1) • Protein identification by MALDI-TOF mass and “label-free” methods. spectrometry using Peptide Mass Fingerprint • Determination of the molecular mass of vari- and Post Source Decay data (LIFT). ous biological molecules (peptides, oligonucle- • Protein identification by LC-MS/MS (Orbitrap) otides, RNA) by MALDI-TOF mass spectrom- mass spectrometry with equip ment located at etry. the ZMBH. • Analysis of posttranslational protein modification by LC-MS/MS (Orbitrap) mass spectrometry. Johannes Lechner Phone: +49 (0)6221-54 4371 • Quantitative mass spectrometry by LC-MS/MS E-mail: [email protected] (Orbitrap) focusing on SILAC (stable isotope

A Lysine and arginine auxotroph strain

Light isotope Heavy isotope

Mix cells 1:1 Purification of protein complex Fractionation by SDS PAGE In-gel digest of 20-40 individual fractions Analysis by LC-MS/MS B light

C

5

105

5 Relative Intensity Relative

heavy 105

Retention (min)

Fig. 1: Quantitative mass spectrometry utilizing SILAC (A) Workflow (B) Survey scan revealing a peptide pair (z=2) with one light or heavy lysine respectively (C) Ion intensities of the light and heavy peptide extracted from the chromatogram. The red squares indicate the individual survey scans that detected the peptide pair. The purple bars indicate product scans that revealed the identity of the peptides. The red area delimits the part of the chromatogram that was used for quantification.

46 Facilities Microscopy

In the BZH researchers have access to a Zeiss automated and is equipped with a piezo drive for LSM 510 META spectral imaging confocal laser all objectives, an automated Z-stage, an emis- scanning system. The system can be used for 3-D sion filter wheel and a sensitive ORCA/ER cooled reconstruction and time-lapse (4D), FLIP, FRAP, CCD camera. dynamic FRET and linear unmixing. It permits the precise separation of fluorophores with highly Finally, our Zeiss Axiovert 200 Fluorescence overlapping emission spectra. Up to 32 channels Microscope is equipped with an Axiocam MRm can be acquired simultaneously in 1,2 seconds. camera and filters for Cy5, Rhodamine, EGFP and DAPI. An Olympus CellR Imaging Station (resources of SFB 638) enables fast 3D multicolor time-lapse Dimitris Liakopoulos Phone: +49 (0)6221-54 4181 fluorescence microscopy. The microscope is fully E-mail: [email protected]

Fig.: Stu1/CLASP is recruited to unat- tached kinetochore and facilitates their capture to the mi- totic spindle. The un- spindle

(CFP-Tub1) attached chromosome 178 state to the attached state is shown in the budding yeast with time lapse fluorescence im- aging performed on an Olympus CellR Imaging Station. Kinetochores

(Ame1-GFP) are shown in green kinetochore (GFP), the white arrows point to unattached 178 kinetochores, whereas the larger GFP signal in- dicates the attached ki- netochores. The mitotic spindle is shown in blue (CFP). The conserved midzone protein Stu1/

Stu1-3mCherry CLASP (shown in red; 3m-cherry) is recruited to the unattached kine- tochore, then proceeds to travel with the cap- tured kinetochore to the Merge mitotic spindle. (Image 0’3’ 12’ courtesy of C. Funk & J. 6’ 9’ Lechner)

Fluorescence Activated Cell Sorting (FACS)

BZH and ZMBH have established a common FACS Calibur system is available at BZH for ana- FACS facility which is operated by a scientist lytical flow cytometry experiments. The FACS fa- funded by the DFG collaborative research center cility has been made available to all scientists of 638. A state of the art Becton-Dickinson FACS the University of Heidelberg. Aria cell sorter is available for cell sorting experi- ments that has been funded by the Dietmar Hopp Walter Nickel Phone: +49 (0)6221-54 5425 foundation. Additionally, a Becton-Dickinson E-mail: [email protected]

Facilities 47 Lipidomics platform

Lipidomics aims at investigating biological func- showed for the first time that two major mem- tions of lipids in health and disease. Although this brane lipids, cholesterol and sphingomyelin, are research field has emerged only recently, it is rap- segregated from COPI vesicles (Brügger et al., idly expanding. Lipids are increasingly recognised 2000). We have employed the methodology in a as important modulators of many intracellular pro- multitude of fundamental cell biological questions cesses, from regulation of protein function to mod- to establish the lipidomes of various subcellular ulation of cellular pathways. Mass spectrometric membrane systems and of viral particles. We will shotgun lipidomics approaches allow to assess continue to use the lipidomics platform in our own the lipid composition of either total membranes studies of lipid sorting in transport processes as or protein-lipid-assemblies directly from extracts well as in our numerous international collabora- of biological samples. Over the last ten years we tions. have continuously expanded our methods and tools towards a comprehensive and quantitative For selected publications see page 43 (Wieland/ analysis of lipids. Employing this technique we Brügger).

Fig.: Mass spectrometric identification of phosphatidylglycerophosphate, an intermediate in cardiolipin synthesis. Mitochondrial lipids of S. cerevisiae (wild type and a mutant defective in cardiolipin biosynthesis) were separated by TLC. The Lipid spot of interest (arrow) was extracted from the TLC, subjected to mass spec analysis, and identified as phosphatidylglycero- phosphate. Analysis of fragment pattern allowed to establish a scan procedure to selectively monitor and quantify phosphatidylgly- cerophosphate from total mitochondrial extracts..

48 Facilities Instrumentation: The Lipidomics facility builds on unique exper- mode, whereas the other two instruments are tise in qualitative and quantitative lipid analysis coupled to Nanomate devises for high throughput by nano-mass spectrometry. Depending on the analyses. In addition, together with the ZMBH, an scientifc question, three complementary nano- Orbitrap mass spectrometer is available. platforms are available: a hybrid quadrupole time- of-flight mass spectrometer, a hybrid triple qua- Britta Brügger Phone: +49 (0)6221-54 5426 drupole linear ion trap mass spectrometer, and a E-mail: [email protected] triple quadrupole mass spectrometer. The triple quadrupole system operates in single injection

Protein Crystallization Platform

In 2008, the CellNetworks Cluster of Excellence allows distinguishing between protein and salt and Prof. Irmgard Sinning have established a state- crystals at a very early stage using the fluore- of-the-art high-throughput crystallization platform scence of tryptophan residues. for biological macromolecules. Dr. Jürgen Kopp is running the facility assisted by Claudia Siegmann. For more information, please visit the platform The platform is equipped with a Phoenix nano- homepage at http://xtals.bzh.uni-heidelberg.de. liter dispensing robot which allows screening of Jürgen Kopp 1000 crystallization conditions with as little as 100 Phone: +49 (0)6221 – 54 4112 microliters of protein sample. The crystallization Email: [email protected] trials are stored under strict temperature control in two Rigaku Minstrel HT incubators with a to- Irmgard Sinning Phone: +49 (0)6221-54 4781 tal capacity of 800 crystallization plates and are E-mail: [email protected] imaged automatically. Images can be viewed and analyzed via a web interface. Standard and user-defined crystallization screens are available for soluble proteins, RNA-protein and other com- plexes as well as for membrane proteins. In 2010, the infrastructure of the platform was enhanced with a Formulatrix MUVIS UV microscope, which

Facilities 49 Funding

Sonderforschungsbereich (SFB) 638: Dynamics of macromolecular complexes in biosynthetic transport Coordinator: Felix Wieland, Biochemie-Zentrum der Universität Heidelberg (BZH)

Cells are highly dynamic structures that can be in a cell. Even if none of the scientists involved compared with factories full of sophisticated ma- is likely to reach the final goal, we believe that chines. In the last decades many individual parts many important lessons can be learned during of these machines have been identified and char- this journey. The expertise existing in Heidelberg acterised. In the years to come the most excit- has led us to focus our research on biosynthetic ing challenge will be to decipher how individual transport. In this context we use the term dynam- building blocks are put together in variable ways ics at two levels: I) dynamics of macromolecular to perform the cell´s dynamic functions. This is complexes (e.g. their conformational change, or done in an iterative way: one first tries to com- their assembly and disassembly), and ii) the dy- bine the single parts to functional assemblies; namics of the interplay of macromolecular com- once an assembly is defined functionally, such plexes during their further assembly or disas- assemblies are combined to even higher aggre- sembly to form functional subcellular structures gates at a next layer of complexity, again func- (e.g. formation and transport of a pre-ribosomal tionally characterised, and so on. With a hundred assembly through the nuclear pore, the formation thousand or so different proteins that build up a or disassembly of a coated membrane carrier, or human cell, and up to 200 parts comprising a the formation and transport of a virus particle). macromolecular complex (a functional unit), it is Biosynthetic transport is a cellular housekeeping evident that there is still a long way to go in order function of special interest with respect to medi- to completely understand not only the composi- cal research, because many congenital diseases tions of all possible functional units, but also their are caused by defects in transport machinery interplay, i.e. their dynamics. With this knowledge and biosynthetic transport is exploited at various complete, we would understand the molecular steps by pathogenic viruses for productive infec- basis of life, and to prove our understanding, we tion and synthesis of viral progeny. Thus, our SFB would have to reconstitute a living cell from its brings together research groups using structural, defined building blocks. This would have to oc- cell biological, biochemical, molecular biological cur not only by adding each component in exactly and virological methods and analysing various the correct concentration, but also in a defined model organisms, from bacteria via yeasts to sequence, because of their dynamics many of the mammalian cells. Our collaborative approach assemblies can only function correctly in a time- allows integration of colleagues coming from dif- dependent manner. Needless to say that such a ferent fields in the life sciences, driven by their task could be solved only by the activity of many common research interest. As a result, exchange scientists worldwide, and final success, if pos- between the groups of a wide range of knowledge sible at all, lies in the far future. and methodology is achieved naturally, and this, Along this way, the SFB 638 “Dynamics of Mac- combined with the common interest, fosters crea- romolecular Complexes in Biosynthetic Transport” tivity and at the same time strengthens a compe- has initiated an interdisciplinary approach to in- tent and critical view to evaluate results. vestigate the structural and dynamic behaviour of complexes of up to 100 or so components with-

50 Funding Sonderforschungsbereich (SFB) 544: Control of Tropical Infectious Diseases Coordinator: Hans-Georg Kräußlich, Department of Virology

This special research program of the German improving quality and utilization of public Research Council (DFG) has been active since health systems. 1999; it will end after a maximal running time in June 2011. Over the years, 4 out of 18 projects Heiner Schirmer and Luise Krauth-Siegel were have been contributed by group leaders of the among the initi ators of the SFB 544 and of BZH (Davioud-Charvet, Krauth-Siegel, Nickel, the close cooperation between the SFB and Schirmer); the other project leaders of SFB 544 the Centre de Recherche en Santé de Nouna are affiliated with five institutions of Heidelberg (CRSN) in Burkina Faso. This cooperation has University and with the European Molecular resulted in the establishment of a biochemical lab- Biology Laboratory. oratory for molecular parasitology at the CRSN. As a synthetic chemist with therapy-focussed The objec tives of the interdisciplinary SFB 544 concepts, Elisabeth Davioud-Charvet is a driving can be sum marized as follows: force for a number of projects in the SFB. Luise • to analyze biological mechanisms of patho- Krauth-Siegel´s elucidation of the trypanothione genic microorganisms (trypanosomatids, HIV, metabolism led to new drug targeting routes in plasmodium and toxo plasma) in order to dis- parasitic protozoa; she has been a member of the cover new targets and targeting mechanisms SFB´s Steering Committee since 1999. Parasite- for drugs and vaccines as well as new vector specific protein transport in trypanosomatids control strategies and was the contribution of Walter Nickel. Using cell- • to understand the functions of health systems biochemical methods, Heiner Schirmer studies that limit the effectiveness of such con- safety and efficacy of affordable drugs against trol strategies and to explore new ways of paediatric malaria.

Sonderforschungsberreich (SFB) / Transregio (TRR) 83: Molecular architecture and cellular functions of lipid/protein assemblies Coordinator: Thomas Söllner, Heidelberg University Biochemistry Center (BZH)

Biological membranes are fundamental cellular physiological functions such as intracellular building blocks and contain about 10,000 and cell to cell signaling, membrane trafficking, different lipid species, which provide unique local protein processing, virus assembly and infectivity, environments for roughly one third of the proteins compartmental morphology, and membrane/lipid encoded in the human genome. In contrast to turnover/storage. Lipid droplets - a unique form of our knowledge of protein-protein interactions a lipid/protein assembly, containing a hydrophobic our understanding of lipid-lipid and protein-lipid core, covered by a monolayer of polar lipids - are interactions is at a rudimentary stage. included as a model system to understand how Thus, the focus of this transregional collaborative proteins ‘surf’ and assemble on the surface of research centre are lipid/protein assemblies, a lipid monolayer/bilayer. The overall goals are which form microdomains within biological to: i) elucidate the molecular composition of membranes and fulfill a growing array of distinct lipid/protein assemblies, ii) determine the

Funding 51 biophysical forces and structural mechanisms novel and profound molecular insights into the that keep these assemblies together, and to iii) structural and functional nature, specificity, and study the dynamic interactions at high spatial and biology of lipid-protein interactions. temporal resolution in reconstituted systems and To achieve these goals, research groups of the living cells. To address these complex topics, the Universities of Heidelberg and Bonn, the University TRR 83 employs advanced technologies, such as of Technology Dresden, the European Molecular quantitative lipid mass spectrometry, fluorescence- Biology Laboratory (EMBL) in Heidelberg, and cross-correlation spectroscopy, atomic force the Max-Planck Institute of Molecular Cell Biology microscopy, single molecule force spectroscopy, and Genetics in Dresden have joined their forces RNAi screening, and click chemistry. Thus, a and expertise. Three research projects (Brügger/ unique combination of selected membrane model Wieland, Nickel, Söllner), the coordination, and systems and technology platforms shall provide the administration are located at the BZH.

Cluster of Excellence, Cellular Networks: A quantitative view of complex cellular processes Coordinator: Hans-Georg Kräußlich; Vice-coordinators: Michael Brunner and Thomas Holstein

CellNetworks is a research cluster funded by of biological processes that ultimately allows the German Excellence Initiative. It is the aim of mathematical modeling and simulation. CellNetworks to develop a systemic understanding Leading research groups of the DKFZ, EMBL, of the regulation of complex biological networks. Max-Planck-Institute for Medical Research This question is addressed by scientists from and University cooperate in CellNetworks. different disciplines at various levels of complexity. Scientists of the BZH are an essential part of According to these levels the CellNetworks research area A of the cluster, which focuses program is structured into four project areas. on the dynamic interaction of macromolecular Research area A investigates intracellular cellular assemblies. At the BZH a crystallization building blocks and macromolecular assemblies platform has been established with the help of and the spatial and temporal dynamics of their CellNetworks and a facility for mass spectrometry interaction within a network. Area B extends this of lipids is supported by the cluster. Furthermore to the higher architecture of the cell with a focus the BZH junior group of Martin Koš and a number on the cytoskeleton and the mitotic spindle, and of PhD students are supported by CellNetworks. the interaction of the cell with the extracellular In addition CellNetworks has established a deep environment. In area C this is expanded to the sequencing facility at Bioquant which is headed supracellular level, studying signal processing and by Michael Brunner (BZH) an Jochen Wittbrodt development. Research area D adds an additional (COS). level of complexity by addressing the changes of cellular networks by viral and parasite pathogens. CellNetworks also supports methodological and technological platforms necessary to address these questions. CellNetworks aims for a detailed quantitative description and understanding

52 Funding External Funding 2008 - 2010

Total Expenditure

2.500.000,00 €

2.000.000,00 €

1.500.000,00 €

1.000.000,00 €

500.000,00 € SFBs 638, 544 and TRR 83 DFG (without SFBs) Cluster of Excellence EU Foundations 0,00 € Other 2008 2009 2010

Total Expenditure 2008 2009 2010 Total SFBs 638, 544 and TRR 83 1.863.895,44 € 2.005.991,90 € 2.270.325,37 € 6.140.212,71 € DFG (without SFBs) 1.110.847,03 € 1.143.187,06 € 1.362.110,46 € 3.616.144,55 € Cluster of Excellence 467.100,15 € 619.281,22 € 678.848,92 € 1.765.230,29 € EU 200.292,68 € 50.643,42 € 34.101,48 € 285.037,58 € Foundations 169.875,43 € 33.664,63 € 158.160,96 € 361.701,02 € Other 163.322,88 € 178.678,21 € 169.562,22 € 511.563,31 € Total 3.975.333,61 € 4.031.446,44 € 4.673.109,41 € 12.679.889,47 €

Funding 53 Theses

2008 Jens Radzimanowski, Structural and functional analysis of the human Amyloid Precursor Protein Rainer Beck, Molecular Mechanisms of COPI (APP) in complex with the cytosolic interaction Vesicle Biogenesis, Group Leader: Group Leader: partner Fe65, Group Leader: Sinning Wieland Philipp Stelter, Structural Insight into how Dynein Kathrin Buchholz, Redoxnetzwerke des Light Chain (Dyn2) and Nic96 organize the Yeast Malariaerregers Plasmodium: Validierung von Nuclear Pore Complex, Group Leader: Hurt Schlüsselenzymen für neue chemotherapeutische Ansätze, Group Leader: Schirmer Katharina Stengel, Structural Characterization of small Membrane Associated G-Proteins and their Jana Eubel, Interaktion von Methylenblau mit Interacting Partners, Group Leader: Sinning antioxidativen Systemen malariaparasitierter Zellen, Group Leader: Schirmer Stella Tournaviti, The Role of Post-translational Modifi cations of SH4-domain-Containing Proteins Stefanie Grund, Analysis of the inner nuclear in Intracellular Traffi cking And Plasma Membrane membrane protein Src1 that functions at Dynamization, Group Leader: Nickel the interface between gene expression and transcription-coupled mRNA export, Group Alexandra Wendler, Zellbiologische und bioche- Leader: Hurt mische Charakterisierung der Glyoxalase II in Trypanosoma brucei, Group Leader: Krauth-Siegel Daniel Herzenstiel, Charakterisierung der calcium-abhängigen Proteinkinasen PfCDPK4 Cornelius Werner, Proteomanalyse gesunder und 5 aus Plasmodium falciparum, Group Leader: und Parkinson-veränderter humaner Substantia Schirmer nigra, Group Leader: Schirmer Haitong Hou, A network of DHHC acyltrans- Wei Yao, A Dual Role of the Transport Receptor ferases promotes protein palmitoylation and func- Mex67-Mtr2 in Nuclear Export of mRNA, Group tion, Group Leader: Ungermann Leader: Hurt Julian Langer, Conformational dynamics of coatomer: functional and structural studies, Group Leader: Wieland 2009 Svea Leendertz, Kinetische Untersuchungen der Trypanosoma cruzi Trypanothionreduktase, Alexander Brodde, Funktion von Etherlipiden: entwickelt anhand strukturbasierten Wirkstoff- Die Rolle von Plasmalogenen bei der präsynapti- designs, Group Leader: Krauth-Siegel schen Neurotransmission, Group Leader: Just Daniel Markgraf, The role of Rab GTPases in the Lucia Cespón Torrado, Protein folding and Endolysosomal System of S. cerevisiae, Group Quality Control during Unconventional Secretion Leader: Ungermann of Fibroblast Growth Factor 2, Group Leader: Nickel Christian Maurer, Analyse der post-trans- lationalen Regulation der CLOCK/Cycle- Stefanie Hubich, Charakterisierung des Guanin- abhängigen circadianen Transkription, Group nukleotid-Austauschfaktors GBF1, Group Leader: Leader: Weber Wieland Christoph Meiringer, Dynamics and palmitoyla- Judith Jacob, Mutagenesestudien zum Mecha- tion of the SNARE Ykt6 in the yeast endomem- nismus der Thioredoxinreduktasen, Group Lead er: brane system, Group Leader: Ungermann Schirmer Johannes Melchers, NMR-Struktur und Patrick König, Receptors of Protein Transport - Katalysemechanismus der Trypanosoma brucei from cyanobacteria to chloroplasts, Group Leader: Tryparedoxin-Peroxidase III, Group Leader: Sinning Krauth-Siegel Susanne Kreye, Funktion von Munc13-1 in der Tobias Müller, Novel 1,4-Naphthoquinones as regulierten Membranfusion: Mechanistische Inhibitors of Human and Plasmodial Glutathione Analyse im rekonstituierten System, Group Reductase and as Antimalarial Drugs. Synthesis, Leader: Söllner Enzymology and Activity Evaluation, Group Leader: Davioud-Charvet Clemens Ostrowicz, Dynamics and architecture of the HOPS tethering complex in yeast vacuole Andrea Neiß, Charakterisierung von white collar fusion, Group Leader: Ungermann 2 und seinen Proteinisoformen in der circadianen Uhr von Neurospora crassa, Group Leader: Christina Querfurth, Die Rolle der Phosphory- Brunner lierung des negativen Elements FREQUENCY

54 Theses in der circadianen Uhr von Neurospora crassa, Vera Seidel, Klonierung, Überexpression und Group Leader: Brunner phänotypische Analyse der 2-Cys-Glutaredoxine in Afrikanischen Trypanosomen sowie Untersuchung Julia Ritzerfeld, Identifi cation of Components of ihrer Rolle im Glutathionstoffwechsel der Parasiten, the Intracellular Transport Machinery of Acylated Group Leader: Krauth-Siegel Proteins by a Genome-wide RNAi Screen, Group Leader: Nickel Florian Seiler, Molekulare Mechanismen der Complexin vermittelten Stimulation der Vesikelfu- Koen Temmerman, The role of membrane lipids sion im neuronalen Modellsystem, Group Leader: in FGF-2 targeting and membrane translocation, Söllner Group Leader: Nickel Cornelia Ulbrich, Analysis of structure, function Carolin Weimer, Funktionelle Charakterisierung and molecular mechanism of the AAA-ATPase von ArfGAP1, ArfGAP2 und ArfGAP3 im COPI- Rea1 during ribosome biogenesis in S. cerevisiae, vermittelten Transport, Group Leader: Wieland Group Leader: Hurt Nicole Wenzel, Synthesis and Mechanism of Antiparasitic Mannich Base Derivatives Affecting the Redox Equilibrium of Trypanosomes and Malaria Parasites, Group Leader: Davioud- Charvet Ke Xiao, Thiol proteins and other targets for bioinformatical strategies against resistance development in malaria, Group Leader: Schirmer Barbara Zschörnig, Die Proteinkinase CK2- vermittelte Phosphorylierung der humanen Histondeacetylase Sirtuin 1 (SIRT1), Group Leader: Schirmer

2010

Günes Bozkurt, Structural characterization of co-translational and post-translational protein targeting components, Group Leader: Sinning Sevgi Ceylan, Die Rolle der Dithiol-Glutaredo- xine im Trypanothion-Stoffwechsel Afrikanischer Trypanosomen, Group Leader: Krauth-Siegel Antje Ebert, Identifi cation and Functional Char- acterization of Tec Kinase as a Direct Factor in Unconventional Secretion of Fibroblast Growth Factor 2 (FGF2), Group Leader: Nickel Mathias Haag, Development of a nano-ESI-MS/ MS approach for the specifi cation and quantifi ca- tion of membrane lipids, Group Leader: Wieland Daniel Kammerer, Regulation of spindle posi- tioning through modifi cation of the protein Kar9 by SUMO and ubiquitin in yeast, Group Leader: Liakopoulos Christoph Klöckner, Role of the protein Sus1 and its interaction with the Sac3CID motif in transcription-coupled mRNA export, Group Leader: Hurt Sheila Lutz, The role of Sus1, Cdc31 and the Sac3-CID motif in transcription and mRNA export, Group Leader: Hurt

Theses 55 Publications 2008 - 2010

2008

Schafmeier T, Diernfellner A, Schäfer A, Dintsis Köhler A, Schneider M, Cabal GG, Nehrbass U, O, Neiss A, Brunner M. Circadian activity and Hurt E. Yeast Ataxin-7 links histone deubiquitina- abundance rhythms of the Neurospora clock tion with gene gating and mRNA export. Nat Cell transcription factor WCC associated with rapid Biol. 2008; 10(6):707-15. nucleo-cytoplasmic shuttling. Genes Dev. 2008; 22(24):3397-402. Schrader N, Stelter P, Flemming D, Kunze R, Hurt E, Vetter IR. Structural basis of the nic96 sub- Neiss A, Schafmeier T, Brunner M. Transcriptional complex organization in the nuclear pore channel. regulation and function of the Neurospora clock Mol Cell. 2008; 29(1):46-55. gene white collar 2 and its isoforms. EMBO Rep. 2008; 9(8):788-94. Yao W, Lutzmann M, Hurt E. A versatile interac- tion platform on the Mex67-Mtr2 receptor creates Brunner M, Káldi K. Interlocked feedback loops an overlap between mRNA and ribosome export. of the circadian clock of Neurospora crassa. Mol EMBO J. 2008; 27(1):6-16. Microbiol. 2008; 68(2):255-62. Review. Filser M, Comini MA, Molina-Navarro MM, Brunner M, Simons MJ, Merrow M. Lego clocks: Dirdjaja N, Herrero E, Krauth-Siegel RL. Cloning, building a clock from parts. Genes Dev. 2008; functional analysis, and mitochondrial localization 22(11):1422-6. of Trypanosoma brucei monothiol glutaredoxin-1. Biol Chem. 2008; 389(1):21-32. Brunner M, Merrow M. The green yeast uses its plant-like clock to regulate its animal-like tail. Krauth-Siegel RL, Comini MA. Redox control in Genes Dev. 2008; 22(7):825-31. trypanosomatids, parasitic protozoa with trypan- othione-based thiol metabolism. Biochim Biophys Viry E, Battaglia E, Deborde V, Müller T, Réau Acta. 2008; 1780(11):1236-48. Review. R, Davioud-Charvet E, Bagrel D. A sugar-mod- Melchers J, Krauth-Siegel RL, Muhle-Goll C. 1H, ified phosphole gold complex with antiprolifera- 13 15 tive properties acting as a thioredoxin reductase C, and N assignment of the oxidized and re- inhibitor in MCF-7 cells. ChemMedChem. 2008; duced forms of T. brucei glutathione peroxidase- 3:1667-70. type tryparedoxin peroxidase. Biomolecular NMR Assignments. 2008; 2:65-68. M ü l l e r T, M ü l l e r TJ J , D a v i o u d - C h a r v e t E . S y n t h e s i s of photo-reactive naphthoquinones for photoaffin- Comini MA, Rettig J, Dirdjaja N, Hanschmann ity labeling of glutathione reductases. Flavins and EM, Berndt C, Krauth-Siegel RL. Monothiol glu- Flavoproteins 2008 (Frago S, Gómez-Moreno C, taredoxin-1 is an essential iron-sulfur protein in Medina M eds) Prensas Universitarias Zaragoza. the mitochondrion of African trypanosomes. J 2008; 16:443–452. Biol Chem. 2008; 283(41):27785-98. Melchers J, Diechtierow M, Fehér K, Sinning Morin C, Besset T, Moutet JC, Fayolle M, Brückner I, Tews I, Krauth-Siegel RL, Muhle-Goll C. M, Limosin D, Becker K, Davioud-Charvet E. The Structural basis for a distinct catalytic mechanism aza-analogues of 1,4-naphthoquinones are po- in Trypanosoma brucei tryparedoxin peroxidase. tent substrates and inhibitors of plasmodial thiore- J Biol Chem. 2008; 283(44):30401-11. doxin and glutathione reductases and of human erythrocyte glutathione reductase. Org Biomol Beig M, Bender F, Oellien F, Rohwer A, Gaßel Chem. 2008; 6(15):2731-42. M, Selzer P, Unden G, Krauth-Siegel RL. Trypanothione reductase: a target protein for Friebolin W, Jannack B, Wenzel N, Furrer J, a combined in silico and in vitro screening ap- Oeser T, Sanchez CP, Lanzer M, Yardley V, proach. Flavins and Flavoproteins 2008 (Frago Becker K, Davioud-Charvet E. Antimalarial dual S, Gómez-Moreno C, Medina M eds) Prensas drugs based on potent inhibitors of glutathione Universitarias Zaragoza. 2008; 5503-8. reductase from Plasmodium falciparum. J Med Chem. 2008; 51(5):1260-77. Stump B, Eberle C, Kaiser M, Brun R, Krauth- Siegel RL, Diederich F. Diaryl sulfide-based in- Hurt E. Der Kernporenkomplex oder das Tor zur hibitors of trypanothione reductase: inhibition po- Welt des Zytoplasmas. Leopoldina Jahrbuch tency, revised binding mode and antiprotozoal ac- 2007(Meulen V ter ed.). 2008; (3)53, 457-460. tivities. Org Biomol Chem. 2008; 6(21):3935-47. Grund SE, Fischer T, Cabal GG, Antúnez O, Leisner C, Kammerer D, Denoth A, Britschi M, Pérez-Ortín JE, Hurt E. The inner nuclear mem- Barral Y, Liakopoulos D. Regulation of mitotic brane protein Src1 associates with subtelomeric spindle asymmetry by SUMO and the spindle- genes and alters their regulated gene expression. assembly checkpoint in yeast. Curr Biol. 2008; J Cell Biol. 2008; 182(5):897-910. 18(16):1249-55.

Kressler D, Roser D, Pertschy B, Hurt E. The Nickel W, Seedorf M. Unconventional mecha- AAA ATPase Rix7 powers progression of ribo- nisms of protein transport to the cell surface of some biogenesis by stripping Nsa1 from pre-60S eukaryotic cells. Annu Rev Cell Dev Biol. 2008; particles. J Cell Biol. 2008; 181(6):935-44. 24:287-308. Review.

56 Publications Wegehingel S, Zehe C, Nickel W. Rerouting of fi- Stengel KF, Holdermann I, Cain P, Robinson C, broblast growth factor 2 to the classical secretory Wild K, Sinning I. Structural basis for specific sub- pathway results in post-translational modifica- strate recognition by the chloroplast signal recog- tions that block binding to heparan sulfate prote- nition particle protein cpSRP43. Science. 2008; oglycans. FEBS Lett. 2008; 582(16):2387-92. 321(5886):253-6.

Temmerman K, Ebert AD, Müller HM, Sinning I, Radzimanowski J, Ravaud S, Schlesinger S, Tews I, Nickel W. A direct role for phosphatidyli- Koch J, Beyreuther K, Sinning I, Wild K. Crystal nositol-4,5-bisphosphate in unconventional se- structure of the human Fe65-PTB1 domain. J Biol cretion of fibroblast growth factor 2. Traffic. 2008; Chem. 2008; 283(34):23113-20. 9(7):1204-17. König P, Oreb M, Rippe K, Muhle-Goll C, Sinning Seelenmeyer C, Stegmayer C, Nickel W. I, Schleiff E, Tews I. On the significance of Unconventional secretion of fibroblast growth fac- Toc-GTPase homodimers. J Biol Chem. 2008; tor 2 and galectin-1 does not require shedding of 283(34):23104-12. plasma membrane-derived vesicles. FEBS Lett. 2008; 582(9):1362-8. Radzimanowski J, Ravaud S, Beyreuther K, Sinning I, Wild K. Mercury-induced crystalliza- tion and SAD phasing of the human Fe65-PTB1 Buchholz K, Schirmer RH, Eubel JK, Akoachere domain. Acta Crystallogr Sect F Struct Biol Cryst MB, Dandekar T, Becker K, Gromer S. Interactions Commun. 2008; 64(Pt 5):382-5. of methylene blue with human disulfide re- ductases and their orthologues from Plasmodium Radzimanowski J, Beyreuther K, Sinning I, Wild K. falciparum. Antimicrob Agents Chemother. 2008; Overproduction, purification, crystallization and 52(1):183-91. preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein in- Zoungrana A, Coulibaly B, Sié A, Walter-Sack I, tracellular domain. Acta Crystallogr Sect F Struct Mockenhaupt FP, Kouyaté B, Schirmer RH, Klose Biol Cryst Commun. 2008; 64(Pt 5):409-12. C, Mansmann U, Meissner P, Müller O. Safety and efficacy of methylene blue combined with arte- Ravaud S, Stjepanovic G, Wild K, Sinning I. The sunate or amodiaquine for uncomplicated falci- crystal structure of the periplasmic domain of parum malaria: a randomized controlled trial from the Escherichia coli membrane protein insertase Burkina Faso. PLoS ONE. 2008; 3(2):e1630. YidC contains a substrate binding cleft. J Biol Chem. 2008; 283(14):9350-8. Buchholz K, Comini MA, Wissenbach D, Schirmer RH, Krauth-Siegel RL, Gromer S. Cytotoxic inter- König P, Oreb M, Höfle A, Kaltofen S, Rippe K, actions of methylene blue with trypanosomatid- Sinning I, Schleiff E, Tews I. The GTPase cycle specific disulfide reductases and their dithiol prod- of the chloroplast import receptors Toc33/Toc34: ucts. Mol Biochem Parasitol. 2008; 160(1):65-9. implications from monomeric and dimeric struc- tures. Structure. 2008; 16(4):585-96. Buchholz K, Rahlfs S, Schirmer RH, Becker K, Matuschewski K. Depletion of Plasmodium ber- Ravaud S, Wild K, Sinning I. Purification, crystalli- ghei plasmoredoxin reveals a non-essential role zation and preliminary structural characterization for life cycle progression of the malaria parasite. of the periplasmic domain P1 of the Escherichia PLoS ONE. 2008; 3(6):e2474. coli membrane-protein insertase YidC. Acta Crystallogr Sect F Struct Biol Cryst Commun. Schirmer RH, Adler H, Zappe HA, Gromer 2008; 64(Pt 2):144-8. S, Becker K, Coulibaly B, Meissner P (2008) Disulfide reductases as drug targets: Methylene Malsam J, Kreye S, Söllner TH. Membrane fu- blue combination therapies for falciparum malar- sion: SNAREs and regulation. Cell Mol Life Sci. ia in African children. Flavins and Flavoproteins 2008; 65(18):2814-32. Review. 2008 (Frago S, Gómez-Moreno C, Medina M eds) Prensas Universitarias Zaragoza. 2008; 481-486. Kreye S, Malsam J, Söllner TH. In vitro assays to measure SNARE-mediated vesicle fusion. Schirmer H. Book Review on KC Nicolaou and T Methods Mol Biol. 2008; 440:37-50. Montagnon. Molecules that changed the world. J Lab Med 2008; 32:382-383. Brunsing R, Omori SA, Weber F, Bicknell A, Friend L, Rickert R, Niwa M. B- and T-cell de- velopment both involve activity of the unfolded Bionda T, König P, Oreb M, Tews I, Schleiff E. pH protein response pathway. J Biol Chem. 2008; Sensitivity of the GTPase Toc33 as a regulatory 283(26):17954-61. circuit for protein translocation into chloroplasts. Plant Cell Physiol. 2008; 49(12):1917-21. Langer JD, Roth CM, Béthune J, Stoops EH, Brügger B, Herten DP, Wieland FT. A conforma- Oreb M, Tews I, Schleiff E. Policing Tic ‘n’ Toc, the tional change in the alpha-subunit of coatomer in- doorway to chloroplasts. Trends Cell Biol. 2008; duced by ligand binding to gamma-COP revealed 18(1):19-27. Review. by single-pair FRET. Traffic. 2008. 9(4):597-607.

Radzimanowski J, Simon B, Sattler M, Beyreuther Kwa LG, Wegmann D, Brügger B, Wieland FT, K, Sinning I, Wild K. Structure of the intracel- Wanner G, Braun P. Mutation of a single residue, lular domain of the amyloid precursor protein in beta-glutamate-20, alters protein-lipid interac- complex with Fe65-PTB2. EMBO Rep. 2008; tions of light harvesting complex II. Mol Microbiol. 9(11):1134- 40. 2008; 67(1):63-77.

Publications 57 Haberkant P, Schmitt O, Contreras FX, Thiele factor Pfa1 promote 20 to 18 S rRNA processing C, Hanada K, Sprong H, Reinhard C, Wieland catalyzed by the endonuclease Nob1.J Biol Chem. FT, Brügger B. Protein-sphingolipid interactions 2009; 284(50):35079-91. within cellular membranes. J Lipid Res. 2008; 49(1):251-62. Ulbrich C, Diepholz M, Bassler J, Kressler D, Pertschy B, Galani K, Böttcher B, Hurt Trajkovic K, Hsu C, Chiantia S, Rajendran L, E. Mechanochemical removal of ribosome Wenzel D, Wieland F, Schwille P, Brügger B, biogenesis factors from nascent 60S ribosomal Simons M. Ceramide triggers budding of exo- subunits.Cell. 2009; 138:911-22. some vesicles into multivesicular endosomes. Science. 2008; 319(5867):1244-7. Ferreira-Cerca S, Hurt E. Cell biology: Arrest by ribosome.Nature. 2009; 459(7243):46-7. Beck R, Sun Z, Adolf F, Rutz C, Bassler J, Wild K, Sinning I, Hurt E, Brügger B, Béthune J, Wieland Flemming D, Sarges P, Stelter P, Hellwig A, F. Membrane curvature induced by Arf1-GTP is Böttcher B, Hurt E. Two structurally distinct essential for vesicle formation. Proc Natl Acad domains of the nucleoporin Nup170 cooperate to Sci U S A. 2008; 105(33):11731-6. tether a subset of nucleoporins to nuclear pores.J Cell Biol. 2009; 185(3):387-95. Superti-Furga G, Wieland F, Cesareni G. Finally: The digital, democratic age of scientific abstracts. Jani D, Lutz S, Marshall NJ, Fischer T, Köhler FEBS Lett. 2008; 582(8):1169. A, Ellisdon AM, Hurt E, Stewart M. Sus1, Cdc31, and the Sac3 CID region form a conserved Zitzler S, Hellwig A, Hartl FU, Wieland F, interaction platform that promotes nuclear pore Diestelkötter-Bachert P. Distinct binding sites for association and mRNA export.Mol Cell. 2009; the ATPase and substrate-binding domain of hu- 33(6):1-11. man Hsp70 on the cell surface of antigen present- ing cells. Mol Immunol. 2008; 45(15):3974-83. Faza MB, Kemmler S, Jimeno S, González- Aguilera C, Aguilera A, Hurt E, Panse VG. Sem1 Krauss M, Jia JY, Roux A, Beck R, Wieland FT, is a functional component of the nuclear pore De Camilli P, Haucke V. Arf1-GTP-induced tubule complex-associated messenger RNA export formation suggests a function of Arf family pro- machinery.J Cell Biol. 2009; 184(6):833-46. teins in curvature acquisition at sites of vesicle budding. J Biol Chem. 2008; 283(41):27717-23. Klöckner C, Schneider M, Lutz S, Jani D, Kressler D, Stewart M, Hurt E, Köhler A. Mutational Weimer C, Beck R, Eckert P, Reckmann I, uncoupling of the role of Sus1 in nuclear pore Moelleken J, Brügger B, Wieland F. Differential complex targeting of an mRNA export complex roles of ArfGAP1, ArfGAP2, and ArfGAP3 in and histone H2B deubiquitination.J Biol Chem. COPI trafficking. J Cell Biol. 2008; 183(4):725-35. 2009; 284(18):12049-56.

Lacombe T, García-Gómez JJ, de la Cruz J, Roser 2009 D, Hurt E, Linder P, Kressler D. Linear ubiquitin fusion to Rps31 and its subsequent cleavage are Diernfellner AC, Querfurth C, Salazar C, Höfer required for the effi cient production and functional T, Brunner M. Phosphorylation modulates rapid integrity of 40S ribosomal subunits.Mol Microbiol. nucleocytoplasmic shuttling and cytoplasmic 2009; 72(1):69-84. accumulation of Neurospora clock protein FRQ on a circadian time scale.Genes Dev. 2009; Katahira J, Inoue H, Hurt E, Yoneda Y. Adaptor 23(18):2192-200. Aly and co-adaptor Thoc5 function in the Tap-p15- mediated nuclear export of HSP70 mRNA.EMBO Sancar G, Sancar C, Brunner M, Schafmeier T. J. 2009; 28(5):556-67. Activity of the circadian transcription factor White Collar Complex is modulated by phosphorylation Skruzný M, Schneider C, Rácz A, Weng J, Tollervey of SP-motifs.FEBS Lett. 2009; 583(12):1833-40. D, Hurt E. An endoribonuclease functionally linked to perinuclear mRNP quality control associates Chavain N, Davioud-Charvet E, Trivelli X, Mbeki L, with the nuclear pore complexes.PLoS Biol. 2009; Rottmann M, Brun R, Biot C. Antimalarial activities 7(1):e8. of ferroquine conjugates with either glutathione reductase inhibitors or glutathione depletors via Garrenton LS, Braunwarth A, Irniger S, Hurt E, a hydrolyzable amide linker.Bioorg Med Chem. Künzler M, Thorner J. Nucleus-specifi c and cell 2009; 17(23):8048-59. cycle-regulated degradation of mitogen-activated protein kinase scaffold protein Ste5 contributes to Kressler D, Hurt E, Baβler J. Driving ribosome the control of signaling competence.Mol Cell Biol. assembly.Biochim Biophys Acta. 2009. 2009; 29(2):582-601.

Batisse J, Batisse C, Budd A, Böttcher B, Hurt Komljenovic D, Sandhoff R, Teigler A, Heid H, Just E. Purifi cation of nuclear poly(A)-binding protein WW, Gorgas K. Disruption of blood-testis barrier Nab2 reveals association with the yeast transcrip- dynamics in ether-lipid-defi cient mice.Cell Tissue tome and a messenger ribonucleoprotein core Res. 2009; 337(2):281-99. structure.J Biol Chem. 2009; 284(50):34911-7. Teigler A, Komljenovic D, Draguhn A, Gorgas Pertschy B, Schneider C, Gnädig M, Schäfer T, K, Just WW. Defects in myelination, paranode Tollervey D, Hurt E. RNA helicase Prp43 and its co- organization and Purkinje cell innervation in the

58 Publications ether lipid-defi cient mouse cerebellum.Hum Mol Torrado LC, Temmerman K, Müller HM, Mayer Genet. 2009; 18(11):1897-908. MP, Seelenmeyer C, Backhaus R, Nickel W. An intrinsic quality-control mechanism ensures Eberle C, Burkhard JA, Stump B, Kaiser M, Brun unconventional secretion of fi broblast growth R, Krauth-Siegel RL, Diederich F.Synthesis, factor 2 in a folded conformation.J Cell Sci. 2009; inhibition potency, binding mode, and antiprotozoal 122(Pt 18):3322-9. activities of fl uorescent inhibitors of trypanothione reductase based on mepacrine-conjugated Merk M, Baugh J, Zierow S, Leng L, Pal U, Lee SJ, diaryl sulfi de scaffolds.ChemMedChem. 2009; Ebert AD, Mizue Y, Trent JO, Mitchell R, Nickel W, 4(12):2034-44. Kavathas PB, Bernhagen J, Bucala R. The Golgi- associated protein p115 mediates the secretion of Comini MA, Dirdjaja N, Kaschel M, Krauth- macrophage migration inhibitory factor.J Immunol. Siegel RL.Preparative enzymatic synthesis of 2009; 182(11):6896-906. trypanothione and trypanothione analogues.Int J Parasitol. 2009; 39(10):1059-62. Fischer MA, Temmerman K, Ercan E, Nickel W, Seedorf M. Binding of plasma membrane lipids Wenzel IN, Wong PE, Maes L, Müller TJ, recruits the yeast integral membrane protein Krauth-Siegel RL, Barrett MP, Davioud-Charvet Ist2 to the cortical ER.Traffi c. 2009; 10(8):1084- E. Unsaturated Mannich bases active against 97. multidrug-resistant Trypanosoma brucei brucei strains.ChemMedChem. 2009; 4(3):339-51. Tournaviti S, Pietro ES, Terjung S, Schafmeier T, Wegehingel S, Ritzerfeld J, Schulz J, Smith Stump B, Eberle C, Schweizer WB, Kaiser M, DF, Pepperkok R, Nickel W. Reversible Brun R, Krauth-Siegel RL, Lentz D, Diederich phosphorylation as a molecular switch to regulate F. Pentafl uorosulfanyl as a novel building block plasma membrane targeting of acylated SH4 for enzyme inhibitors: trypanothione reductase domain proteins.Traffi c. 2009; 10(8):1047-60. inhibition and antiprotozoal activities of diarylamines.Chembiochem. 2009; 10(1):79-83. Maass K, Fischer MA, Seiler M, Temmerman K, Nickel W, Seedorf M. A signal comprising a basic Wendler A, Irsch T, Rabbani N, Thornalley cluster and an amphipathic alpha-helix interacts PJ, Krauth-Siegel RL. Glyoxalase II does not with lipids and is required for the transport of Ist2 support methylglyoxal detoxifi cation but serves to the yeast cortical ER.J Cell Sci. 2009; 122(Pt as a general trypanothione thioesterase in African 5):625-35. trypanosomes.Mol Biochem Parasitol. 2009; 163(1):19-27. Temmerman K, Nickel W. A novel fl ow cytometric assay to quantify interactions between proteins Ramos EI, Garza KM, Krauth-Siegel RL, Bader and membrane lipids.J Lipid Res. 2009; J, Martinez LE, Maldonado RA. 2,3-diphenyl-1,4- 50(6):1245-54. naphthoquinone: a potential chemotherapeutic agent against Trypanosoma cruzi.J Parasitol. Nickel W, Rabouille C. Mechanisms of regulated 2009; 95(2):461-6. unconventional protein secretion.Nat Rev Mol Cell Biol. 2009; 10(2):148-55. Review. Erratum: Cavalli A, Lizzi F, Bongarzone S, Brun R, Krauth- Nat Rev Mol Cell Biol. 2009; 10(3):234. Siegel RL, Bolognesi ML. Privileged structure- guided synthesis of quinazoline derivatives as Munte CE, Becker K, Schirmer RH, Kalbitzer inhibitors of trypanothione reductase.Bioorg Med HR. NMR assignments of oxidised thioredoxin Chem Lett. 2009; 19(11):3031-5. from Plasmodium falciparum.Biomol NMR Assign. 2009; 3(2):159-61. Krauth-Siegel RL. Redox signaling and regulation in biology and medicine.(editors: Jacob BC and Müller O, Sié A, Meissner P, Schirmer RH, Kouyaté Winyard PG), book review in ChemMedChem. B. Artemisinin resistance on the Thai-Cambodian 2009; 4:2123-2127. border.Lancet. 2009; 374(9699):1419.

Ortiz J, Funk C, Schäfer A, Lechner J. Stu1 inversely Gallo V, Schwarzer E, Rahlfs S, Schirmer RH, van regulates kinetochore capture and spindle stability. Zwieten R, Roos D, Arese P, Becker K. Inherited Genes Dev. 2009; 23(23):2778-91. glutathione reductase defi ciency and Plasmodium falciparum malaria--a case study.PLoS One. 2009; Kemmler S, Stach M, Knapp M, Ortiz J, Pfannstiel 4(10):e7303. J, Ruppert T, Lechner J. Mimicking Ndc80 phosphorylation triggers spindle assembly Xiao K, Jehle F, Peters C, Reinheckel T, checkpoint signalling.EMBO J. 2009; 28(8):1099- Schirmer RH, Dandekar T. CA/C1 peptidases of 110. the malaria parasites Plasmodium falciparum and P. berghei and their mammalian hosts- Barral Y, Liakopoulos D. Role of spindle asymmetry -a bioinformatical analysis.Biol Chem. 2009; in cellular dynamics.Int Rev Cell Mol Biol. 2009; 390(11):1185-97. 278:149-213. Review. Coulibaly B, Zoungrana A, Mockenhaupt FP, Ercan E, Momburg F, Engel U, Temmerman K, Schirmer RH, Klose C, Mansmann U, Meissner Nickel W, Seedorf M. A conserved, lipid-mediated PE, Müller O. Strong gametocytocidal effect sorting mechanism of yeast Ist2 and mammalian of methylene blue-based combination therapy STIM proteins to the peripheral ER.Traffi c. 2009; against falciparum malaria: a randomised 10(12):1802-18. controlled trial.PLoS One. 2009; 4(5):e5318.

Publications 59 Rahlfs S, Koncarevic S, Iozef R, Mailu BM, Sav- signal transduction in the circadian clockworks. vides SN, Schirmer RH, Becker K. Myristoylated Naturwissenschaften. 2009; 96(3):321-37. Review. adenylate kinase-2 of Plasmodium falciparum forms a heterodimer with myristoyltransferase. Emr S, Glick BS, Linstedt AD, Lippincott-Schwartz Mol Biochem Parasitol. 2009; 163(2):77-84. J, Luini A, Malhotra V, Marsh BJ, Nakano A, Pfeffer SR, Rabouille C, Rothman JE, Warren G, Wieland Schirmer H. Essay on the Medical History of FT. Journeys through the Golgi--taking stock in a Methylene Blue. http://www.alzforum.org/new/ new era.J Cell Biol. 2009; 187(4):449-53. Review. Schirmer.aspAlzheimer Research Forum 2009. Beck R, Rawet M, Wieland FT, Cassel D. The Bozkurt G, Stjepanovic G, Vilardi F, Amlacher COPI system: molecular mechanisms and S, Wild K, Bange G, Favaloro V, Rippe K, Hurt function.FEBS Lett. 2009; 583(17):2701-9. Review. E, Dobberstein B, Sinning I. Structural insights Erratum in: FEBS Lett. 2009; 583(21):3541. into tail-anchored protein binding and membrane insertion by Get3.Proc Natl Acad Sci U S A. 2009; Lorizate M, Brügger B, Akiyama H, Glass B, 106(50):21131-6. Müller B, Anderluh G, Wieland FT, Kräusslich HG. Probing HIV-1 membrane liquid order by Kock I, Bulgakova NA, Knust E, Sinning I, Laurdan staining reveals producer cell-dependent Panneels V. Targeting of Drosophila rhodopsin differences.J Biol Chem. 2009; 284(33):22238- requires helix 8 but not the distal C-terminus. 47. PLoS One. 2009; 4(7):e6101. Rutz C, Satoh A, Ronchi P, Brügger B, Warren Grudnik P, Bange G, Sinning I. Protein targeting G, Wieland FT. Following the fate in vivo of by the signal recognition particle.Biol Chem. 2009; COPI vesicles generated in vitro.Traffi c. 2009; 390(8):775-82. Review. 10(8):994-1005.

Neuwirth M, Strohmeier M, Windeisen V, Wallner Osman C, Haag M, Potting C, Rodenfels J, S, Deller S, Rippe K, Sinning I, Macheroux P, Dip PV, Wieland FT, Brügger B, Westermann Tews I. X-ray crystal structure of Saccharomyces B, Langer T. The genetic interactome of cerevisiae Pdx1 provides insights into the prohibitins: coordinated control of cardiolipin oligomeric nature of PLP synthases.FEBS Lett. and phosphatidylethanolamine by conserved 2009; 583(13):2179-86. regulators in mitochondria.J Cell Biol. 2009; 184(4):583-96. Cross BC, Sinning I, Luirink J, High S. Delivering proteins for export from the cytosol. Nat Rev Mol Beck R, Adolf F, Weimer C, Brügger B, Wieland Cell Biol. 2009; 10(4):255-64. Review. FT. ArfGAP1 activity and COPI vesicle biogenesis. Traffi c. 2009; 10(3):307-15. Sinning I, Wild K, Bange G. Signal sequences get active.Nat Chem Biol. 2009; 5(3):146-7. Saher G, Quintes S, Möbius W, Wehr MC, Krämer- Albers EM, Brügger B, Nave KA. Cholesterol Wallner S, Neuwirth M, Flicker K, Tews I, Macheroux regulates the endoplasmic reticulum exit of P. Dissection of contributions from invariant amino the major membrane protein P0 required for acids to complex formation and catalysis in the peripheral myelin compaction.J Neurosci. 2009; heteromeric pyridoxal 5-phosphate synthase 29(19):6094-104. complex from Bacillus subtilis.Biochemistry. 2009; 48(9):1928-35. 2010 Seiler F, Malsam J, Krause JM, Söllner TH. A role of complexin-lipid interactions in membrane Tataroğlu O, Schafmeier T. Of switches and fusion.FEBS Lett. 2009; 583(14):2343-8. hourglasses: regulation of subcellular traffi c in circadian clocks by phosphorylation. EMBO Rep. Malsam J, Seiler F, Schollmeier Y, Rusu P, Krause 2010; 11(12):927-35. JM, Söllner TH. The carboxy-terminal domain of complexin I stimulates liposome fusion.Proc Natl Malzahn E, Ciprianidis S, Káldi K, Schafmeier T, Acad Sci U S A. 2009; 106(6):2001-6. Brunner M. Photoadaptation in Neurospora by competitive interaction of activating and inhibitory Hung HC, Maurer C, Zorn D, Chang WL, LOV domains. Cell 2010; 142(5):762-72. Weber F. Sequential and compartment-specifi c phosphorylation controls the life cycle of the Smith KM, Sancar G, Dekhang R, Sullivan CM, circadian CLOCK protein. J Biol Chem. 2009; Li S, Tag AG, Sancar C, Bredeweg EL, Priest 284(35):23734-42. HD, McCormick RF, Thomas TL, Carrington JC, Stajich JE, Bell-Pedersen D, Brunner M, Freitag Hung HC, Kay SA, Weber F. HSP90, a capacitor M. Transcription factors in light and circadian of behavioral variation.J Biol Rhythms. 2009; clock signaling networks revealed by genomewide 24(3):183-92. mapping of direct targets for neurospora white collar complex. Eukaryot Cell 2010; 9(10):1549- Maurer C, Hung HC, Weber F. Cytoplasmic 56. interaction with CYCLE promotes the post- translational processing of the circadian CLOCK Haid S, Gentzsch J, Jannack B, Bailleul F, protein. FEBS Lett. 2009; 583(10):1561-6. Davioud-Charvet E, Pietschmann T. Inhibition of hepatitis C virus entry by a plant-derived fl avone. Weber F. Remodeling the clock: coactivators and J. Hepatol. Suppl. 1, 52:S293.

60 Publications Wenzel NI, Chavain N, Wang Y, Friebolin W, of african trypanosomes have distinct roles and Maes L, Pradines B, Lanzer M, Yardley V, Brun R, are closely linked to the unique trypanothione Herold-Mende C, Biot C, Tóth K, Davioud-Charvet metabolism. J Biol Chem. 2010; 285(45):35224- E. Antimalarial versus cytotoxic properties of 37. dual drugs derived from 4-aminoquinolines and Mannich bases: interaction with DNA. J Med Bakker BM, Krauth-Siegel RL, Clayton C, Chem. 2010; 53(8):3214-26. Matthews K, Girolami M, Westerhoff HV, Michels PA, Breitling R, Barrett MP. The silicon Bassler J, Kallas M, Pertschy B, Ulbrich C, Thoms trypanosome. Parasitology. 2010; 137(9):1333- M, Hurt E. The AAA-ATPase Rea1 drives removal 41. Review. of biogenesis factors during multiple stages of 60S ribosome assembly. Mol Cell. 2010; 38(5):712-21. Muhle-Goll C, Füller F, Ulrich AS, Krauth-Siegel RL. The conserved Cys76 plays a crucial role Flemming D, Thierbach K, Stelter P, Böttcher B, for the conformation of reduced glutathione Hurt E. Precise mapping of subunits in multiprotein peroxidase-type tryparedoxin peroxidase. FEBS complexes by a versatile electron microscopy Lett. 2010; 584(5):1027-32. label. Nat Struct Mol Biol. 2010; 17(6):775-8. Kammerer D, Stevermann L, Liakopoulos D. Köhler A, Zimmerman E, Schneider M, Hurt Ubiquitylation regulates interactions of astral E, Zheng N. Structural basis for assembly microtubules with the cleavage apparatus. Curr and activation of the heterotetrameric SAGA Biol. 2010; 20(14):1233-43. histone H2B deubiquitinase module. Cell 2010; 141(4):606-17. Siden-Kiamos I, Schüler H, Liakopoulos D, Louis C. Arp1, an actin-related protein, in Plasmodium Köhler A, Hurt E. Gene regulation by nucleoporins berghei. Mol Biochem Parasitol. 2010; 173(2):88- and links to cancer. Mol Cell. 2010; 38(1):6-15. 96. Review Nickel W. Pathways of unconventional protein Ellisdon AM, Jani D, Köhler A, Hurt E, Stewart M. secretion. Curr Opin Biotechnol. 2010; 21(5):621- Structural basis for the interaction between yeast 6. Review Spt-Ada-Gcn5 acetyltransferase (SAGA) complex components Sgf11 and Sus1. J Biol Chem. 2010; Ebert AD, Laussmann M, Wegehingel S, Kaderali 285(6):3850-6. L, Erfl e H, Reichert J, Lechner J, Beer HD, Pepperkok R, Nickel W. Tec-kinase-mediated Kressler D, Hurt E, Bassler J. Driving ribosome phosphorylation of fi broblast growth factor 2 is assembly. Biochim Biophys Acta 2010; essential for unconventional secretion. Traffi c. 1803(6):673-83. 2010; 11(6):813-26.

Schollenberger L, Gronemeyer T, Huber CM, Lay Buchholz K, Putrianti ED, Rahlfs S, Schirmer D, Wiese S, Meyer HE, Warscheid B, Saffrich R, RH, Becker K, Matuschewski K. Molecular Peränen J, Gorgas K, Just WW. RhoA regulates genetics evidence for the in vivo roles of the two peroxisome association to microtubules and major NADPH-dependent disulfi de reductases the actin cytoskeleton. PLoS One 2010; in the malaria parasite. J Biol Chem. 2010; 5(11):e13886. 285(48):37388-95.

Alexander RD, Barrass JD, Dichtl B, Koš M, Bountogo M, Zoungrana A, Coulibaly B, Klose Obtulowicz T, Robert MC, Koper M, Karkusiewicz C, Mansmann U, Mockenhaupt FP, Burhenne I, Mariconti L, Tollervey D, Dichtl B, Kufel J, Mikus G, Walter-Sack I, Schirmer RH, Sié J, Bertrand E, Beggs JD. RiboSys, a high- A, Meissner P, Müller O. Effi cacy of methylene resolution, quantitative approach to measure the blue monotherapy in semi-immune adults with in vivo kinetics of pre-mRNA splicing and 3'-end uncomplicated falciparum malaria: a controlled processing in Saccharomyces cerevisiae. RNA trial in Burkina Faso. Trop Med Int Health 2010; 2010; 16(12):2570-80. 15(6):713-7.

Boon KL, Koš M. Deletion of Swm2p Falk S, Sinning I. The C terminus of Alb3 interacts selectively impairs trimethylation of snRNAs by with the chromodomains 2 and 3 of cpSRP43. J trimethylguanosine synthase (Tgs1p). FEBS Lett. Biol Chem. 2010; 285(53):le25-6; 2010; 584(15):3299-304. Erez E, Stjepanovic G, Zelazny AM, Brügger B, Koš M, Tollervey D. Yeast pre-rRNA processing Sinning I, Bibi E. Genetic evidence for functional and modifi cation occur cotranscriptionally. Mol interaction of the Escherichia coli signal recognition Cell 2010; 37(6):809-20. particle receptor with acidic lipids in vivo. J Biol Chem. 2010; 285(52):40508-14. Eberle C, Lauber BS, Fankhauser D, Kaiser M, Brun R, Krauth-Siegel RL, Diederich F. Derrer B, Windeisen V, Guédez Rodríguez G, Improved Inhibitors of Trypanothione Reductase Seidler J, Gengenbacher M, Lehmann WD, Rippe by Combination of Motifs: Synthesis, Inhibitory K, Sinning I, Tews I, Kappes B. Defi ning the Potency, Binding Mode, and Antiprotozoal structural requirements for ribose 5-phosphate- Activities. ChemMedChem. 2010; 16. binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase. FEBS Lett. Ceylan S, Seidel V, Ziebart N, Berndt C, Dirdjaja 2010; 584(19):4169-74. N, Krauth-Siegel RL. The dithiol glutaredoxins

Publications 61 Bange G, Kümmerer N, Engel C, Bozkurt G, thaliana. Acta Crystallogr Sect F Struct Biol Cryst Wild K, Sinning I. FlhA provides the adaptor for Commun. 2010; 66(Pt 1):12-4. coordinated delivery of late fl agella building blocks to the type III secretion system. Proc Natl Acad Falk S, Ravaud S, Koch J, Sinning I. The C Sci U S A. 2010; 107(25):11295-300. terminus of the Alb3 membrane insertase recruits cpSRP43 to the thylakoid membrane. J Biol Chem. Falk S, Sinning I. cpSRP43 is a novel chaperone 2010; 285(8):5954-62. specifi c for light-harvesting chlorophyll a,b-binding proteins. J Biol Chem. 2010; 285(28):21655-61. Kögel T, Rudolf R, Hodneland E, Hellwig A, Kuznetsov SA, Seiler F, Söllner TH, Barroso Schott A, Ravaud S, Keller S, Radzimanowski J, J, Gerdes HH. Distinct roles of myosin Va in Viotti C, Hillmer S, Sinning I, Strahl S. Arabidopsis membrane remodeling and exocytosis of secretory stromal-derived Factor2 (SDF2) is a crucial granules. Traffi c 2010; 11(5):637-50. target of the unfolded protein response in the endoplasmic reticulum. J Biol Chem. 2010; Beck R, Brügger B, Wieland FT. Membrane 285(23):18113-21. deformation and separation. F1000 Biol Rep. 2010; 2. pii: 35. Koenig P, Mirus O, Haarmann R, Sommer MS, Sinning I, Schleiff E, Tews I. Conserved properties Osman C, Haag M, Wieland FT, Brügger B, of polypeptide transport-associated (POTRA) Langer T. A mitochondrial phosphatase required domains derived from cyanobacterial Omp85. J for cardiolipin biosynthesis: the PGP phosphatase Biol Chem. 2010; 285(23):18016-24. Gep4. EMBO J. 2010; 29(12):1976-87.

Bozkurt G, Wild K, Amlacher S, Hurt E, Lavieu G, Orci L, Shi L, Geiling M, Ravazzola M, Dobberstein B, Sinning I. The structure of Get4 Wieland F, Cosson P, Rothman JE. Induction of reveals an alpha-solenoid fold adapted for multiple cortical endoplasmic reticulum by dimerization interactions in tail-anchored protein biogenesis. of a coatomer-binding peptide anchored to FEBS Lett. 2010; 584(8):1509-14. endoplasmic reticulum membranes. Proc Natl Acad Sci U S A. 2010;107(15):6876-81. Schrul B, Kapp K, Sinning I, Dobberstein B. Signal peptide peptidase (SPP) assembles with Pewzner-Jung Y, Park H, Laviad EL, Silva LC, substrates and misfolded membrane proteins into Lahiri S, Stiban J, Erez-Roman R, Brügger B, distinct oligomeric complexes. Biochem J. 2010; Sachsenheimer T, Wieland F, Prieto M, Merrill AH Jr, 427(3):523-34. Futerman AH. A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic Wild K, Bange G, Bozkurt G, Segnitz B, Hendricks pathways. J Biol Chem. 2010; 285(14):10902-10. A, Sinning I. Structural insights into the assembly of the human and archaeal signal recognition Ernst AM, Contreras FX, Brügger B, Wieland F. particles. Acta Crystallogr D Biol Crystallogr. FEBS Determinants of specifi city at the protein- 2010; 66(Pt 3):295-303. lipid interface in membranes. FEBS Lett. 2010; 584(9):1713-20. Review. Panneels V, Sinning I. Membrane protein expression in the eyes of transgenic fl ies. Methods Mora R, Dokic I, Kees T, Hüber CM, Keitel Mol Biol. 2010; 601:135-47. D, Geibig R, Brügger B, Zentgraf H, Brady NR, Régnier-Vigouroux A. Sphingolipid rheostat Radzimanowski J, Ravaud S, Schott A, Strahl alterations related to transformation can be S, Sinning I. Cloning, recombinant production, exploited for specifi c induction of lysosomal cell crystallization and preliminary X-ray diffraction death in murine and human glioma. Glia. 2010; analysis of SDF2-like protein from Arabidopsis 58(11):1364-83.

62 Publications 63 Staff (* only part of the time reported)

Michael Brunner Group

Secretary PhD Students Gencer Sancar* Technical Assistants Martina Franke-Schaub Francois Cesbron* Christoph Schneider* Julia Kaim* Stilianos Ciprianidis* Julia Stefanski* Juliane Payk* Postdocs Orfeas Dintsis* Özgür Tataroglu Thomas Pils Axel Diernfellner Felix Heise* Johanna Scholz* Christian Maurer* Linda Lauinger* Research Assistant Sabine Schultz Christina Querfurth Erik Malzahn Cigdem Sancar Tobias Schafmeier Christian Nahstoll* Claudia Seelenmeyer Andrea Neiß*

Elisabeth Davioud-Charvet Group Postdoc PhD Students Tobias Müller* Technical Assistants Arnaud Pailot* Thibault Gendron* Alexandra Novodomska* Margit Brückner* Laure Johann* Nicole Wenzel* Beate Jannack Don Antoine Lanfranchi*

Tamás Fischer Group*

PhD Students Technical Assistant Jessica Fuhrmeister* Jutta Worsch* Bianca Hennig*

Ed Hurt Group Secretary Postdocs PhD Students Karsten Thierbach Andrea Schliwa Jochen Baßler Stefan Amlacher Matthias Thoms Julien Batisse* Lyudmila Dimitrova Cornelia Ulbrich* Bettina Bradatsch Jessica Fischer Julan Weng Sébastien Ferreira-Cerca Stefanie Grund* Dirk Flemming Christoph Klöckner* Technical Assistants Alwin Köhler* Sheila Lutz* Marén Gnädig Dieter Kressler* Rizos Georgios Manikas Sabine Griesel* Yoshitaka Matsuo Helge Paternoga* Martina Kallas Brigitte Pertschy* Monika Radwan* Ruth Kunze Michal Skruzny* Phillip Sarges* Daniela Strauß* Philipp Stelter Anshuk Sarkar* Emma Thomson* Johannes Schwarz*

Wilhelm Just Group*

Postdoc PhD Students Technical Assistant Dorothee Lay* Alexander Brodde* Susanne Reusing* Lukas Schollenberger* André Teigler*

64 Staff Martin Koš Group* Postdocs PhD Students Technical Assistants Kum Loong Boon* Juliane Benz* Anne-Kathrin Enke* Isabelle Koš* Andriana Halacheva* Ilona Jung* Daniel George Maeda*

Luise Krauth-Siegel Group

Postdocs PhD Students Vera Seidel* Technical Assistants Florian Füller* Mathias Beig* Alexandra Wendler* Natalie Dirdjaja Alejandro Leroux* Sevgi Ceylan* Edith Röckel Michael Diechtierow Johannes Melchers* Angela Maria Roldan*

Hans Lechner Group Postdoc PhD Students Manuel Stach* Technical Assistant Jennifer Ortiz Caroline Funk* (MD) Ana Stelkic Maria Knapp de Lechner Stephan Kemmler* Verena Schmeiser*

Dimitris Liakopoulos Group PhD Students Lea Stevermann Technical Assistant Hauke Drechsler Ann Na Tan Petra Hubbe* Daniel Kammerer* Marisa Kirchenbauer

Walter Nickel Group Postdocs PhD Students Koen Temmerman* Master Student Hans-Michael Müller Helena Andreas* Styliani Tournaviti* Özgen Deniz* Julia Ritzerfeld Lucia Cespón Torrado* Paulina Turcza* Antje Ebert* Tao Wang* Technical Assistant Giuseppe La Venuta* Georg Weidmann* Sabine Wegehingel Mareike Laußmann* Sonja Zacherl* Julia Steringer

Heiner Schirmer Group PhD Students Technical Assistants Kathrin Buchholz* Heike Adler Ke Xiao* Ursula Göbel

Staff 65 Irmgard Sinning Group Secretary PhD Students Goran Stjepanovic Crystallization Platform Anja Weber Günes Bozkurt* Volker Windeisen Jürgen Kopp Sebastian Falk Claudia Siegmann Postdocs Jan Timo Grotwinkel* Master Students Gert Bange Przemyslaw Grudnik Ajay Aravind* Protein Expression Katja Kapp* Gabriela Guédez- Katja Deselaers* Gunter Stier* Esther Lenherr* Rodríguez* Christoph Engel* Domenico Lupo* Iris Holdermann Hamed Kooshapur* Computer Support Valerie Panneels Annemarie Horn* Stefan Weber* Lutz Nücker Stephaníe Ravaud* Bhalchandra R. Jadhav* Simon Reitz* Wilfried Klug* Technical Assistants Meriem Rezgaoui* Ines Kock* (MD) Silke Adrian Ivo Tews* Patrick König* Astrid Hendricks Klemens Wild Nico Kümmerer Elke Herwig Yin-Yuin Pang* Gabriela Müller Jens Radzimanowski* Bernd Segnitz Katharina Stengel*

Thomas Söllner Group

Secretary PhD Students Florian Seiler* SFB TRR 83 Office* Martina Franke-Schaub Bernhard Dörr* Rostislav Veselinov* Gabriella Kälin* Susanne Kreye* Heike Lorenzen-Schmidt* Postdoc Simone Paulsen Technical Assistants Jörg Malsam Patricia Rusu Jean Michel Krause Julia Schneider* Andrea Scheutzow* Yvette Schollmeier

Frank Weber Group PostDoc PhD Students Hsiu-Cheng Hung Ines Metzger* (MD) Daniela Zorn*

Felix Wieland / Britta Brügger Group Secretary PhD Students Master Student SFB 638 Office Barbara Schröter Frank Adolf Myriam Trausch* Margot Ruland Support Frank Anderl* Jutta Wiech Monika Bertram* Rainer Beck* Technical Assistants Support Gifta Martial* Andreas Max Ernst* Priska Eckert Carmen Monasterio* Iva Ganeva* Alexia Herrmann* Postdocs Michael Geiling* Iris Leibrecht FEBS-Letters Alexander Brodde* Basak Gönen* Ingrid Meißner Editorial Manager Xabier Contreras Mathias Haag Ingeborg Reckmann Patricia McCabe Petra Diestelkötter- Kathrin Höhner* Bachert Stefanie Hubich* Lipidomics Platform Assistant Editors Vincent Popoff* Julian Langer* Timo Sachsenheimer* Aleksander Benjak Oliver Schmitt* Cagakan Özbalci* Daniela Ruffell* Jeroen Strating Simone Röhling* Christoph Rutz* Reviews Editor Carolin Weimer* Wilhelm Just*

Editorial Assistant Anne Müller

66 Staff Mass Spectrometry Group Technical Assistants Susanne Eisel Jürgen Reichert

Teaching

Coordinator Assistant Secretaries Technical Assistants and Lecturer and Lecturer Evelyn Hartmann Evelyn Bauer* Cordula Harter Petra Schling Barbara Schneider Gera Breypohl* Martina Gruß Tanja Schlüter

Central Services Central Computer Support Technician Cell Culture, Administration Lutz Nücker Peter Böhm Technical Assistant Theresa Schaub Gabi Weiß Administrator Janitor Catarina Vill-Härtlein Secretary 1. Floor Josef Back Media Kitchen Petra Krapp-Meiser Selene Cordeiro Assistants Jutta Müller Barbara Bohne Claudia Schönwiese- Dish Washing Service M‘Bengue Linda Boelsen Heiderose Stahl Andrea Zuber

Staff 67 Scientific Advisory Board

In order to maintain the highest standard of research, the BZH uses a process of review and feedback: The Scientific Advisory Board, composed of internationally recognized scientists, meets every three years at the BZH. We very much appreciate the engagement and support of our current advisory board members.

Members of the Scientific Advisory Board in 2010:

Prof. Dr. Elena Conti Max-Planck-Institut für Biochemie, Martinsried, Germany

Prof. Dr. Ulrike Kutay ETH Zürich, Switzerland

Prof. Dr. Dr. Walter Neupert Ludwigs-Maximilians-Universität München, Germany

Prof. Dr. Graham Warren Max F. Perutz Laboratories, Wien, Austria

Prof. Dr. Alfred Wittinghofer Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany

68 Scientific Advisory Board 69 lab time

transport time

summit time

dinner time

night time

party time! How to get to the BZH...

Biochemie-Zentrum der Universität Heidelberg Im Neuenheimer Feld 328 D-69120 Heidelberg

© ZENTRALBEREICH Neuenheimer Feld · Print + Medien

How to get to the BZH 71 Biochemie-Zentrum der Universität Heidelberg Im Neuenheimer Feld 328 D-69120 Heidelberg

BZH_Report_U4_RZ.indd 1 01.03.2011 10:30:24 Uhr