REPORT 2008–2010
RESEARCH AND EDUCATION IN MOLECULAR LIFE SCIENCES
Biochemie-Zentrum der Universität Heidelberg
BZH_Report_U1_RZ.indd 3
RESEARCH AND EDUCATION IN MOLECULAR LIFE SCIENCES
HEIDELBERG UNIVERSITY BIOCHEMISTRY CENTER
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: barbara.bohne@bzh.uni-heidelberg.de
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 molecular 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 unconventional 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 concerned 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 recruited. 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 molecular 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 engages 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 graduate 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-MaximiliansUniversität 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
6
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
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 findings 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
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
nuclo-cytoplasmic
distribution
of
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 degradation 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 dephosphorylation 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 control 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 nucleocytoplasmic 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
activity rhythm of WCC.
Light entrainment of the clock
Light
responses
and
photoadaptation
of
Neurospora are dependent on the photosensory
light-oxygen-voltage
(LOV)
domains
of
the
circadian transcription factor WCC and its negative
8
Michael Brunner
Fig. 2: Molecular mechanism of light-dependent activation
of the WCC and photoadaptation. (A) Light treatment of monomeric 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, thereby competing with the assembly of the WCC light complex.
Box: WCC-dependent gene expression (black line) rapidly increases upon light induction. Transcription adapts to steady
state levels after an initial peak. Increase of light intensity
(red line) results in a second peak of transcription followed by
adaptation of gene expression on slightly elevated levels. (B)
VVD provides a memory of the preceding daylight intensity,
thereby protecting the circadian clock from getting reset by
disturbing light cues during the night, e.g. by the moon.
Fig. 3: Genome-wide identification of WCC-controlled genes. Chromatin immunoprecipitation followed by deep sequencing revealed more than 400 binding sites of the WCC in the Neurospora genome. Genes near WCC binding sites fall into diverse functional 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
responses to light cues of lower intensity (Fig 2B).
We found that VVD is essential to discriminate
between day and night, in particular in naturally
ambiguous photoperiods with moonlight.
Gemome-wide analysis of WCC-controlled
genes
Light signaling has profound effects on the
development and behavior of Neurospora. We
used ChIP-sequencing to uncover direct targets of
the WCC. We found that the light-activated WCC
binds to hundreds of regions, including promoters
of known clock and light-regulated genes.
Amongst the genes activated by the WCC are 28
transcription factor genes (Fig 3). Transcription of
most, but not all, WCC target genes is induced
by light. Our findings provide links between WC-2
and effectors in downstream regulatory pathways
Selected Publications 2008 - 2010
Tataroğlu, O. and Schafmeier, T. (2010) Of switches and
hourglasses: regulation of subcellular traffic in circadian clocks
by phosphorylation. EMBO Rep 11, 927-935.
Malzahn, E., Ciprianidis S., Kaldi, K., Schafmeier, T. and
Brunner, M. (2010) Photoadaptation in Neurospora by
competitive interaction of activating and inhibitory LOV
domains. Cell 142, 762-772.
Smith, K.M., Sancar, G., Dekhang, R., Sullivan, C.M., Li, S.,
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,
D., Brunner, M. and Freitag, M. (2010) Transcription factors
in light and circadian clock signaling networks revealed by
genome-wide mapping of direct targets for neurospora white
collar complex. Eukaryot Cell 9, 1549-1556.
Diernfellner, A., Querfurth, C., Salazar, C., Höfer, T., Brunner, M.
(2009) Phosphorylation modulates rapid nucleo-cytoplasmic
shuttling and cytoplasmic accumulation of Neurospora clock
protein FRQ on a circadian time scale. Genes Dev 23, 21922200.
Sancar, G, Sancar, C, Brunner M and Schafmeier, T (2009)
Activity of the circadian transcription factor White Collar
Complex is modulated by phosphorylation of SP-motifs. FEBS
Lett 583, 1833-1840.
Schafmeier, T, Diernfellner, A, Schäfer, A, Dintsis, O, Neiss,
A, and Brunner, M (2008) Circadian activity and abundance
rhythms of the Neurospora clock transcription factor WCC
associated with rapid nucleo-cytoplasmic shuttling. Genes
Dev 22, 3397-3402.
Neiss, A, Schafmeier, T and Brunner, M (2008) Transcriptional
regulation and function of the Neurospora clock gene white
collar-2 and its isoforms. EMBO Rep 9, 788-794.
for light-induced behavior. Our data suggest
a "flat" hierarchical network in which 20% of all
annotated Neurospora transcription factors are
Michael Brunner
Phone: +49 (0)6221-54 4207
E-mail: michael.brunner@bzh.uni-heidelberg.de
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 Scientifique (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, photoreactive inhibitors (as tools for photoaffinity la-
GR
beling studies) of the selected targets, namely the
glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, the thioredoxin reductases (TrxR) of P. falciparum and man,
the trypanothione reductase (TR) from Trypanosoma cruzi, and the thioredoxin-glutathione reductase (TGR) of Schistosoma mansoni.
Research Highlights
Our strategy for inhibitor optimization is based on
the design and the synthesis of dual drugs that
10
Elisabeth Davioud-Charvet
Fig. 1: “Drowning Plasmodium in Redox” was achieved by
3-benzylmenadione derivatives with potent antimalarial
effects both in vitro and in vivo. Compounds with GR redoxcycling activity displaying the ability to reduce ironIII to ironII
from haemoglobin and heme species.
NADPH, H+
NADP+
GR
O
O
OH
[O]
R
O
benzylNQ
O
RedOx
Cycle
R
O
OH O
benzoylNQ =
biometabolite I
inhibition of
hemozoin formation
and
trophozoite
development arrest
R
reduced benzoylNQ
= biometabolite II
Hb(Fe2+)
2+
PFIX(Fe )
3+
metHb(Fe )
3+
PFIX(Fe )
hemozoin
formation
and
growth of
the parasite
Fig. 2: Proposed cascade of redox reactions for bioactivation of antimalarial 3-benzyl menadione derivatives (benzylNQ). The reaction 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
reductases from the Plasmodium-infected erythrocyte was proposed to be involved in the action
mechanism of the 3-benzylmenadione series.
The biometabolites were shown to act, in oxidized
form, as the most efficient subversive substrates
of both glutathione reductases of Plasmodiuminfected erythrocytes described so far, and, in
reduced form, to redox-cycle methemoglobin to
hemoglobin. Ultimately, the antimalarial naphthoquinones are suggested to affect the redox
equilibrium of target cells resulting in trophozoite
development arrest, by drowning the parasite in
its own metabolic products. For leishmania and
trypanosomes various unsaturated ketones derivatives acting as trypanothione-reactive agents
were produced and revealed potent trypanocidal
effects against pentamidine-sensitive and -resistant strains of Trypanosoma and Leishmania species in vitro. Current efforts include the chemistry of prodrugs of 1,4-naphthoquinones and bis
Wenzel, N. I., Chavain, N., Wang, Y., Friebolin, W., Maes,
L., Pradines, B., Lanzer, M., Yardley, V., Brun, R., HeroldMende, C., Biot, C., Katalin Tóth, Davioud-Charvet, E. (2010)
Antimalarial versus Cytotoxic Properties of Dual Drugs
Derived From 4-Aminoquinolines and Mannich Bases:
Interaction with DNA. J. Med. Chem. 53, 3214–3226.
Chavain, N., Davioud-Charvet, E., Trivelli, X., Mbeki,
L., Rottmann, M., Brun, R., Biot, C. (2009) Antimalarial
Antimalarial activities of ferroquine conjugates with either
glutathione reductase inhibitors or glutathione depletors via
a hydrolyzable amide linker. Bioorg. Med. Chem. 17, 8048–
8059.
Wenzel, I. N., Wong, P. E., Maes, L., Müller, T. J. J., KrauthSiegel, L. R., Barrett, M., Davioud-Charvet, E. (2009)
Antitrypanosomal unsaturated Mannich bases active against
multidrug-resistant T. brucei brucei strains. ChemMedChem.
4,339-351.
Davioud-Charvet E., Müller, T., Bauer, H., Schirmer, H.
1,4-Naphthoquinones as Inhibitors of Glutathione Reductases
and Antimalarial Agents. European Patent EP 08290278.4
(March 26, 2008). PCT/EP2009/053483 (25-03-2009).
WO/2009/118327 (01.10.2009).
Viry, E., Battaglia, E., Deborde, V., Müller, T., Réau, R.,
Davioud-Charvet, E., Bagrel, D. (2008) A sugar-modified
phosphole gold complex with antiproliferative properties
acting as a thioredoxin reductase inhibitor in MCF-7 cells.
ChemMedChem. 3, 1667-1670.
Müller, T., Müller, T.J.J., Davioud-Charvet, E. (2008) Synthesis
of photo-reactive naphthoquinones for photoaffinity labeling
of glutathione reductases. In Flavins and Flavoproteins 2008,
16, 443–452. Frago, S., Gómez-Moreno, C., Medina, M., Eds.
Prensas Universitarias de Zaragoza, Spain, 2008.
Morin, C., Besset, T., Moutet, J.-C., Fayolle, M., Brückner M,
Limosin, D., Becker, K., Davioud-Charvet, E. (2008) The AzaAnalogues of 1,4-Naphthoquinones are potent Substrates
and Inhibitors of Disulfide Reductases. Org. Biomol. Chem.
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,
Germany
zymic studies on the mechanism and the regulation of disulfide reductase in vivo.
Phone: +49 (0)6221-54 4293
E-mail: elisabeth.davioud@bzh.uni-heidelberg.de
Selected Publications 2008 - 2010
Our work is supported by:
Davioud-Charvet, E., Lanfranchi, D. A. Subversive substrates of glutathione reductases from P. falciparum-infected
red blood cells as antimalarial agents. In K. Becker and P.
Selzer (Eds.) Drug Discovery against Apicomplexan parasites – 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, maintenance of genomic stability and chromosome
segregation.
modifying activities in genomic indexing;
• to understand the link between cryptic transcript
accumulation and genomic instability and how
it contributes to cancer development.
Background
12
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
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
with RNAi and heterochromatin factors to eliminate antisense RNAs.” Nature 461(7262):419-22 (*These authors
contributed equally).
suggest that ncRNAs form DNA-RNA hybrids
with their DNA template, which can lead to replication fork collapses and consequently to DNA
lesions and genomic instability. We would like to
further study the molecular mechanisms leading
to genomic instability, and the in vivo function of
RNase H in eukaryotic genome organization.
Fischer, T., B. Cui, J. Dhakshnamoorthy, M. Zhou, C. Rubin,
M. Zofall, T.D. Veenstra, S.I. Grewal. (2009). “Diverse roles
of HP1 proteins in heterochromatin assembly and functions
in fission yeast.” PNAS 106(22):8998-9003.
Roguev, A., S. Bandyopadhyay, M. Zofall, K. Zhang K, T.
Fischer, S.R. Collins, H. Qu, M. Shales, H.O. Park, J. Hayles,
K.L. Hoe, D.U. Kim, T. Ideker, S.I. Grewal, J.S. Weissman, N.J.
Krogan. (2008). “Conservation and Rewiring of Functional
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 studies will increase our general understanding of ge-
Tamás Fischer
Phone: +49 (0)6221-54 4728
E-mail: tamas.fischer@bzh.uni-heidelberg.de
nomic organization, transcriptional regulation and
the biological significance of ncRNAs in the eukaryotic cell. Mutations leading to genomic instability 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) composed of factors, which fucntion in transcription
14
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
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, Purified 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-Ubp8Sgf73 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 endonuclease 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).
Fig. 4: . Model of Rea1 function during 60S ribosome biogenesis.
ribosome.
Rea1 consists of an AAA-ATPase
head and a long flexible tail, both of which can
dock to the pre-ribosomal particle. Subsequently
the molecular motor uses ATP to build up a tensile force. This force can be compared to a spiral
spring and is transmitted to the ribosome precursor via the tail (Figure 4). This force could release
late biogenesis factors (such as Rsa4 or the Rix1subcomplex) from the pre-ribosomal particles in
the nucleus, which makes the pre-ribosome competent for export to the cytoplasm.
Recent work revealed that this same mechanoenzyme Rea1 is used twice in the ribosome
biogenesis pathway, acting also in the nucleolus
to pull off other biogenesis factors from an earlier
pre-60S particle. Overall, these studies revealed
mechanistic insight into the complex pathway of
ribosome biogenesis and clarified the function of
some of the participating factors.
Selected Publications 2008 - 2010
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
(2010).
Alwin Köhler, Eric Zimmermann, Maren Schneider, Ed Hurt
and Ning Zheng: Structural basis for assembly and activation
of the heterotetrameric SAGA histone H2B deubiquitinase
module. Cell 141, 606-617 (2010).
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
mRNA Export Complex and Histone
H2B deubiquitination. J. Biol. Chem.
284, 12049-12056 (2009).
Divyang Jani, Sheila Lutz, Neil J. Marshall, Tamas Fischer,
Alwin Köhler, Andrew M. Ellisdon, Ed Hurt and Murray
Stewart: Sus1, Cdc31 and the Sac3 CID region form a conserved interaction platform that promotes nuclear pore association and mRNA export. Mol. Cell 33, 727-737 (2009).
Michal Skruzny, Claudia Schneider, Attila Rácz, Julan Weng,
David Tollervey and Ed Hurt: An endoribonuclease functionally linked to perinuclear mRNP quality control associates
with the nuclear pore complexes.
PLoS Biology 7, e8 (2009).
Dirk Flemming, Philipp Sarges, Philipp Stelter, Andrea
Hellwig, Bettina Boettcher and Ed Hurt: Two structurally distinct domains of the nucleoporin Nup170 cooperate to tether
a subset of nucleoporins to nuclear pores. J. Cell Biol. 185,
387-395 (2009).
Stefanie Grund, Tamas Fischer, Ghislain G. Cabal, Oreto
Antúnez, José E. Pérez-Ortín and Ed Hurt: The inner nuclear
membrane protein Src1 associates with subtelomeric genes
and alters their regulated gene expression. J. Cell Biol. 182,
897-910 (2008).
Dieter Kressler, Daniela Roser, Brigitte Pertschy and Ed Hurt:
The AAA-ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles. J. Cell Biol.
181, 835-844 (2008).
Alwin Köhler, Maren Schneider, Ghislain Cabal, Ulf Nehrbass
and Ed Hurt: An integrative role of Sgf73 in multiple steps of
SAGA-dependent gene gating.
Nat. Cell Biol. 10, 707-15 (2008).
Nils Schrader, Philipp Stelter, Dirk Flemming, Ruth Kunze, Ed
Hurt* and Ingrid Vetter* (*corresponding authors): Structural
basis of the Nic96 subcomplex organization in the nuclear
pore channel.
Mol. Cell 29, 46-55 (2008).
Wei Yao, Malik Lutzmann and Ed Hurt: A versatile interaction platform on the Mex67-Mtr2 receptor creates an overlap between mRNA and ribosome export. EMBO J. 27, 6–16
(2008).
Awards and Honors
2007
Feldberg Prize
2001
Gottfried Wilhelm Leibniz Prize
Since 2010 Editorial Board of EMBO Journal
Dirk Flemming, Karsten Thierbach, Philipp Stelter, Bettina
Boettcher and Ed Hurt: Precise mapping of subunits in multiprotein complexes by a versatile EM-label
Nat. Struct. Mol. Biol.17,775-778 (2010).
Since 2007 Member of ACADEMIA EUROPAEA
Julien Batisse, Claire Batisse, Aidan Budd, Bettina Böttcher
and Ed Hurt: Purification of poly(A)-binding protein Nab2 reveals association with the yeast transcriptome and a messenger ribonucleoprotein (mRNP) core structure. J. Biol.
Chem. 284, 34911-34917 (2009).
Since 1994 Member of EMBO
Brigitte
Pertschy,
Claudia
Schneider,
Maren
Gnädig,
Since 2005 Member of LEOPOLDINA
Ed Hurt
Phone: +49 (0)6221-54 4173
E-mail: ed.hurt@bzh.uni-heidelberg.de
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. Investigating 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 constrictions 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
new components associated with peroxisomes
(Schollenberger et al, 2010). Other components
18
Wilhelm Just
Fig. 2: The model describes molecules implicated in
vesicular transport between peroxisomes and the ER
(steps 1and 2) and autonomous peroxisome division
(steps 3 and 4).
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 deficiency in CNS and testis (Teigler et al, 2009;
Komljenovic et al 2009). In CNS we found: (i) defects 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 organization by extending the Caspr distribution and
disrupting axo-glial septate-like junctions, (iv) impaired 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 structures (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 ensuring the sluice mechanism for leptotene translocation into the adluminal compartment (Komljenovic
et al 2009).
Selected Publications 2008 - 2010
Fig. 3: In mutants, CF synapses (green) reside on PC
(red) somata (curved arrows) and proximal dendritic
trunk (straight arrows) and occupy a severely restricted
area of PC innervation.
and (v) formation of axon swellings by the accumulation of inositol-tris-phosphate receptor 1-containing smooth ER-like tubuli (Fig. 4) (Teigler et
al, 2009).
Grunau S, D. Lay, S. Mindthoff, H. W. Platta, W. Girzalsky,
W.W. Just, and R. Erdmann. 2010. The Phosphoinositide-3Kinase Vps34p is required for pexophagy in Saccharomyces
cerevisiae. Biochem J. in press.
Schollenberger L, T. Gronemeyer, C. M. Huber, D. Lay, S.
Wiese, H. E. Meyer, B. Warscheid, R. Saffrich, J. Peranen,
K. Gorgas, W.W. Just. 2010. RhoA Regulates Peroxisome
Association to Microtubules and the Actin Cytoskeleton.
PLoS One, 5, e13886.
Teigler A, D. Komljenovic, A. Draguhn, K. Gorgas, W.W.Just.
2009. Defects in myelination, paranode organization and
Purkinje cell innervation in the ether lipid-deficient mouse
cerebellum. Hum Mol Genet. 18, 1897-1908.
Komljenovic D, R. Sandhoff, A. Teigler, H. Heid, W.W. Just
and K. Gorgas. 2009. Disruption of blood-testis barrier dynamics 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
down-regulation and mis-targeting of claudin-3
Wilhelm Just
Phone: +49 (0)6221-54 4286
E-mail: wilhelm.just@bzh.uni-heidelberg.de
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 approximately 100 sites in a process that requires base-
20
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
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.,
Tollervey,D., Dichtl,B., Kufel,J., Bertrand,E. and Beggs,J.D.
(2010). RiboSys, a high-resolution, quantitative approach to
measure the in vivo kinetics of pre-mRNA splicing and 3’end processing in Saccharomyces cerevisiae. RNA 16, 25702580.
cellular stress response and cell growth regulation. We are characterizing the link between ribosome biogenesis and stress response.
Boon,K.-L. and Koš,M. (2010). Deletion of Swm2p selectively
impairs trimethylation of snRNAs by Trimethylguanosine synthase (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 release 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: martin.kos@bzh.uni-heidelberg.de
Fig. 2. Identification of Rcm1 as a methylase of the cytosine 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
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,
thiol
redox
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
and other tropical diseases. All these parasitic
peroxiredoxins
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
trypanothione T(SH)2 which is involved
with Claudia Muhle-Goll (EMBL), Ivo Tews and
in a wide variety of metabolic pathways
(Fig. 1; Krauth-Siegel and Comini 2008).
trypanosomes
and
possess
glutathione
2-Cys-
peroxidase-
Fig. 1
The trypanothione metabolism
Trypanothione disulfide
Research Highlights
Trypanothione
Protein biosynthesis
reductase
as
potential drug target molecule
Trypanothione reductase (TR) which
catalyzes
reduction
the
of
NADPH-dependent
trypanothione
disulfide
(TS2) to the dithiol T(SH)2 is essential
and represents a key enzyme for the
parasite antioxidant defense (Figs. 1 and
22
Luise Krauth-Siegel
Trypanothione
reductase
a
Ascorbate
homeostasis
Thioredoxin-S2
Thioredoxin-(SH)2
Trypanothione
Glutaredoxin-S2
NDP
Ribonucleotide
reductase
Glutaredoxin-(SH)2
dNDP
Glyoxalase system
ROH
Export/
Sequestration
Conjugation of
Metals and drugs
ROOH
Reduction of
hydroperoxides
Fig. 2: Detoxification
of hydroperoxides
(ROOH)
by
the
trypanothione cascade.
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 Pxtype enzymes are responsible for protecting the
parasite specifically against lipid peroxidation
(Michael Diechtierow, unpublished results).
The role of parasite glutaredoxins
Despite the lack of a GR, the genome of African
trypanosomes encodes several genes for monoand dithiol glutaredoxins (Grx). The 1-Cys-Grx1
proved to be an essential iron-sulfur cluster protein
in the single mitochondrion of these parasites
(Comini et al. 2008). The cytosolic 2-Cys-Grx1
also forms an iron sulfur cluster although the
protein contains the canonical CPYC active site
motif previously claimed not to allow complex
formation. 2-Cys-Grx2 is an essential protein in
the mitochondrial intermembrane space (Ceylan
et al. 2010). The oxidized form of both 2-Cys-Grxs
containing an intramolecular disulfide is reduced
by T(SH)2 at rate constants that are three orders
of magnitude higher than those with GSH which
underlines the close link between the Grx and
T(SH)2 metabolism.
Large scale synthesis of trypanothione
A prerequisite for the analysis of the parasite
thiol redox metabolism is the availability of
trypanothione. For this purpose we developed
Fig. 3: Superposition of the structures of oxidized Px
III obtained by X-ray diffraction (gray) and NMR (black)
analysis and the NMR-structure of reduced Px III (light
blue). L1, L2, and L3 represent the most extended loops.
Cys47 and Cys95 are involved in catalysis.
Selected Publications 2008 - 2010
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
unique trypanothione metabolism, J. Biol. Chem. 285, 3522435237.
Wenzel, N. I., Wong, P. E., Maes, L., Müller, T. J. J., KrauthSiegel, R. L., Barrett, M. P. and Davioud-Charvet, E. (2009)
Unsaturated Mannich bases active against multidrug-resistant
Trypanosoma strains. Chem. Med. Chem. 4, 339-351.
Comini, M. A., Dirdjaja, N., Kaschel, M. and Krauth-Siegel, R.
L. (2009) Preparative enzymatic synthesis of trypanothione
and trypanothione analogues. Intl J. Parasitol. 39, 1059-1062.
Eberle, C., Burkhard, J. A., Stump, B., Kaiser, M., Brun, R.,
Krauth-Siegel, R. L. and Diederich, F. (2009) Synthesis,
inhibition potency, binding mode, and antiprotozoal activities
of fluorescent inhibitors of trypanothione reductase based on
mepacrine-conjugated diaryl sulfide scaffolds. Chem. Med.
Chem. 4, 2034-2044.
Krauth-Siegel, R. L. and Comini, M. A. (2008) Redox control in
trypanosomatids, parasitic protozoa with trypanothione-based
thiol metabolism. Biochim. Biophys. Acta, 1780, 1236-1248.
Comini, M., Rettig, J., Dirdjaja, N., Hanschmann, E. M., Berndt,
C. and Krauth-Siegel, R. L. (2008) Monothiol glutaredoxin-1 is
an essential, iron sulfur protein in the mitochondrion of African
trypanosomes. J. Biol. Chem. 283, 27785-27798.
Melchers, J., Diechtierow, M., Fehér, C., Sinning, I., Tews,
I., Krauth-Siegel, R. L. and Muhle-Goll, C. (2008) Structural
basis for a distinct catalytic mechanism in Trypanosoma brucei
tryparedoxin peroxidase. J. Biol. Chem. 283, 30401-30411.
an enzymatic system that allows the large
scale production of both reduced and oxidized
trypanothione (Comini et al. 2009).
Luise Krauth-Siegel
Phone: +49 (0)6221 54 4187
E-mail: luise.krauth-siegel@bzh.uni-heidelberg.de
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 kinetochorespindle attachment but does not actively enhance
Background
Organized by centrosomes (or spindle pole bodies in yeast) the mitotic spindle executes chromosome segregation. It is composed of interpolar
this step. Consequently, unattached kinetochores
are likely to direct further schemes designed to
increase their chance of being captured by a microtubule.
microtubules that overlap in the spindle midzone
24
and by kinetochore microtubules that attach to
Research Highlights
chromosomes via the kinetochore. The S. cer-
The
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.
Johannes Lechner
CLASP
homolog
Stu1
regulates
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.
Genes Dev 23(23): 2778-2791.
learn whether there is a cross talk between Stu1
oligomerization and the SAC, since both are controlled by kinetochore—microtubule interaction.
the centromeric chromatin.
Mps1, Ndc80 and SAC regulation
The kinase Mps1 is a component of the SAC sig-
Kemmler, S., Stach, M., Knapp, M., Ortiz, J., Pfannstiel,
J., Ruppert, T., and Lechner, J. 2009. Mimicking Ndc80
phosphorylation triggers spindle assembly checkpoint
signalling. EMBO J 28(8): 1099-1110.
Maekawa, H., Priest, C., Lechner, J., Pereira, G., and Schiebel,
E. 2007. The yeast centrosome translates the positional
information of the anaphase spindle into a cell cycle signal. J
Cell Biol 179(3): 423-436.
naling pathway. Ndc80 is a subunit of the Ndc80
complex that not only is directly involved in kinetochore-microtubule interaction but also is essential for kinetochores to execute SAC function.
Johannes Lechner
Phone +49 (0)6221-54 4371
E-mail: johannes.lechner@bzh.uni-heidelberg.de
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 cytoskeleton 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 complexes 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 Kar9 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
with the cleavage apparatus. Curr. Biol. 27, 1233-43.
Our aim in the future is to elucidate how sumoylation regulates function of Kar9 and interactions of
aMTs with cortical actin filaments.
We also showed that budding yeast GSK-3 phosphorylates the kinesin Kip2 and reduces its af-
Leisner,C.,
Kammerer,D.,
Denoth,A.,
Barral,Y.
and
Liakopoulos,D. (2008) Regulation of Mitotic-Spindle
Asymmetry by SUMO and the Spindle-Assembly Checkpoint
in Yeast, Curr. Biol. 16, 1249-55.
Barral, Y. and Liakopoulos, D. Role of spindle asymmetry in
cellular dynamics (2009) Int. Rev. Cell Mol Biol. 278, 149213.
finity for aMTs and transport of dynein and Kar9
on aMTs. Interestingly, Kip2 is able to stabilize
aMTs. Very little is known on the mechanisms of
Dimitris Liakopoulos
Phone: +49 (0)6221-54 4181
E-mail: dimitris.liakopoulos@bzh.uni-heidelberg.de
MT-stabilizing kinesins and their regulation. In the
future, we will investigate the mechanism through
which Kip2 stabilizes aMTs in vitro, in collaboration 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-
28
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
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
FGF2 translocates across plasma membranes in
a fully folded conformation (Cespon-Torrado et al.
2009, J. Cell Sci.). To complete membrane translocation, on the extracellular side of the plasma
membrane, heparan sulfate proteoglycans are
required to extract FGF2 from the membrane resulting in its storage on cell surfaces.
Using a genome-wide RNAi screening approach,
a third component of the FGF2 secretion machinery was revealed to be Tec kinase (Ebert et
al., 2010), an enzyme that contains a PH domain
and, alike FGF2, is recruited to the inner leaflet
by phosphoinositides (Fig. 1; Nickel, 2010). FGF2
has been demonstrated a target of Tec kinase resulting in its phosphorylation at tyrosine 82. This
modification is essential for FGF2 secretion and
Ebert AD, Laussmann M, Wegehingel S, Kaderali L, Erfle H,
Reichert J, Lechner J, Beer HD, Pepperkok R, Nickel W. Teckinase-mediated phosphorylation of fibroblast growth factor
2 is essential for unconventional secretion. Traffic. 2010;
11(6):813-26.
Torrado LC, Temmerman K, Müller HM, Mayer MP,
Seelenmeyer C, Backhaus R, Nickel W. An intrinsic qualitycontrol mechanism ensures unconventional secretion of fibroblast growth factor 2 in a folded conformation.
J Cell Sci. 2009; 122(Pt 18):3322-9.
Nickel W, Rabouille C. Mechanisms of regulated unconventional protein secretion. Nat Rev Mol Cell Biol. 2009;
10(2):148-55.
Temmerman K and Nickel W, A novel flow cytometric assay
to quantify interactions between proteins and membrane lipids. J. Lipid Res. 2009; 50:1245-1254.
Tournaviti S, Pietro ES, Terjung S, Schafmeier T, Wegehingel
S, Ritzerfeld J, Schulz J, Smith DF, Pepperkok R, Nickel W.
Reversible phosphorylation as a molecular switch to regulate
plasma membrane targeting of acylated SH4 domain proteins. Traffic. 2009; 10(8):1047-60.
Nickel W, Seedorf M. Unconventional mechanisms of protein
transport to the cell surface of eukaryotic cells. Annu Rev
Cell Dev Biol. 2008; 24:287-308.
Temmerman K, Ebert AD, Müller HM, Sinning I, Tews I, Nickel
W. A direct role for phosphatidylinositol-4,5-bisphosphate in
unconventional secretion of fibroblast growth factor 2. Traffic.
2008; 9(7):1204-17.
may play a role in PI(4,5)P2-induced FGF2 oligomerization and membrane insertion.
Based on the components illustrated in Fig. 1
as well as additional factors identified through
Walter Nickel
Phone: +49 (0)6221-54 5425
E-mail: walter.nickel@bzh.uni-heidelberg.de
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-
30
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-
Heiner Schirmer
Fig. 1: Cytosolic human glutathione reductase homodimer
with bound pyocyanin (PYO). Pyocyanin
(blue) and FAD (yellow) 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 accommodated 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,
Schirmer RH, Sié A, Meissner P, Müller O (2010) Efficacy
of methylene blue monotherapy in semi-immune adults with
uncomplicated falciparum malaria: a controlled trial in Burkina
Faso Trop Med Int Health 15, 713-717
from less than € 150 to € 30000 (http://www.alzforum.org/new/Schirmer.asp). If this situation does
not change MB has no future as a drug for malaria
as a disease of the poor. As a consequence, we
study the cell biochemistry of azure B, the major
metabolite of MB in man. Azure B (see legends
Buchholz K, Putrianti ED, Rahlfs S, Schirmer RH, Becker K,
Matuschewski K (2010) Molecular genetics evidence for the
in vivo roles of the two major NADPH-dependent disulfide
reductases in the malaria parasite. J Biol Chem 285: 3738837395
Buchholz K, Schirmer RH, Eubel JK, Akoachère MB, Dandekar
T, Becker K, Gromer S (2008) Interactions of methylene blue
with human disulfide 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) Disulfide reductases as drug
targets: Methylene blue combination therapies for falciparum
malaria in African children. Flavins and Flavoproteins 16, 481486
Awards and Honors
1976
2002-2009
Fig. 2 Methylene blue as an H2O2-producing subversive redox-cycler. The enzyme glutathione reductase and
other disulfide reductases of the malaria parasite catalyze
the reduction of methylene blue to leucomethylene blue.
Leucomethylene blue auto-oxidizes instantaneously regenerating MB and producing parasitocidal H2O2. Pyocyanin and
azure B can undergo the same redox-cycling as MB.
Appointment as a Bicentennial
Lecturer in Philadelphia and Boston
Dream Action Award of the Dutch
chemical company DSM
Heiner Schirmer
Phone: +49 (0)6221-54 4165
E-mail: heiner.schirmer@bzh.uni-heidelberg.de
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 crystallography as our key method. Our data provide
32
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.
Irmgard Sinning
Although the SRP system is conserved in evolution, it can be adapted for specific requirements.
The post-translational function of SRP in chloroplasts is particularly interesting as it guides
nuclear encoded light-harvesting chlorophyll a,b
binding proteins (LHCPs) to the thylakoid membrane. LHCPs serve as antenna complexes in
photosynthesis and are the most abundant membrane 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
cpSRP43, a novel component of cpSRP, in order
to understand its role in LHCP biogenesis.
Fig. 1: Recognition of targeting signals by SRP and Get
pathways.
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 chromodomains (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
clamped together by a Zn ion and comprises two subdomains
(blue, green).
in the ATPase fold. Interestingly, the TA protein
binding site shares the enrichment in methionine
residues with the signal sequence binding site in
34
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
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 membrane 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 domain.
targeted to the rhabdomeres. We could localize
the targeting signal of Drosophila rhodopsin in
the distal part of helix 8 which might be a useful
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:
21131-6.
Kock, I., Bulgakova, N.A., Knust, E., Sinning, I. & Panneels, V.
(2009) Targeting of Drosophila rhodopsin requires helix 8 but
not the distal C-terminus, PLoSOne 4: e6101.
tool to improve heterologous expression of
Grudnik, P., Bange, G. & Sinning, I. (2009) Protein targeting
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.
Cell. Biol. 10: 255-64.
transporters produced in fly eyes have entered into
crystallization trials. Ivo Tews studies the molecular
mechanisms of Toc GTPases in chloroplast import,
mycobacterial adenylylcyclases and Vitamin B6
biosynthesis. Structural studies of PLP synthase
provided insights into the assembly mechanism
of this huge molecular machine and highlighted
Sinning, I., Wild, K. & Bange, G. (2009) Signal sequences get
active. Nat Chem Biol. 5: 146-7.
Stengel, K., Holdermann, I., Cain, P., Robinson, C., Wild, K. &
Sinning, I. (2008) Structural basis for specific substrate recognition by the chloroplast signal recognition particle protein
cpSRP43, Science 321: 253-6.
Ravaud, S., Stjepanovic, G., Wild, K. & Sinning, I. (2008) The
crystal structure of the periplasmic domain of the Escherichia
coli membrane protein insertase YidC contains a substrate
binding cleft, J. Biol. Chem. 283: 9350-8.
a number of key intermediates in PLP synthesis.
Chloroplasts contain a majority of proteins that
are nuclear encoded, synthesized in the cytosol
and imported into the stroma across the outer
and inner envelope. Import is regulated by the
GTPases Toc33 and Toc159. Structure analyses
Awards and Honors
2010
Member of EMBO
2010
Member of LEOPOLDINA
2010
Heidelberg Molecular Life Sciences
(HMLS) Investigator Award
and biochemical data clarified the role of Toc33
dimerization for protein import. Klemens Wild
analyses the structure and function of amyloid
precursor protein (APP) complexes. APP is the
Irmgard Sinning
Phone: +49 (0)6221-54 4781
E-mail: irmi.sinning@bzh.uni-heidelberg.de
central player in Alzheimer Disease pathogenesis.
Structures were determined of the APP intracellular
domain (AICD) in complex with a physiologically
and pathologically important phosphotyrosinebinding 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
36
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-
Thomas Söllner
Rab3
Synaptotagmin
Rim
complexin
syntaxin 1
Munc13
SNAP-25
VAMP2
Munc18
SNAREpin-independent
vesicle tethering
t-SNARE complex
formation
vesicle docking
by SNAREpins
SV
SV
priming
priming
calcium
fusion pore
opening
SV
SV
Fig. 1: Model of the cascade of events controlling exocytosis at the neuronal synapse. Only key regulatory components are indicated 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 exocytosis 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 complexin 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
membrane depolarization (addition of KCl) and detected by a
transient increase of NPY-pHluorin-fluorescence. (pHluorin,
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 fusion events (red circles) detected by a custom-made MatLab
application (Kögel et al., 2010).
Kreye, S., Malsam, J., Söllner, T.H. (2008). In vitro assays
to measure SNARE mediated membrane fusion. Methods in
Molecular Biology 440, 37-50.
Thomas Söllner
Phone: +49 (0)6221-54 5342
E-mail: thomas.soellner@bzh.uni-heidelberg.de
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.
2. How cellular and circadian signalling cross-
of biological timing that are similarly important for
accurate cell cycle and developmental regulation.
talk in order to temporally coordinate genome-wide transcription and physiology.
3. How neuronal and cellular signalling networks control behaviour.
Research Highlights
1) The timing of transcription factors
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 oscillation. Circadian regulation provides a vital advan-
CBP
P
CYC
P
CLK
CLK
SUMO?
tage by allowing a temporal separation and coCBP
ordination of homeostatic functions, such as an
P
P
up-regulation of apoptotic and DNA-repair genes
is associated with diseases, such as sleeping-,
bipolar-, and depressive-disorder, diabetes, Alzheimer disease and increased tumorigenesis.
38
Frank Weber
per, tim
E E
prior to sunrise or of metabolic enzymes prior to
food uptake. Malfunction of the circadian system
CLK CYC
P
P
P
CLK P
P P
P
P
CLK P
P P
cytoplasm
P
P
CLK P
P
nucleus
Fig.1: A post-translational interval-timer of the Drosophila
circadian clock based on sequential and compartmentspecific modification of the CLK protein.
compartment-specific phosphorylation controls the life
cycle of the CLK protein, uncovering a post-translational
timing mechanisms of the
circadian clock. Our results
indicate that every step of
the CLK life cycle is precisely controlled by co-factor and
DNA interactions, as well as
by a cascade of specific posttranslational
modifications
that include phosphorylation,
Fig. 2: Distinct sets of neurons assemble a network that controls circadian behaviour
(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 understand 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-specific phosphorylation
controls the life cycle of the circadian CLOCK protein. J. Biol.
Chem. 284:23734-23742.
as metabolism, cell proliferation, and neuronal
activity. We found that cyclic-nucleotide/PKA,
calcium/CaMKII and Ras/MAPK pathways contribute to the regulation of circadian transcription,
partially by direct phosphorylation of the CLK protein and partially through regulation of the CREBbinding protein (CBP), which we showed to act
as a co-activator and regulatory factor of CLK/
CYC-dependent transcription. These signalling
pathways are likely involved in the regulation of
C. Maurer, H-C. Hung and F. Weber (2009) Cytoplasmic
interaction with CYCLE promotes the post-translational
processing of the circadian CLOCK protein. FEBS letters
583:1561-1566.
H-C. Hung, S. Kay and F. Weber (2009) HSP90, a capacitor of
behavioural variation. J. Biol. Rhythms. 24:183-192.
R. Brunsing, S.A. Omori, F. Weber, A. Bicknell, L. Friend,
R. Rickert, M. Niwa (2008) B- and T-cell development both
involve activity of the unfolded protein response pathway. J.
Biol. Chem. 283:17954-17961.
H-C. Hung, C. Maurer, S.A. Kay and F. Weber (2007) Circadian
transcription depends on limiting amounts of the transcription
co-activator nejire/CBP. J. Biol. Chem. 282:31349-31357.
circadian transcription by metabolic and behavioural activity.
3) Neurobiology of circadian behaviour
Frank Weber
Phone: +49 (0)6221-54 8573
E-mail: frank.weber@bzh.uni-heidelberg.de
In order to gain insights into neuronal network
Frank Weber
39
1978
1978 - 1986
1986 - 1988
1988 - 1997
since 1991
1997 - 2002
since 2001
2003 - 2005
2005 - 2007
Ph.D. - Ludwig-Maximilians-Universität München, Germany
(Max Planck Institute of Biochemistry, Martinsried)
PostDoc and Group Leader - University of Regensburg,
Germany
Visiting Scientist - Dept. of Biochemistry, Stanford University,
USA
Full Professor and Chairman of Biochemistry I - University of
Heidelberg, Germany
Chairman of SFB 352 and of SFB 638
Director - BZH
Managing Editor FEBS Letters
President elect German Society of Biochemistry and
Molecular Biology (GBM)
President GBM
Felix Wieland
Molecular mechanisms of COPI transport
the donor membrane, vesicle fission and initiation
Goal
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
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
We
are
their
interested
coordinate
in
the
action
that
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-
40
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.
Felix Wieland / Britta Brügger
Ph.D. - University of Frankfurt, Germany
1998
PostDoc - Memorial Sloan Kettering Cancer Center,
New York, USA (Prof. James E. Rothman)
1998 - 2000
PostDoc - BZH (Prof. Felix T. Wieland)
2000 - 2002
Research Fellow - BZH
Habilitation in Biochemistry, University of Heidelberg,
Medical Faculty
since 2002
2007
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
A
B
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
42
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
Felix Wieland / Britta Brügger
to quantify membrane lipids has allowed us to
probe the boundary lipids of all subunits of the
membrane protein protease complex -secretase.
As a result we can forward a model in which
-secretase is organized within the membrane
between microdomains with its substrate binding
site oriented towards a liquid-ordered phase, implicating liquid-ordered phase as entrance doors
to proteolytic degradation.
These investigations are based on a wide range of
methods, including live cell imaging (with Rainer
Osman C, Haag M, Potting C, Rodenfels J, Dip PV, Wieland
FT, Brügger B, Westermann B, Langer T. The genetic
interactome of prohibitins: coordinated control of cardiolipin
and phosphatidylethanolamine by conserved regulators in
mitochondria. J Cell Biol. 2009 Feb 23;184(4):583-96.
Weimer C, Beck R, Eckert P, Reckmann I, Moelleken J,
Brügger B, Wieland F. Differential roles of ArfGAP1, ArfGAP2,
and ArfGAP3 in COPI trafficking. J Cell Biol. 2008 Nov
17;183(4):725-35.
Beck R, Sun Z, Adolf F, Rutz C, Bassler J, Wild K, Sinning I,
Hurt E, Brügger B, Béthune J, Wieland F. Membrane curvature
induced by Arf1-GTP is essential for vesicle formation. Proc
Natl Acad Sci U S A. 2008 Aug 19;105(33):11731-6.
Trajkovic K, Hsu C, Chiantia S, Rajendran L, Wenzel D,
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.
Haberkant P, Schmitt O, Contreras FX, Thiele C, Hanada
K, Sprong H, Reinhard C, Wieland FT, Brügger B. Proteinsphingolipid interactions within cellular membranes. J Lipid
Res. 2008 Jan;49(1):251-62.
Pepperkok; EMBL), bioinformatics (with Gunnar
von Heijne and Arne Elofsson, Stockholm), molecular dynamics simulations (with Erik Lindahl,
Awards and Honors Felix Wieland
Stockholm) microinjection studies (together with
1993
Graham Warren, Vienna), in vivo and in vitro FRET
Honorary Member of Charité,
Medical Faculty of the
Humboldt University, Berlin
studies, cryo-electron microscopy (with John
Since 2000 EMBO Member
Briggs, EMBL), protein chemistry, molecular biol-
2001
ogy, and quantitative nano-mass spectrometry of
since 2003 Member of Deutsche Akademie der
Naturforscher Leopoldina
lipids, as well as chemical biology approaches.
2006
Heinrich-Wieland Award
Feldberg Foundation Award
Our research is supported by the German
Research Council (SFB 638: Dynamics of macromolecular complexes in biosynthetic transport,
SFB/TRR83: Molecular architecture and cellular
functions of protein/lipid assemblies, GRK 1188:
Quantitative analysis of dynamic processes in
membrane transport and translocation, SPP1175:
Felix Wieland
Phone: +49 (0)6221-54 4150
E-mail: felix.wieland@bzh.uni-heidelberg.de
Britta Brügger
Phone: +49 (0)6221-54 5426
E-mail: britta.bruegger@bzh.uni-heidelberg.de
Dynamics of cellular membranes and their exploitation 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.
Traffic. 2009 Aug;10(8):994-1005.
Beck R, Adolf F, Weimer C, Brügger B, Wieland FT.
ArfGAP1 activity and COPI vesicle biogenesis. Traffic. 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
44
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).
Teaching at the BZH
In the biosciences, bachelor and master programs were gradually introduced between 2004
and 2008 with a major revision of the bachelor
program in 2009. The curricula consist of modules, with basic biochemistry subdivided in theoretical and practical courses in the second or third
semester and advanced courses in the fourth or
fifth semester. The BZH has restructured the basic lecture in 2009 (which was transferred from
the third to the second semester in the revised
curriculum) and introduced a new form of interdisciplinary multiple-choice examination together
with cell and molecular biology. In addition, lecturers of the BZH offer new seminars which attract many new students. In the major “Molecular
and Cellular Biology” of the international master
program “Molecular Biosciences” the BZH contributes to various modules with lectures, tutorials, practical courses and offers lab rotations and
master theses to selected students.
Practical training is at the heart our teaching activities:
Here, medical school students analyse the lipid
composition of blood samples.
In the last 3 years, more than 30% of the class
registered for biochemistry (although the students
can choose among more than 20 electives) and
many students are attracted to biochemistry. The
elective modules provide training in state-of-theart biochemistry and molecular biology as well as
a specific introduction into structural biology.
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 supervision and to promote discussions and scientific
interactions. To this end, each student discusses
the project on a regular basis with a thesis advisory committee and presents his or her work in a
weekly seminar series. In addition, opportunity is
Work in small groups in an undergraduate seminar
promotes active learning and problem solving.
With the introduction of bachelor and master programs in chemistry, the BZH has reorganized the
basic, obligatory module and established new,
elective modules for chemistry students. The
basic, obligatory module consists of two parts: A
lecture and a one week practical with accompanying seminars. To support the students in their
given to discuss science issues in guest speakers’
seminars and at an annual retreat, both of which
are organized by the graduate students themselves. Students are also encouraged to participate in activities of other programs on campus,
like the Hartmut Hoffmann-Berling international
graduate school (HBIGS) and the DFG-funded
research training group 1188, to foster inspiring
discussions and scientific collaborations.
preparation for the examinations at the end of
each part, tutorials are offered parallel to the lectures and the students are intensely supervised
Cordula Harter
Phone: +49 (0)6221 / 54 6758
E-mail: cordula.harter@bzh.uni-heidelberg.de
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
and Post Source Decay data (LIFT).
• Determination of the molecular mass of various biological molecules (peptides, oligonucle-
• Protein identification by LC-MS/MS (Orbitrap)
mass spectrometry with equipment located at
otides, RNA) by MALDI-TOF mass spectrometry.
the ZMBH.
• Analysis of posttranslational protein modification
Johannes Lechner
by LC-MS/MS (Orbitrap) mass spectrometry.
• Quantitative mass spectrometry by LC-MS/MS
Phone: +49 (0)6221-54 4371
E-mail: johannes.lechner@bzh.uni-heidelberg.de
(Orbitrap) focusing on SILAC (stable isotope
A
Lysine and arginine
auxotroph strain
Heavy isotope
Light isotope
B
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
light
Relative Intensity
C
5
105
heavy
5
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
Dimitris Liakopoulos
of SFB 638) enables fast 3D multicolor time-lapse
Phone: +49 (0)6221-54 4181
E-mail: dimitris.liakopoulos@bzh.uni-heidelberg.de
(CFP-Tub1)
178
kinetochore
Stu1-3mCherry (Ame1-GFP)
spindle
fluorescence microscopy. The microscope is fully
Merge
178
0’
3’
6’
9’
12’
Fig.: Stu1/CLASP is
recruited
to
unattached
kinetochore
and facilitates their
capture to the mitotic spindle. The unattached chromosome
state to the attached
state is shown in the
budding yeast with time
lapse fluorescence imaging performed on an
Olympus CellR Imaging
Station. Kinetochores
are shown in green
(GFP), the white arrows
point to unattached
kinetochores, whereas
the larger GFP signal indicates the attached kinetochores. The mitotic
spindle is shown in blue
(CFP). The conserved
midzone protein Stu1/
CLASP (shown in red;
3m-cherry) is recruited
to the unattached kinetochore, then proceeds
to travel with the captured kinetochore to the
mitotic spindle. (Image
courtesy of C. Funk & J.
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 experiments that has been funded by the Dietmar Hopp
foundation.
Additionally,
a
Becton-Dickinson
Walter Nickel
Phone: +49 (0)6221-54 5425
E-mail: walter.nickel@bzh.uni-heidelberg.de
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 phosphatidylglycerophosphate. Analysis of fragment pattern allowed to establish a scan procedure to selectively monitor and quantify phosphatidylglycerophosphate 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 timeof-flight mass spectrometer, a hybrid triple quadrupole linear ion trap mass spectrometer, and a
Britta Brügger
Phone: +49 (0)6221-54 5426
E-mail: britta.bruegger@bzh.uni-heidelberg.de
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
1000 crystallization conditions with as little as 100
microliters of protein sample. The crystallization
Jürgen Kopp
Phone: +49 (0)6221 – 54 4112
Email: juergen.kopp@bzh.uni-heidelberg.de
trials are stored under strict temperature control
in two Rigaku Minstrel HT incubators with a total capacity of 800 crystallization plates and are
imaged automatically.
Irmgard Sinning
Phone: +49 (0)6221-54 4781
E-mail: irmi.sinning@bzh.uni-heidelberg.de
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 complexes 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 initiators 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.
The objectives of the interdisciplinary SFB 544
can be summarized as follows:
•
•
As a synthetic chemist with therapy-focussed
concepts, Elisabeth Davioud-Charvet is a driving
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 toxoplasma) 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
biological
to: i) elucidate the molecular composition of
membranes and fulfill a growing array of
distinct lipid/protein assemblies, ii) determine the
form
microdomains
within
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
This question is addressed by scientists from
and
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
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).
area
A
investigates
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
52
Funding
description
and
understanding
University
for
cooperate
Medical
in
Research
CellNetworks.
External Funding 2008 - 2010
Total Expenditure
2.500.000,00 €
2.000.000,00 €
1.500.000,00 €
1.000.000,00 €
SFBs 638, 544 and TRR 83
DFG (without SFBs)
500.000,00 €
Cluster of Excellence
EU
Foundations
0,00 €
2008
Total Expenditure
SFBs 638, 544 and TRR 83
DFG (without SFBs)
Cluster of Excellence
EU
Foundations
Other
Total
Other
2009
2010
2008
1.863.895,44 €
1.110.847,03 €
467.100,15 €
200.292,68 €
169.875,43 €
163.322,88 €
3.975.333,61 €
2009
2.005.991,90 €
1.143.187,06 €
619.281,22 €
50.643,42 €
33.664,63 €
178.678,21 €
4.031.446,44 €
2010
Total
2.270.325,37 € 6.140.212,71 €
1.362.110,46 € 3.616.144,55 €
678.848,92 € 1.765.230,29 €
34.101,48 €
285.037,58 €
158.160,96 €
361.701,02 €
169.562,22 €
511.563,31 €
4.673.109,41 € 12.679.889,47 €
Funding
53
Theses
2008
Rainer Beck, Molecular Mechanisms of COPI
Vesicle Biogenesis, Group Leader: Group Leader:
Wieland
Kathrin Buchholz, Redoxnetzwerke des
Malariaerregers Plasmodium: Validierung von
Schlüsselenzymen für neue chemotherapeutische
Ansätze, Group Leader: Schirmer
Jana Eubel, Interaktion von Methylenblau mit
antioxidativen Systemen malariaparasitierter
Zellen, Group Leader: Schirmer
Stefanie Grund, Analysis of the inner nuclear
membrane protein Src1 that functions at
the interface between gene expression and
transcription-coupled mRNA export, Group
Leader: Hurt
Daniel Herzenstiel, Charakterisierung der
calcium-abhängigen Proteinkinasen PfCDPK4
und 5 aus Plasmodium falciparum, Group Leader:
Schirmer
Philipp Stelter, Structural Insight into how Dynein
Light Chain (Dyn2) and Nic96 organize the Yeast
Nuclear Pore Complex, Group Leader: Hurt
Katharina Stengel, Structural Characterization of
small Membrane Associated G-Proteins and their
Interacting Partners, Group Leader: Sinning
Stella Tournaviti, The Role of Post-translational
Modifications of SH4-domain-Containing Proteins
in Intracellular Trafficking And Plasma Membrane
Dynamization, Group Leader: Nickel
Alexandra Wendler, Zellbiologische und biochemische Charakterisierung der Glyoxalase II in
Trypanosoma brucei, Group Leader: Krauth-Siegel
Cornelius Werner, Proteomanalyse gesunder
und Parkinson-veränderter humaner Substantia
nigra, Group Leader: Schirmer
Haitong Hou, A network of DHHC acyltransferases promotes protein palmitoylation and function, Group Leader: Ungermann
Wei Yao, A Dual Role of the Transport Receptor
Mex67-Mtr2 in Nuclear Export of mRNA, Group
Leader: Hurt
Julian Langer, Conformational dynamics of
coatomer: functional and structural studies, Group
Leader: Wieland
2009
Svea Leendertz, Kinetische Untersuchungen
der Trypanosoma cruzi Trypanothionreduktase,
entwickelt anhand strukturbasierten Wirkstoffdesigns, Group Leader: Krauth-Siegel
Alexander Brodde, Funktion von Etherlipiden:
Die Rolle von Plasmalogenen bei der präsynaptischen Neurotransmission, Group Leader: Just
Daniel Markgraf, The role of Rab GTPases in the
Endolysosomal System of S. cerevisiae, Group
Leader: Ungermann
Christian Maurer, Analyse der post-translationalen
Regulation
der
CLOCK/Cycleabhängigen circadianen Transkription, Group
Leader: Weber
Lucia Cespón Torrado, Protein folding and
Quality Control during Unconventional Secretion
of Fibroblast Growth Factor 2, Group Leader:
Nickel
Stefanie Hubich, Charakterisierung des Guaninnukleotid-Austauschfaktors GBF1, Group Leader:
Wieland
Christoph Meiringer, Dynamics and palmitoylation of the SNARE Ykt6 in the yeast endomembrane system, Group Leader: Ungermann
Judith Jacob, Mutagenesestudien zum Mechanismus der Thioredoxinreduktasen, Group Leader:
Schirmer
Johannes
Melchers,
NMR-Struktur
und
Katalysemechanismus der Trypanosoma brucei
Tryparedoxin-Peroxidase III, Group Leader:
Krauth-Siegel
Patrick König, Receptors of Protein Transport from cyanobacteria to chloroplasts, Group Leader:
Sinning
Tobias Müller, Novel 1,4-Naphthoquinones as
Inhibitors of Human and Plasmodial Glutathione
Reductase and as Antimalarial Drugs. Synthesis,
Enzymology and Activity Evaluation, Group
Leader: Davioud-Charvet
Andrea Neiß, Charakterisierung von white collar
2 und seinen Proteinisoformen in der circadianen
Uhr von Neurospora crassa, Group Leader:
Brunner
54
Jens Radzimanowski, Structural and functional
analysis of the human Amyloid Precursor Protein
(APP) in complex with the cytosolic interaction
partner Fe65, Group Leader: Sinning
Theses
Susanne Kreye, Funktion von Munc13-1 in der
regulierten Membranfusion: Mechanistische
Analyse im rekonstituierten System, Group
Leader: Söllner
Clemens Ostrowicz, Dynamics and architecture
of the HOPS tethering complex in yeast vacuole
fusion, Group Leader: Ungermann
Christina Querfurth, Die Rolle der Phosphorylierung des negativen Elements FREQUENCY
in der circadianen Uhr von Neurospora crassa,
Group Leader: Brunner
Julia Ritzerfeld, Identification of Components of
the Intracellular Transport Machinery of Acylated
Proteins by a Genome-wide RNAi Screen, Group
Leader: Nickel
Koen Temmerman, The role of membrane lipids
in FGF-2 targeting and membrane translocation,
Group Leader: Nickel
Carolin Weimer, Funktionelle Charakterisierung
von ArfGAP1, ArfGAP2 und ArfGAP3 im COPIvermittelten Transport, Group Leader: Wieland
Vera Seidel, Klonierung, Überexpression und
phänotypische Analyse der 2-Cys-Glutaredoxine in
Afrikanischen Trypanosomen sowie Untersuchung
ihrer Rolle im Glutathionstoffwechsel der Parasiten,
Group Leader: Krauth-Siegel
Florian Seiler, Molekulare Mechanismen der
Complexin vermittelten Stimulation der Vesikelfusion im neuronalen Modellsystem, Group Leader:
Söllner
Cornelia Ulbrich, Analysis of structure, function
and molecular mechanism of the AAA-ATPase
Rea1 during ribosome biogenesis in S. cerevisiae,
Group Leader: Hurt
Nicole Wenzel, Synthesis and Mechanism of
Antiparasitic Mannich Base Derivatives Affecting
the Redox Equilibrium of Trypanosomes and
Malaria Parasites, Group Leader: DavioudCharvet
Ke Xiao, Thiol proteins and other targets for
bioinformatical strategies against resistance
development in malaria, Group Leader:
Schirmer
Barbara Zschörnig, Die Proteinkinase CK2vermittelte 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-Glutaredoxine im Trypanothion-Stoffwechsel Afrikanischer
Trypanosomen, Group Leader: Krauth-Siegel
Antje Ebert, Identification and Functional Characterization 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 specification and quantification of membrane lipids, Group Leader: Wieland
Daniel Kammerer, Regulation of spindle positioning through modification 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
O, Neiss A, Brunner M. Circadian activity and
abundance rhythms of the Neurospora clock
transcription factor WCC associated with rapid
nucleo-cytoplasmic shuttling. Genes Dev. 2008;
22(24):3397-402.
Neiss A, Schafmeier T, Brunner M. Transcriptional
regulation and function of the Neurospora clock
gene white collar 2 and its isoforms. EMBO Rep.
2008; 9(8):788-94.
Brunner M, Káldi K. Interlocked feedback loops
of the circadian clock of Neurospora crassa. Mol
Microbiol. 2008; 68(2):255-62. Review.
Brunner M, Simons MJ, Merrow M. Lego clocks:
building a clock from parts. Genes Dev. 2008;
22(11):1422-6.
Brunner M, Merrow M. The green yeast uses
its plant-like clock to regulate its animal-like tail.
Genes Dev. 2008; 22(7):825-31.
Viry E, Battaglia E, Deborde V, Müller T, Réau
R, Davioud-Charvet E, Bagrel D. A sugar-modified phosphole gold complex with antiproliferative properties acting as a thioredoxin reductase
inhibitor in MCF-7 cells. ChemMedChem. 2008;
3:1667-70.
Müller T, Müller TJJ, Davioud-Charvet E. Synthesis
of photo-reactive naphthoquinones for photoaffinity labeling of glutathione reductases. Flavins and
Flavoproteins 2008 (Frago S, Gómez-Moreno C,
Medina M eds) Prensas Universitarias Zaragoza.
2008; 16:443–452.
Morin C, Besset T, Moutet JC, Fayolle M, Brückner
M, Limosin D, Becker K, Davioud-Charvet E. The
aza-analogues of 1,4-naphthoquinones are potent substrates and inhibitors of plasmodial thioredoxin and glutathione reductases and of human
erythrocyte glutathione reductase. Org Biomol
Chem. 2008; 6(15):2731-42.
Friebolin W, Jannack B, Wenzel N, Furrer J,
Oeser T, Sanchez CP, Lanzer M, Yardley V,
Becker K, Davioud-Charvet E. Antimalarial dual
drugs based on potent inhibitors of glutathione
reductase from Plasmodium falciparum. J Med
Chem. 2008; 51(5):1260-77.
56
Köhler A, Schneider M, Cabal GG, Nehrbass U,
Hurt E. Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export. Nat Cell
Biol. 2008; 10(6):707-15.
Schrader N, Stelter P, Flemming D, Kunze R, Hurt
E, Vetter IR. Structural basis of the nic96 subcomplex organization in the nuclear pore channel.
Mol Cell. 2008; 29(1):46-55.
Yao W, Lutzmann M, Hurt E. A versatile interaction platform on the Mex67-Mtr2 receptor creates
an overlap between mRNA and ribosome export.
EMBO J. 2008; 27(1):6-16.
Filser M, Comini MA, Molina-Navarro MM,
Dirdjaja N, Herrero E, Krauth-Siegel RL. Cloning,
functional analysis, and mitochondrial localization
of Trypanosoma brucei monothiol glutaredoxin-1.
Biol Chem. 2008; 389(1):21-32.
Krauth-Siegel RL, Comini MA. Redox control in
trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Biochim Biophys
Acta. 2008; 1780(11):1236-48. Review.
Melchers J, Krauth-Siegel RL, Muhle-Goll C. 1H,
C, and 15N assignment of the oxidized and reduced forms of T. brucei glutathione peroxidasetype tryparedoxin peroxidase. Biomolecular NMR
Assignments. 2008; 2:65-68.
13
Comini MA, Rettig J, Dirdjaja N, Hanschmann
EM, Berndt C, Krauth-Siegel RL. Monothiol glutaredoxin-1 is an essential iron-sulfur protein in
the mitochondrion of African trypanosomes. J
Biol Chem. 2008; 283(41):27785-98.
Melchers J, Diechtierow M, Fehér K, Sinning
I, Tews I, Krauth-Siegel RL, Muhle-Goll C.
Structural basis for a distinct catalytic mechanism
in Trypanosoma brucei tryparedoxin peroxidase.
J Biol Chem. 2008; 283(44):30401-11.
Beig M, Bender F, Oellien F, Rohwer A, Gaßel
M, Selzer P, Unden G, Krauth-Siegel RL.
Trypanothione reductase: a target protein for
a combined in silico and in vitro screening approach. Flavins and Flavoproteins 2008 (Frago
S, Gómez-Moreno C, Medina M eds) Prensas
Universitarias Zaragoza. 2008; 5503-8.
Hurt E. Der Kernporenkomplex oder das Tor zur
Welt des Zytoplasmas. Leopoldina Jahrbuch
2007(Meulen V ter ed.). 2008; (3)53, 457-460.
Stump B, Eberle C, Kaiser M, Brun R, KrauthSiegel RL, Diederich F. Diaryl sulfide-based inhibitors of trypanothione reductase: inhibition potency, revised binding mode and antiprotozoal activities. Org Biomol Chem. 2008; 6(21):3935-47.
Grund SE, Fischer T, Cabal GG, Antúnez O,
Pérez-Ortín JE, Hurt E. The inner nuclear membrane protein Src1 associates with subtelomeric
genes and alters their regulated gene expression.
J Cell Biol. 2008; 182(5):897-910.
Leisner C, Kammerer D, Denoth A, Britschi M,
Barral Y, Liakopoulos D. Regulation of mitotic
spindle asymmetry by SUMO and the spindleassembly checkpoint in yeast. Curr Biol. 2008;
18(16):1249-55.
Kressler D, Roser D, Pertschy B, Hurt E. The
AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S
particles. J Cell Biol. 2008; 181(6):935-44.
Nickel W, Seedorf M. Unconventional mechanisms of protein transport to the cell surface of
eukaryotic cells. Annu Rev Cell Dev Biol. 2008;
24:287-308. Review.
Publications
Wegehingel S, Zehe C, Nickel W. Rerouting of fibroblast growth factor 2 to the classical secretory
pathway results in post-translational modifications that block binding to heparan sulfate proteoglycans. FEBS Lett. 2008; 582(16):2387-92.
Stengel KF, Holdermann I, Cain P, Robinson C,
Wild K, Sinning I. Structural basis for specific substrate recognition by the chloroplast signal recognition particle protein cpSRP43. Science. 2008;
321(5886):253-6.
Temmerman K, Ebert AD, Müller HM, Sinning I,
Tews I, Nickel W. A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2. Traffic. 2008;
9(7):1204-17.
Radzimanowski J, Ravaud S, Schlesinger S,
Koch J, Beyreuther K, Sinning I, Wild K. Crystal
structure of the human Fe65-PTB1 domain. J Biol
Chem. 2008; 283(34):23113-20.
Seelenmeyer C, Stegmayer C, Nickel W.
Unconventional secretion of fibroblast growth factor 2 and galectin-1 does not require shedding of
plasma membrane-derived vesicles. FEBS Lett.
2008; 582(9):1362-8.
Buchholz K, Schirmer RH, Eubel JK, Akoachere
MB, Dandekar T, Becker K, Gromer S. Interactions
of methylene blue with human disulfide reductases and their orthologues from Plasmodium
falciparum. Antimicrob Agents Chemother. 2008;
52(1):183-91.
Zoungrana A, Coulibaly B, Sié A, Walter-Sack I,
Mockenhaupt FP, Kouyaté B, Schirmer RH, Klose
C, Mansmann U, Meissner P, Müller O. Safety and
efficacy of methylene blue combined with artesunate or amodiaquine for uncomplicated falciparum malaria: a randomized controlled trial from
Burkina Faso. PLoS ONE. 2008; 3(2):e1630.
Buchholz K, Comini MA, Wissenbach D, Schirmer
RH, Krauth-Siegel RL, Gromer S. Cytotoxic interactions of methylene blue with trypanosomatidspecific disulfide reductases and their dithiol products. Mol Biochem Parasitol. 2008; 160(1):65-9.
Buchholz K, Rahlfs S, Schirmer RH, Becker K,
Matuschewski K. Depletion of Plasmodium berghei plasmoredoxin reveals a non-essential role
for life cycle progression of the malaria parasite.
PLoS ONE. 2008; 3(6):e2474.
Schirmer RH, Adler H, Zappe HA, Gromer
S, Becker K, Coulibaly B, Meissner P (2008)
Disulfide reductases as drug targets: Methylene
blue combination therapies for falciparum malaria in African children. Flavins and Flavoproteins
2008 (Frago S, Gómez-Moreno C, Medina M eds)
Prensas Universitarias Zaragoza. 2008; 481-486.
Schirmer H. Book Review on KC Nicolaou and T
Montagnon. Molecules that changed the world. J
Lab Med 2008; 32:382-383.
Bionda T, König P, Oreb M, Tews I, Schleiff E. pH
Sensitivity of the GTPase Toc33 as a regulatory
circuit for protein translocation into chloroplasts.
Plant Cell Physiol. 2008; 49(12):1917-21.
König P, Oreb M, Rippe K, Muhle-Goll C, Sinning
I, Schleiff E, Tews I. On the significance of
Toc-GTPase homodimers. J Biol Chem. 2008;
283(34):23104-12.
Radzimanowski J, Ravaud S, Beyreuther K,
Sinning I, Wild K. Mercury-induced crystallization and SAD phasing of the human Fe65-PTB1
domain. Acta Crystallogr Sect F Struct Biol Cryst
Commun. 2008; 64(Pt 5):382-5.
Radzimanowski J, Beyreuther K, Sinning I, Wild K.
Overproduction, purification, crystallization and
preliminary X-ray analysis of human Fe65-PTB2
in complex with the amyloid precursor protein intracellular domain. Acta Crystallogr Sect F Struct
Biol Cryst Commun. 2008; 64(Pt 5):409-12.
Ravaud S, Stjepanovic G, Wild K, Sinning I. The
crystal structure of the periplasmic domain of
the Escherichia coli membrane protein insertase
YidC contains a substrate binding cleft. J Biol
Chem. 2008; 283(14):9350-8.
König P, Oreb M, Höfle A, Kaltofen S, Rippe K,
Sinning I, Schleiff E, Tews I. The GTPase cycle
of the chloroplast import receptors Toc33/Toc34:
implications from monomeric and dimeric structures. Structure. 2008; 16(4):585-96.
Ravaud S, Wild K, Sinning I. Purification, crystallization and preliminary structural characterization
of the periplasmic domain P1 of the Escherichia
coli membrane-protein insertase YidC. Acta
Crystallogr Sect F Struct Biol Cryst Commun.
2008; 64(Pt 2):144-8.
Malsam J, Kreye S, Söllner TH. Membrane fusion: SNAREs and regulation. Cell Mol Life Sci.
2008; 65(18):2814-32. Review.
Kreye S, Malsam J, Söllner TH. In vitro assays
to measure SNARE-mediated vesicle fusion.
Methods Mol Biol. 2008; 440:37-50.
Brunsing R, Omori SA, Weber F, Bicknell A,
Friend L, Rickert R, Niwa M. B- and T-cell development both involve activity of the unfolded
protein response pathway. J Biol Chem. 2008;
283(26):17954-61.
Oreb M, Tews I, Schleiff E. Policing Tic ‘n’ Toc, the
doorway to chloroplasts. Trends Cell Biol. 2008;
18(1):19-27. Review.
Langer JD, Roth CM, Béthune J, Stoops EH,
Brügger B, Herten DP, Wieland FT. A conformational change in the alpha-subunit of coatomer induced by ligand binding to gamma-COP revealed
by single-pair FRET. Traffic. 2008. 9(4):597-607.
Radzimanowski J, Simon B, Sattler M, Beyreuther
K, Sinning I, Wild K. Structure of the intracellular domain of the amyloid precursor protein in
complex with Fe65-PTB2. EMBO Rep. 2008;
9(11):1134-40.
Kwa LG, Wegmann D, Brügger B, Wieland FT,
Wanner G, Braun P. Mutation of a single residue,
beta-glutamate-20, alters protein-lipid interactions of light harvesting complex II. Mol Microbiol.
2008; 67(1):63-77.
Publications
57
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 Res. 2008;
49(1):251-62.
Trajkovic K, Hsu C, Chiantia S, Rajendran L,
Wenzel D, Wieland F, Schwille P, Brügger B,
Simons M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes.
Science. 2008; 319(5867):1244-7.
Beck R, Sun Z, Adolf F, Rutz C, Bassler J, Wild K,
Sinning I, Hurt E, Brügger B, Béthune J, Wieland
F. Membrane curvature induced by Arf1-GTP is
essential for vesicle formation. Proc Natl Acad
Sci U S A. 2008; 105(33):11731-6.
Superti-Furga G, Wieland F, Cesareni G. Finally:
The digital, democratic age of scientific abstracts.
FEBS Lett. 2008; 582(8):1169.
Zitzler S, Hellwig A, Hartl FU, Wieland F,
Diestelkötter-Bachert P. Distinct binding sites for
the ATPase and substrate-binding domain of human Hsp70 on the cell surface of antigen presenting cells. Mol Immunol. 2008; 45(15):3974-83.
Krauss M, Jia JY, Roux A, Beck R, Wieland FT,
De Camilli P, Haucke V. Arf1-GTP-induced tubule
formation suggests a function of Arf family proteins in curvature acquisition at sites of vesicle
budding. J Biol Chem. 2008; 283(41):27717-23.
Weimer C, Beck R, Eckert P, Reckmann I,
Moelleken J, Brügger B, Wieland F. Differential
roles of ArfGAP1, ArfGAP2, and ArfGAP3 in
COPI trafficking. J Cell Biol. 2008; 183(4):725-35.
2009
Diernfellner AC, Querfurth C, Salazar C, Höfer
T, Brunner M. Phosphorylation modulates rapid
nucleocytoplasmic shuttling and cytoplasmic
accumulation of Neurospora clock protein FRQ
on a circadian time scale.Genes Dev. 2009;
23(18):2192-200.
Sancar G, Sancar C, Brunner M, Schafmeier T.
Activity of the circadian transcription factor White
Collar Complex is modulated by phosphorylation
of SP-motifs.FEBS Lett. 2009; 583(12):1833-40.
Chavain N, Davioud-Charvet E, Trivelli X, Mbeki L,
Rottmann M, Brun R, Biot C. Antimalarial activities
of ferroquine conjugates with either glutathione
reductase inhibitors or glutathione depletors via
a hydrolyzable amide linker.Bioorg Med Chem.
2009; 17(23):8048-59.
Kressler D, Hurt E, Baβler J. Driving ribosome
assembly.Biochim Biophys Acta. 2009.
Batisse J, Batisse C, Budd A, Böttcher B, Hurt
E. Purification of nuclear poly(A)-binding protein
Nab2 reveals association with the yeast transcriptome and a messenger ribonucleoprotein core
structure.J Biol Chem. 2009; 284(50):34911-7.
Pertschy B, Schneider C, Gnädig M, Schäfer T,
Tollervey D, Hurt E. RNA helicase Prp43 and its co-
58
Publications
factor Pfa1 promote 20 to 18 S rRNA processing
catalyzed by the endonuclease Nob1.J Biol Chem.
2009; 284(50):35079-91.
Ulbrich C, Diepholz M, Bassler J, Kressler
D, Pertschy B, Galani K, Böttcher B, Hurt
E. Mechanochemical removal of ribosome
biogenesis factors from nascent 60S ribosomal
subunits.Cell. 2009; 138:911-22.
Ferreira-Cerca S, Hurt E. Cell biology: Arrest by
ribosome.Nature. 2009; 459(7243):46-7.
Flemming D, Sarges P, Stelter P, Hellwig A,
Böttcher B, Hurt E. Two structurally distinct
domains of the nucleoporin Nup170 cooperate to
tether a subset of nucleoporins to nuclear pores.J
Cell Biol. 2009; 185(3):387-95.
Jani D, Lutz S, Marshall NJ, Fischer T, Köhler
A, Ellisdon AM, Hurt E, Stewart M. Sus1, Cdc31,
and the Sac3 CID region form a conserved
interaction platform that promotes nuclear pore
association and mRNA export.Mol Cell. 2009;
33(6):1-11.
Faza MB, Kemmler S, Jimeno S, GonzálezAguilera C, Aguilera A, Hurt E, Panse VG. Sem1
is a functional component of the nuclear pore
complex-associated messenger RNA export
machinery.J Cell Biol. 2009; 184(6):833-46.
Klöckner C, Schneider M, Lutz S, Jani D, Kressler
D, Stewart M, Hurt E, Köhler A. Mutational
uncoupling of the role of Sus1 in nuclear pore
complex targeting of an mRNA export complex
and histone H2B deubiquitination.J Biol Chem.
2009; 284(18):12049-56.
Lacombe T, García-Gómez JJ, de la Cruz J, Roser
D, Hurt E, Linder P, Kressler D. Linear ubiquitin
fusion to Rps31 and its subsequent cleavage are
required for the efficient production and functional
integrity of 40S ribosomal subunits.Mol Microbiol.
2009; 72(1):69-84.
Katahira J, Inoue H, Hurt E, Yoneda Y. Adaptor
Aly and co-adaptor Thoc5 function in the Tap-p15mediated nuclear export of HSP70 mRNA.EMBO
J. 2009; 28(5):556-67.
Skruzný M, Schneider C, Rácz A, Weng J, Tollervey
D, Hurt E. An endoribonuclease functionally linked
to perinuclear mRNP quality control associates
with the nuclear pore complexes.PLoS Biol. 2009;
7(1):e8.
Garrenton LS, Braunwarth A, Irniger S, Hurt E,
Künzler M, Thorner J. Nucleus-specific and cell
cycle-regulated degradation of mitogen-activated
protein kinase scaffold protein Ste5 contributes to
the control of signaling competence.Mol Cell Biol.
2009; 29(2):582-601.
Komljenovic D, Sandhoff R, Teigler A, Heid H, Just
WW, Gorgas K. Disruption of blood-testis barrier
dynamics in ether-lipid-deficient mice.Cell Tissue
Res. 2009; 337(2):281-99.
Teigler A, Komljenovic D, Draguhn A, Gorgas
K, Just WW. Defects in myelination, paranode
organization and Purkinje cell innervation in the
ether lipid-deficient mouse cerebellum.Hum Mol
Genet. 2009; 18(11):1897-908.
Eberle C, Burkhard JA, Stump B, Kaiser M, Brun
R, Krauth-Siegel RL, Diederich F.Synthesis,
inhibition potency, binding mode, and antiprotozoal
activities of fluorescent inhibitors of trypanothione
reductase based on mepacrine-conjugated
diaryl sulfide scaffolds.ChemMedChem. 2009;
4(12):2034-44.
Comini MA, Dirdjaja N, Kaschel M, KrauthSiegel RL.Preparative enzymatic synthesis of
trypanothione and trypanothione analogues.Int J
Parasitol. 2009; 39(10):1059-62.
Wenzel IN, Wong PE, Maes L, Müller TJ,
Krauth-Siegel RL, Barrett MP, Davioud-Charvet
E. Unsaturated Mannich bases active against
multidrug-resistant Trypanosoma brucei brucei
strains.ChemMedChem. 2009; 4(3):339-51.
Stump B, Eberle C, Schweizer WB, Kaiser M,
Brun R, Krauth-Siegel RL, Lentz D, Diederich
F. Pentafluorosulfanyl as a novel building block
for enzyme inhibitors: trypanothione reductase
inhibition and antiprotozoal activities of
diarylamines.Chembiochem. 2009; 10(1):79-83.
Wendler A, Irsch T, Rabbani N, Thornalley
PJ, Krauth-Siegel RL. Glyoxalase II does not
support methylglyoxal detoxification but serves
as a general trypanothione thioesterase in African
trypanosomes.Mol Biochem Parasitol. 2009;
163(1):19-27.
Ramos EI, Garza KM, Krauth-Siegel RL, Bader
J, Martinez LE, Maldonado RA. 2,3-diphenyl-1,4naphthoquinone: a potential chemotherapeutic
agent against Trypanosoma cruzi.J Parasitol.
2009; 95(2):461-6.
Cavalli A, Lizzi F, Bongarzone S, Brun R, KrauthSiegel RL, Bolognesi ML. Privileged structureguided synthesis of quinazoline derivatives as
inhibitors of trypanothione reductase.Bioorg Med
Chem Lett. 2009; 19(11):3031-5.
Krauth-Siegel RL. Redox signaling and regulation
in biology and medicine.(editors: Jacob BC and
Winyard PG), book review in ChemMedChem.
2009; 4:2123-2127.
Ortiz J, Funk C, Schäfer A, Lechner J. Stu1 inversely
regulates kinetochore capture and spindle stability.
Genes Dev. 2009; 23(23):2778-91.
Kemmler S, Stach M, Knapp M, Ortiz J, Pfannstiel
J, Ruppert T, Lechner J. Mimicking Ndc80
phosphorylation triggers spindle assembly
checkpoint signalling.EMBO J. 2009; 28(8):1099110.
Barral Y, Liakopoulos D. Role of spindle asymmetry
in cellular dynamics.Int Rev Cell Mol Biol. 2009;
278:149-213. Review.
Ercan E, Momburg F, Engel U, Temmerman K,
Nickel W, Seedorf M. A conserved, lipid-mediated
sorting mechanism of yeast Ist2 and mammalian
STIM proteins to the peripheral ER.Traffic. 2009;
10(12):1802-18.
Torrado LC, Temmerman K, Müller HM, Mayer
MP, Seelenmeyer C, Backhaus R, Nickel W. An
intrinsic quality-control mechanism ensures
unconventional secretion of fibroblast growth
factor 2 in a folded conformation.J Cell Sci. 2009;
122(Pt 18):3322-9.
Merk M, Baugh J, Zierow S, Leng L, Pal U, Lee SJ,
Ebert AD, Mizue Y, Trent JO, Mitchell R, Nickel W,
Kavathas PB, Bernhagen J, Bucala R. The Golgiassociated protein p115 mediates the secretion of
macrophage migration inhibitory factor.J Immunol.
2009; 182(11):6896-906.
Fischer MA, Temmerman K, Ercan E, Nickel W,
Seedorf M. Binding of plasma membrane lipids
recruits the yeast integral membrane protein
Ist2 to the cortical ER.Traffic. 2009; 10(8):108497.
Tournaviti S, Pietro ES, Terjung S, Schafmeier
T, Wegehingel S, Ritzerfeld J, Schulz J, Smith
DF, Pepperkok R, Nickel W. Reversible
phosphorylation as a molecular switch to regulate
plasma membrane targeting of acylated SH4
domain proteins.Traffic. 2009; 10(8):1047-60.
Maass K, Fischer MA, Seiler M, Temmerman K,
Nickel W, Seedorf M. A signal comprising a basic
cluster and an amphipathic alpha-helix interacts
with lipids and is required for the transport of Ist2
to the yeast cortical ER.J Cell Sci. 2009; 122(Pt
5):625-35.
Temmerman K, Nickel W. A novel flow cytometric
assay to quantify interactions between proteins
and membrane lipids.J Lipid Res. 2009;
50(6):1245-54.
Nickel W, Rabouille C. Mechanisms of regulated
unconventional protein secretion.Nat Rev Mol
Cell Biol. 2009; 10(2):148-55. Review. Erratum:
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Munte CE, Becker K, Schirmer RH, Kalbitzer
HR. NMR assignments of oxidised thioredoxin
from Plasmodium falciparum.Biomol NMR Assign.
2009; 3(2):159-61.
Müller O, Sié A, Meissner P, Schirmer RH, Kouyaté
B. Artemisinin resistance on the Thai-Cambodian
border.Lancet. 2009; 374(9699):1419.
Gallo V, Schwarzer E, Rahlfs S, Schirmer RH, van
Zwieten R, Roos D, Arese P, Becker K. Inherited
glutathione reductase deficiency and Plasmodium
falciparum malaria--a case study.PLoS One. 2009;
4(10):e7303.
Xiao K, Jehle F, Peters C, Reinheckel T,
Schirmer RH, Dandekar T. CA/C1 peptidases of
the malaria parasites Plasmodium falciparum
and P. berghei and their mammalian hosts-a bioinformatical analysis.Biol Chem. 2009;
390(11):1185-97.
Coulibaly B, Zoungrana A, Mockenhaupt FP,
Schirmer RH, Klose C, Mansmann U, Meissner
PE, Müller O. Strong gametocytocidal effect
of methylene blue-based combination therapy
against falciparum malaria: a randomised
controlled trial.PLoS One. 2009; 4(5):e5318.
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Rahlfs S, Koncarevic S, Iozef R, Mailu BM, Savvides SN, Schirmer RH, Becker K. Myristoylated
adenylate kinase-2 of Plasmodium falciparum
forms a heterodimer with myristoyltransferase.
Mol Biochem Parasitol. 2009; 163(2):77-84.
Schirmer H. Essay on the Medical History of
Methylene Blue. http://www.alzforum.org/new/
Schirmer.aspAlzheimer Research Forum 2009.
Bozkurt G, Stjepanovic G, Vilardi F, Amlacher
S, Wild K, Bange G, Favaloro V, Rippe K, Hurt
E, Dobberstein B, Sinning I. Structural insights
into tail-anchored protein binding and membrane
insertion by Get3.Proc Natl Acad Sci U S A. 2009;
106(50):21131-6.
Kock I, Bulgakova NA, Knust E, Sinning I,
Panneels V. Targeting of Drosophila rhodopsin
requires helix 8 but not the distal C-terminus.
PLoS One. 2009; 4(7):e6101.
Grudnik P, Bange G, Sinning I. Protein targeting
by the signal recognition particle.Biol Chem. 2009;
390(8):775-82. Review.
Neuwirth M, Strohmeier M, Windeisen V, Wallner
S, Deller S, Rippe K, Sinning I, Macheroux P,
Tews I. X-ray crystal structure of Saccharomyces
cerevisiae Pdx1 provides insights into the
oligomeric nature of PLP synthases.FEBS Lett.
2009; 583(13):2179-86.
Cross BC, Sinning I, Luirink J, High S. Delivering
proteins for export from the cytosol. Nat Rev Mol
Cell Biol. 2009; 10(4):255-64. Review.
Sinning I, Wild K, Bange G. Signal sequences get
active.Nat Chem Biol. 2009; 5(3):146-7.
Wallner S, Neuwirth M, Flicker K, Tews I, Macheroux
P. Dissection of contributions from invariant amino
acids to complex formation and catalysis in the
heteromeric pyridoxal 5-phosphate synthase
complex from Bacillus subtilis.Biochemistry. 2009;
48(9):1928-35.
Seiler F, Malsam J, Krause JM, Söllner TH. A
role of complexin-lipid interactions in membrane
fusion.FEBS Lett. 2009; 583(14):2343-8.
Malsam J, Seiler F, Schollmeier Y, Rusu P, Krause
JM, Söllner TH. The carboxy-terminal domain of
complexin I stimulates liposome fusion.Proc Natl
Acad Sci U S A. 2009; 106(6):2001-6.
Hung HC, Maurer C, Zorn D, Chang WL,
Weber F. Sequential and compartment-specific
phosphorylation controls the life cycle of the
circadian CLOCK protein. J Biol Chem. 2009;
284(35):23734-42.
Hung HC, Kay SA, Weber F. HSP90, a capacitor
of behavioral variation.J Biol Rhythms. 2009;
24(3):183-92.
Maurer C, Hung HC, Weber F. Cytoplasmic
interaction with CYCLE promotes the posttranslational processing of the circadian CLOCK
protein. FEBS Lett. 2009; 583(10):1561-6.
Weber F. Remodeling the clock: coactivators and
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signal transduction in the circadian clockworks.
Naturwissenschaften. 2009; 96(3):321-37. Review.
Emr S, Glick BS, Linstedt AD, Lippincott-Schwartz
J, Luini A, Malhotra V, Marsh BJ, Nakano A, Pfeffer
SR, Rabouille C, Rothman JE, Warren G, Wieland
FT. Journeys through the Golgi--taking stock in a
new era.J Cell Biol. 2009; 187(4):449-53. Review.
Beck R, Rawet M, Wieland FT, Cassel D. The
COPI system: molecular mechanisms and
function.FEBS Lett. 2009; 583(17):2701-9. Review.
Erratum in: FEBS Lett. 2009; 583(21):3541.
Lorizate M, Brügger B, Akiyama H, Glass B,
Müller B, Anderluh G, Wieland FT, Kräusslich
HG. Probing HIV-1 membrane liquid order by
Laurdan staining reveals producer cell-dependent
differences.J Biol Chem. 2009; 284(33):2223847.
Rutz C, Satoh A, Ronchi P, Brügger B, Warren
G, Wieland FT. Following the fate in vivo of
COPI vesicles generated in vitro.Traffic. 2009;
10(8):994-1005.
Osman C, Haag M, Potting C, Rodenfels J,
Dip PV, Wieland FT, Brügger B, Westermann
B, Langer T. The genetic interactome of
prohibitins: coordinated control of cardiolipin
and phosphatidylethanolamine by conserved
regulators in mitochondria.J Cell Biol. 2009;
184(4):583-96.
Beck R, Adolf F, Weimer C, Brügger B, Wieland
FT. ArfGAP1 activity and COPI vesicle biogenesis.
Traffic. 2009; 10(3):307-15.
Saher G, Quintes S, Möbius W, Wehr MC, KrämerAlbers EM, Brügger B, Nave KA. Cholesterol
regulates the endoplasmic reticulum exit of
the major membrane protein P0 required for
peripheral myelin compaction.J Neurosci. 2009;
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2010
Tataroğlu O, Schafmeier T. Of switches and
hourglasses: regulation of subcellular traffic in
circadian clocks by phosphorylation. EMBO Rep.
2010; 11(12):927-35.
Malzahn E, Ciprianidis S, Káldi K, Schafmeier T,
Brunner M. Photoadaptation in Neurospora by
competitive interaction of activating and inhibitory
LOV domains. Cell 2010; 142(5):762-72.
Smith KM, Sancar G, Dekhang R, Sullivan CM,
Li S, Tag AG, Sancar C, Bredeweg EL, Priest
HD, McCormick RF, Thomas TL, Carrington JC,
Stajich JE, Bell-Pedersen D, Brunner M, Freitag
M. Transcription factors in light and circadian
clock signaling networks revealed by genomewide
mapping of direct targets for neurospora white
collar complex. Eukaryot Cell 2010; 9(10):154956.
Haid S, Gentzsch J, Jannack B, Bailleul F,
Davioud-Charvet E, Pietschmann T. Inhibition of
hepatitis C virus entry by a plant-derived flavone.
J. Hepatol. Suppl. 1, 52:S293.
Wenzel NI, Chavain N, Wang Y, Friebolin W,
Maes L, Pradines B, Lanzer M, Yardley V, Brun R,
Herold-Mende C, Biot C, Tóth K, Davioud-Charvet
E. Antimalarial versus cytotoxic properties of
dual drugs derived from 4-aminoquinolines and
Mannich bases: interaction with DNA. J Med
Chem. 2010; 53(8):3214-26.
Bassler J, Kallas M, Pertschy B, Ulbrich C, Thoms
M, Hurt E. The AAA-ATPase Rea1 drives removal
of biogenesis factors during multiple stages of 60S
ribosome assembly. Mol Cell. 2010; 38(5):712-21.
Flemming D, Thierbach K, Stelter P, Böttcher B,
Hurt E. Precise mapping of subunits in multiprotein
complexes by a versatile electron microscopy
label. Nat Struct Mol Biol. 2010; 17(6):775-8.
Köhler A, Zimmerman E, Schneider M, Hurt
E, Zheng N. Structural basis for assembly
and activation of the heterotetrameric SAGA
histone H2B deubiquitinase module. Cell 2010;
141(4):606-17.
Köhler A, Hurt E. Gene regulation by nucleoporins
and links to cancer. Mol Cell. 2010; 38(1):6-15.
Review
Ellisdon AM, Jani D, Köhler A, Hurt E, Stewart M.
Structural basis for the interaction between yeast
Spt-Ada-Gcn5 acetyltransferase (SAGA) complex
components Sgf11 and Sus1. J Biol Chem. 2010;
285(6):3850-6.
of african trypanosomes have distinct roles and
are closely linked to the unique trypanothione
metabolism. J Biol Chem. 2010; 285(45):3522437.
Bakker BM, Krauth-Siegel RL, Clayton C,
Matthews K, Girolami M, Westerhoff HV,
Michels PA, Breitling R, Barrett MP. The silicon
trypanosome. Parasitology. 2010; 137(9):133341. Review.
Muhle-Goll C, Füller F, Ulrich AS, Krauth-Siegel
RL. The conserved Cys76 plays a crucial role
for the conformation of reduced glutathione
peroxidase-type tryparedoxin peroxidase. FEBS
Lett. 2010; 584(5):1027-32.
Kammerer D, Stevermann L, Liakopoulos D.
Ubiquitylation regulates interactions of astral
microtubules with the cleavage apparatus. Curr
Biol. 2010; 20(14):1233-43.
Siden-Kiamos I, Schüler H, Liakopoulos D, Louis
C. Arp1, an actin-related protein, in Plasmodium
berghei. Mol Biochem Parasitol. 2010; 173(2):8896.
Nickel W. Pathways of unconventional protein
secretion. Curr Opin Biotechnol. 2010; 21(5):6216. Review
Kressler D, Hurt E, Bassler J. Driving ribosome
assembly. Biochim Biophys Acta 2010;
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Ebert AD, Laussmann M, Wegehingel S, Kaderali
L, Erfle H, 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; 11(6):813-26.
Schollenberger L, Gronemeyer T, Huber CM, Lay
D, Wiese S, Meyer HE, Warscheid B, Saffrich R,
Peränen J, Gorgas K, Just WW. RhoA regulates
peroxisome association to microtubules and
the actin cytoskeleton. PLoS One 2010;
5(11):e13886.
Buchholz K, Putrianti ED, Rahlfs S, Schirmer
RH, Becker K, Matuschewski K. Molecular
genetics evidence for the in vivo roles of the two
major NADPH-dependent disulfide reductases
in the malaria parasite. J Biol Chem. 2010;
285(48):37388-95.
Alexander RD, Barrass JD, Dichtl B, Koš M,
Obtulowicz T, Robert MC, Koper M, Karkusiewicz
I, Mariconti L, Tollervey D, Dichtl B, Kufel
J, Bertrand E, Beggs JD. RiboSys, a highresolution, quantitative approach to measure the
in vivo kinetics of pre-mRNA splicing and 3'-end
processing in Saccharomyces cerevisiae. RNA
2010; 16(12):2570-80.
Bountogo M, Zoungrana A, Coulibaly B, Klose
C, Mansmann U, Mockenhaupt FP, Burhenne
J, Mikus G, Walter-Sack I, Schirmer RH, Sié
A, Meissner P, Müller O. Efficacy of methylene
blue monotherapy in semi-immune adults with
uncomplicated falciparum malaria: a controlled
trial in Burkina Faso. Trop Med Int Health 2010;
15(6):713-7.
Boon KL, Koš M. Deletion of Swm2p
selectively impairs trimethylation of snRNAs by
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2010; 584(15):3299-304.
Falk S, Sinning I. The C terminus of Alb3 interacts
with the chromodomains 2 and 3 of cpSRP43. J
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Koš M, Tollervey D. Yeast pre-rRNA processing
and modification occur cotranscriptionally. Mol
Cell 2010; 37(6):809-20.
Eberle C, Lauber BS, Fankhauser D, Kaiser
M, Brun R, Krauth-Siegel RL, Diederich F.
Improved Inhibitors of Trypanothione Reductase
by Combination of Motifs: Synthesis, Inhibitory
Potency, Binding Mode, and Antiprotozoal
Activities. ChemMedChem. 2010; 16.
Ceylan S, Seidel V, Ziebart N, Berndt C, Dirdjaja
N, Krauth-Siegel RL. The dithiol glutaredoxins
Erez E, Stjepanovic G, Zelazny AM, Brügger B,
Sinning I, Bibi E. Genetic evidence for functional
interaction of the Escherichia coli signal recognition
particle receptor with acidic lipids in vivo. J Biol
Chem. 2010; 285(52):40508-14.
Derrer B, Windeisen V, Guédez Rodríguez G,
Seidler J, Gengenbacher M, Lehmann WD, Rippe
K, Sinning I, Tews I, Kappes B. Defining the
structural requirements for ribose 5-phosphatebinding and intersubunit cross-talk of the malarial
pyridoxal 5-phosphate synthase. FEBS Lett.
2010; 584(19):4169-74.
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Bange G, Kümmerer N, Engel C, Bozkurt G,
Wild K, Sinning I. FlhA provides the adaptor for
coordinated delivery of late flagella building blocks
to the type III secretion system. Proc Natl Acad
Sci U S A. 2010; 107(25):11295-300.
Falk S, Sinning I. cpSRP43 is a novel chaperone
specific for light-harvesting chlorophyll a,b-binding
proteins. J Biol Chem. 2010; 285(28):21655-61.
Schott A, Ravaud S, Keller S, Radzimanowski J,
Viotti C, Hillmer S, Sinning I, Strahl S. Arabidopsis
stromal-derived Factor2 (SDF2) is a crucial
target of the unfolded protein response in the
endoplasmic reticulum. J Biol Chem. 2010;
285(23):18113-21.
Koenig P, Mirus O, Haarmann R, Sommer MS,
Sinning I, Schleiff E, Tews I. Conserved properties
of polypeptide transport-associated (POTRA)
domains derived from cyanobacterial Omp85. J
Biol Chem. 2010; 285(23):18016-24.
Bozkurt G, Wild K, Amlacher S, Hurt E,
Dobberstein B, Sinning I. The structure of Get4
reveals an alpha-solenoid fold adapted for multiple
interactions in tail-anchored protein biogenesis.
FEBS Lett. 2010; 584(8):1509-14.
Schrul B, Kapp K, Sinning I, Dobberstein B.
Signal peptide peptidase (SPP) assembles with
substrates and misfolded membrane proteins into
distinct oligomeric complexes. Biochem J. 2010;
427(3):523-34.
Wild K, Bange G, Bozkurt G, Segnitz B, Hendricks
A, Sinning I. Structural insights into the assembly
of the human and archaeal signal recognition
particles. Acta Crystallogr D Biol Crystallogr.
2010; 66(Pt 3):295-303.
Panneels V, Sinning I. Membrane protein
expression in the eyes of transgenic flies. Methods
Mol Biol. 2010; 601:135-47.
Radzimanowski J, Ravaud S, Schott A, Strahl
S, Sinning I. Cloning, recombinant production,
crystallization and preliminary X-ray diffraction
analysis of SDF2-like protein from Arabidopsis
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thaliana. Acta Crystallogr Sect F Struct Biol Cryst
Commun. 2010; 66(Pt 1):12-4.
Falk S, Ravaud S, Koch J, Sinning I. The C
terminus of the Alb3 membrane insertase recruits
cpSRP43 to the thylakoid membrane. J Biol Chem.
2010; 285(8):5954-62.
Kögel T, Rudolf R, Hodneland E, Hellwig A,
Kuznetsov SA, Seiler F, Söllner TH, Barroso
J, Gerdes HH. Distinct roles of myosin Va in
membrane remodeling and exocytosis of secretory
granules. Traffic 2010; 11(5):637-50.
Beck R, Brügger B, Wieland FT. Membrane
deformation and separation. F1000 Biol Rep.
2010; 2. pii: 35.
Osman C, Haag M, Wieland FT, Brügger B,
Langer T. A mitochondrial phosphatase required
for cardiolipin biosynthesis: the PGP phosphatase
Gep4. EMBO J. 2010; 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;107(15):6876-81.
Pewzner-Jung Y, Park H, Laviad EL, Silva LC,
Lahiri S, Stiban J, Erez-Roman R, Brügger B,
Sachsenheimer T, Wieland F, Prieto M, Merrill AH Jr,
Futerman AH. A critical role for ceramide synthase 2
in liver homeostasis: I. alterations in lipid metabolic
pathways. J Biol Chem. 2010; 285(14):10902-10.
Ernst AM, Contreras FX, Brügger B, Wieland F.
FEBS Determinants of specificity at the proteinlipid interface in membranes. FEBS Lett. 2010;
584(9):1713-20. Review.
Mora R, Dokic I, Kees T, Hüber CM, Keitel
D, Geibig R, Brügger B, Zentgraf H, Brady NR,
Régnier-Vigouroux A. Sphingolipid rheostat
alterations related to transformation can be
exploited for specific induction of lysosomal cell
death in murine and human glioma. Glia. 2010;
58(11):1364-83.
63
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Bhalchandra R. Jadhav*
Wilfried Klug*
Ines Kock* (MD)
Patrick König*
Nico Kümmerer
Yin-Yuin Pang*
Jens Radzimanowski*
Katharina Stengel*
Goran Stjepanovic
Volker Windeisen
Master Students
Ajay Aravind*
Katja Deselaers*
Christoph Engel*
Hamed Kooshapur*
Stefan Weber*
Crystallization Platform
Jürgen Kopp
Claudia Siegmann
Protein Expression
Gunter Stier*
Computer Support
Lutz Nücker
Technical Assistants
Silke Adrian
Astrid Hendricks
Elke Herwig
Gabriela Müller
Bernd Segnitz
Thomas Söllner Group
Secretary
Martina Franke-Schaub
Postdoc
Jörg Malsam
PhD Students
Bernhard Dörr*
Susanne Kreye*
Simone Paulsen
Patricia Rusu
Julia Schneider*
Yvette Schollmeier
Florian Seiler*
Rostislav Veselinov*
SFB TRR 83 Office*
Gabriella Kälin*
Heike Lorenzen-Schmidt*
Technical Assistants
Jean Michel Krause
Andrea Scheutzow*
Frank Weber Group
PostDoc
Hsiu-Cheng Hung
PhD Students
Ines Metzger* (MD)
Daniela Zorn*
Felix Wieland / Britta Brügger Group
Secretary
Barbara Schröter
Support
Monika Bertram*
Gifta Martial*
Postdocs
Alexander Brodde*
Xabier Contreras
Petra DiestelkötterBachert
Vincent Popoff*
Oliver Schmitt*
Jeroen Strating
PhD Students
Frank Adolf
Frank Anderl*
Rainer Beck*
Andreas Max Ernst*
Iva Ganeva*
Michael Geiling*
Basak Gönen*
Mathias Haag
Kathrin Höhner*
Stefanie Hubich*
Julian Langer*
Cagakan Özbalci*
Simone Röhling*
Christoph Rutz*
Carolin Weimer*
Master Student
Myriam Trausch*
Technical Assistants
Priska Eckert
Alexia Herrmann*
Iris Leibrecht
Ingrid Meißner
Ingeborg Reckmann
Lipidomics Platform
Timo Sachsenheimer*
SFB 638 Office
Margot Ruland
Jutta Wiech
Support
Carmen Monasterio*
FEBS-Letters
Editorial Manager
Patricia McCabe
Assistant Editors
Aleksander Benjak
Daniela Ruffell*
Reviews Editor
Wilhelm Just*
Editorial Assistant
Anne Müller
66
Staff
Mass Spectrometry Group
Technical Assistants
Susanne Eisel
Jürgen Reichert
Teaching
Coordinator
and Lecturer
Cordula Harter
Assistant
and Lecturer
Petra Schling
Secretaries
Evelyn Hartmann
Barbara Schneider
Technical Assistants
Evelyn Bauer*
Gera Breypohl*
Martina Gruß
Tanja Schlüter
Computer Support
Lutz Nücker
Theresa Schaub
Technician
Peter Böhm
Cell Culture,
Technical Assistant
Gabi Weiß
Central Services
Central
Administration
Administrator
Catarina Vill-Härtlein
Assistants
Barbara Bohne
Claudia SchönwieseM‘Bengue
Secretary 1. Floor
Petra Krapp-Meiser
Janitor
Josef Back
Media Kitchen
Selene Cordeiro
Jutta Müller
Dish Washing Service
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