INTERNATIONAL RESEARCH TRAINING GROUP 6th Joint Symposium Annweiler August 28-31, 2017
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INTERNATIONAL RESEARCH TRAINING GROUP
6th Joint Symposium
Annweiler
August 28-31, 2017
2
Compendium
Monday, 08/28/17 Tuesday, 08/29/17 Wednesday, 08/30/17 Thursday, 08/31/17
8.00 Arrival/Registration
Begin of Meeting 10.25 am
Breakfast Breakfast Breakfast
9.00 Eva Zöller Regine Stutz
Free time 9.30 Julian Oesterreicher Ruiqi Cai
10.00 Shahid Ullah Laine Lysyk
10.30 Lisa Ohler Coffee break Coffee break
Departure to Basel (Lunch packages)
11.00 Pratiwi Prananingrum Nilam Yadao Sabrina Marz
11.30 Alka Kumari Rawad Lashhab Swai Khaing
12.00
Lunch Lunch Lunch
12.30
13.00
Hike & guided tour to Trifels Castle
Poster Session II
Hasib Sarder
Visit of Novartis
13.30 Xiaobing Li
14.00 Coffee break
Trainee & PI Meeting 14.30 Coffee break
15.00
Guided tour “Museum unterm Trifels”
Concluding remarks
15.30
End of Meeting
Departure/Free time
16.00
16.30
17.00 Anne Grethen
Departure to Annweiler & KL
17.30 Bartholomäus Danielczak
18.00
Dinner Dinner Dinner
18.30
19.00
Poster Session I Meetings of Guidance
Committees Free time 20.00
20.30
20 min talk & 10 min discussion
3
Day One (Monday, August 28, 2017)
Arrival and Registration until 10 am
Session 1 (Chair: Kathrin Patzke)
10:25-10:30 Ekkehard Neuhaus Welcome
10:30-11:00 Lisa Ohler Pyrimidine salvage and related transport processes across the chloroplast envelope
11:00-11:30 Pratiwi Prananingrum The characterization of the plastidic homolog of the Vacuolar Glucose Transporter1 (VGT1)
11:30-12:00 Alka Kumari Rescue of corneal dystrophy-causing SLC4A11 mutants by ophthalmological non-steroidal anti-inflammatory drugs
12:00 – 13:00 Lunch
13:00 – 17:30 Hike & guided tour to the Trifels Castle
18:00 – 19:00 Dinner
19:00 – 20:30 Poster Session I
Day Two (Tuesday, August 29, 2017)
08:00 – 09:00 Breakfast
Session 2 (Chair: Daniel Hickl)
09:00-09:30 Eva Zöller Cellular stress response mechanisms during inhibited protein import in mitochondria in yeast
09:30-10:00 Julian Oesterreicher Identification and characterization of a novel intracellular glutathione transporter
10:00-10:30 Shahid Ullah
Characterizing the relationship between claudin-4 and kidney anion exchanger-1 (kAE1) in the collecting duct in the kidneys
10:30 – 11:00 Coffee break
4
Session 3 (Chair: Azkia Khan)
11:00-11:30 Nilam Yadao tba
11:30-12:00 Rawad Lashhab The basolateral kidney anion exchanger 1 regulates tight junction integrity by interacting with claudin-4
12:00 – 13:00 Lunch
13:00 – 14:30 Poster Session II
14:30 – 15:00 Coffee break
15:00 – 17:00 Guided tour “Museum unterm Trifels”
Session 4 (Chair: Praneeth Chitirala)
17:00-17:30 Anne Grethen A close look at polymer-bounded nanodiscs using multi-detection size exclusion chromatography
17:30-18:00 Bartholomäus Danielczak Membrane-protein solubilisation by amphiphilic copolymers
18:00 – 19:00 Dinner
19:00 - 20:35 Meetings of Guidance Committees
Time Wintergarten Hofrat-Krafft-Raum
Adelberg (OG) Förlenberg (OG) Großer Saal
19.15–19.35
A. Blum M. Schmitt J. Casey
B. Danielczak S. Keller T. Möhlmann
G. Khandpur B. Morgan M. van der Laan N. Touret
A. Russo S. Lang R. Zimmermann X-Z. Chen
*New Trainees E. Neuhaus K. Philippar J. Engel B. Niemeyer H. Herrmann (consultation about Guidance committees for the new trainees )
19.35–19.55
P. Chitirala V. Flockerzi J. Casey
A. Grethen S. Keller M. van der Laan
X. Li M. Schmitt B. Morgan E. Cordat
L. Ohler T. Möhlmann E. Neuhaus
19.55 –20.15
H. Sarder M. Schmitt E. Neuhaus E. Cordat
D. Hickl T. Möhlmann S. Keller
N. Yadao M. van der Laan J. Herrmann
20.15-20.35
R. Stutz R. Zimmermann B. Niemeyer E. Cordat
E. Zöller J. Herrmann M. van der Laan T. Alexander
K. Patzke T. Möhlmann E. Neuhaus
* New Trainees: Azkia Khan, Pratiwi Prananingrum, Pauline Schepsky, Anne Könnel, Wassilina Bugaeva, Janina Laborenz , Cristina Martins Rodrigues, Mona Schöppe
5
Day Three (Wednesday, August 30, 2017)
08:00 – 09:00 Breakfast
(Meetings of remaining Guidance Committees anytime in the course of the day)
T. Bentrcia V. Flockerzi E. Friauf T. Alexander
S. Marz E. Friauf V. Flockerzi T. Alexander
D.P. Vu T. Möhlmann E. Neuhaus
*New trainees
Session 5 (Chair: Sarah Haßdenteufel)
09:00-09:30 Regine Stutz Client spectrum of the translocon-associated protein complex
09:30-10:00 Ruiqi Cai Binding between the N- and C-termini of TRP channels mediates functional regulation by PIP2
10:00-10:30 Laine Lysyk
10:30 – 11:00 Coffee break
Session 6 (Chair: Gurleen Kaur Khandpur)
11:00-11:30 Sabrina Marz Shotgun proteomics of Glycine transporter 2 (GlyT2) immunoprecipitations revealed 64 GlyT2-interacting proteins: the calcium-dependent secretion activator 1 (CAPS1) reduced glycine uptake by enhanced endocytosis of GlyT2
11:30-12:00 Swai Khaing Regulation of CD36 signal transduction by F-Actin and lipid nanodomains
12:00 – 13:00 Lunch
Session 7 (Chair: Duc Phuong Vu)
13:00-13:30 Hasib Sarder Dissecting intracellular trafficking and mis-trafficking of human kidney AE1 in
yeast and mammalian cells
13:30-14:00 Xiaobing Li Yeast overexpression screen for cellular components restoring plasma membrane trafficking of human kidney anion exchanger 1
14:00 – 15:00 Coffee break / Trainee & PI Meeting
(The Trainee meeting will be organized by Kathrin Patzke and Anne Grethen (Trainee Spokespersons), the PI meeting will be organized by Ekkehard Neuhaus (IRTG-Spokesperson).
15:00 - 15:05 Concluding remarks / End of Meeting
6
Thursday, August 31, 2017 (Novartis trip for IRTG Trainees)
08:00 – 09:00 Breakfast
9:30 Departure to Basel
13:00 – 16:30 Visit of Novartis (details on program in the appendix)
17:00 Departure in Basel
(Arrival in Annweiler at about 20.00; Arrival in Kaiserslautern (main station) at about 21:00)
Attendees Novartis trip:
1 Amoroso, Gabriele TU KL
2 Bugaeva, UdS
3 Cai, Ruiqi UofA
4 Chitirala, Praneeth UdS
5 Danielczak, Bartholomäus TU KL
6 Grethen, Anne TU KL
7 Güneri, Illayda TU KL
8 Hickl, Daniel TU KL
9 Hofmann, Laura UdS
10 Khaing, Swai UofA
11 Khan, Azkia TU KL
12 Khandpur, Gurleen TU KL
13 Klostermann, Viola UdS
14 Könnel, Anne UdS
15 Kumari, Alka UofA
16 Laborenz, Janina TU KL
17 Li, Xiaobing UdS
18 Lysyk, Laine UofA
19 Mahler, Florian TU KL
20 Martins Rodrigues, Cristina TU KL
21 Marz, Sabrina TU KL
22 Ohler, Lisa TU KL
23 Patzke, Kathrin TU KL
24 Prananingrum, Pratiwi TU KL
25 Russo, Antonietta UdS
26 Sarder, Hasib UdS
27 Schepsky, Pauline UdS
28 Ullah, Shahid UofA
29 Yadao, Nilam UdS
30 Zöller, Eva TU KL
31 Babysitter Pratiwi`s daughter KL
7
Talk abstracts in chronological order
8
Pyrimidine salvage and related transport processes across the chloroplast envelope
L. Ohler1 & T. Möhlmann
1
1Department of Plant Physiology, University of Kaiserslautern, Germany
Pyrimidine nucleotides are of high importance for plants as they are components of DNA and
RNA and play a role in many primary and secondary metabolic pathways. As the de novo
synthesis is highly energy consuming, uracil and uridine/cytidine can be recycled into
UTP/CTP via the salvage pathway. These reactions mainly take place in the cytosol and
occur via uridine and cytidine kinases. Uracil salvage seems to be less important, but
surprisingly knock-out plants of the uracil phosphoribosyl transferase (UPP), a key enzyme in
this process, show a severe phenotype. The same phenomenon can be observed for the
further enzyme of the uracil salvage – the nucleoside monophosphate kinase - which is
described to interact with the transcript of photosystem I. As this additional function of the
plastidic salvage enzymes might be more important, the chloroplasts need an alternative way
to obtain nucleotides. For this, corresponding nucleotide transporters must be localized in the
chloroplast envelope. During proteome analysis, several candidates were found, which I
want to further investigate regarding subcellular localization and transport activity.
The characterization of the plastidic homolog of the Vacuolar Glucose Transporter1 (VGT1)
P. Prananingrum1, K. Patzke
1, B. Bölter
2, I. Heferkamp
1 & H.E. Neuhaus
1*
1 Plant Physiology, University of Kaiserslautern, Kaiserslautern, Germany
2 Plant Biochemistry, Ludwig-Maximilians University, Munich, Germany
The major carbohydrates in plants are glucose, fructose, sucrose, cellulose and starch. The
transport of sugars across membrane barriers is essential for higher plants, since sugar
represent transport and storage units of cellular energy generation and thus play a
fundamental role during developmental processes and stress responses. In addition to
transport across the plasma membrane, carrier- mediated sugar transport has also been
demonstrated across organellar membranes, such as the inner plastid envelope or the
vacuolar membrane, named tonoplast. The monosaccharide transporter family is diverse and
contains seven distinct clades. In this study, I will focus on VGT-like protein family of
monosaccharide transporter in which comprised of three genes, At3g03090, At5g17010 and
At5g59250. Recently, two genes from this family, AtVGT1 (At3g03090) and AtVGT2
(At5g17010), have been shown to localize to the vacuolar membrane of Arabidopsis thaliana
(A. thaliana) (Aluri and Büttner, 2006) and it has been shown that VGT1 transports glucose
and a proton-coupled antiport. In contrast, here I show that the protein encoded by
At5g5925, the third member of this sub-group, locates to the chloroplast membrane. In the
present study, we want to further characterize the function of this protein in the sugar
transport mechanism in plants.
9
Rescue of corneal dystrophy-causing SLC4A11 mutants by ophthalmological non-steroidal anti-inflammatory drugs
A. Kumari & J R. Casey Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Purpose: Mutations of the membrane transport protein, SLC4A11, cause genetic endothelial
corneal dystrophies: congenital hereditary endothelial corneal dystrophy (CHED), Harboyan
syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). Out of 58 SLC4A11 point
mutants, 36 were identified as candidates for folding correction therapy based on their
endoplasmic reticulum (ER)-retained phenotype. Glafenine, which is a non-steroidal anti-
inflammatory drug, was earlier found to be promising in rescuing cell surface functional
activity of ER-retained mutants of SLC4A11. Here, we tested topical FDA- approved
ophthalmic NSAIDs for their efficacy in moving ER-retained SLC4A11 mutants to the cell
surface to restore functional activity.
Methods: A bioluminescence resonance energy transfer (BRET)-based assay was
established to identify SLC4A11 mutants amenable to folding correction therapy. With this
assay FDA-approved ophthalmological non-steroidal anti-inflammatory drugs (NSAIDs) were
screened to identify those enabling diseased SLC4A11 to target to the plasma membrane.
Confocal immunofluorescence was also used to measure cell-surface localization of mutant
SLC4A11. Functional activity of these mutants, upon drug treatment, was measured by
monitoring rates of cell swelling in hypo-osmotically challenged transfected HEK293 cells.
Results: The BRET assay enabled rapid, sensitive and accurate screening of
ophthalmological NSAIDs. Ketorolac tromethamine, nepafenac, bromfenac, diclofenac, and
flurbiprofen were tested for their ability to correct SLC4A11 ER-retained mutants (E143K and
G709E). Nepafenac and diclofenac treatment gave rise to statistically significant increases in
cell surface abundance of SLC4A11 mutants (BRET assay), which was confirmed by an
increased cell surface abundance measured by confocal immunofluorescence. Ophthalmic
NSAID-rescued mutants also retained the water flux function. The ability of nepafenac and
diclofenac to rescue ER-retained SLC4A11 point mutants establishes the therapeutic
potential of these drugs, when directed to individuals with particular SLC4A11 lesions.
Conclusions: The BRET assay measured total cell surface SLC4A11 abundance.
Nepafenac and diclofenac rescued the folding defect of E143K and G709E SLC4A11. These
drugs were tested for their ability to correct the folding of the 36 identified ER-retained
mutants of SLC4A11.
Cellular stress response mechanisms during inhibited protein import in mitochondria in yeast
E. Zöller Department of Cellular Biology, University of Kaiserslautern, Kaiserslautern, Germany
99 % of mitochondrial proteins are initially synthesized by cytosolic ribosomes as precursor-
proteins. Guided by chaperones the precursors are transported to the outer membrane of the
mitochondria. The import into the matrix is regulated by the translocase of the outer
mitochondrial membrane (TOM) complex and subsequent the translocase of the inner
mitochondrial membrane (TIM)-23 complex. To inhibit mitochondrial import, a fusion protein
(clogger), consisting of a mitochondrial targeting signal, a cytochrome b2-, and a
dihydrofolate reductase (DHFR)-domain, can be used. RNA-Seq analysis have surprisingly
shown that in case of clogger expression in a wild type yeast strain, genes of the oxidative
phosphorylation machinery are downregulated. Interestingly, chaperones and various
10
proteasome components are strongly upregulated at the same time. So far we do not know
how the signaling towards the nucleus looks like and how it is regulated. We hypothesize the
activation of a signaling cascade similar to the unfolded protein response (UPR) in the
endoplasmic reticulum (ER). First experiments on RNA (RT-PCR) and protein level (western
blot) could successfully verify those findings. Additionally, a genome wide screen will be
conducted to identify proteins that play an important role within the stress response.
Identification and characterization of a novel intracellular glutathione trans-porter
J. Oesterreicher & B. Morgan Department of Cellular Biology, University of Kaiserslautern, Kaiserslautern, Germany
Glutathione fulfils multiple roles in the cell, including acting as an important redox co-factor
and playing an essential role in FE-S cluster biogenesis.
The introduction of genetically encoded sensors, which enable measurements of the
glutathione redox potential inside living cells has changed our view of cellular glutathione.
Cellular glutathione appears to be highly compartmentalized. We now know that the cytosolic
glutathione pool is extremely reduced and robustly regulated, any glutathione disulphide
(GSSG) that is formed is either quickly reduced, actively transported to the vacuole or
excreted from the cell. Thus, we can infer that any GSSG observed in whole cell lysates
must have been located in a non-cytosolic cellular compartment. Consequently, transporter
expression level-dependent increases or decreases in cellular GSSG content can serve as
an indirect indicator of GSSG transport between the cytosol and other cellular compartments.
Building upon our recently acquired insights we now employ new techniques to screen for
novel intracellular GSH and GSSG transporters. By targeting glutathione biosynthetic
pathway enzymes, Gsh1 and Gsh2 to alternative cellular compartments we can employ
growth assays and biochemical analyses of cellular GSH and GSSG content to identify
putative intracellular glutathione transporters. We have identified a strong candidate for an
ER GSSG exporter.
Characterizing the relationship between claudin-4 and kidney anion exchanger-1 (kAE1) in the collecting duct cells in the kidneys
AKM. S. Ullah & E. Cordat Department of Physiology, University of Alberta, Edmonton, Canada
Basolateral kidney anion exchanger 1 (kAE1) is expressed in α-intercalated cells in the
collecting duct (CD) of the kidney and maintains pH homeostasis by exchanging Cl- for
HCO3-. Mutations in kAE1 gene cause distal renal tubular acidosis (dRTA), a disease that we
still do not fully understand. In a yeast two-hybrid assay, we unveiled a physical interaction
between kAE1 and claudin-4. Claudin-4, a protein expressed at the tight junctions of the CD
cells, allows paracellular transport of Cl-. Expression of kAE1 in immortalized inner medullary
collecting duct cells that endogenously express claudin-4 resulted in a significantly reduced
transepithelial electrical resistance, supporting a role of kAE1 in tight junction properties.
Importantly, this effect was mediated by claudin-4. We thus hypothesized that the
kAE1/claudin-4 interaction is important to maintain both pH and electrolyte homeostasis. Our
major aim is to delve into the physiological role of claudin-4 and kAE1 interaction for pH and
electrolyte homeostasis in vivo. To pursue this aim we generated α-intercalated cell specific
claudin-4 knockout mice. The KO mice developed normally and did not present obvious
11
phenotypic aberration. No difference in plasma composition was detected at the steady-
state. Three and 6 months old mice have been fed a low or normal NaCl diet for 2 weeks.
Examination of plasma pH and bicarbonate content of 3 month old mice showed that these
mice have not developed dRTA at this age. Examination of urinary composition is on-going.
These mice will be challenged with an acidic diet to investigate how they maintain normal
plasma and urine pH and electrolytes. These experiments will provide a physiological insight
into the role of the physical claudin-4/kAE1 interaction and pH homeostasis.
TBA
N. Yadao Department of Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany
The basolateral kidney anion exchanger 1 regulates tight junction integrity by interacting with claudin-4
Lashhab R#, Arutyunov D
#, Alexander RT
#, Cordat E
#
# Department of Physiology, University of Alberta, Edmonton, T6G 2H7, Canada
Patients with distal renal tubular acidosis (dRTA) have impaired renal acid secretion and, as
a consequence, abnormal bicarbonate reabsorption from their distal nephron. dRTA patients
develop kidney stones, hypokalemia, hyperchloremia, nephrocalcinosis, metabolic acidosis
and difficulties to thrive. Mutations in the SLC4A1 gene encoding the anion exchanger 1 can
cause dRTA. Kidney anion exchanger 1 (kAE1) is a transmembrane Cl-/HCO3- exchanger
that is expressed in α-intercalated cells in the collecting duct. Using a membrane yeast two-
hybrid assay, we found that kAE1 interacts with Claudin-4 (Cldn-4). Cldn-4 is a tight junction
protein, which is expressed in many tissues including intercalated cells. Cldn-4 forms a
paracellular Cl- selective pore and has been implicated in Cl- reabsorption from the collecting
duct. We therefore hypothesized that a kAE1/Cldn-4 interaction regulates pH and electrolyte
homeostasis in the distal nephron. To confirm a physical association, we performed
immunofluorescence and proximity ligation assays, which demonstrated co-localization
between kAE1 and Cldn-4 in polarized murine inner medullary collecting duct cells.
Immunoprecipitations confirmed the physical interaction. BCECF-based functional assays
assessing AE1 activity did not demonstrate alterations when Cldn-4 was over-expressed.
However, Ussing chamber experiments revealed a decrease in transepithelial electrical
resistance and an increase in paracellular Cl- & Na+ permeability upon kAE1 expression,
indicating that expression of the basolateral anion exchanger altered the tight junction
integrity. Our data support that kAE1 alters tight junction properties independent of changes
in intracellular pH. Our results demonstrate a physical interaction between kAE1 and Cldn-4
and have uncovered an un-expected role of a basolateral anion exchanger on tight junction
integrity, and possibly further on electrolyte homeostasis and blood pressure regulation.
Supported by CIHR, the Canadian Foundation for Innovation, the Kidney Foundation of
Canada & the NSERC CREATE Program.
12
A close look at polymer-bounded nanodiscs using multi-detection size
exclusion chromatography A. Grethen, J. Klingler, & S. Keller Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany
To study membrane proteins in vitro, they need to be extracted from their native lipid
environment while maintaining their integrity. Styrene/maleic acid (SMA) [1] and
diisobutylene/maleic acid (DIBMA) [2] copolymers self-insert into artificial or native
membranes to form polymer-bounded nanodiscs containing membrane proteins and lipids.
Without conventional detergents, this approach enables high-resolution structural and
functional characterization of membrane proteins down to the single-
molecule level.
The composition and architecture of polymer-bounded nanodiscs remain
poorly understood but are of outstanding importance for their judicious
use in membrane-protein research. Therefore, we analyzed the influence
of copolymer composition and concentration on phospholipid vesicle
solubilization by multi-detection size exclusion chromatography (SEC). In
particular, we characterized the morphology and composition of
fluorescently labelled polymer-bounded nanodiscs using size exclusion
chromatography simultaneously monitored by absorbance,
refractometry, viscometry, and light scattering. [1] Knowles et al. J. Am. Chem. Soc. 2009, 131, 7484 [2] Oluwole et al. Angew. Chem. Int. Ed. 2017, 56, 1919 [3] Vargas et al. Biospektrum, 2016, 22, 140
Membrane-protein solubilisation by amphiphilic copolymers
B. Danielczak Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany
To stay in a native and functional conformation, integral membrane proteins (MPs) rely on a
heterogeneous and complex environment, namely, a lipid-bilayer membrane. The isolation
and the purification of MPs are challenging but crucial steps to enable in vitro investigations
of these essential components of each living cell. For decades, MP extraction has been
accomplished by the use of detergents, which solubilise MPs into micelles. However,
detergents tend to be harsh, that is, they provide only limited stability and insufficient
membrane-mimicking properties.
Recently, a promising approach without classical detergents has emerged that is based on
the use of styrene/maleic acid (SMA)1 or diisobutylene/maleic acid (DIBMA)2 copolymers.
SMA and DIBMA solubilise biological and model membranes into nanodiscs by forming
patches of intact lipid bilayers bounded by a polymer belt.
Our work focuses on the ability of these two copolymers to solubilise a wide variety of
biological membranes while retaining the native functionality of the solubilised proteins. On
the one hand, the solubilisation efficiency of various protein classes is determined with the
aid of proteomics approaches relying on quantitative mass spectrometry. On the other hand,
we examine the ability of SMA and DIBMA to retain the functionality of particular membrane
proteins by utilising a simple enzyme-activity assay of mitochondrial respiratory chain
complexes.
1Knowles et al. J. Am. Chem. Soc. 2009, 131, 7484
2Oluwole et al. Angew. Chem. Int. Ed. 2017, 56, 1919
[3]
13
Client spectrum of the translocon-associated protein complex
R. Stutz Department Of Medical Biochemistry And Molecular Biology, Saarland University
Thirty percent of all polypeptides synthesized in mammalian cells are inserted into or
translocated across the membrane of the endoplasmic reticulum (ER) via the polypeptide-
conducting Sec61 channel. The ribosome-associated Sec61 complex is stably associated
with the TRAP complex which assists amino-terminal signal peptides (sp) or transmembrane
helices (tmh) of a sub-population of precursor polypeptides in their productive insertion into
the Sec61 channel recently, mutations in the human TRAP subunits were observed to result
in congenital disorders of glycosylation (CDG). Nevertheless, the exact function(s) and
mechanism(s) of the TRAP complex have not been understood in detail yet.
Our current studies on the translocation machinery combine structural elucidation of the
translocon complex with the functional characterization of the human TRAP complex. Here
we combined siRNA-mediated TRAP depletion in human cells, label-free quantitative
proteomic analysis, and differential `expression´ analysis in an unbiased strategy to identify
TRAP-dependent polypeptides or clients as `down-regulated´ or negatively affected in living
human cells. Analysis of their sp points to a lower over-all hydrophobicity and demonstrates
a higher than average glycine plus proline content, i.e. lower helix propensity, as the
distinguishing features for TRAP dependence.
We suggest that both features are detrimental to the process of insertion of sp into the Sec61
channel. Strikingly, global analysis of sp revealed that these features are found in a sub-
population of human sp, but not in those of precursors from yeast which lacks TRAP. In light
of recent insights into TRAP architecture, these results suggest TRAP as potential sp
receptor on the cytosolic face of the ER membrane and an information relay from the
ribosome via cytosolic and ER lumenal domains of TRAP to the ER lumenal loop 5 of
Sec61α, allowing TRAP to assist insertion of certain sp and tmh into the Sec61 channel in a
precursor specific manner.
Binding between the N- and C-termini of TRP channels mediates functional regulation by PIP2
R. Cai MSB 7-29, Department of Physiology, University of Alberta, Canada
TRP channels are regulated by diverse stimuli comprising thermal, chemical and mechanical
modalities, which underlies their implications in numerous physiological processes. However,
the structural bases of these regulation mechanisms remain largely unclear. Further, while
most TRPs are known to be either activated or inhibited by phospholipid phosphatidylinositol
4,5-bisphosphate (PIP2) it is unknown as to whether there is a shared underlying
mechanism. Here, we first identified by Xenopus oocyte electrophysiology that the human
TRPP3 aromatic residue W81 in the N-terminal pre-S1 domain and the cationic residue K568
in the C-terminal TRP-like domain are functionally critical. By pull-down, co-
immunoprecipitation and co-immunofluorescence assays, we then showed that the W81-
K568 pair mediates the N- to C-termini (N-C) interaction, presumably through forming a π-
cation bond. N-terminal peptide I40-L95, but not the one carrying the W81A mutation, inhibits
TRPP3 channel activity, presumably through disrupting the N-C binding in TRPP3. The W81
and K568 are highly conserved in most TRPs and we indeed found that the N-C binding and
its functional importance are conversed in TRPP2/-M8/-V1/C4 as well using mammalian cells
14
and oocytes. Next, we found that PIP2 binds with TRPP3 through C-terminal K568 and
cationic residues in 594-RLRLRK-599 and inhibits its channel function through disrupting the
N-C binding. The negative regulation of the N-C binding by PIP2 was also seen in TRPP2.
Interestingly, the N-C binding in TRPM8 and -V1 was enhanced by PIP2, which was
concurrent with a stimulating effect of PIP2. PIP2 regulates the function of different TRP
channels through modulating their N-C binding, which represents a shared mechanism of
regulation that should serve as a molecular relay/switch to mediate the activation of TRP
channels by extracellular agonists.
TBA
L. Lysyk
Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta , Edmonton, Alberta, Canada T6G 2H7
Shotgun proteomics of Glycine transporter 2 (GlyT2) immunoprecipitations revealed 64 GlyT2-interacting proteins: the calcium-dependent secretion activator 1 (CAPS1) reduced glycine uptake by enhanced endocytosis of GlyT2
S. Marz, M. Jones, C. Fecher-Trost, M. Jung, RT. Alexander & E. Friauf Department of Animal Physiology, University of Kaiserslautern, Kaiserslautern, Germany
Glycine is an essential inhibitory neurotransmitter in mammals. Its homeostasis depends on
the neuronal glycine transporter 2 (GlyT2), which mediates glycine uptake from the synaptic
cleft. Modulation of GlyT2’s functionality appears to be beneficial for the treatment of the
startle disease Hyperekplexia, which is associated with mutations in human GlyT2, as well as
pain. We hypothesize that a molecular network of proteins exists, which regulates GlyT2
activity. To this end, we screened for novel GlyT2-interacting proteins via co-
immunoprecipitations (co-IPs) followed by ultra-sensitive mass spectrometry. In three
biological replicates, 357 proteins were identified. To extract putative interactors, we applied
several sensitivity filters. 64 GlyT2-interacting proteins fulfilled these criteria. Functional
clustering revealed that 27% of them regulate ion transport/homeostasis and 40% protein
transport. One candidate of the latter group was the calcium-dependent secretion activator 1
(CAPS1). Western blots of co-IPs and reverse co-IPs with endogenous protein verified the
mass spectrometry data proposed interaction. In an independent experiment, a peptide spot
array, CAPS1 specifically bound to the conserved GlyT2 carboxy-terminus, also suggesting a
physical interaction in vivo. Therefore, we co-expressed GlyT2 and CAPS1 in HEK-293 cells.
In the presence of CAPS1, GlyT2 abundance was higher but shifted to intracellular
compartments. Moreover, glycine uptake experiments indicated a lower maximal transport
rate by unchanged substrate affinity when CAPS1 is co-expressed, suggesting a decreased
surface abundance of GlyT2. Certainly, biotinylation of surface proteins confirmed a lower
relative amount of biotinylated GlyT2 in the presence of CAPS1, most likely due to enhance
endocytosis. In sum, our data provide evidence for a functional interaction between GlyT2
and CAPS1, decreasing GlyT2 surface abundance by increased endocytosis. Additionally,
the identification of 26 other proteins regulating protein transport propose a molecular
network modulating GlyT2 trafficking.
15
Regulation of CD36 signal transduction by F-actin and Lipid nanodomains
S. Khaing, & N. Touret. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7.
CD36, a multi-ligand plasma membrane receptor, has been implicated in immunity,
metabolism and angiogenesis. We have recently demonstrated that CD36 nanoclustering at
the plasma membrane is key to the initiation of CD36 signaling. In endothelial cells (ECs),
the binding of thrombospondin-1 (TSP-1, an endogenous extracellular matrix anti-angiogenic
factor) to CD36 nanoclusters activates an associated Src family kinase, Fyn, leading to ECs
apoptosis, hence, inhibiting angiogenesis. We are interested in elucidating the mechanisms
of CD36-Fyn enrichment in lipid nanodomains and in F-actin rich area during TSP-1 induced
signaling in ECs.
We hypothesize that lipid nanodomains play a role in bringing together CD36-Fyn to F-actin
regions through adaptor molecules which forms a signaling platform. We undertook to
characterize the lipid nanodomains in which CD36 nanoclusters are enriched and to identify
adaptor proteins enabling F-actin localization. Using microscopy methods on HeLa cells co-
transfected with Fyn and various fluorescent lipid biosensors and stained for F-actin
(Phalloidin-AF647), we determined that Fyn is enriched on F-actin area at sites of
phosphatidylinositol 4,5-bisphosphate enrichment (PIP2). During TSP-1 stimulation on
Human Microvascular Endothelial Cells (HMEC), the CD36-Fyn-F-actin enrichment shift to
domains containing PI(3,4,5)P3, suggesting a role for the phosphoinositide 3-kinase in
signaling. The role of this kinase is further investigated using inhibitor targeting PI3-Kinase
subunits for signal transduction. We’ve determined that PI3K is important for the activation of
Fyn kinase. Furthermore, to characterize the adaptor molecules involved in connecting F-
actin to lipid nanodomains and/or CD36 nanoclusters, we conducted BioID proximity-
dependent labelling and fractionation approaches followed by mass spectrometry (MS)
analysis. The MS screen was narrowed down to 9 potential proteins that are biotinylated,
adjacent to CD36 and enriched in F-actin fraction. Altogether, our investigation will provide
insights into understanding the activity of plasma membrane receptor nanoclustering and
signaling.
Dissecting Intracellular trafficking and mis-trafficking of human kidney AE1 in yeast and mammalian cells
H A M. Sarder, B. Becker & MJ. Schmitt Molecular & Cell Biology, Department of Biosciences, Center of Human and Molecular Biology (ZHMB), Saarland University, Saarbrücken, Germany.
Kidney anion exchanger 1 (kAE1) is a bicarbonate exchange protein in the basolateral
membrane of α-intercalated cells of the human kidney that is responsible for the reabsorption
of bicarbonate ions (HCO3-) by exchange with Cl- ions, thereby ensuring acid excretion in the
urine [1]. Various genetically inherited mutations in the kAE1 encoding gene have been
reported to negatively affect HCO3-/Cl- exchange and to ultimately result in clinical disorders
known as distal renal tubular acidosis (dRTA). Until now, autosomal dominant (AD) and
recessive (AR) mutations have been identified that are either linked to false kAE1 localization
or mistrafficking [2]. Since the underlying molecular mechanisms for proper kAE1 targeting
are still poorly understood, we are using yeast as simple eukaryotic model organism to
dissect the intracellular targeting and trafficking of wild-type kAE1 and its mutant variants.
Since yeast Bor1p is proposed to be the homologue of mammalian kAE1 [3], we are
currently focusing on three major aspects: (i) Expression of codon-optimized wild-type kAE1
16
and its clinically relevant mutant variants in a yeast ∆bor1 knock-out mutant for functional
complementation; (ii) Analysis of intracellular kAE1 trafficking by life cell imaging (CLSM) and
determination of kAE1 plasma membrane localization through mRAS recruitment and cell
surface biotinylation; (iii) Genetic screen of selected yeast knock-out mutants to identify
cellular components involved in proper kAE1 trafficking. The results obtained from the yeast
screen will be translated into the situation of mammalian cells to get a deeper mechanistic
understanding of kAE1 mis-targeting/trafficking in dRTA associated clinical disorders.
Yeast overexpression screen for cellular components restoring plasma membrane trafficking of human kidney anion exchanger 1
X. Li Department of Molecular & Cell Biology, Saarland University, Saarbrücken, Germany
Human kidney anion exchanger 1 (kAE1) represents a bicarbonate transporter in the
basolateral membrane of renal epithelial cells that participates in the fine-tuning of acid-base
homeostasis by mediating electroneutral Cl-/HCO3- exchange. Several autosomal mutations
in the kAE1 encoding gene (SLC4A1) can cause clinical disorders known as distal renal
tubular acidosis (dRTA) which are linked to kAE1 mis-folding, ER/Golgi retention, and/or
premature degradation. Despite that some proteins involved in kAE1 trafficking could be
identified, the precise mechanism(s) resulting in dRTA still remain unclear. Since wild-type
kAE1 can be expressed in yeast and is correctly targeted to the plasma membrane, we are
going to use yeast as experimental system to identify proteins which affect intracellular kAE1
trafficking to the plasma membrane and/or its turnover which is vital for proper kidney
function. By using a yeast ORF expression library (~ 6,000 ORFs), we will initially establish a
pH-sensor-, Western- and FACS-based screening approach in S. cerevisiae to test which
yeast proteins, when overexpressed, modulate the cytosolic pH as well as the cellular
expression and plasma membrane localization of kAE1. In further experiments we want to
analyse the identified proteins to understand how these proteins are capable to increase
and/or restore plasma membrane transport of wild-type kAE1 as well as clinically relevant
kAE1 mutant variants.
Key words: kAE1; dRTA; S. cerevisiae; overexpression screen; cellular expression; plasma
membrane trafficking
17
Poster Sessions
Session I
Monday, August 28, 7 pm - 8.30 pm
1 Bentrcia, Teqiyya TRPC6 function - continuation
2 Blum, Andrea In vivo localization and function of eukaryotic KDEL receptors
3 Bugaeva, Wassilina Molecular decoding of plastid fatty acid export
4 Chitirala, Praneeth Role of V-ATPase in mouse cytotoxic T lymphocytes cytotoxicity
5 Haßdenteufel, Sarah Chaperone mediated Sec61 channel gating during import of small presecretory proteins into the human ER
6 Hickl, Daniel Equilibrative nucleoside transporter - Physiology and attempts to gain structural insights
7 Kaur Khandpur, Gurleen Respiratory chain components regulate cell growth in response to changing amino acid availability
8 Khan, Azkia Characterization of the putative vacuolar sugar transporter Aterdl4
Session II
Tuesday, August 29, 1 pm - 2.30 pm
9 Laborenz, Janina J-Proteins are involved in the targeting of mitochondrial precursor proteins
10 Mahler, Florian Membrane interactions of conventional and fluorinated surfactants
11 Martins Rodriguez, Cristina
Chilling lessons: Teaching the sugar beet how to cope with cold
12 Patzke, Kathrin Chloroplast sugar metabolism and its influence on cellular signaling
13 Russo, Antonietta Structure and mechanisms of TRAP complex
14 Schepsky, Pauline The Na
+-activated K
+ channel Slack (Slo2.2) in the mouse
auditory system
15 Schöppe, Mona tba
16 Sicking, Mark hSnd2: One component of a new targeting pathway to the human ER
17 Vu, Duc Phuong Analyses of sucrose compartmentation in Arabidopsis thaliana
18
Poster abstracts in alphabetical order
19
TRPC6 function - continuation
T. Bentrcia & V. Flockerzi Department of Experimental & Clinical Pharmacology & Toxicology, Saarland University, Homburg., Germany
Transient receptor potential (TRP) channels play important roles in many cellular processes
and they may also be essential for platelet function. We identified the TRPC6 protein in
human platelets; it might act as the subunit of a tetrameric TRPC6 channel. In order to
understand and characterize TRPC6 function in human platelets and to identify potential
subunits of the channel, we generated antibodies for TRPC6 which allow detecting the
protein in tissue homogenates and in Western blots. Initially, we establish an antibody-based
affinity purification scheme to enrich solubilized TRPC6 protein from platelets, in the
presence of 1% Digitonin. The bound-TRPC6 protein was eluted under non denaturing
condition, run on blue native gels and followed by mass spectrometry. By the latter approach
we wanted to identify proteins associated and enriched with the TRPC6 protein. By mass
spectrometry, the G-protein–coupled receptor kinase interacting protein-1 (GIT1) and some
additional proteins were found to be associated with TRPC6 but not with the non-specified
control. The interaction between TRPC6 and GIT1 was confirmed by coimmunoprecipitation
and in vitro pull down assay, indicating a direct interaction. With the aim to identify the
molecular domain mediating TRPC6-GIT1 interaction, pull down assays were conducted
using different recombinant fragments of GIT1 fused to GST. We could show that the GIT1
protein bind TRPC6 channel via its Ankyrin repeat domain. In calcium imaging, the TRPC6
channel can be activated by diacylglycerol or its derivative OAG. The OAG-induced calcium
entry in HEK293 cells stably expressing TRPC6 cDNA was reduced in the presence of GIT1
protein indicating a potential inhibitory effect of GIT1 on TRPC6-mediated calcium entry.
Next we want to map the TRPC6 domain required for GIT1 binding and to identify the amino
acids responsible for TRPC6-GIT1 interaction.
In vivo localization and function of eukaryotic KDEL receptors
A. Blum Department of Molecular & Cell Biology, Saarland University, Saarbrücken, Germany
A/B toxins such as cholera toxin, Pseudomonas exotoxin and yeast killer toxin K28 contain a
KDEL-like motif at either subunit which ensures retrograde toxin transport through the
secretory pathway of a target cell. Intoxication and host cell entry is initiated by toxin binding
to plasma membrane (PM) receptors. We recently identified Erd2p, the yeast KDEL receptor
(KDELR), as PM receptor of the viral K28 toxin carrying a C-terminal HDEL motif at its cell
binding β-subunit. Consistent with its function at the cell surface, immunogold labelling and
electron microscopy (TEM) demonstrated PM colocalization of Erd2p. All experiments in
yeast were performed with a wild-type strain and an endocytosis mutant (∆end3) expressing
a C-terminal (HA)10 -tagged ERD2 variant from its natural chromosomal ERD2 locus. In order
to identify how a cell ensures that a major KDELR fraction is retained in the ER and Golgi
while a minor fraction resides in the PM, we analysed the C-terminal lysine cluster of
KDELRs for a potential function as ER retention signal in both yeast and mammalian cells
and investigated subcellular localization of PM markers and red fluorescent KDELR variants
either extended by classical retrieval motifs or by the natural C-terminus of KDELR or its K/R
substituted variant. PM localization of the yeast arginine permease Can1p could be
prevented by the addition of the Erd2p C-terminus, supporting our hypothesis that the C-
terminal KDELR sequence functions as lysine-based retention motif. As KDELRs have
recently been shown to function in intra-Golgi/ER signalling and maintenance of Golgi
20
homeostasis, we assume a similar signalling function of KDELRs after cargo binding at the
cell surface. To address such novel functions, we are currently focusing on a CRISPR/Cas9-
mediated KDELR knock-out (k/o) in HeLa/HEK293 cells. By the initial use of a homology-
directed repair (HDR) based CRISPR/Cas9 knock-out system, only monoallelic k/o clones
were obtained. To improve the k/o efficiency, we used a non homologous end joining system
which leads to small insertions or deletions at the double strand break position. By using an
expression system in which the Cas9 ribonuclease is coupled to eGFP via a 2A-peptide,
subsequent cell sorting for a strong green fluorescence ensures the selection of cells with
strong Cas9 expression. In this way, however, only a few single cell colonies could be
recovered that were subsequently identified of wild-type genotype. By sorting cells for either
a strong, medium or weak green fluorescence, we could demonstrate that the level of Cas9
expression correlates with the observed prominent cell dying. In parallel to the ongoing
screening, we are characterizing a commercial KDELR1 k/o HAP1 cell line.
Molecular decoding of plastid fatty acid export
W. Bugaeva Department of Plant Biology, Saarland University, Saarbrücken, Germany
Fatty acids (FAs) are building blocks for the majority of cellular lipids, which are essential
throughout life of all organisms. Besides their role as constituents of biological membranes,
plant acyl-lipids are used for diverse functions at different destinations and tissues. Thus,
membrane transport and distribution of FAs and lipids is crucial for plant growth and
development (Li et al. 2016, TIPS 21, 145-158). Further, plant-derived lipid compounds are
of biotechnological importance, e.g. for production of biodiesel or improvement of nutrient
quality. Since plant de novo FA synthesis essentially takes place in plastids, export to the ER
for acyl editing and lipid assembly is necessary. Although, it is generally agreed that free FAs
are shuttled across plastid envelope membranes, the mode of export simple diffusion or
protein-mediated still is a matter of debate. The identification of FAX1 (Li et al. 2015, PLOS
Biology 13), a novel protein for FA-export across the inner envelope of chloroplasts, gave an
answer to this question. FA-transport function of FAX1 demonstrated in yeast cells is crucial
for plant biomass production, male fertility and synthesis of FA-derived compounds such as
lipids, ketone waxes, or pollen cell wall material. ER-derived lipids decrease when FAX1 is
missing, but levels of plastid-produced species increase. FAX1 over-expressing lines show
the opposite, including a pronounced increase of TAG oils in flowers and leaves. In
Arabidopsis, 7 proteins belong to the FAX family and since besides FAX1 also FAX2 and
FAX3 are predicted to be plastid targeted, heteromer formation and/or tissue-specific
expression and function is likely. Our current research therefore focusses on the distinct
subcellular localisation of FAX2, FAX3 proteins as well as the characterisation of fax1/fax3
double mutant lines.
Role of V-ATPase in mouse cytotoxic T lymphocytes cytotoxicity
P. Chitirala Department of Physiology, Saarland University, Homburg / Saar, Germany
Cytotoxic T lymphocytes (CTLs) play an important role in our body's immune system. Their
main effector function is to recognize and destroy viral-infected and tumorigenic target cells.
They contain cytotoxic proteins such as granzymes and perforin in specialized secretory
21
granules termed cytotoxic granules (CGs). CG contents are released upon target cell
recognition at the highly dynamic CTL: target cell interface called the immune synapse. Ca2+
signalling inside the CTL is essential for its activation, effector function, tolerance of self-
antigens and homeostasis. Despite it being such an important parameter, the Ca2+
concentration inside CGs is unknown. We aim to estimate the Ca2+ concentration inside CGs
and examine how this concentration regulates CG function. To this end, we have generated
organelle-specific Ca2+and pH sensors, which we attempt to use as a tool to calculate the
absolute calcium concentration and pH of CGs. We selected ratiometric, FRET based
Troponin C Ca2+ sensors (Twitch calcium sensors), which have a Kd ranging from 150 nM to
9.5 µM (Griesbeck et al., 2014). The Ca2+ sensors were fused to the C-terminus of the CG
membrane protein Synaptobrevin2 (Syb2) targeting the sensor into the lumen of the CG. The
Ca2+ concentration inside the CG was measured by generating in vivo calibration curves at
pH 7.3 using Ionomycin. However, since the pH of CGs is acidic, knowledge of the absolute
pH inside CGs is required for the calibration of the Ca2+ sensor. Therefore, the ratiometric pH
sensor ClopHensorN (Q69M) was generated as a fusion protein to the C-terminus of
granzymeB (GraB-ClopHensorN (Q69M)). The correct localization was verified by co-staining
with an antibody and by co-transfection with either Syb2-Twitch or granzymeB-mTFP. Using
GraB-ClopHensorN (Q69M), the pH in CGs was found to be 5.9 ± 0.2. RT-PCR in CTLs
showed that the V0 domain subunits a1, a2 and a3 are expressed. Next, we aim to
investigate which a-isoform of the V-ATPase is functional for CGs by down-regulating the
expression of the three subunit isoforms using RNAi and then measuring CG pH using GraB-
clopHensorN (Q69M). After Knockdown (KD) of a3 subunit alone increased the pH in CGs
from 5.7 to 6.8 with out affecting the localization of granzymeB. Staining of a3 subunit with
antibody showed that it localizes to CGs. Further, a3 subunit KD showed reduced killing of
p815 target cells by mouse CTLs. Wild type CTLs were transfected with a3 siRNA and
granzymeB-mcherry construct and secretion of granzymeB was analysed with TIRF
microscopy. a3 KD cells showed 75% reduction in CG secretion. Further we would like to
investigate the effect of V-ATPase on CG fusion by patching Synaptobrevin2-mRFP CTLs
with a 12 amino acid peptide, which binds to c-Subunit of V-ATPase, where VAMP2
interacts.
Chaperone mediated Sec61 channel gating during import of small presecretory proteins into the human ER
S. Haßdenteufel1, P-H. Lee
2, S. High
3, J. Paton
4, A. Paton
4, V. Helms
2 & R. Zimmermann
1
1 Department of Medical Biochemistry, Saarland University, Germany; /
2 Department of Bioinformatics, Saarland University,
Germany / 3
Faculty of Life Sciences, University of Manchester, Manchester, UK / 4
Research Centre for Infectious Disease, University of Adelaide, Australia
Following targeting, efficient translocation of short presecretory proteins across the ER
membrane is mediated by a complex machinery of membrane-embedded and associated
proteins. These auxiliary components are supposed to regulate the open-closed equilibrium
of the central highly dynamic Sec61 protein-conducting channel, thereby allowing protein
translocation across the membrane. Using either siRNA or subtilase AB toxin for proteolytic
cleavage, the role of Sec63 and the ER luminal Hsp70-chaperone BiP was analyzed. The
results suggest multiple substrate-specific functions of Sec63 in protein translocation. In
addition, it is demonstrated that the Sec62-Sec63 interaction is not essential for all
substrates. More precisely, Sec63 itself shows an intrinsic function, related to the signal
peptide (SP) of the precursor polypeptide. Indeed, the data strongly suggest that the Hsp70-
22
type chaperone, BiP, and its Hsp40-type co-chaperone Sec63, facilitate Sec61 channel
gating to the open state in SRP-dependent and -independent transport when precursor
polypeptides with interfering features in the mature part were targeted. Therefore, it is
proposed that after targeting of a precursor polypeptide to the Sec61 complex, gating of the
Sec61 channel to the open conformation either occurs spontaneously or has to be facilitated
by co-chaperone mediated chaperone action on the channel. Interestingly, the mature
domain-specific BiP depletion phenotype was mimicked by the SP-selective Sec61-inhibitor
CAM741. Molecular docking suggested that heptadepsipeptides strengthen the same barrier
for opening of the Sec61 channel, which is overcome by BiP binding to Sec61α1-loop 7.
Thus, using CAM741 allows to operationally define “weak” or “difficult to translocate”
precursor polypeptides depending on auxiliary BiP.
Equilibrative nucleoside transporter - Physiology and attempts to gain structural insights
D. Hickl1, L. Ohler
1, C. Girke
1, M. Daumann
1, J. Casey
2, M. J. Lemieux
3 & T. Möhlmann
1
1Department of Plant Physiology, University of Kaiserslautern, Germany /
2Deptment of Physiology and Biochemistry, University
of Alberta, Canada / 3Deptment of Biochemistry, University of Alberta, Canada
Nucleoside transporters play an important role in many organisms as they transport
hydrophilic nucleosides across membranes. In human, these transporters can either act as
equilibrative (ENT) or concentrative (CNT) nucleoside transporters and are possible targets
for the treatment of cancers and viral diseases. In the prokaryotic organisms Vibrio cholerae
and Neisseria wadsworthii the first crystal structures of CNT proteins were unraveled,
indicating an elevator-like transport mode of nucleosides with sodium. In contrast, no ENT
structure was resolved so far. However, recombinant protein could be functionally purified
from yeast, human and plants and the ENT topology was predicted containing 11
transmembrane domains. Arabidopsis thaliana harbors eight ENT isoforms, whereof AtENT7
works like mammalian ENTs. All others act as nucleoside/proton symporter. With the aim to
obtain deeper insights in ENT properties, a mutant lacking all endogenous cysteins, but
containing extra cysteins at critical positions, was created for functional and topological
analysis. Furthermore AtENT7 can be highly expressed in Pichia pastoris, which gives the
opportunity for Cryo EM and/or crystallization studies. Both approaches shall be followed in
future work.
Respiratory chain components regulate cell growth in response to changing amino acid availability
G. Kaur Khandpur Department of Cellular Biology, University of Kaiserslautern, Kaiserslautern, Germany
Changes in amino acid handling have been observed in a wide-range of human pathologies
including diabetes and cancer. We used Saccharomyces cerevisiae as a model to
investigate how changes in amino acid availability influences cell growth and fitness.
Intriguingly, we observe that increasing the general availability of amino acids relative to the
availability of leucine leads to striking growth defects on glucose containing media.
Surprisingly, the amino acid-dependent growth phenotypes are completely absent when cells
are grown in media containing non-fermentable carbon sources. We found that deletion of
the mitochondrial external NADH dehydrogenase-1 (Nde1) in combination with Cox6 (an
essential component of complex IV) partially rescued amino acid-dependent growth
23
phenotypes. However, deletion of the Nde1 homolog, Nde2, in combination with Cox6 had
the opposite effect, further decreasing growth rate. Furthermore, in all cells grown in
conditions of increased amino acids/normal leucine we observe a 5-fold increase in oxidized
glutathione levels, suggesting significant cross-talk between amino acid metabolism and
homeostasis of cellular redox species. We speculate that specific respiratory chain
components, but not respiratory chain function per se, can play an important role in
‘buffering’ cells against these changes, although the mechanism remains to be determined.
Characterization of the putative vacuolar sugar transporter Aterdl4
A. Khan, P. Klemens, & H.E. Neuhaus Department of Plant Physiology, University of Kaiserslautern, Kaiserslautern, Germany
In plants, the central vacuole is the largest organelle and essential for plant growth. Within a
cell, the vacuole can serve as temporary storage for many metabolites and signaling
compounds. Both the availability of sugars and the accumulation of macro- and
micronutrients ensure proper plant growth. Sugars in plants provide energy for metabolic
processes as well as act as precursors in the synthesis of starch and amino acids. The
movements of sugar to and from the vacuole rely on numerous vacuolar transporters.
Vacuolar sugar transport is mediated by sucrose transporter family, the monosaccharide
transporters (MST) and members of a family called SWEET. Early response to dehydration
(ERD) 6–like1 (ESL1), is a member of the ERD-6-like clade, is a vacuolar protein and a
member of MST family. Other members of this clade are also targeted to the tonoplast. The
current study is on AtERDL4 which we have identified as a vacuolar located sugar carrier,
induced by cold and drought stress. The overexpressor and knockout plants also exhibit
variation in sugar accumulation under different stress conditions.
J-Proteins are involved in the targeting of mitochondrial precursor proteins
J. Laborenz Department of Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany
Cells contain different compartments such as mitochondria, peroxisomes and ER. Almost all
proteins are synthesized in the cytosol and have to be targeted subsequently to their cellular
destination. While the membrane-located translocation machineries of these compartments
were studied extensively in the past, we know only little about factors which help to usher
precursor proteins from the ribosome to surface receptors on their target compartments. To
identify novel cytosolic components involved in the mitochondrial import process we
established a genome-wide screen to select mutants in which mitochondrial precursor
proteins accumulate in the cytosol. Two factors found in this screen were Djp1 and Pex21,
both proteins involved in the import process of peroxisomal proteins. In the absence of Djp1
or Pex21 we observed defects in the biogenesis of mitochondria and of peroxisomes.
Overexpression of the mitochondrial protein Oxa1 in ∆djp1 cells caused a strong
accumulation of Oxa1 precursor in the cytosol. Djp1 is an abundant cytosolic DnaJ protein of
poorly characterized function. We are currently trying to identify the client proteins bound by
Djp1 in order to better understand its role in mitochondrial and peroxisomal biogenesis. Our
results point to a role of the cytosolic chaperone network in coordination of the early step of
intercellular protein targeting.
24
Membrane interactions of conventional and fluorinated surfactants
F. Mahler Department of Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany
Membrane proteins (MPs) play a vital role in all kinds of cells and biological processes.
Therefore, MPs are also important drug targets. However, in general, only little is known
about their structures and mechanisms. This is because MPs are embedded in lipid bilayers
and particularly spectroscopic techniques are seriously limited by the large aggregates thus
formed. One way to overcome this problem is to extract MPs from their native membrane
with the aid of amphiphilic molecules such as detergents. Detergents are able to solubilize
lipid membranes and form mixed lipid–detergent micelles which can accommodate MPs. In
addition to conventional hydrocarbon-based surfactants, fluorinated surfactants have
emerged, which contain partially or fully fluorinated carbon chains. Fluorocarbon groups
show a larger cross-section than hydrocarbon groups; moreover, as fluorocarbon and
hydrocarbon chains are poorly miscible, fluorinated detergents interact less avidly with lipid
bilayers. Hence, fluorinated detergents are less harsh towards native interactions of MPs.
This makes them a promising tool for sustaining MP structure and activity in solution. Here,
we present a biophysical characterization and biological applications to MPs of homologous
series of hydrogenated and fluorinated surfactants. We investigated the thermodynamics of
micellization by isothermal titration calorimetry (ITC) and the size of the micelles by dynamic
light scattering (DLS). With DLS we were also able to show that both surfactant series can
solubilize POPC vesicles, thus displaying detergent-like behavior. For the hydrogenated
surfactants, we could construct pseudophase diagrams to access the thermodynamics of
lipid-membrane solubilization. Further, we investigated the solubilization of MPs from native
Escherichia coli membranes to quantitatively compare the amounts of MPs extracted by the
different surfactants. We found that, contrary to current textbook knowledge, some of the
fluorinated surfactants extract MPs with similar efficiencies as their hydrogenated
counterparts, which opens new avenues for the mild solubilization of labile target proteins.
Chilling lessons: Teaching the sugar beet how to cope with cold
C. Martins Rodrigues Department of Plant Physiology, University of Kaiserslautern, Kaiserslautern, Germany
Sugar beet (Beta vulgaris) is an important industrial crop of temperate climate zones (Europe
and North America) which contributes to nearly 30% of the world´s annual sugar production.
Although a biannual plant species, sugar beet is grown as an annual crop due to its
prominent sensitivity to freezing. A protective mechanism of higher plants against low
temperatures is the accumulation of soluble solutes, especially sugars. An accurate
compartmentalization of sugars into the vacuole, the cellular sugar storage organelle, is
guided by several sugar carriers. To identify ´cold´-associated carriers RNA-seq, proteomic
and metabolic analyses will be performed with total beet extract and isolated vacuoles.
Possible beneficial or unfavourable effects of the candidate proteins for cold tolerance will be
checked by heterologous expression in the model plant Arabidopsis thaliana. Furthermore,
investigations including 14C-metabolit and electrophysiological uptake experiments should
provide insights concerning their transport mechanism. Engineering of sugar transport
activity, e.g. by genome editing of the respective sugar transporter genes may, therefore,
alter the sugar composition of young tap roots. Consequently, an increase of vacuolar sugar
25
in young sugar beet tap roots would result in elevated frost tolerance allowing the growth of
elite lines as biannual crop species.
Chloroplast sugar metabolism and its influence on cellular signaling
K. Patzke, L. Wenner, I. Haferkamp & E. Neuhaus. Department of Plant Physiology, University of Technology, Kaiserslautern, Germany
Carbohydrates represent the main energy source in plants and are produced via
photosynthesis in chloroplasts. These carbohydrates remain as transitory starch in the
chloroplast in order to be remobilized at night, or are directly transported to the cytosol to
serve as precursors for sucrose synthesis. Sucrose is exclusively synthesized in the cytosol,
but accumulates during cold in the plastidial stroma to protect the chloroplasts i.a. against
ROS (reactive oxygen species). Stromal sugars do not only act as protectants but are also
involved in cellular signaling processes. The chloroplast-located Invertase (INVE) (Vargas et
al. 2008) is known for catalyzing the hydrolysis of sucrose to glucose and fructose and data
suggest that it takes part in such signaling processes. A protein with a point-mutation
(C294Y) tend to be more stable and to show an increased catalytic activity (Tamoi et al.
2010). Moreover, exogenously applied sugars, particularly sucrose, result in yellow
cotyledons in the early developmental stage of Arabidopsis thaliana. This gain-of-function
mutant is called sicy-192 (sugar induced cotyledon yellow-192). Sugar feeding associated
molecular and physiological changes in the sicy-192 plants led to the assumption that INVE
participates in inter-organellar communication by retrograde signaling (Tamoi et al. 2010).
We use different approaches to get more insights into the physiological function of INVE and
its possible role in sugar signaling, e.g. transcriptional analyses, metabolic profiling and
EMS-mutagenesis. Our current results support the hypothesis that INVE is involved in
carbon metabolism and signaling. This Project is supported by the DFG (T-SFB 175, The
Green Hub).
Structure and mechanisms of TRAP complex
A. Russo
Department of Medical Biochemistry, Saarland University, Homburg/Saar, Germany
The evolutionary conserved Sec61 complex is a heterotrimeric protein-conducting channel (PCC)
which translocates secretory proteins, post- and co-translationally, across the membrane of the
endoplasmatic reticulum (ER) in eukaryotes. During cotranslational protein translocation, Sec61
complex can be associated with the ribosome (via two cytosolic loops), and with a large number
of accessory proteins or protein complexes (RCC, ribosome-channel complexes). The eukaryotic
TRAP complex comprises 4 subunits: α, β, γ, δ which are always present in mammals. The α, β
and δ subunits comprise single spanning transmembrane domains (TM) with prominent luminal
domains, whilst the γ subunit has four transmembrane domains with prominent cytosolic domain.
TRAPα subunit has a conserved negatively charged N-terminal sequence. Structural analyses by
cryo-electrontomography show contacts between luminal domain of TRAPα/β and Sec61α and
between the cytosolic domain of TRAPγ and the ribosome (Pfeffer et al. BBA 2017). However,
the function(s) of TRAP is/are not fully understood, it/they may include: 1) formation of a seal
between the nascent polypeptide and the Sec61 translocon; 2) interaction with Sec61 leading to
opening of PCC and consequently influencing translocation efficiency; 3) directing chaperones to
26
the nascent chain, Ca2+ binding by the subunit α, assembly/folding of the nascent polypeptide
precursor, or even degradation of the said nascent protein.
We are cloning and purifying the luminal domains of TRAP α, β and δ and the cytosolic domain of
γ as fusion proteins (GST, 6HIS). Then, in order to understand which interactions take place, we
will carry out “peptide arrays” between TRAP α, β and Sec61, and analysis of ribosome binding of
TRAP γ by pull down. Furthermore, we will perform “circular dichroism” analyses in collaboration
with Prof. Sandro Keller to address more precisely the structure of TRAP complex subunits. The
poster will report on progress of the project.
Keywords: ER, protein translocation, TRAP, protein interactions, Ca2+ binding.
The Na+-activated K+ channel slack (Slo2.2) in the mouse auditory system
P. Schepsky1, F. Stephani
1, K. Blum
1, K. Sorg
2, D. Hecker
2 & J. Engel
1
1 Saarland University, Department of Biophysics and CIPMM
2 Saarland University, Department of Otorhinolaryngology
The sodium-activated K+ channel (synonyms: Slack, Slo2.2; gene: Kcnt1) is a Na+- and
voltage-activated K+ channel and closely related to the big conductance, Ca2+-activated K+
channel (BK, Slo1.1). In contrast to BK channels, very little is known about Slack channels so
far. Slack channels are expressed in various central neurons, especially in neurons of the
auditory pathway (Bhattacharjee et al., J Comp Neurol 2002) and in dorsal root ganglion
neurons where they play a role in neuropathic pain (Lu et al., J. Neurosci. 2015). We have
shown protein expression in neonatal inner hair cells (IHCs) and peripheral dendrites of
spiral ganglion neurons. Currently, we are confirming these findings using different anti-Slack
antibodies and Slack knockout mice as a control, which were kindly provided by Dr. Bausch,
Prof. Lukowski and Prof. Ruth, University of Tübingen. We have also started to analyze
Slack protein expression in the auditory brainstem, inferior colliculus and auditory cortex.
Immunolabeling was very prominent in fibers and at somata of auditory neurons and of
cerebellar Purkinje cells.
Hearing measurements at 4 weeks and 12 weeks of age revealed normal auditory brainstem
response (ABR) thresholds and normal distortion product otoacoustic emissions of Slack
knockout mice compared with their wild type littermates, pointing to normal function of inner
and outer hair cells. A detailed analysis of ABR waveforms, highlighting potential aberrations
of specific auditory nuclei, will be performed when sufficient numbers of wild type and
knockout mice will have undergone hearing measurements.
Another on-going project is phenotyping of inner hair cells of Slack knockout mice, including
quantitative analysis of ribbon synapses with presynaptic Ca2+ channels, postsynaptic PSD-
95 clusters and ribbons.
TBA
M. Schöppe Department of Biophysics, Saarland University, Homburg/Saar, Germany
27
hSnd2: One component of a new targeting pathway to the human ER
M. Sicking Department of Medical Biochemistry, Saarland University, Homburg/Saar, Germany
For one third of synthesized polypeptides in eukaryotic cells protein translocation into the
endoplasmic reticulum is a basic step in their biogenesis. To allow for efficient translocation,
it appears, cells established different targeting mechanisms that vary in substrate recognition
and the mode of transport. In 2016 a new pathway to the endoplasmic reticulum was
described in S. cerevisiae (Aviram et al. 2016). This SND targeting route (SRP independent)
is described as an alternative/ back-up pathway to the previously characterized SRP and
GET targeting routes in yeast. Interestingly, all three protein targeting pathways seem to be
constituted of three core proteins including a cytosolic mobile mediator and a heterodimeric
ER-membrane located receptor. For the SND pathway the three identified core proteins were
termed Snd1 (mediator) and Snd2/Snd3 (heterodimeric receptor).
Our recent efforts show, that the human ortholog to the Snd2 protein, hSnd2, shows genetic
interaction with components of established targeting pathways such as SRP and that hSnd2
is a component of a previously unknown targeting machinery for proteins into the human ER.
Furthermore, we identified some proteins as potential interaction partners of the hSnd2
protein, which leads to suppositions about hSnd3, localization, and structural composition of
the membrane located receptor of this novel targeting pathway called SND.
In summary, our data demonstrate that hSnd2 is the first component of a human targeting
mechanism which corresponds to the novel yeast SND Pathway (Haßdenteufel et al. under revision).
Analyses of sucrose compartmentation in Arabidopsis thaliana
D.P. Vu, E. Neuhaus Department of Plant Physiology, University of Kaiserslautern, Kaiserslautern, Germany.
Sugars fulfill plenty functions in plants. They are e. g. involved as signal molecules in stress
response, they regulate photosynthesis and moreover sugars modulate plant development.
To achieve these functions, cellular sugar homeostasis must be regulated, inter alia via
membrane transporters. Among these sugar transporters are the Tonoplast Sugar
Transporter (TST), which locates to the vacuolar membrane.
The present study examines the impact of an altered sugar homeostasis in Arabidopsis
thaliana by increasing the vacuolar sucrose concentration. To accumulate a higher vacuolar
sucrose level in Arabidopsis, the sucrose importer BvTST2.1 from the sugar beet Beta
vulgaris was heterologously overexpressed.
However, an increase of the vacuolar sucrose concentration in Arabidopsis by BvTST2.1
overexpressors compared to the control could surprisingly not be observed, while higher
glucose and fructose contents were detected. These results imply that in mutants the
vacuolar invertase converts sucrose into its monomers glucose and fructose.
To study the impact of an increased vacuolar sucrose concentration on Arabidopsis
development and stress resistance, the aim is thus to create further mutants with a knock
down of the vacuolar invertase (so that the sucrose conversion might be decreased). The
alternative option for achieving an accumulation of sucrose in the vacuole could be an
overexpression of an invertase inhibitor protein, which might prevent the cleavage of
sucrose.
28
List of Participants
29
Alexander, Todd Department of Pediatrics
Division of Nephrology & Physiology 2B2.42 Walter Mackenzie Centre University of Alberta Edmonton, Alberta, Canada, T6G 2R7 [email protected]
Amoroso, Gabriele Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 67663 Kaiserslautern Germany [email protected]
Becker, Holger Institut für Physiologische Chemie Stiftung Tierärztliche Hochschule Hannover Bünteweg 17, Gebäude 218 30559 Hannover Germany [email protected]
Bentrcia, Teqiyya Department of Experimental & Clinical Pharmacology & Toxicology Faculty of Medicine Saarland University University Hospital Building 46 66421 Homburg Germany [email protected]
Blum, Andrea Department of Molecular & Cell Biology Faculty of Natural Sciences & Technology III Saarland University Campus Saarbrücken, Building A 1.5 P.O. Box 151150 66041 Saarbrücken Germany [email protected]
Bugaeva, Wassilina Department of Plantbiology Faculty of Natural Sciences & Technology Saarland University University Hospital, Building A 2.4 66421 Homburg /Saar Germany [email protected]
Cai, Ruiqi Department of Physiology Faculty of Medicine & Dentistry University of Alberta 7-29A Medical Sciences Building Edmonton, Alberta, Canada, T6G 2H7 [email protected]
Casey, Joe Department of Biochemistry Faculty of Medicine & Dentistry University of Alberta 4020E Katz Group Rexall Building Edmonton, Alberta, Canada, T6G 2E1 [email protected]
Chen, Xing-Zhen Department of Physiology Faculty of Medicine & Dentistry University of Alberta 7-29A Medical Sciences Building Edmonton, Alberta, Canada, T6G 2H7 [email protected]
30
Chitirala, Praneeth Department of Physiology Faculty of Medicine Saarland University CIPMM, Building 48 66421 Homburg / Saar Germany [email protected]
Cordat, Emmanuelle Department of Physiology School of Moleclar & Systems Medicine Medical Sciences Building/Room 7-34 University of Alberta Edmonton, Alberta, Canada, T6G 2H7 [email protected]
Danielczak, Bartholomäus Department of Molecular Biophysics Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
Engel, Jutta Department of Biophysics Faculty of Medicine Saarland University CIPMM , Building 48 66421 Homburg/Saar Germany [email protected]
Feik, Samira (Babysitter)
Germany [email protected]
Fliegel, Larry Department of Biochemistry Faculty of Medicine & Dentistry University of Alberta 347 Medical Sciences Building Edmonton, Alberta, Canada, T6G 2H7 [email protected]
Flockerzi, Veit Department of Experimental & Clinical Pharmacology & Toxicology Faculty of Medicine Saarland University University Hospital, Building 46 66424 Homburg/Saar Germany [email protected]
Friauf, Eckhard Department of Animal Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
Grethen, Anne Department of Molecular Biophysics Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
31
Groh, Carina Department of Cellular Biology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
Haßdenteufel, Sarah Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University University Hospital, Building 44 66424 Homburg Germany [email protected]
Herrmann, Johannes Department of Cellular Biology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
Hickl, Daniel Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Hofmann, Laura Department of Experimental & Clinical Pharmacology & Toxicology Faculty of Medicine Saarland University University Hospital, Building 46 66424 Homburg/Saar Germany [email protected]
Illaydal, Güneri Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany
John, Annalisa Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Keller, Sandro Department of Molecular Biophysics Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
32
Khaing, Swai Department of Biochemistry
School of Translational Medicine Faculty of Medicine & Dentistry University of Alberta 4-020H Katz Group-Rexall Centre for Pharmacy & Health Research Edmonton, Alberta, Canada T6G 2E1 [email protected]
Khan, Azkia Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Khandpur, Gurleen Kaur Department of Cellular Biochemistry Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
Klostermann, Viola Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University Building 45.2 D-66421 Homburg/Saar Germany [email protected]
Krämer, Lena Department of Cellular Biology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
Kumari, Alka Department of Biochemistry Faculty of Medicine & Dentistry University of Alberta 4020E Katz Group Rexall Building Edmonton, Alberta, Canada, T6G 2E1 [email protected]
Laborenz, Janina Department of Cellular Biology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
Lang, Sven Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University University Hospital, Building 44 66424 Homburg Germany [email protected]
33
Lashhab, Rawad Department of Physiology
School of Moleclar & Systems Medicine Medical Sciences Building/Room 7-34 University of Alberta Edmonton, Alberta, Canada, T6G 2H7 [email protected]
Li, Xiaobing Department of Molecular & Cell Biology Faculty of Natural Sciences & Technology III Saarland University Campus Saarbrücken, Building A 1.5 P.O. Box 151150 66041 Saarbrücken Germany [email protected]
Lysyk, Laine Department of Biochemistry School of Translational Medicine Faculty of Medicine & Dentistry University of Alberta 451 Medical Sciences Building Edmonton, Alberta, Canada T6G 2H7 [email protected]
Mahler, Florian Department of Cellular Biology Falculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 6763 Kaiserslautern Germany [email protected]
Martins Rodrigues, Cristina Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Marz, Sabrina Department of Animal Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
Miederer, Anna-Maria Department of Biophysics Faculty of Medicine Saarland University University Hospital, Building 58 66424 Homburg Germany [email protected]
Möhlmann, Torsten Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
34
Morgan, Bruce Department of Cellular Biology
Falculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 6763 Kaiserslautern Germany [email protected]
Neuhaus, Ekkehard Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Niemeyer, Barbara Department of Biophysics Faculty of Medicine Saarland University University Hospital, Building 58 66424 Homburg Germany [email protected]
Oesterreicher, Julian Department of Cellular Biology Falculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 6763 Kaiserslautern Germany [email protected]
Ohler, Lisa Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Patzke, Kathrin Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Prananingrum, Pratiwi Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Philippar, Katrin Department of Plant Biology Faculty of Medicine Saarland University Building A 2.4, Room 3.03 66421 Homburg / Saar Germany [email protected]
35
Riemer, Jan Institute for Biochemistry
University of Cologne Zuelpicher Str. 47a/R. 3.49 50674 Cologne Germany [email protected]
Russo, Antonietta Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University University Hospital, Building 44 66424 Homburg Germany [email protected]
Sarder, Hasib Department of Molecular & Cell Biology Faculty of Natural Sciences & Technology III Saarland University Campus Saarbrücken, Building A 1.5 P.O. Box 151150 66041 Saarbrücken Germany [email protected]
Schepsky, Pauline Department of Biophysics Faculty of Medicine Saarland University CIPMM , Building 48 66421 Homburg/Saar Germany [email protected]
Schmitt, Manfred Department of Molecular & Cell Biology Faculty of Natural Sciences & Technology III Saarland University Campus Saarbrücken, Building A 1.5 P.O. Box 151150 66041 Saarbrücken Germany [email protected]
Schöppe, Mona Department of Biophysics Faculty of Medicine Saarland University University Hospital, Building 58 66424 Homburg Germany [email protected]
Sicking, Mark Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University Building 45.2 D-66421 Homburg/Saar Germany [email protected]
Stutz, Regine Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University Building 45.2 D-66421 Homburg/Saar Germany [email protected]
36
Touret, Nicolas Department of Biochemistry
School of Translational Medicine Faculty of Medicine & Dentistry University of Alberta 4-020H Katz Group-Rexall Centre for Pharmacy & Health Research Edmonton, Alberta, Canada T6G 2E1 [email protected]
Ullah, Shahid Department of Physiology School of Moleclar & Systems Medicine Medical Sciences Building/Room 7-34 University of Alberta Edmonton, Alberta, Canada, T6G 2H7 [email protected]
Van der Laan, Martin Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University Building 45.2 D-66421 Homburg/Saar Germany [email protected]
Vu, Duc Phuong Department of Plant Physiology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 22 67663 Kaiserslautern Germany [email protected]
Yadao, Nilam Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University Building 45.2 D-66421 Homburg/Saar Germany [email protected]
Zimmermann, Richard Department of Medical Biochemistry & Molecular Biology Faculty of Medicine Saarland University University Hospital, Building 44 66424 Homburg Germany [email protected]
Zöller, Eva Department of Cellular Biology Faculty of Biology University of Kaiserslautern Erwin-Schrödinger-Straße 13 67663 Kaiserslautern Germany [email protected]
37
Location Kurhaus Trifels Seminarhotel GmbH Kurhausstraße 25 76855 Annweiler Tel: 06346 - 30 88 60 Fax: 06346 - 30 88 633 E-Mail: [email protected] Website: http://www.kurhaus-trifels.de/ Conference phone 0151 18234824 Taxi-Service 06346 8133 (Taxi Zeller) 06346 8133 (Taxi Götz)
Venue by train:
Please use www.deutschebahn.com for suitable train connections from your origin to the station “Annweiler am Trifels”. From there, you may either walk to the meeting location (about 30 min) or take a bus to “Bindersbach Ortsmitte” (from there it takes a 5 min walk to the Kurhaus Trifels).
Venue by car (google maps):
from Kaiserslautern from Frankfurt
from Saarbrücken/Homburg
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