international research training group · 7:00 pm-9:00 pm poster session and cash bar max bell rooms...

INTERNATIONAL RESEARCH TRAINING GROUP

7th Joint SymposiumBanff Center

Banff, Alberta, CanadaApril 8th to 10th, 2018

GCCANADIANGERMAN

COMPLEX MEMBRANE PROTEINSIN CELLULAR DEVELOPMENT AND DISEASE

PROGRAM INDEXpage

INTERNATIONAL RESEARCH TRAINING GROUP

GCCANADIANGERMAN

COMPLEX MEMBRANE PROTEINSIN CELLULAR DEVELOPMENT AND DISEASE

7th Joint SymposiumBanff Center

Banff, Alberta, CanadaApril 8th to 10th, 2018

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Quick Schedule

Maps & Information

Program

Guidance Committee

Speaker Abstracts

Poster Presentations

Poster Abstracts

Contacts

Notes

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Monday April 9th Tuesday April 10th Wednesday April 11thSunday April 8th

Banff Centre

Max Bell Central Foyer

Hotel Check-inProfessional Develp-

ment Building

Meeting Registration

Dinner

page 1

107, Tunnel Mountain DriveBanff, Alberta1.403.762.6100 or1.800.422.2633www.banffcentre.ca

for any question contact Rohit Rothore: 780-886-7179 or [email protected]

Dinner

Vistas Dining Room

Dinner

TraineeDinner

Trainee

socialevent

PIDinner

Vistas Dining Room

Breakfast

Vistas Dining

Cash Bar

Max Bell BuildingElder Tom Crane Bear Room

Max Bell Auditorium

Session 1

Max Bell Auditorium

Session 2

Coffee Break

Brown Bag Lunch

Breakfast

Vistas Dining RoomBreakfast

Vistas Dining Room

Max Bell Auditorium

Session 3

CSMBsatellite meeting

CSMBMeeting

Max Bell Auditorium

Max Bell BuildingSession 4

Max Bell Auditorium

Session 5

Coffee Break

Lunch

Hike - Banff Visitmeet oustide Profes-sional Development

Centre@12:20

meet oustide

Professional

Development Centre

@ 5 pm

Poster Session

all the posters can be set at once for the 2 days

Max Bell-252Max Bell-253

Cash Bar

Poster Session

Cash Bar

Guidance Committee Isee page 11

Guidance Committee IIsee page 11

Elder Tom Crane Bear

Elder Tom Crane Bear

PosterSession

Cash Bar

please take down

poster at session end

Vistas Dining Room

Lunch

Vistas Dining Room

Vistas Dining

Meeting RegistrationMax Bell Foyer

Max Bell-252Max Bell-253

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page 4

The Max Bell Building

MB154

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Washrooms Washrooms

StairsElev.

pillar

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MB 251MB 253

Stairs

Walkway to Corbett Hall

Elev.Auditorium

Husky Energy Foyer

Entrance

Entrance

The Elder Tom Crane BearRoom

Balcony

Audio Visual

Max Bell Building Lower Floor

Max Bell Building Main Floor

POSTERS

POSTERS

POSTERS

Plenary Sessions

page 5

IRTG Meeting Schedule Monday April 9th page 6

MEETING SCHEDULE

IRTG BANFF SYMPOSIUM – THE BANFF CENTRE

APRIL 8-10, 2018

Sunday 8 April

9:00 am Travel to Banff

3:00 pm Hotel Check-in and Registration

3:00 pm-6:00 pm IRTG Meeting Registration Desk Max Bell Central Foyer

5:30 pm-7:30 pm Dinner Vistas Dining Room

7:00 pm-9:00 pm Mixer with Cash Bar Elder Tom Crane Bear Room (MB Lounge)

Monday April 9

7:00 am-8:00 am Breakfast Vistas Dining Room

8:15 am-10:00 am Session 1 Membrane Proteins in Adaptation- I

Chair: Ruiqi Cai Max Bell Auditorium

8:15 am-8:30 am Introductory Remarks: Joe Casey

8:30 am-9:00 am Larry Fliegel, “Structural and Functional Characterization of Different

Families of Na+/H+ Exchangers”

9:00 am-9:30 am Eva Zöller (Ph.D. student, Johannes Herrmann’s Lab), “Cuz1- a possible

new player in dealing with cytosolic stress due to inhibition of mitochondrial

protein import”

9:30 am-10:00 am Cristina Martins Rodrigues (Ph.D. student, Ekkehard Neuhaus’ Lab),

“Chilling Lessons: Teaching the sugar beet how to cope with cold”

10:00 am-10:30 am Coffee Break Max Bell Foyer


10:30 am-12:00 pm Session 2 Membrane Proteins in Adaptation- II

Chair: Hasib Sarder Max Bell Auditorium

10:30 am-11:00 am Wassilina Bugaeva (Ph.D. student, Katrin Philippar’s Lab), “Physiological

analysis of plastid fatty acid export proteins”

11:00 am-11:30 am Gurleen Kaur Khandpur (Ph.D. student, Bruce Morgan’s Lab), “Respiratory

chain components regulate cell growth in response to changing amino acid

availability”

11:30 am-12:00 pm Julian Oestreicher (Ph.D. student, Bruce Morgan’s Lab), “Identification and

characterization of a novel intracellular glutathione transporter”

12:00 pm Pick up Brown Bag Lunches outside Max Bell

12:20-3:30 Meet outside Professional Development Centre for Hike or walk into

Banff

3:30 pm-5:00 pm Poster Session Max Bell Rooms 252, 253

3:30 pm-5:00 pm Cash Bar Max Bell Central Foyer

6:00 pm-7:00 pm Dinner Main Dining Room

7:00 pm-8:00 pm

(7:00 – 7:20 PM)

(7:20 – 7:40 PM)

(7:40 – 8:00 PM)

Supervisory Committee Meetings- Session 1 Max Bell Lounge Room PhD student Main Supervisor Co-Supervisor (D) Co-Supervisor (C) Andrea Blum Manfred Schmitt Jens Rettig Joe Casey

Kathrin Patzke Ekkehard Neuhaus Torsten Mohlmann -- Gurleen Khandpur Bruce Morgan Martin van der Laan Nicolas Touret

Anne Konnel Katrin Philippar Johannes Herrmann Joanne Lemieux PhD student Main Supervisor Co-Supervisor (D) Co-Supervisor (C) Sabrina Marz Eckhard Friauf Viet Flockerzi Todd Alexander

Antonietta Russo Sven Lang Richard Zimmerman Xing-Zhen Chen Hasib Sarder Manfred Schmitt Ekkehard Neuhaus --

Florian Wollweber Martin van der Laan Markus Hoth Joe Casey

PhD student Main Supervisor Co-Supervisor (D) Co-Supervisor (C)

Eva Zoller Johannes Hermann Martin van der Laan Todd Alexander

Regine Stutz Richard Zimmerman Barbara Niemer -- Daniel Hickl Torsten Mohlmann Sandro Keller Joanne Lemieux

Wassilina Bugaeva Katrin Philippar Ekkehard Neuhaus Joe Casey

7:00 pm-9:00 pm Poster Session and Cash Bar Max Bell Rooms 252, 253



8:00 pm-9:00 pm

(8:00 – 8:20 PM)

(8:20 – 8:40 PM)

(8:40 – 9:00 PM)

Supervisory Committee - Session 2 Max Bell Lounge Room PhD student Main Supervisor Co-Supervisor (D) Co-Supervisor (C)

Janina Laborenz Johannes Hermann Sven Lang Nicolas Touret Duc Phuong Vu Ekkehard Neuhaus Torsten Mohlmann Joanne Lemieux

Pauline Schepsky Jutta Engel Viet Flockerzi - Xiaobing Li Manfred Schmitt Bruce Morgan -

PhD student Main Supervisor Co-Supervisor (D) Co-Supervisor (C) Lisa Ohler Torsten Mohlmann Ekkehard Neuhaus Joanne Lemieux

Anne Grethen Sandro Keller Martin van der Laan Howard Young Julian Oestreicher Bruce Morgan - -

Mark Sicking Sven Lang - - PhD student Main Supervisor Co-Supervisor (D) Co-Supervisor (C)

Cristina Martins-Rodrigues

Ekkehard Neuhaus Katrin Philippar Xing-Zhen Chen

Nilam Yadao Martin van der Laan Johannes Herrmann Joanne Lemieux Mona Schoppe Barbara Niemer Sven lang Larry Fliegel Bartholomaus

Danielczak Sandro Keller Torsten Mohlmann Howard Young

IRTG Meeting Schedule Tuesday April 10h page 9

Tuesday April 10

7:00 am-8:15 am Breakfast Vistas Dining Room

8:30 am– 10:00 am Session 3 – What’s up with the SLC4 Family?

Chair: Andrea Blum Max Bell Auditorium

8:30 am-9:00 am Katherine Badior (Ph.D. Student, Joe Casey’s Lab), “Band 3

conformational Dynamics in Red Blood Cell Senescence”

9:00 am-9:30 am Rawad Lashab (Ph.D. student, Emmanuelle Cordat’s lab), “The basolateral

kidney anion exchanger 1 regulates tight junction integrity by interacting

with Claudin 4”

9:30 am-10:00 am Darpan Malhotra, (Ph.D. student, Joe Casey’s Lab), “Cell adhesion role of

SLC4A11 in endothelial corneal dystrophy pathology and therapeutics”

10:00 am-10:30 am Coffee Break Max Bell Foyer

10:30 am-12:30 pm Session 4 – Membrane Transport

Chair: Nada Alshumaimeri Max Bell Auditorium

10:30 am-11:00 am Lisa Ohler (Ph.D. student, Torsten Möhlmann’s Lab), “Pyrimidine salvage

and related transport processes across the chloroplast envelope”

11:00 am-11:30 am Duc Phuong Vu (Ph.D. student, Ekkehard Neuahus’Lab), “Sugar homeostasis in higher plants. Transporters and enzymes”

11:30 am-12:00 pm Sabrina Marz (Ph.D. student, Eckhard Friauf’s Lab), “Glycine transporter 2

surface abundance is reduced by the calcium-activated protein for secretion

1”

12:00 pm-12:30 pm Anne Könnel (Ph.D. student, Katrin Philippar’s Lab), “Molecular analysis

of fatty acid transporters in plant cells”

12:30 pm-1:30 pm Lunch Vistas Dining Room

IRTG Meeting Schedule Tuesday April 10h page 10

1:30 pm-3:30 pm Session 5 – Calcium Homeostasis

Chair: Allen Plain Max Bell Auditorium

1:30 pm-2:00 pm Justin Lee, (Ph.D. student, Todd Alexander’s Lab), “Novel functional role

of intestinal calcium sensing receptor in regulation of calcium homeostasis”

2:00 pm-2:30 pm Mona Schoppe (Ph.D. student, Barbara Niemeyer’s Lab), “Characterization

of a novel splice variant of the stromal interaction molecular 1 (STIM1)”

2:30 pm-3:00 pm Gareth Armanious (Ph.D. student, Howard Young’s Lab), “Newly

identified human phospholamban mutations display similarities with the

disease-causing fingerprint”

3:00 pm-3:15 pm Concluding Remarks – Joe Casey & Ekkehard Neuhaus

3:30 pm-5:00 pm Poster Session Max Bell Rooms 252, 253

Please take down posters at session end.


5:00 pm-6:30 pm Trainee Dinner Vistas Dining Room

5:00 pm-??? Principal Investigator Group Dinner- Meet outside Professional

Development Centre for travel

6:30 pm- to

breakfast

Trainees Visit Banff Pubs- Meet outside Professional Development

Centre to walk together into Banff

Wednesday April 11

7:00 am-9:30am Breakfast Vistas Dining Room

Satellite Meetings of the CSMB Conference Max Bell Room 152

11:30am-1:30pm Lunch Vistas Dining Room

GUIDANCE COMMITTEES page 11

GUIDANCE COMMITTEES Monday | 7:00 pm-8:00 pm

Supervisory Committee Meetings- Session 1 Max Bell Lounge Room PhD student Main Supervisor Co-Supervisor (D) Co-Supervisor (C)

7:00- 7:20

Andrea Blum Manfred Schmitt Jens Rettig Joe Casey Kathrin Patzke Ekkehard Neuhaus Torsten Mohlmann --

Gurleen Khandpur Bruce Morgan Martin van der Laan Nicolas Touret Anne Konnel Katrin Philippar Johannes Herrmann Joanne Lemieux


7:20- 7:40

Sabrina Marz Eckhard Friauf Viet Flockerzi Todd Alexander Antonietta Russo Sven Lang Richard Zimmerman Xing-Zhen Chen

Hasib Sarder Manfred Schmitt Ekkehard Neuhaus --

Florian Wollweber Martin van der Laan Markus Hoth Joe Casey


7:40- 8:00

Eva Zoller Johannes Hermann Martin van der Laan Todd Alexander

Regine Stutz Richard Zimmerman Barbara Niemer -- Daniel Hickl Torsten Mohlmann Sandro Keller Joanne Lemieux

Wassilina Bugaeva Katrin Philippar Ekkehard Neuhaus Joe Casey Monday | 8:00 pm – 9:00 pmSupervisory Committee - Session 2 Max Bell Lounge Room


8:00- 8:20

Janina Laborenz Johannes Hermann Sven Lang Nicolas Touret Duc Phuong Vu Ekkehard Neuhaus Torsten Mohlmann Joanne Lemieux

Pauline Schepsky Jutta Engel Viet Flockerzi - Xiaobing Li Manfred Schmitt Bruce Morgan -


8:20- 8:40

Lisa Ohler Torsten Mohlmann Ekkehard Neuhaus Joanne Lemieux Anne Grethen Sandro Keller Martin van der Laan Howard Young

Julian Oestreicher Bruce Morgan - - Mark Sicking Sven Lang - -


8:40- 9:00

Cristina Martins-Rodrigues

Ekkehard Neuhaus Katrin Philippar Xing-Zhen Chen

Nilam Yadao Martin van der Laan Johannes Herrmann Joanne Lemieux Mona Schoppe Barbara Niemer Sven lang Larry Fliegel Bartholomaus

Danielczak Sandro Keller Torsten Mohlmann Howard Young

SPEAKER ABSTRACTS page 12

SPEAKER ABSTRACTS MONDAY, APRIL 9 Session 1 8:30 am – 9:00 am

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF DIFFERENT FAMILIES OF

Na+/H+ EXCHANGERS

Larry Fliegel, Debajyoti Dutta, Kyungsoo Shin and Jan K. Rainey. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH ubiquitously in mammalian cells. It functions to regulate intracellular pH by exchanging one external Na+ for an internal proton. The protein is involved in myocardial ischemia/reperfusion damage and is a trigger for metastatic behaviour in triple negative breast cancer. Presently the structure of four bacterial plasma membrane antiporters are known, E. coli, NhaA, NapA of Thermus thermophilus, MjNhaP1 of Methanocaldoccocus jannaschii and PaNhaP of Pyrococcus abyssi. EcNhaA contains 12 transmembrane helices per protomer with both N- and C-terminal cytoplasmic tails. It has two distinct subdomains: a dimerization or scaffolding subdomain and a cylindrical or 6-helix bundle transport subdomain. The structures of three other plasma membrane Na+/H+ antiporters PaNhaP, TthNapA, and MjNhaP1 show diversity from EcNhaA. These structures contain 13 transmembrane helices with the extra transmembrane helix at the N-terminal believed to act as an uncleaved signal sequence that also affects allosteric regulation of antiporter activity. For TthNapA it was shown that the topology is N-terminal out and C-terminal in. The structures have led to the development of the idea that Na+/H+ antiporters have a novel fold (NhaA fold) or assembly, consisting of two pairs of short helices that are connected by crossed, extended non-helical chains. The structures of fragments of the human NHE1 membrane domain have been examined by nuclear magnetic resonance. Several transmembrane segments also have a general structure of an extended central region flanked by helical segments. The extended regions in the middle of the helix contain amino acids that are important in protein function and possibly in cation coordination and transport. The structure of TM XI of the Na+/H+ exchanger of S. pombe was recently deduced. Structural analysis suggested that it had a helical propensity over amino acids 360-368, an extended region from 369-378 and was helical over amino acids 379-386. TM XI was sensitive to side chain alterations. The results suggest that different members of the Na+/H+ exchanger family have a similar helix-extended region-helix conformation, though the general overall structure of the protein varies within families somewhat. Supported by CIHR and NSERC.

9:00 am – 9:30 am

CUZ1 – A POSSIBLE NEW PLAYER IN DEALING WITH CYTOSOLIC STRESS DUE TO

INHIBITION OF MITOCHONDRIAL PROTEIN IMPORT

Eva Zöller, Felix Boos, Clara Stiefel, Johannes M. Herrmann, Technical University Kaiserslautern Yeast cells synthesize about 99% of their mitochondrial proteins by cytosolic ribosomes, which then get imported into the mitochondria through the TOM- and TIM-23-complex. Various severe diseases are


known, which show defects in the mitochondrial import machinery, which at the end leads to the development of neurodegenerative effects. We use yeast as an experimental system to study the molecular mechanism of underlying signal pathways of mitochondrial stress responses. We engineered a „clogger“-protein that allows to inhibit mitochondrial protein import without interfering with the functionality of mitochondrial proteins. Inhibiting the mitochondrial import leads to the accumulation of precursor proteins in the cytosol, called mitochondrial precursor overaccumulation stress (mPOS). The induction of this clogger construct also leads to a severe growth phenotype. Additionally, we found by using RNA-Seq analysis that in case of clogger expression in a wild type yeast strain, chaperones and various proteasome components are significantly upregulated. So far, we do not know how the signaling from the mitochondria towards the nucleus looks like and how it is regulated. Experiments on RNA (qRT-PCR) and protein level (western blot) could successfully reproduce the RNA-Seq data. Additionally, the RNA-Seq analysis showed a significant upregulation of Cuz1, a novel cytosolic zink-finger protein, which exhibits an ubiquitin-like domain and interacts with Cdc48. After conducting first experiments, we suppose this protein plays a role in removing non-productive import intermediates or even the clogger constructs itself. Due to the lack of knowledge about the exact role of the protein, different experiments are planned to further investigate this topic.

9:30 am – 10:00 am

CHILLING LESSONS: TEACHING THE SUGAR BEET HOW TO COPE WITH COLD

Cristina Martins-Rodrigues, Technical University Kaiserslautern In temperate climate zones (Europe and North America) sugar beet (Beta vulgaris) is the exclusive source of sugar (sucrose) for the food industry and a source for bio-energy generation. Sugar beet tap roots accumulate sucrose to as much as 20% of their fresh weight at maturity. Although a bi-annual plant species, sugar beet is grown as an annual crop in temperate climate zones due to its prominent sensitivity to freezing. This project aims to improve the frost tolerance of frost-sensitive young tap roots in elite sugar beet lines, thus allowing their growth as a bi-annual crop species and improving the tap root sugar yield per hectare. To understand sugar beet cold response and to identify key genes/proteins critical for achieving frost tolerance in the tap root tissue, we have conducted comparative proteomic, RNAseq, and metabolic analyses of sugar beet genotypes with contrasting cold sensitivities. Temperature- and genotype-dependent regulated factors involved in subcellular sugar homeostasis and transport are in the focus of in-depth characterization. Accumulation of sugars is critical for frost tolerance as sugars do not only act as solutes (thereby lowering the freezing temperature of a cell), but also protect membranes and proteins during the onset of freezing temperatures.

Session 2 10:30 am – 11:00 am

PHYSIOLOGICAL ANALYSIS OF PLASTID FATTY ACIDS EXPORT PROTEINS

Wassilina Bugaeva, Anne Könnel and Katrin Philippar Molecular Plant Biology, Center for Human- and Molecular Biology, Saarland University Saarbrücken, Germany In plants, fatty acids (FAs) are synthesized in the plastid stroma and become available for lipid assembly mainly in the form of long-chain FAs (C16–18). Some of these FAs are integrated into lipids inside plastids (prokaryotic pathway), but the majority is exported to the ER for further elongation, acyl editing,


and lipid assembly (eukaryotic pathway). The identification of FAX, a novel membrane fatty acid export protein in the inner envelope membrane of chloroplasts (Li et al. 2015), significantly contributes to the understanding of the FA transport mechanism and the importance of FAX1 in plant development, biomass formation and fertility. Fax1 knockout mutants show a strong defect in the outer pollen cell wall most likely due to an impaired assembly of lipid derived components. This defect results in male sterility, which makes it impossible to breed homozygous knockout mutants. In Arabidopsis thaliana, 7 proteins belong to the FAX family and besides FAX1 also FAX2 and FAX3 are shown to be integrated into the inner envelope of chloroplasts. FAX2 might be associated in a protein complex playing an important role in lipid remodeling between mitochondria and chloroplasts during phosphate starvation. FAX3 is assumed to partly complement FAX1 function due to transcript levels in fax1 knockouts and segregation analysis of fax1/fax3 double mutant lines. Our current research focuses on the characterization of fax2/fax3 double mutant lines as well as respective RNAi lines and a functional analysis in heterologous yeast cells. Publication Li et al. (2015) PLoS Biology. 13(2): e1002053

11:00 am – 11:30 am

RESPIRATORY CHAIN COMPONENTS REGULATE CELL GROWTH IN RESPONSE TO

CHANGING AMINO ACID AVAILABILITY

Gurleen Kaur Khandpur, Martin Van der Laan, Nicolas Touret and Bruce Morgan Department of Cellular Biochemistry, University of Kaiserslautern, Germany Faculty of Medicine Medical Biochemistry and Molecular Biology, University of Saarland, Germany Department of Biochemistry, University of Alberta, Canada 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. We also observed that when cells grown in conditions of increased amino acids/normal leucine, exhibited a 6-fold increase in oxidized glutathione levels. Furthermore, either establishment of the functional leucine biosynthetic pathway or provision of extra leucine in the growth media, rescued these oxidized glutathione levels suggesting significant cross-talk between amino acid metabolism and homeostasis of cellular redox species. Surprisingly, the amino acid-dependent growth phenotypes are completely absent when cells 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 phenotypes. However, deletion of the Nde1 homolog, Nde2, in combination with Cox6 had the opposite effect, further decreasing growth rate. We speculate that specific respiratory chain components, but not the respiratory chain function per se, can play an important role in ‘buffering’ cells against these changes, although the mechanism remains to be determined. Supported by IRTG1830.


11:30 am – 12:00 pm

IDENTIFICATION AND CHARATERIZATION OF A NOVEL INTRACELLULAR

GLUTATHIONE TRANSPORTER

Julian Oestreicher, Bruce Morgan. Department of Cellular Biochemistry, Technical University Kaiserslautern Glutathione fulfils multiple roles in the cell, including acting as an important redox co-factor and playing an essential role in iron-sulphur 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.

Session 3

8:30 am – 9:00 am BAND 3 CONFORMATIONAL DYNAMICS IN RED BLOOD CELL SENESCENCE Katherine Badior and Joe Casey. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7 Essential to respiration, red blood cells (RBCs) transport oxygen and carbon dioxide throughout the body. During their circulating lifetime of 120 days, RBCs are exposed to extreme physical and chemical stresses. As oxidative and physical damages accumulate, aged RBCs transport function becomes less efficient, and they must be removed from circulation. Circulating auto-antibodies (IgG) are highly enriched on membranes of senescent RBCs, and are necessary for recognition by macrophages for phagocytosis and removal. Senescent cell IgG is specific for Band 3, a membrane protein in the SLC4 family of anion transporters. We propose that rare conformational events in Band 3 lead to the formation of the senescence antigen and IgG binding, marking RBCs for clearance. The Band 3 senescence epitope is formed by residues 538-553 and 812-827. Decades of Band 3 research controversially supports both extracellular and intracellular localization of residues 812-827. Recent advances in membrane protein structural biology led to the crystal structure of Band 3 membrane domain, which places residues 812-827 in intracellular loop 6 (ICL6), inaccessible to sera auto-antibodies. We propose a consolidated mechanism for Band 3 as a molecular clock for RBC senescence. We present a dynamic model of Band 3, where residues 812-827 can access both the intracellular and extracellular environments of RBCs. In order to assess the localization of Band 3 ICL6, substituted cysteine accessibility assays were performed. Band 3 cysteine point mutants were tested for their reactivity with membrane-impermeant LYIA, to assess extracellular accessibility. Time course assays of accessibility to LYIA were also performed. In addition, accessibility of ICL6 was analyzed using immunofluorescence, in both RBCs and transiently


transfected HEK293 cells, using an antibody raised against ICL6. Supported by Canadian Institutes of Health Research

9:00 am – 9:30 am

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 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 �-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. Immuno precipitations confirmed the physical interaction. BCECF-based functional assays assessing AE1 activity did not demonstrate alterations when Cldn-4 was over-expressed. However, Using chamber experiments revealed a decrease in trans epithelial 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.

9:30 am – 10:00 am

CELL ADHESION ROLE OF SLC4A11 IN ENDOTHELIAL CORNEAL DYSTROPHY PATHOLOGY AND THERAPEUTICS Darpan Malhotra1, Martin Jung2, Claudia Fecher-Trost3, Sergei Noskov4, Richard Zimmermann2 and Joseph R. Casey1* 1Department of Biochemistry, Membrane Protein Disease Research Group, University of Alberta. 2Department of Medical Biochemistry and Molecular Biology, Saarland University. 3Department of Experimental Pharmacology, Saarland University.

4Centre for Molecular Simulations, Department of Biological Sciences, University of Calgary.

Endothelial Corneal Dystrophies (ECD) are the most common cause of corneal blindness worldwide with complex genetic etiology and unclear pathophysiology. In ECD, the basis for clinical presentation of corneal edema is well understood, but no explanation exists for the painful erosions of corneal endothelial cells (CEC) from their basement membrane, the Descemet’s membrane (DM). Here we show that SLC4A11, a plasma membrane transport protein, functions as a cell adhesion molecule (CAM) to promote direct interaction between CEC with DM. An antibody directed against a portion of


third extracellular loop (EL3) blocked SLC4A11-mediated adhesion to DM, indicating a key role of this region. ECD-causing missense mutations in SLC4A11 mapping to EL3 lead to ablation of its cell adhesion function, with no effect on cell surface trafficking, or membrane transport function. Energy-minimized 3D molecular model of SLC4A11-EL3 refined by replica-exchange molecular dynamics simulations shows that these mutations cluster together and are buried within the structure of the loop, suggesting that they do not directly form the binding site of the loop. Cell adhesion function of SLC4A11 is pan-mammal as human, bovine and murine orthologues promoted cell adhesion to a similar extent. GST pull-down combined with mass spectrometry and peptide arrays confirmed COL8A2 (another FECD gene) and COL8A1 as the DM proteins interacting with SLC4A11. A chimeric protein of SLC4A11 EL3 on Glycophorin-A precursor (GPA) promoted cell-adhesion to similar levels as full length SLC4A11, thereby holding potential in therapeutic strategies for treatment of ECDs. SLC4A11 is the first solute carrier (SLC) protein reported to be a CAM. Together, these data support SLC4A11-mediated cell adhesion to DM as a cell biological pathway defective in ECD, suggesting additional avenues for therapeutic intervention.

Session 4

10:30 am – 11:00 am

PYRIMIDINE SALVAGE AND RELATED TRANSPORT PROCESSES ACROSS THE CHLOROPLAST ENVELOPE Lisa Ohler and Torsten Möhlmann Department 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. Uracil and uridine/cytidine can be recycled into UTP/CTP via the salvage pathway, which mainly takes place in the cytosol via uridine and cytidine kinases. Uracil salvage seems to be less important, but surprisingly knockout 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. Knock-out plants lacking the only known plastidic uracil transporter PLUTO, supporting the chloroplast with an important intermediate, show no phenotype. In contrast, plants which are impaired in uracil degradation – which also takes place in the plastid - show a retarded growth. We want to gain further insight into the importance of the plastidic salvage, as well as the uracil import by analysis various mutant plants. My work is supported by the DFG and the IRTG1830. 11:00 am – 11:30 am

SUGAR HOMEOSTASIS IN HIGHER PLANTS. TRANSPORTERS AND ENZYMES.

Duc Phuong Vu and Ekkehard Neuhaus. Plant Physiology, University of Kaiserslautern, Erwin Schrödinger-Str. 22, D-67659 Kaiserslautern, Germany. The present study examines the impact of an altered sugar homeostasis in Arabidopsis thaliana by increasing the vacuolar sugar concentration. 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 tightly regulated, inter alia via control of membrane transporters and enzyme activities. Among these sugar


transporters are the Tonoplast Sugar Transporters (TSTs), which locate to the vacuolar membrane (tonoplast). The TST1 and TST2 in Arabidopsis have been identified as glucose and fructose importers. Double knockout of mutants AtTST1 and AtTST2 (Attst1-2), are nearly any more to accumulate monosaccharides in the vacuole. The agronomical highly important sugar beet plants (Beta vulgaris) comprise four isoforms of the TST-family: The BvTST1, BvTST2.1, BvTST2.2 and BvTST3. The heterologous overexpression of the BvTST1, BvTST2.1 and BvTST3 gene in the background of the Arabidopsis Attst1-2 knockout mutant restored sugar accumulation. During cold acclimation, BvTST1 and BvTST3 overexpressors showed an accumulation of glucose and fructose which indicates that they function as monosaccharide transporters. For the BvTST2.1 it was furthermore revealed that this transporter is responsible for the vacuolar sucrose loading in sugar beet taproots. However, the BvTST2.1 overexpressors accumulated surprisingly only slightly higher amounts of sucrose and the concomitant increase of glucose and fructose in BvTST2.1 overexpressors can be explained by a high activity of the vacuolar invertase which cleaves sucrose into its monomers. Thus, it is not as simple to concert Arabidopsis to a sucrose storing plant like sugar beet. To study the impact of an altered sugar homeostasis on Arabidopsis development and stress resistance, new results on mutants with increased activities of selected sugar beet TST-proteins will be presented.

11:30 am – 12:00 pm

GLYCINE TRANSPORTER 2 SURFACE ABUNDANCE IS REDUCED BY THE CALCIUM-ACTIVATED PROTEIN FOR SECRETION 1 Sabrina Marz1, Mattson Jones1, Claudia Fecher-Trost2, Martin Jung3, R. Todd Alexander4 and Eckhard Friauf1. 1Animal Physiology Group, Department of Biology, University of Kaiserslautern; 2Institute for Experimental and Clinical Pharmacology and Toxicology, Saarland University; 3Medical Biochemistry and Molecular Biology, Saarland University; 4Department of Physiology, Canada Department of Pediatrics, University of Alberta.

Glycine is an essential inhibitory neurotransmitter in the CNS of vertebrates. Active recapture of glycine from the synaptic cleft depends on the neuronal glycine transporter 2 (GlyT2). GlyT2 received growing attention as target for the treatment of hyperekplexia and pain. Therefore, we investigated the molecular network of proteins regulating GlyT2 activity. Shotgun proteomics of GlyT2 co-IPs with brainstem and spinal cord lysate of GlyT2+/+ mice identified 64 putative GlyT2-interacting proteins. Among these was the calcium-activated protein for secretion 1 (CAPS1), which was detected in all three biological replicates with 7, 14 and 5 exclusive unique peptides. CAPS1 drew our specific attention because it promotes vesicle exocytosis and regulates large-dense core vesicle trafficking. Therefore, the interaction of GlyT2 with CAPS1 may alter transporter levels at the cell surface. Western Blots of GlyT2 co-IPs and CAPS1 co-IPs with brainstem and spinal cord lysate of GlyT2+/+ mice verified the binding of CAPS1 to GlyT2, suggesting a physical interaction in vivo. Additionally, we demonstrated CAPS1 binding to the GlyT2-carboxy-terminus and GlyT2 binding to the CAPS1 C2 and Munc-homology domain in a peptide spot array. To investigate the physiological relevance of the interaction, we co-expressed GlyT2 and CAPS1 in HEK-293 cells. CAPS1 increased GlyT2 abundance, albeit shifting GlyT2 location to intracellular compartments. Thus, biotinylation of surface proteins showed that the relative surface abundance of GlyT2 is decreased in the presence of CAPS1. Glycine uptake studies exhibited that the maximal transport velocity of GlyT2 is also reduced by CAPS1, but the substrate affinity is unchanged. Moreover, internalization assays and live-cell imaging indicated that the endocytosis may be facilitated in the presence of CAPS1, as the proportion of endocytosis is higher than exocytosis. Together, our findings


provide evidence for a physical and functional interaction of CAPS1 and GlyT2, reducing GlyT2 surface abundance.

Supported by DFG (Fr 1784/181)

12:00 pm - 12:30 pm

MOLECULAR ANALYSIS OF FATTY ACID TRANSPORTERS IN PLANT CELLS

Anne Könnel, Wassilina Bugaeva & Katrin Philippar. Molecular Plant Biology, Center for Human- and Molecular Biology, Saarland University Fatty acid (FA) de novo synthesis in plants occurs in plastids. These FAs are building blocks for acyl lipids, in biomembranes or for triaglycerol oils, which represent an important form of carbon storage in plants. Assembly and modification of acyl lipids happens in plastids (prokaryotic pathway), in the endoplasmic reticulum (ER, eukaryotic pathway) or in mitochondria. Therefore intracellular transport and distribution of FAs and lipids is an important issue for plant growth and development. The transport of free FAs across plastid envelope membranes was enlightened by the identification of FAX1, a novel protein for FA-export across the inner envelope of chloroplasts (Li et al., 2015). Functionality of FAX1 is crucial for biomass production, male fertility and synthesis of FA-derived compounds. Seven proteins, belonging to the FAX family are present in the model plant Arabidopsis thaliana. While FAX1, 2, 3 and 4 are predicted in the chloroplast, FAX5, 6 and 7 seem to be located in membranes of the secretory pathway. The inner envelope insertion of FAX1-3 has been shown experimentally, FAX4-7 are under current investigation regarding subcellular localization, function and physiology. FAX4 RNAi lines in Arabidopsis show preliminary defects in growth and development, similar to fax1 knockouts. T-DNA insertion lines of FAX5, FAX6 and FAX7 are in analysis. Further, yeast assays and structure/function analysis of peptide domains within the transporters should validate FA/lipid-transport function of this protein family. Reference Li et al., (2015) PLoS Biology. 13(2): e1002053

Session 5

1:30 pm – 2:00 pm

NOVEL FUNCTIONAL ROLE OF INTESTINAL CALCIUM SENSING RECEPTOR IN REGULATION OF CALCIUM HOMEOSTASIS Justin J. Lee, Henrik Dimke, and R. Todd Alexander Department of Physiology, University of Alberta Membrane Protein Disease Research Group Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark Department of Pediatrics, University of Alberta Calcium concentration is tightly regulated in plasma. To do so, the extracellular calcium sensing receptor (CaSR) detects the blood calcium levels and signals to alter renal calcium excretion and parathyroid hormone release. The CaSR is expressed along the intestine. However, its role in regulating intestinal calcium absorption is unknown. We therefore set out to assess whether activation of the CaSR alters intestinal calcium absorption. Mice fed the CaSR agonist, cinacalcet, for 5-days had decreased expression of the transcellular calcium absorption mediators Trpv6, calbindin-D9K, and Pmca1b in duodenum, cecum and proximal colon – the sites of significant transcellular calcium absorption. The greatest effect was in the proximal colon. To assess the functional response to CaSR activation, radioactive calcium fluxes in Ussing chambers were conducted across proximal colon of 3-month old, wild type mice in the


absence of a transepithelial electrochemical gradient for calcium. The CaSR was activated by changing 0.5 mM calcium buffer to 2.5 mM calcium buffer on both sides of the epithelium. This caused a decrease in net calcium flux (Jca), -17.9 nmol h-1cm-2, while exchanging buffers from a high to low calcium concentration increased Jca (+22.5 nmol h-1cm-2). To determine whether apical or basolateral CaSR activation mediated the effect, each side of the epithelium was exposed independently to cinacalcet. Basolateral application of cinacalcet decreased Jca (-9.65 nmol h-1cm-2 before vs after drug addition); an effect not observed when apically added (+8.92 nmol h-1cm-2). The experiments were repeated on genetically modified mice expressing non-functional Trpv6, an apical membrane calcium channel. Jca significantly decreased in the wild type mice (-11.1 nmol h-1cm-2 before vs after cinacalcet treatment), however, Jca did not decrease in the knock out mice (+9.66 nmol h-1cm-2). Thus, basolateral activation of the CaSR in proximal colon inhibits transcellular calcium absorption through Trpv6. Supported by CIHR and NSERC.

2:00 pm – 2:30 pm

CHARACTERIZATION OF A NOVEL SPLICE VARIANT OF THE STROMAL INTERACTION MOLECULE 1 (STIM 1) Mona Schöppe, Maik Konrad, Alina Gilson, Markus Grimm, Annette Lis, Barbara Niemeyer Molecular Biophysics, Saarland University, Homburg, Germany / Biophysics, Saarland University, Homburg, Germany / Exerimental Neurology, Saarland University, Homburg, Germany Changes in intracellular free calcium concentration [Ca2+] probably represent the most widespread and important signaling event in cellular physiology, since transient elevations of Ca2+ directly or indirectly control and regulate a plethora of cellular responses. Therefore cells must be able to react to minor changes in [Ca2+]i and changes must be tightly regulated. The major Ca2+ pathway in electrically nonexcitable cells is the store operated calcium entry (SOCE) via calcium release activated calcium channels. The Ca2+ selective channel is located in the plasma membrane and formed by Orai-family proteins. Stromal interaction molecule (STIM1 and STIM2) proteins activate SOCE by sensing changes in the luminal Ca2+ concentration in the endoplasmic reticulum via their N-terminal EF hand motif. Upon store depletion, STIM molecules change conformation, multimerize and trigger SOCE by directly gating Orai channels within ER-PM junctional regions. Here, we report the identification and characterization of a novel STIM1 splice variant, STIM1A, which retains an additional 31 amino acid long exon within its C-terminal cytosolic region. The so called exon A is spliced into the mRNA downstream of the channel activating region and also downstream of a region encoding an acidic inhibitory domain (ID) that mediates fast Ca2+ inactivation of Orai1. On mRNA level the variant is ubiquitously expressed, but its abundance relative to the more common STIM1 variant varies upon cell type. In contrast to the RNA analysis, STIM1A could be detected only in murine testis on Western blots. Transient overexpression of the splice variant leads to an overall reduced SOCE and ICRAC when compared with STIM1. Future experiments aim to understand the physiological role of STIM1A in Testis and to identify splice-specific interaction partners. Supported by IRTG 1830 and SFB 894

2:30 pm – 3:00 pm

NEWLY IDENTIFIED HUMAN PHOSPHOLAMBAN MUTATIONS DISPLAY SImiLARITIIES

WITH The DISEASE-CAUSING FINGERPRINT

Gareth Armanious, and Howard Young. Department of Biochemistry, University of Alberta SERCA achieves the majority of the calcium removal from the cytosol of cardiomyocytes by actively transporting calcium ions from the cytosol into the sarcoplasmic reticulum during diastole. During


systole, the efflux of the stored calcium from the SR results in the activation of the contractile apparatus of the cardiomyocyte. Reversible inhibition of SERCA by the 52 amino acid SR membrane protein phospholamban (PLN) is crucial to controlling the rate of calcium sequestration, as well as the magnitude of the calcium gradient between the sarcoplasm and cytoplasm. This in turn determines heart rate and the force of the subsequent contraction. Unphosphorylated PLN decreases the apparent calcium affinity of SERCA, while β-Adrenergic-mediated phosphorylation of PLN at S16 by PKA restores SERCA activity and increases cardiac output. New mutations in PLN have been recently identified in patients with heart failure, and are being discovered at any increasing rate. For example, an A15T mutation was identified in a 4 year old female DCM patient, and a P21T mutation in a 60 year old female patient. Both patients have a family history of DCM. The effects that these variants of PLN have on the kinetics of SERCA, as well as their implications to the regulation of PLN via phosphorylation by PKA is discussed. Recombinantly expressed PLN was purified and co-reconstituted in the presence of SERCA and spectroscopic techniques were used to assess the calcium dependent specific activity of SERCA. The secondary structure of PLN variants was assessed by circular dichroism (CD) in order to correlate structural changes of PLN with altered PLN-SERCA regulatory complex kinetics. Lastly, the ability for these mutants to be phosphorylated by the catalytic subunit of PKA and then dephosphorylated by PP1 was assessed. In addition to altered structure not seen before in human PLN mutation, a small number of PLN mutations identified show hallmarks of known disease-causing PLN mutations. Supported by NSERC CREATE IRTG, CIHR, and the Heart and Stroke Foundation.

3:00 pm – 3:15 pm Concluding Remarks

Ekkehard Neuhaus & Joe Casey

POSTER LIST page 22

POSTER LIST

Poster Board Number Poster First Author Room

1 Nada Alshumaimeri Max Bell Room 252 2 Gareth Armanious Max Bell Room 252 3 Katherine Badior Max Bell Room 252 4 Andrea Blum Max Bell Room 252 5 Wassilina Bugaeva Max Bell Room 252 6 Ruiqi Cai/Xiong Liu Max Bell Room 252 7 Bartholomaus Danielczak Max Bell Room 252 8 M’Lynn Fisher Max Bell Room 252 9 Anne Grethen Max Bell Room 252 10 Daniel Hickl Max Bell Room 252 11 Swai Mon Khaing Max Bell Room 252 12 Janina Laborenz Max Bell Room 252 13 Rawad Lashhab Max Bell Room 252 14 Xiaobing Li Max Bell Room 252 15 Darpan Malhotra Max Bell Room 252 16 Kathrin Patzke Max Bell Room 253 17 Allen Plain Max Bell Room 253 18 Antonietta Russo Max Bell Room 253 19 Hasib Sarder Max Bell Room 253 20 Pauline Schepsky Max Bell Room 253 21 Mona Schoppe Max Bell Room 253 22 Mark Sicking Max Bell Room 253 23 Regine Stutz Max Bell Room 253 24 JingFeng Tang Max Bell Room 253 25 Shahid Ullah Max Bell Room 253 26 Florian Wollweber Max Bell Room 253 27 Nilam Yadao Max Bell Room 253 28 Cefan Zho Max Bell Room 253

POSTER ABSTRACTS page 23

POSTER ABSTRACTS

Poster Board #1

CATEGORIZATION OF CORNEAL DYSTROPHY MUTANTS OF SLC4A11: TRANSPORT OR CELL ADHESION DEFECTIVE Nada Alshumaimeri, and Joseph Casey. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada. SLC4A11 is a membrane transport protein found at the basolateral surface of corneal endothelial cells that facilitates transmembrane movement of H2O, NH3, and OH-/H+. 56 point mutations has been identified in SLC4A11 that cause some cases of three different posterior endothelial corneal dystrophies: late-onset Fuch’s Endothelial Corneal Dystrophy (FECD), pediatric-onset Congenital Hereditary Endothelial Dystrophy (CHED) and Harboyan Syndrome (HS). ECDs caused by SLC4A11 mutations are marked by endothelial cell loss and corneal stromal edema. SLC4A11 mutants are categorized into two different molecular phenotypes: 1- Endoplasmic reticulum (ER) retained protein due to incorrect folding. 2- Defect in the functional activity of the protein. ER retained mutants can be rescued by the treatment with some non-steroidal anti-inflammatory drugs. Amongst SLC4A11 mutants 80% of FECD, 59% of CHED and 40% of HS mutants process to the cell surface. To identify the molecular defect in normally trafficked SLC4A11 mutants, the function of SLC4A11 water movement was monitored in HEK293 cells co-expressing eGFP and either WT-SLC4A11 or SLC4A11 plasma membrane targeted mutants. Cultured cells were perfused in an isotonic medium and shifted to a hypotonic medium. The hypotonic solution causes swelling of the cells, which leads to dilution of cytosolic eGFP thus the rate of fluorescence decreases representing the water flux activity. Amongst the seventeen cell surface targeted SLC4A11 mutants, some had a functional water flux functional activity and some did not. We will further categorize mutants by determining affecting the recently identified cell adhesion role of the protein in the mutants. Endothelial corneal dystrophies negatively affect the quality of vision. Categorization of corneal dystrophy mutants of SLC4A11 provides a better understanding of SLC4A11 physiological role in endothelial corneal dystrophies. Supported by CIHR.

Poster Board # 2

NEWLY IDENTIFIED HUMAN PHOSPHOLAMBAN MUTATIONS DISPLAY

SIMILARITIIES WITH THE DISEASE-CAUSING FINGERPRINT

Gareth Armanious, and Howard Young. Department of Biochemistry, University of Alberta SERCA achieves the majority of the calcium removal from the cytosol of cardiomyocytes by actively transporting calcium ions from the cytosol into the sarcoplasmic reticulum during diastole. During systole, the efflux of the stored calcium from the SR results in the activation of the contractile apparatus of the cardiomyocyte. Reversible inhibition of SERCA by the 52 amino acid SR membrane protein phospholamban (PLN) is crucial to controlling the rate of calcium sequestration, as well as the magnitude of the calcium gradient between the sarcoplasm and cytoplasm. This in turn determines heart rate and the force of the subsequent contraction. Unphosphorylated PLN decreases the apparent calcium affinity of SERCA, while β-Adrenergic-


mediated phosphorylation of PLN at S16 by PKA restores SERCA activity and increases cardiac output. New mutations in PLN have been recently identified in patients with heart failure, and are being discovered at any increasing rate. For example, an A15T mutation was identified in a 4 year old female DCM patient, and a P21T mutation in a 60 year old female patient. Both patients have a family history of DCM. The effects that these variants of PLN have on the kinetics of SERCA, as well as their implications to the regulation of PLN via phosphorylation by PKA is discussed. Recombinantly expressed PLN was purified and co-reconstituted in the presence of SERCA and spectroscopic techniques were used to assess the calcium dependent specific activity of SERCA. The secondary structure of PLN variants was assessed by circular dichroism (CD) in order to correlate structural changes of PLN with altered PLN-SERCA regulatory complex kinetics. Lastly, the ability for these mutants to be phosphorylated by the catalytic subunit of PKA and then dephosphorylated by PP1 was assessed. In addition to altered structure not seen before in human PLN mutation, a small number of PLN mutations identified show hallmarks of known disease-causing PLN mutations. Supported by NSERC CREATE IRTG, CIHR, and the Heart and Stroke Foundation.

Poster Board #3

BAND 3 CONFORMATIONAL DYNAMICS IN RED BLOOD CELL SENESCENCE Katherine Badior and Joe Casey. Department of Biochemistry, University of Alberta Essential to respiration, red blood cells (RBCs) transport oxygen and carbon dioxide throughout the body. During their circulating lifetime of 120 days, RBCs are exposed to extreme physical and chemical stresses. As oxidative and physical damages accumulate, aged RBCs transport function becomes less efficient, and they must be removed from circulation. Circulating auto-antibodies (IgG) are highly enriched on membranes of senescent RBCs, and are necessary for recognition by macrophages for phagocytosis and removal. Senescent cell IgG is specific for Band 3, a membrane protein in the SLC4 family of anion transporters. We propose that rare conformational events in Band 3 lead to the formation of the senescence antigen and IgG binding, marking RBCs for clearance. The Band 3 senescence epitope is formed by residues 538-553 and 812-827. Decades of Band 3 research controversially supports both extracellular and intracellular localisation of residues 812-827. Recent advances in membrane protein structural biology led to the crystal structure of Band 3 membrane domain, which places residues 812-827 in intracellular loop 6 (ICL6), inaccessible to sera auto-antibodies. We propose a consolidated mechanism for Band 3 as a molecular clock for RBC senescence. We present a dynamic model of Band 3, where residues 812-827 can access both the intracellular and extracellular environments of RBCs. In order to assess the localisation of Band 3 ICL6, substituted cysteine accessibility assays were performed. Band 3 cysteine point mutants were tested for their reactivity with membrane-impermeant LYIA, to assess extracellular accessibility. Time course assays of accessibility to LYIA were also performed. In addition, accessibility of ICL6 was analyzed using immunofluorescence, in both RBCs and transiently transfected HEK293 cells, using an antibody raised against ICL6.

Supported by Canadian Institutes of Health Research


Poster Board #4

LOCALIZATION AND FUNCTION OF KDEL RECEPTORS IN YEAST AND

MAMMALIAN CELLS

Andrea Blum, Manfred J. Schmitt; Molecular & Cell Biology, Saarland University, D-66123 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. Consistent with its function at the cell surface, immunogold labelling and electron microscopy (TEM) demonstrated PM colocalization of Erd2p in a yeast wild-type strain and an endocytosis mutant (Δend3) expressing a C-terminal (HA)10 -tagged ERD2 variant from its natural chromosomal ERD2 locus. In order to identify how a major KDELR fraction is retained in the ER and Golgi, we analysed the C-terminal lysine cluster of KDELRs for a potential function as ER retention signal in yeast and mammalian cells and investigated subcellular localization of PM markers and 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 the 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 homeostasis, we assume a similar signalling function of KDELRs after cargo binding at the cell surface. To address such novel functions, we are focusing on a CRISPR/Cas9-mediated KDELR knock-out (k/o). 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. By sorting cells for either a strong, medium or weak green fluorescence, we could demonstrate that the level of Cas9 expression correlates with an observed prominent cell dying and we succeed in generating a KDELR2 and KDELR3 k/o.

Poster Board #5

PHYSIOLOGICAL ANALYSIS OF PLASTID FATTY ACIDS EXPORT PROTEINS Wassilina Bugaeva1, Anne Könnel and Katrin Philippar Molecular Plant Biology, Center for Human- and Molecular Biology, Saarland University In plants, fatty acids (FAs) are synthesized in the plastid stroma and become available for lipid assembly mainly in the form of long-chain FAs (C16–18). Some of these FAs are integrated into lipids inside plastids (prokaryotic pathway), but the majority is exported to the ER for further elongation, acyl editing, and lipid assembly (eukaryotic pathway). The identification of FAX, a novel membrane fatty acid export protein in the inner envelope membrane of chloroplasts (Li et al. 2015), significantly contributes to the understanding of the FA transport mechanism and the importance of FAX1 in plant development, biomass formation and fertility. Fax1 knockout mutants show a strong defect in the outer pollen cell wall most likely due to an impaired assembly of lipid derived components. This defect results in male sterility, which makes it impossible to breed homozygous knockout mutants. In Arabidopsis thaliana, 7 proteins belong to the FAX family and besides FAX1 also FAX2 and FAX3 are shown to be integrated into the inner envelope of


chloroplasts. FAX2 might be associated in a protein complex playing an important role in lipid remodeling between mitochondria and chloroplasts during phosphate starvation. FAX3 is assumed to partly complement FAX1 function due to transcript levels in fax1 knockouts and segregation analysis of fax1/fax3 double mutant lines. Our current research focuses on the characterization of fax2/fax3 double mutant lines as well as respective RNAi lines and a functional analysis in heterologous yeast cells. Publication Li et al. (2015) PLoS Biology. 13(2): e1002053

Poster Board #6

INTRAMOLECULAR INTERACTIONS IN TRpv6 CHANNEL MODULATED BY PIP2

Ruiqi Cai1, Xiong Liu1, Wang Zheng1, Laura Hoffmann2, Qiaolin Hu1, Veit Flockerzi2 and Xing-Zhen Chen1 1Department of Physiology, University of Alberta 2Experimentelle und Klinische Pharmakologie und Toxikologie, Universitat des Saarlandes

Transient receptor potential (TRP) vanilloid 6 (TRPV6), a calcium selective channel, plays crucial roles in human physiology such as calcium absorption in epithelia and bone. Abnormal expression or function of TRPV6 contributes to a series of human diseases, such as vitamin D-deficiency rickets, kidney stone and osteoporosis. Elevated TRPV6 expression has also been reported to be associated with pathological stages of cancer, including human breast, prostate and colon cancer. Membrane-anchored phospholipid known as phosphatidylinositol 4,5-bisphosphate (PIP2) has been proposed to activate TRPV6 channel. However, the mechanism underlying how PIP2 regulates TRPV6 channel function and gating remains largely unclear. In the present study, we identified four functionally critical residues in the pre-S1 (Trp361), S4-S5 linker (Arg510) or TRP helix (Trp633 and Ile637) domain. By electrophysiology and co-immunoprecipitation, we found that Trp361 in pre-S1 pairs with Ile637 in TRP helix to mediate a direct interaction between the two domains, which is functionally critical. We found that Arg510 in S4-S5 linker interacts with Trp633 in TRP helix, which we found stabilizes the pre-S1/TRP helix interaction. By functional and immunoprecipitation experiments we identified four cationic amino acid residues as part of the PIP2 binding pocket. We found that PIP2 disrupts the pre-S1/TRP helix and S4-S5 linker/TRP helix interactions through direct binding with TRPV6. In summary, this study demonstrated that intramolecular interactions between pre-S1, TRP helix and S4-S5 linker are critical to maintain TRPV6 channel in the closed state and that PIP2 directly binds with TRPV6, which disrupts or reduces these interactions thereby activating the channel. Supported by NSERC Discovery Grant (to XZC), DFG (to VF), IRTG Studentship (to RC and QH), AIGSS (to RC and WZ).

Poster Board #7

COLLISIONAL LIPID TRANSFER AMONG DIBMA-BOUNDED NANODISCS Bartholomäus Danielczak and Sandro Keller. Molecular Biophysics, Technische Universität Kaiserslautern. The Amphiphilic copolymers of styrene/maleic acid (SMA)1 or diisobutylene/maleic acid (DIBMA)2 can solubilise membrane proteins and surrounding lipids directly from artificial and biological membranes to assemble into polymer-bounded nanodiscs. Although the latter preserve a lipid-bilayer core, they are much more dynamic than other membrane mimics. Unlike other bilayer


systems, they exchange lipids not only by monomer diffusion but also by fast collisional transfer, as we demonstrated recently for nanodiscs bounded by SMA(3:1).3 The latter is an aromatic and rather hydrophobic copolymer and, thus, is relatively harsh toward the lipid bilayer core. By contrast, aliphatic DIBMA perturbs the bilayer core to a much lesser extent4 but has a higher negative charge density due to its high maleic acid content. By using time-resolved Förster resonance energy transfer (FRET) spectroscopy, we quantified the lipid-transfer kinetics among DIBMA-bounded nanodiscs (DIBMALPs). Moreover, we demonstrated the role of Coulombic repulsion and the role of nanodisc diameter in the transfer kinetics. Our experiments show that lipid transfer among DIBMALPs is relatively slow, with diffusional and collisional rate constants being several orders of magnitude slower than among SMALPs. Moreover, the kinetics of lipid transfer kinetics increases dramatically with the ionic strength of the solution. Although collisional transfer is slowed down by polyanionic DIBMA, lipids still exchange orders of magnitude faster than among MSP-bounded nanodiscs or vesicles. In summary, DIBMALPs are dynamic equilibrium rather than kinetically trapped assemblies that exchange lipids on the timescale of minutes to hours. Supported by IRTG 1830. 1Knowles et al. J. Am. Chem. Soc. 2009, 131, 7484 2Oluwole et al. Angew. Chem. Int. Ed. 2017, 56, 1919 3Cuevas Arenas et al. Sci. Rep. 2017, 7, 45875 4Grethen et al. Sci. Rep. 2017, 7, 11517

Poster Board #8

DWORF: A SMALL PEPTIDE WITH A LOT OF HEART M’Lynn Fisher, and Howard Young. Department of Biochemistry, University of Alberta Muscle and cardiac contraction is controlled by the release and reuptake of calcium ions from the Sarco Endoplasmic Reticulum (SR). SERCA pumps calcium against its concentration gradient into the SR to initiate relaxation of the contractile element. Dysfunction of this system leads to dilated cardiomyopathy and potentially heart failure, so regulation of this system must be tightly controlled. Historically, Phospholamban and Sarcolipin have been labelled as key inhibitory regulators of SERCA, however, small open reading frames previously thought to be noncoding have been found to actually code for regulatory micropeptides. One of the pioneers of these recently discovered micropeptides is a 35 amino acid membrane peptide named “dwarf open reading frame” (DWORF). DWORF has unique regulatory properties in that it is currently the only known activator of SERCA, while all other known regulators are inhibitors, effectively increasing calcium re-uptake and muscle relaxation. Increased calcium re-uptake has been shown to be due to displacement of phospholamban from SERCA’s inhibitory groove as well as interacting with SERCA directly. This adds an elaborate new level of regulating calcium homeostasis as well as a provocative new view on calcium regulation in the cell.


Poster Board #9

ROLE OF COULOMBIC REPULSION IN COLLISIONAL LIPID TRANSFER AMONG SMA(2:1) NANODISCS Anne Grethen, David Glueck, and Sandro Keller. Molecular Biophysics, University of Kaiserslautern Styrene/maleic acid (SMA) copolymers are attracting great interest because they are able to solubilise membrane proteins and lipids from native or artificial membranes to form polymer-bounded nanodiscs1. These nanodiscs preserve a native-like lipid-bilayer core that is surrounded by a polymer shell and can harbour a membrane protein or a membrane-protein complex. SMA exists in various styrene/maleic acid molar ratios, which results in different charge densities, hydrophobicities, and thus, solubilisation properties. We have recently reported fast collisional lipid transfer among nanodiscs bounded by the relatively hydrophobic copolymer SMA(3:1)2. Herein, we employed time-resolved Förster resonance energy transfer to quantify the kinetics of lipid transfer among nanodiscs encapsulated by SMA(2:1), a less hydrophobic copolymer that is more efficient in terms of lipid and protein solubilisation3. Furthermore, we assessed the role of ionic strength and, thereby, how Coulombic repulsion affects the transfer of lipid molecules among these polyanionic nanodiscs. Collisional lipid transfer is slower among SMA(2:1) nanodiscs (kcol = 5.9 M−1 s−1) as compared with SMA(3:1) nanodiscs (kcol = 222 M−1 s−1) but still four to five orders of magnitude faster than diffusional lipid transfer among protein-encapsulated nanodiscs or vesicles. Increasing ionic strength further accelerates lipid exchange among SMA(2:1) nanodiscs in a manner predicted by either the Davies equation, an empirical extension of the Debye–Hückel limiting law, or a modified form of the Debye–Hückel law that accounts for the finite size of nanodiscs. 1Knowles et al. J. Am. Chem. Soc. 2009, 131, 7484 2Cuevas Arenas et al. Sci. Rep. 2017, 7, 45875 3Grethen et al. Sci. Rep. 2017, 7, 11517

Poster Board #10

EQUILIBRATIVE NUCLEOSIDE TRANSPORTER - PHYSIOLOGY AND ATTEMPTS TO GAIN STRUCTURAL INSIGHTS Daniel Hickl1, Lisa Ohler1, M. Joanne Lemieux2 and Torsten Möhlmann1. 1Department of Plant Physiology, University of Kaiserslautern. 2Dept. of Biochemistry, University of Alberta 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 solved 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. This approaches shall be realized by cobalt based IMAC, which allows plenty clean protein purification, with subsequently reconstitution into liposomes. On the one hand proteoliposomes shall be used to reveal AtENT7 functionality on the other hand proteoliposomes are also suitable for Cryo EM observations, in case the purified AtENT7 cannot be applied for traditionally Cryo EM. Supported by IRTG1830 and DFG. Poster Board #11

ROLE OF LIPID NANODOMAINS IN CD36 SIGNAL TRANSDUCTION Swai Mon Khaing, and Nicolas Touret. Department of Biochemistry, University of Alberta

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. Our project centralized in elucidating the mechanisms of CD36-Fyn enrichment on the lipid nanodomains and actin cytoskeleton during TSP-1 induced signaling in ECs.

We hypothesized that lipid nanodomains play a role in bringing together CD36-Fyn to F-actin regions through adaptor molecules which forms a signaling platform. We have 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. To test the role of PI3K in Fyn activation and in CD36 nanocluster enhancements, we employed pharmacological inhibition of PI3K (LY294002) to arrest the production of PIP3 on the plasma membrane and depletion of PI(4,5)P2, a precursor for PI(3,4,5)P3 using ionomycin. Using Immunoblotting and super resolution fluorescence microscopy (TIRF-PALM), we determined that PI3K is important for Fyn activation and in CD36 nanocluster enhancements in CD36-Fyn signaling upon stimulation with TSP-1. Additionally, we employed a unique optogenetic tool (LARIAT) to facilitate in understanding the role of lipid nanodomains in CD36-Fyn signaling. Upon clustering of CD36 molecules using LARIAT, Fyn activation enhanced within these clusters and this activation is reduced by treatment with LY294002 which further supported our hypothesis that engagement with PI3K (lipid nanodomains PI(4,5)P2 and PI(3,4,5)P3) plays a significant role in Fyn activation and CD36 nanoclustering. With this, we proposed a model in which CD36 nanoclusters are located within PI(4,5)P2 domains and upon TSP-1 stimulation, PI3K is engaged, producing PI(3,4,5)P3 within the CD36 nanoclusters and enhances the nanoclusters and downstream Fyn activity. Supported by CIHR, NSERC-IRTG. Poster Board #12

ER MEDIATED IMPORT OF MITOCHONDRIAL MEMBRANE PROTEINS Janina Laborenz, Katja Hansen, Naama Aviram, Maren Meyer, Maya Schuldiner and Johannes M. Herrmann, Department of Cell Biology, University of Kaiserslautern, Germany. Weizmann Institute of Sciences, Rehovot, Israel


Most mitochondrial proteins are initially synthesized in the cytosol as precursor proteins and imported into mitochondria. While the import of soluble mitochondrial proteins was well studied in the past, only little is known how cells manage to translocate the many hydrophobic membrane proteins of the inner membrane. We developed a genetic screen in yeast cells to identify genes that are critical for the efficient translocation of the hydrophobic inner membrane protein Oxa1 into mitochondria. Surprisingly, in this screen we identified several so far uncharacterized, though conserved ER proteins that are crucial for mitochondrial targeting if Oxa1. By combining biochemical and genetic analyses we characterized the function of the protein Djp1 in this process, which belongs to the family of J-domain cochaperones of the Hsp70 system. We found that a large fraction of the newly synthesized Oxa1 precursor associates with the ER surface from where it is recognized by Djp1 to be directed to the mitochondrial outer membrane translocase. We propose that the ER surface can serve as a collection system that facilities intracellular protein transport to mitochondria. We called this import route the ER-SURF pathway. In addition to Djp1, we identified three further ER membrane proteins, which we named Ema17, Ema19 and Ema35. First results about the function of these proteins in the context of mitochondrial preprotein sorting will be presented.

Poster Board #13

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

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.


Poster Board # 14

YEAST OVEREXPRESSION SCREEN FOR CELLULAR COMPONENTS RESTORING PLASMA MEMBRANE TRAFFICKING OF HUMAN KIDNEY ANION EXCHANGER 1 Xiaobing Li, Björn Becker, and Manfred J. Schmitt. Molecular and Cell Biology, Department of Biosciences and Center of Human and Molecular Biology (ZHMB), Saarland University, D-66123 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 could be successfully expressed in yeast and partially colocalizes in the plasma membrane, we are going to use yeast as experimental model 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 Western- and FACS-based screening approach in S. cerevisiae to test which yeast proteins, when overexpressed, modulate the cellular expression and plasma membrane localization of kAE1. To date, we finished a FLAG-tagged kAE1 expression construct which was successfully integrated into yeast genome by homologous recombination and confirmed its in vivo expression and localization by western analyses and immunofluorescence microscopy. In further experiments we want to analyze the cellular proteins that have been identified in the ORF screen 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. This study is kindly supported by the Deutsche Forschungsgemeinschaft (IRTG 1830).

Poster Board #15

CELL ADHESION ROLE OF SLC4A11 IN ENDOTHELIAL CORNEAL DYSTROPHY PATHOLOGY AND THERAPEUTICS Darpan Malhotra1, Martin Jung2, Claudia Fecher-Trost3, Sergei Noskov4, Richard Zimmermann2 and Joseph R. Casey1* 1Department of Biochemistry, Membrane Protein Disease Research Group, University of Alberta 2Department of Medical Biochemistry and Molecular Biology, Saarland University 3Department of Experimental Pharmacology, Saarland University 4Centre for Molecular Simulations, Department of Biological Sciences, University of Calgary Endothelial Corneal Dystrophies (ECD) are the most common cause of corneal blindness worldwide with complex genetic etiology and unclear pathophysiology. In ECD, the basis for clinical presentation of corneal edema is well understood, but no explanation exists for the painful erosions of corneal endothelial cells (CEC) from their basement membrane, the Descemet’s


membrane (DM). Here we show that SLC4A11, a plasma membrane transport protein, functions as a cell adhesion molecule (CAM) to promote direct interaction between CEC with DM. An antibody directed against a portion of third extracellular loop (EL3) blocked SLC4A11-mediated adhesion to DM, indicating a key role of this region. ECD-causing missense mutations in SLC4A11 mapping to EL3 lead to ablation of its cell adhesion function, with no effect on cell surface trafficking, or membrane transport function. Energy-minimized 3D molecular model of SLC4A11-EL3 refined by replica-exchange molecular dynamics simulations shows that these mutations cluster together and are buried within the structure of the loop, suggesting that they do not directly form the binding site of the loop. Cell adhesion function of SLC4A11 is pan-mammal as human, bovine and murine orthologues promoted cell adhesion to a similar extent. GST pull-down combined with mass spectrometry and peptide arrays confirmed COL8A2 (another FECD gene) and COL8A1 as the DM proteins interacting with SLC4A11. A chimeric protein of SLC4A11 EL3 on Glycophorin-A precursor (GPA) promoted cell-adhesion to similar levels as full length SLC4A11, thereby holding potential in therapeutic strategies for treatment of ECDs. SLC4A11 is the first solute carrier (SLC) protein reported to be a CAM. Together, these data support SLC4A11-mediated cell adhesion to DM as a cell biological pathway defective in ECD, suggesting additional avenues for therapeutic intervention.

Supported by NSERC IRTG, CIHR, DFG

Poster Board #16

NEW INSIGHTS INTO PLASTIDIAL SUGAR TRANSPORT Kathrin Patzke1, Pratiwi Prananingrum1, Patrick Klemens1, Ilka Haferkamp1, Bettina Bölter2 and Ekkehard Neuhaus1. 1Department of Plant Physiology, Technische Universität Kaiserslautern. 2Ludwig-Maximilians-Universität München Carbohydrates represent the main energy source in plants and are produced during photosynthesis in chloroplasts. These carbohydrates are stored 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 transport across membranes is essential for the plant, since sucrose is the main transport unit and plays a fundamental role in developmental processes and responses to various stressors, including cold. Apart from translocation across the plasma membrane, sugar transport occurs at organelle membranes such as the plastidial inner envelope or the vacuolar membrane, named tonoplast. To gain more insights into the importance of sugar transport across the plastidial inner envelope especially under stress conditions, we are focusing on the characterization of VGT3 (vacuolar glucose transporter 3). VGT3 is one of three members of the VGT-like family. VGT1 as well as VGT2 are known to localize in the tonoplast of Arabidopsis thaliana and VGT1 is apparently involved in glucose transport across the vacuolar membrane (Aluri and Büttner, 2007). By contrast GFP-fusion analyses and protein import assays prove the localization of VGT3 in the chloroplast inner envelope. Non aqueous fractionation analyses as well as a delay in flowering and silique development in vgt3 null mutants, suggest that VGT3 is involved in sucrose transport. This project is supported by DFG (T-SFB 175, The Green Hub).


Poster Board #17

GENE EXPRESSION OF CALCIUM TRANSPORT RELATED PROTEINS ON THE INTESTINE OF CLAUDIN-12 KO MOUSE Allein Plain2, Megan Beggs2, Wanling Pan2, Emmanuelle Cordat2, R. Todd Alexander1,2 1 Department of Pediatrics & 2 Physiology, The University of Alberta Claudin-12 is a tight junction protein expressed along the intestine and in the kidney. Claudins confer to the tight junction permeability properties. The function of claudin-12 is not yet well elucidated, though it appears to be involved in calcium transport. To investigate the role of claudin-12 in vivo we generated a claudin-12 null mouse by replacing the coding exon with beta galactosidase from E. coli. Small bowel from wild-type (WT) and claudin-12 KO (KO) mice were similar by light and electron microscopy. The KO mice had reduced voltage sensitive calcium fluxes compared to WT. We therefore studied the mRNA expression of genes known to be involved in calcium transport in all the segments of the intestine to determine if there where compensatory adjustment to counteract the lack of claudin-12. All animals were healthy. KO and WT mice grew similarly, ate and drunk comparably, had corresponding values in all plasma parameters, and had no differences in volumes or composition of urinary and fecal excretions. Duodenum was analyzed for expression of claudin-2, claudin-15, plasma membrane Ca2+ ATPase (PMCA1b), calbindin D9k (Calb9K), and the transient receptor potential cation channel subfamily V member 6 (TRPV6). Jejunum and Ileum were analyzed for claudin-2, claudin-15 and for the calcium voltage-dependent channel Cav1.3. Cecum was examined for Calb9K, PMCA1b, TRPV6 and Cav1.3, while Proximal Colon was studied for expression of TRPV6, Calb9K, Cav1.3, and for the sodium-calcium exchanger (NCX). Our results show no differences in the mRNA expression of any of the genes studied in duodenum, jejunum and ileum. Cecum of KO mice expressed approximately 25% more PMCA1 and Proximal Colon almost doubled its amount of Calb9K. These results are consistent with claudin-12 deletion decreasing calcium transport in the small intestines which is compensated by an increase in transcellular transport of calcium in the proximal large bowel. Supported by Canadian Institutes of Health Research

Poster Board #18

THE SIGNAL PEPTIDE: A MULTIFUNCTIONAL ZIP CODE Antonietta Russo, Medical Biochemistry and Molecular Biology, University of Saarland. An accurate transport and localization of the proteins is essential for cell viability. The “signal hypothesis” was proposed by Günter Blobel in 1970s (Rockefeller University) to explain how proteins - by intrinsic signals – regulate these pathways at several levels. The signal peptides (in average 16-30 amino acid residues) have not homology, the only common characteristic is a tripartite structure: a central hydrophobic h-region, a hydrophilic N- and C-terminal flanking regions. The N-region is extremely variable in terms of length and composition (one or several positively charged basic amino acids), its elimination compromises the post-translocation of short secretory proteins (Guo et al. 2017); the hydrophobic region (h-region) is still not completely definable, probably the residues from 7 to 15 are the most important, a threshold level of hydrophobicity is necessary for the signal recognition particle (SRP) binding (Peterson et al. 2003; Nilsson et al., 2014); the C- region (5-6 residues) defines the cleavage site, recognized by the cleavage peptidase. In both prokaryotes and eukaryotes, many proteins that are exported from the cytoplasm contain the signal peptide (SP); a cleavable N-terminal sequence which can specify the


targeting pathway, the efficiency of translocation, the timing of cleavage and even post-cleavage events. In fact, it is also involved in antigen presentation to the major histocompatibility complex class I (MHC I) during immune response; in addition, it could play a role in virus-host interactions (Powers and at., 2008). The full functional potential of these signal sequences, with many intrinsic codes, is still to be determined. I will present some characteristics of SP composition and structure and how these aspects can underlie its functionality. Supported by IRTG 1830, SFB 894 and DAAD. Poster Board #19

DISSECTING INTERCELLULAR TRAFFICKING AND MIS-TRAFFICKING OF HUMAN KIDNEY AE1 IN YEAST AND MAMMALIAN CELLS Hasib A. M. Sarder, Björn Becker and Manfred J. Schmitt. Molecular & Cell Biology, Department of Biosciences, Center of Human and Molecular Biology (ZHMB), Saarland University, D-66123 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 chloride ions (Cl-), 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 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 mis-trafficking [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. So far, a yeast codon optimized kAE1 variant had been successfully expressed as full-length protein in yeast. Proper kAE1 localization at the cell periphery was confirmed by indirect immunofluorescence microscopy, co-localization with the yeast plasma membrane marker Pma1p, as well as by cell surface biotinylation experiments. Furthermore, in vivo functionality of kAE1 was determined by using a pH sensitive dye. Changes in cellular pH homeostasis were observed in kAE1 expressing cells compared to negative control cells providing indirect hints of kAE1 functionality in yeast. The overall aim of this thesis is the establishment of a genetic screening system to identify cellular components involved in proper kAE1 trafficking. The results obtained from the yeast screen with selected deletion mutants should be translated into the mammalian situation to get a deeper mechanistic understanding of kAE1 mis-targeting/trafficking in dRTA associated clinical disorders.

This study is kindly supported by grants from the Deutsche Forschungsgemeinschaft thorough IRTG 1830 and by a PhD fellowship from the DAAD.

Poster Board #20

ROLE OF THE Na+-ACTIVATED K+ CHANNEL SLACK (Slo2.2) FOR DEVELOPMENT AND FUCNTION OF THE MAMMALIAN COCHLEA AND AUDITORY SYSTEM Pauline Schepsky1, Friederike Stephani1, Anne Bausch2, Robert Lukowski2, Katharina Sorg3, Dietmar Hecker3, Peter Ruth2, Jutta Engel1 1Saarland University, Department of Biophysics and CIPMM; 2University of Tübingen, Department Clinical Pharmacy; 3Saarland University, Department of Otorhino-laryngology


Slack (Slo2.2, Kcnt1) is a Na+- and voltage-activated potassium channel that reduces neuronal excitability in response to neuronal activation and subsequent Na+ influx. It is strongly expressed in those neurons of the central auditory pathway, which operate at very high firing rates. Slack mRNA expression has also been reported in peripheral auditory neurons (spiral ganglion neurons, SGNs) in the cochlea. Nonetheless, the subcellular localization and function of Slack in the mammalian cochlea and auditory pathway remains to be elucidated. Immunolabeling Slack protein in cochlear whole mounts, SGNs and auditory brainstem sections using different anti-Slack antibodies (two polyclonal and one monoclonal) yielded differential results. Unfortunately, the labeling patterns were non-specific as tested in tissue of Slack-/- mice. For recording Slack currents in SGNs we established a dissociated primary culture of apical and basal cochlear halves of three week-old (hearing) mice. After three days in vitro, immunolabeling for beta III-tubulin was performed to assess the number of type I SGNs isolated from either Slack+/+ or Slack-/- mice. Next, these primary SGN cultures will be used for whole-cell patch clamp recordings of Slack currents. Hearing function, which was determined by First cclick and frequency-dependent auditory brainstem response (ABR) measurements and recordings of distortion product otoacoustic emissions (DPOAE), recordings indicatedwas normal in 12 – 14 week-old hearing thresholds in Slack-/- mice compared with wild type littermates at the age of 4 and 12 weeks.

Poster Board #21

CHARACTERIZATION OF A NOVEL SPLICE VARIANT OF THE STROMAL INTERACTION MOLECULE 1 (STIM 1) Mona Schöppe1, Maik Konrad1, Alina Gilson1, Markus Grimm3, Annette Lis2, Barbara Niemeyer1 1Molecular Biophysics, Saarland University / 2Biophysics, Saarland University/ 3Exerimental Neurology, Saarland University Changes in intracellular free calcium concentration [Ca2+] probably represent the most widespread and important signaling event in cellular physiology, since transient elevations of Ca2+ directly or indirectly control and regulate a plethora of cellular responses. Therefore cells must be able to react to minor changes in [Ca2+]i and changes must be tightly regulated. The major Ca2+ pathway in electrically nonexcitable cells is the store operated calcium entry (SOCE) via calcium release activated calcium channels. The Ca2+ selective channel is located in the plasma membrane and formed by Orai-family proteins. Stromal interaction molecule (STIM1 and STIM2) proteins activate SOCE by sensing changes in the luminal Ca2+ concentration in the endoplasmic reticulum via their N-terminal EF hand motif. Upon store depletion, STIM molecules change conformation, multimerize and trigger SOCE by directly gating Orai channels within ER-PM junctional regions. Here, we report the identification and characterization of a novel STIM1 splice variant, STIM1A, which retains an additional 31 amino acid long exon within its C-terminal cytosolic region. The so called exon A is spliced into the mRNA downstream of the channel activating region and also downstream of a region encoding an acidic inhibitory domain (ID) that mediates fast Ca2+ inactivation of Orai1. On mRNA level the variant is ubiquitously expressed, but its abundance relative to the more common STIM1 variant varies upon cell type. In contrast to the RNA analysis, STIM1A could be detected only in murine testis on Western blots. Transient overexpression of the splice variant leads to an overall reduced SOCE and ICRAC when compared with STIM1. Future experiments aim to understand the physiological role of STIM1A in Testis and to identify splice-specific interaction partners. Supported by IRTG 1830 and SFB 894


Poster Board #22

SENDING PROTEINS TO AND CALCIUM IONS THROUGH THE SEC61 COMPLEX OF HUMAN CELLS Mark Sicking1, Sarah Haßdenteufel1, Martina Zivna2, Martin Jung1, Richard Zimmermann1, Sven Lang1 1 Department of Medical Biochemistry and Molecular Biology, Saarland University 2 Institute of Inherited Metabolic Disorders, Charles University, Prague For up to one third of synthesized polypeptides in eukaryotic cells protein translocation across or insertion into the membrane of the endoplasmic reticulum (ER) is required for their biogenesis. This process is organized by different machineries, two of which we address in more detail. Project 1 focuses on the targeting of newly synthesized proteins to the ER membrane of human cells. In 2016 a new targeting pathway to the ER was described in S. cerevisiae (Aviram et al. 2016). The SND route was described as alternative pathway able to complement the known SRP and GET targeting routes and is constructed by the yeast proteins Snd1, Snd2 and Snd3. Our recent work in HeLa cells characterized the human protein hSnd2 as the functional ortholog of Snd2 (Haßdenteufel et al. 2017). In the ongoing work we want to identify the remaining mammalian counterparts of Snd1 and Snd3 as wells as define the first “substratome” of the SND pathway by quantitative proteomics. Project 2 focuses on the Sec61 complex, the molecular machine designed for the transport of targeted proteins across or into the ER membrane. The main component of the trimeric Sec61 complex is the Sec61α protein, forming a dynamically regulated, aqueous pore. However, imperfect sealing of the Sec61 complex during the transport of proteins causes a leak of calcium ions following the calcium gradient from the lumen of the ER to the cytosol. Interestingly, different mutations of Sec61α are associated with both dysfunction and disease. Two such Sec61α mutations cause Autosomal-dominant tubulo-interstital kidney disease (Bolar et al. 2016). To determine the underlying pathogenic mechanism and further the genotype-phenotype correlation of the Sec61 complex, we started to investigate the influence of the two Sec61α mutations on the calcium homeostasis and the transport functionality in HEK293 cells. Supported by: IRTG1830; DFG; HomFor

Poster Board #23

CLIENT SPECTRUM OF THE TRANSLOCON-ASSOCIATED PROTEIN (TRAP) COMPLEX Regine 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. Supported by IRTG1830

Poster Board #24

STYK1 404 TYROSINE RESIDUE MUTATION ENHANCES STYK1 MEDIATED PI3K SIGNALING TRANSDUCTIONS AND HUMAN BREAST CANCER CELLS MIGRATION

Miao Hu1, Cefan Zhou1,2, Xuehong Qian1, Wenying Qin1, Xing-Zhen Chen1,3, Jingfeng Tang1*

1 National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, China 2 The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, China 3 Membrane Protein Disease Research Group, Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta STYK1 (putative serine/threonine and tyrosine kinase, also called NOK) is a newly identified novel oncogene which belongs to the receptor protein-tyrosine kinase family (PRTK), and distinctly promotes several types of tumor tumorigenesis, proliferation and metastasis. The most important signal-transduction activated by STYK1 was the PI3K signaling pathway previously reported. Like many other PRTK kinases, STYK1 contains a single transmembrane domain and an intracellular tyrosine kinase domain. Due to the shortened extracellular domain, the tyrosine kinase domain was considered to play the mainly functional role in the tumor progression. It’s been reported that specific tyrosine residue point mutation in the kinase domain of STYK1 could dramatically block tumor progression mediated by STYK1. Alternatively, in this study, we found STYK1 Y404F mutation strongly enhanced STYK1 mediated tumor progression in human breast cancer cells. Methods: The level of AKT, p-AKT, E-cadherin, N-cadherin and Snail were detected by Western blot analysis; Partial length of STYK1 cDNA and STYK1 Y404F were prepared by PCR amplification. The recombinant GST-tagged STYK1 was purified by using pGEX-4 system and glutathione sepharose-4B according to the protocols. The activity of STYK1 and STYK1 Y404F were evaluated by an in vitro kinase ELISA kit. Cell proliferation, colony formation and trans-well analysis were used to assess the tumor progression of human breast cancer cells. Results: In the present study, we successfully generated a tyrosine to phenylalanine mutation of the carboxyl terminal tyrosine 404 residue of STYK1 (Y404F), and surprisingly, STYK1 Y404F


has much more in vitro kinase activity than wild type STYK1. We found that STYK1 Y404F significantly improved AKT phosphorylation, and the level of phosphorylated 4EBP1. N-cadherin and Snail were up-regulated after STYK1 Y404 expression, and in contrast, E-cadherin was down-regulated. We further showed that exogenous expression of STYK1 Y404F in human MCF-7 and MDA-MB-231 breast cancer cells significantly promotes cell proliferation, migration, and invasion. Conclusion: Above all, our results demonstrate that STYK1 404 tyrosine residue mutation enhances STYK1 mediated PI3K signaling transductions and human breast cancer cell migration Acknowledgement: This work was supported by the National Natural Science Foundation of China (31701228 to C.F.Z., 81602448 to J.F.T., 81570648 to X.Z.C.), Hubei Chenguang Talented Youth Development Foundation (2015106 to J.F.T).

Poster board #25

TIGHT JUNCTION PROTEIN CLAUDIN-4 AND KIDNEY ANION EXCHANGER-1: DECIPHERING THEIR RELATIONSHIP IN INTERCALATED CELL SPECIFIC CLAUDIN-4 KNOCKOUT MICE AKM Shahid Ullah#, Daphne Fernandes#, Todd Alexander#*, Emmanuelle Cordat# #Department of Physiology, *Department of Pediatrics, University of Alberta The kidney Cl-/HCO3

- exchanger-1 (kAE1) is expressed at the basolateral membrane of α-intercalated cells in the collecting duct (CD) and maintains pH homeostasis. Distal renal tubular acidosis (dRTA) is caused by mutations in kAE1 encoding gene via mechanisms that are not understood as yet. A yeast-two hybrid assay revealed a physical interaction between kAE1 and claudin-4, which is expressed at tight junctions of the CD cells and regulates paracellular transport of Cl- and Na+. Immortalized inner medullary collecting duct cells expressing kAE1 and claudin-4 demonstrated a significantly reduced transepithelial electrical resistance compared to cells expressing kAE1 only, suggesting a role of kAE1 in tight junction properties. Therefore, our hypothesis is that kAE1 and claudin-4 are both essential to maintain pH and electrolyte homeostasis. Our primary aim is to explore the role(s) of claudin-4 and kAE1 interaction in regulation of pH and electrolyte homeostasis in vivo. We generated an α-intercalated cell specific claudin-4 knockout (KO) mouse model. The KO mice develop normally and are healthy. The plasma composition is not different in the KO compared to the wild type at the steady state. Three and 6 months old mice were fed a normal or low NaCl diet for 2 weeks. Plasma and urinary electrolyte composition that have been analyzed up to this point do not show abnormal Na+ or Cl-. Our results so far indicate that claudin-4 in intercalated cells is not involved in Na+ or Cl- homeostasis. Supported by: Canadian Institute of Health Research (CIHR), Kidney Foundation Canada (KFC), and International Research Training Group (IRTG)

Poster Board #26

REGULATION OF MICOS ACTIVITY DURING MITOCHONDRIAL CRISTAE REMODELLING Florian Wollweber1,2, Heike Rampelt3, Ralf Zerbes3, Maria Bohnert3,4, Carolin Gerke3, Katja Noll1,2, Michael T. Ryan5, Nikolaus Pfanner3 & Martin van der Laan1,2 1Medical Biochemistry and Molecular Biology, Saarland University Medical Centre


2Centre for Molecular Signalling (PZMS), Saarland University Medical Centre 3Institute of Biochemistry and Molecular Biology, University of Freiburg 4Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel 5Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia Due to their double-membrane architecture mitochondria show a remarkable structural complexity, which is directly linked to many essential functions in energy metabolism and cell-fate decisions. The mitochondrial outer membrane connects mitochondria to the cytosol and other organelles. The inner membrane harbours the oxidative phosphorylation machinery for ATP synthesis and consists of a boundary region as well as tubular or disc-shaped protrusions, termed cristae. One of the main organisers of cristae architecture is the mitochondrial contact site and cristae organising system (MICOS), which can be found in all cristae-containing eukaryotes. MICOS is essential for maintaining crista junction structure and creates a central hub for mitochondrial biogenesis. Recent studies found that it can be divided into two subcomplexes: A membrane-bridging module which connects MICOS to a variety of mitochondrial machineries and a membrane-shaping module that stabilises the membrane curvature at crista junctions. As cristae are highly dynamic structures that have to adapt to e.g. altered metabolic demands of the cell, the regulation of MICOS activity plays a pivotal role. We are investigating how the membrane-shaping activity of MICOS is influenced by regulatory MICOS components. Our current work aims to elucidate the function of the two lipid-binding subunits Mic26 and Mic27 and their role in coordinating distinct MICOS activities for cristae maintenance and remodelling. Funding: BIF, IRTG1830

Poster Board #27

DIFFERENTIAL SORTING OF MITOCHONDRIAL PREPROTEINS VIA THE TIM23 MACHINERY Nilam Yadao. Department of Medical Biochemistry and Molecular Biology, Saarland University The majority of mitochondrial proteins is encoded by nuclear genes and synthesized as precursors in the cytosol. Mitochondrial preproteins contain a variety of import and sorting signals that guide them to their destined locations within the organelles. The focus of my project is the translocation and membrane-insertion of preproteins with amino-terminal targeting signals. These proteins pass the outer membrane via the TOM complex and are then taken over by the presequence translocase of the inner mitochondrial membrane, the TIM23 complex. Depending on the physicochemical properties and sorting information of the preproteins, they are either translocated completely into the matrix or integrated into the inner membrane. Matrix import requires the interaction and close cooperation of TIM23 with the presequence translocase-associated import motor. Membrane insertion of hydrophobic preprotein segments via a stop-transfer mechanism is supported by the direct physical coupling of proton-pumping respiratory chain complexes. It is unknown how transmembrane segments are recognized by the TIM23 machinery and how they laterally escape from the translocon into the lipid bilayer. Recent studies from my laboratory have shown that the small membrane-integral TIM23 subunit Mgr2 controls the lateral release of preproteins, likely via interactions with charged amino acid residues flanking transmembrane segments. Site-specific photo-crosslinking data suggest that Tim17 differentially interacts with hydrophilic and hydrophobic preprotein segments within the protein-conducting pore of the TIM23 complex. In my studies, I use purified mitochondria from knock-out and conditional tim mutants of the baker's yeast (Saccharomyces cerevisiae) to unravel how Tim17 and Mgr2 cooperate in the decoding of


inner membrane sorting signals and the release of preprotein segments into the phospholipid bilayer, a process referred to as "lateral gating".

Poster Board #28

PYGOPUS2 PROMOTES AUTOPHAGY THROUGH AMPK AND mTOR SIGNALING PATHWAY Xuehong Qian1, Cefan Zhou1, 2, Miao Hu1, Wenying Qin1, Yuan Huang1, Xing-Zhen Chen1, 3, Jingfeng Tang1*

1 National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, China 2 The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, China 3 Membrane Protein Disease Research Group, Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta Autophagy (macroautophagy) is a highly regulated evolutionarily conserved process, which captures and delivers the intracellular constituents such as aged or damaged organelles, aggregated proteins engulfed in autophagosome to lysosomes for digestion and degradation. It’s robust activated under nutrient starvation, hypoxia and other metabolic stress. Up-regulated autophagy has been reported to promote human cancer progress, which accompanied by nutrition and oxygen insufficient, providing the substrates for tumor cellular biosynthesis and energy generation. Activated AMPK induced Beclin1 phosphorylation and abrogated mTOR mediated inhibition of ULK1, were the two major cause of autophagy initiation. Pygopus2 (Pygo2) is a newly identified component of Wnt signaling pathway, and initiates transcription of target genes involved in cell growth and proliferations. Pygo2 has been reported to up-regulated in several types of malignant tumors, which contains breast cancer, human glioblastoma and primary hepatic carcinoma. However, the relationship between elevated Pygo2 level and enhanced autophagy in human breast cancer cells was rarely reported. In this study, we set out to investigate the potential role of Pygo2 in activating autophagy process and their contributions to human breast cancer progress. Methods: The level of Pygo2, LC3, p62, pAMPK-T172, pULK1-S312 and p4EBP1 was detected by Western blot. The LC3 puncta was evaluated by confocal microscope. Partial length of Pygo2 cDNA was prepared by PCR amplification. RNA interference technology was used to knockdown of Pygo2. Results: In the present study, we found that the ratio of LC3-II/I and degradation of p62, which were the two major indicator of autophagy, were drastically decreased in Pygo2 deficient human breast cancer MCF-7 and MDA-MB-213 cells. We also found that autophagy activity was significantly blunted by Pygo2 deficiency treated with rapamycin and serum free starvation. In contrast, exogenous expression of Pygo2 effectively stimulates the activity of autophagy in MCF-7 and MDA-MB-213 cells. We next tested whether Pygo2 mediated autophagy initiation by activating AMPK or inhibiting mTOR activity. As expected, the level of AMPK T172 and ULK1 S317 phosphorylation was improved, and the level of phosphorylated 4EBP1 was decreased. Conclusion: Our results indicated that Pygo2 promotes autophagy through AMPK and mTOR signaling pathway. Acknowledgement: This work was supported by the National Natural Science Foundation of


China (31701228 to C.F.Z., 81602448 to J.F.T., 81570648 to X.Z.C.), Hubei Chenguang Talented Youth Development Foundation (2015106 to J.F.T).

List of attendees page 42

List of Attendees

Name Email Department University

Amoroso, Gabriele [email protected] Plant Physiology University of Kaiserslautern

Herrmann, Johannes [email protected] Cell Biology University of

Kaiserslautern

Keller, Sandro [email protected]

Molecular Biophysics

University of Kaiserslautern

Lang, Sven [email protected] Medical Biochemistry & Molecular Biology

Saarland University

Möhlmann, Torsten [email protected] Plant Physiology University of Kaiserslautern

Morgan, Bruce [email protected] Cellular Biochemistry


Neuhaus, Ekkehard [email protected] Plant Physiology University of Kaiserslautern

Niemeyer, Barbara [email protected] Molecular Biophysics Saarland University

Philippar, Katrin [email protected] Plant Biology Saarland University

van der Laan, Martin [email protected] Physiological Biochemistry Saarland University

Blum, Andrea [email protected] Molecular & Cell Biology Saarland University

Bugaeva, Wassilina [email protected] Plant Biology Saarland University

Danielczak, Bartho [email protected] Molecular Biophysics


Grethen, Anne [email protected] Molecular Biophysics


Hickl, Daniel [email protected] Plant Physiology University of Kaiserslautern

Khandpur, Gurleen [email protected] Cellular Biochemistry




Könnel, Anne [email protected] Plant Biology Saarland University

Laborenz, Janina [email protected] Cell Biology University of Kaiserslautern

Li, Xiaobing [email protected] Molecular & Cell Biology Saarland University

Martins-Rodrigues [email protected] Plant Physiology University of Kaiserslautern

Marz, Sabrina [email protected] Animal Physiology University of Kaiserslautern

Oestreicher, Julian [email protected] Cellular Biochemistry


Ohler, Lisa [email protected] Plant Physiology University of Kaiserslautern

Patzke, Kathrin [email protected] Plant Physiology University of Kaiserslautern

Russo, Antonietta [email protected] Medical Biochemistry & Molecular Biology

Saarland University

Sarder, Hasib [email protected] Molecular & Cell Biology Saarland University

Schepsky, Pauline [email protected] Biophysics Saarland University

Schöppe, Mona [email protected] Biophysics Saarland University

Sicking, Mark [email protected] Medical Biochemistry & Molecular Biology

Saarland University

Stutz, Regine [email protected] Medical Biochemistry & Molecular Biology

Saarland University

Vu, Duc Phuong [email protected] Plant Physiology University of Kaiserslautern

Wollweber, Florian [email protected] Medical Biochemistry & Molecular Biology

Saarland University

Yadao, Nilam [email protected] Medical Biochemistry & Saarland University



Molecular Biology

Zöller, Eva [email protected] Cellular Biology University of Kaiserslautern

Joanne Lemieux [email protected] Biochemistry University of Alberta

Howard S. Young [email protected] Biochemistry University of Alberta

Shahid Ullah [email protected] Physiology University of Alberta

Rawad Lashhab [email protected] Physiology University of Alberta

Xing-Zhen Chen [email protected] Physiology University of Alberta

Joe Casey [email protected] Biochemistry University of Alberta

Katie Badior [email protected] Biochemistry University of Alberta

Darpan Malhotra [email protected] Biochemistry University of Alberta

Nicolas Touret [email protected] Biochemistry University of Alberta

Ruiqi Cai [email protected] Physiology University of Alberta

xiong liu [email protected] Physiology University of Alberta

Nada alshumaimeri [email protected] Biochemistry University of Alberta

Justin Lee [email protected] Physiology University of Alberta

Allein Plain [email protected] Physiology University of Alberta

Gareth Armanious [email protected] Biochemistry University of Alberta



Swai Mon Khaing [email protected] Biochemistry University of Alberta

Larry Fliegel [email protected] Biochemistry University of Alberta

Rohit Rathore [email protected] Biochemistry University of Alberta

Laine Lysyk [email protected] Biochemistry University of Alberta

M'lynn Fisher [email protected] Biochemistry University of Alberta

Jingfeng Tang [email protected] Hubei University of Technology, China

Cefan Zho [email protected] Hubei University of Technology, China

Notes page 46

Notes

Notes page 47

international research training group · 7:00 pm-9:00 pm poster session and cash bar max bell rooms...

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