5th Joint Symposium University of Alberta Edmonton, Alberta, … · 2016. 8. 29. · 5th Joint Symposium University of Alberta Edmonton, Alberta, Canada September 6th to 8th, 2016

INTERNATIONAL RESEARCH TRAINING GROUP

5th Joint SymposiumUniversity of Alberta

Edmonton, Alberta, CanadaSeptember 6th to 8th, 2016

GCCANADIANGERMAN

COMPLEX MEMBRANE PROTEINSIN CELLULAR DEVELOPMENT AND DISEASE

PROGRAM INDEXpage

INTERNATIONAL RESEARCH TRAINING GROUP

GCCANADIANGERMAN

COMPLEX MEMBRANE PROTEINSIN CELLULAR DEVELOPMENT AND DISEASE

5th Joint SymposiumUniversity of Alberta

Edmonton, Alberta, CanadaSeptember 6th to 8th, 2016

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Quick Schedule

Maps & Information

Program

Guidance Committee

Speaker Abstracts

Poster Presentations

Poster Abstracts

Contacts

Appendix

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Tuesday Sept 6th Wednesday Sept 7th Thursday Sept 8thMonday Sept 5th

Quick Shedule

Lister Hall

Varscona Hotel

Mixer

Devaney’s Irish PubLower level

(see map page 2)

Mixer

O’Byrne’sIrish Pub

(see map page 2)

Arrival

RegistrationLi Ka Shing Building

Lobby

Session IOborowski Degner Seminar Hall

Session IIIOborowski Degner Seminar Hall

Session IIIOborowski Degner Seminar Hall

Session IV

Session V

Concluding Remarks

Trainee & SupervisorDiscussions

Oborowski Degner Seminar Hall

Session II

Activities

Activities

Oborowski Degner Seminar Hall

coffee break

coffee break

coffee break

coffee break

Lunch and Poster SessionKatz Atrium



Dinner BBQKatz AtriumEmily Murphy Park

site 1(map page 4)

Coffee / BreakfastLi Ka Shing Lobby

Coffee / BreakfastLi Ka Shing Lobby8:20

Larry Fliegel

Bruce Morgan

Katrin Philippar

Rashmi PanigrahiLaura Hoffmann

Debajyoti DuttaArmin Melnyk

Rawad Lashhab

Andrea Blum

Chitirala PraneethSwai Mon Khaing

Alka KumariShahid Ullah

Megan BeggsSabrina Marz

Wang Zheng

Anna-Maria Miederer

page 1

11613, 87 AvenueEdmonton T6G 2H6see page 3

8208, 106 StreetEdmonton T6E 6R9see page 3

Visit West Edmonton Mall

(see map page 2)

or River Valley Walk(see map page 2 or 4)

for any question contact Skirmante: 780 200 4254 or [email protected]

Trip to Elk Island National Park

Depart fromKatz Atrium

(with car pooling)

or free time

Guidance Committees10 - 11:30

(see page 13)

Maps - overview

Devaney’s Irish Pub 1113 87 Avenue - 780 433 6364

O’Byrne’s Irish Pub 10616 82 Ave NW - 780 414-6766

Overview

Campuspage 3

Emily Murphy

Whyte Ave = 82 Ave

WestEdmonton

Mall

Lister Centre

Varscona Hotel

Varscona Hotel

page 2

O’Byrne’s

Maps - accomodations

from Varscona Hotel to Li Ka Shing20 min walk to University

Shuttle service Diamond Limo: 780-465-4002

from Lister Centre to Li Ka Shing

page 3

MapsLi Ka Shing & Katz Centre Building

Walk to Emily Murphy Park

Li Ka ShingKatz Centre

Atrium

Tom Hortons Seminar room

lobby

page 4

Maps

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page 5

5th IRTG Meeting Program page 7

PROGRAM IRTG in Membrane Biology

Fifth Annual Joint Meeting

University of Alberta, Edmonton Canada

September 6-8, 2016


Monday, September 5, 2016 Arrival in Edmonton, Taxi or shuttle from Edmonton Airport to Hotel Shuttle: http://edmontonskyshuttle.com/ (18 CAD/person), Taxi: (55 CAD)

Accommodations: Lister Centre 11613 87 Ave NW, Edmonton AB T6G 2H6 Lister is 5 minutes away from the Li Ka Shing Building, refer to page 3 for directions to the meeting venues. Contact person for our group is: Sheri Penner Guest Services Coordinator, Reservations 1-044 Lister Centre University of Alberta

Varscona Hotel

8208 106 St NW, Edmonton AB T6E 6R9 The hotel Varscona is approx. 20 min walking distance to the meeting venue, Li Ka Shing Building (see the map page 3 of this booklet). There is also a shuttle service to the university operated by Diamond Limo until 9 am from Varscona hotel. The service is on first come, first served basis, book in advance at 780-465-4002 (ask for Diamond). Other option would be a taxi, which the hotel can book for the guests. Contact person for the IRTG group at Varscona hotel is Ankush Chodvadiya. 19:30 - 21:30 Mixer in Devaney's Irish Pub, 1113 87 Ave NW, Edmonton, 780-433-6364


Day One - Tuesday, September 6, 2016

07:30-08:20 Arrival and Registration Coffee/Breakfast

Li Ka Shing Building lobby

Session 1 (Chair: Gareth Armanious), Li Ka Shing Building, Oborowsky Degner Seminar Hall

08:20-08:30 Dr. Larry Fliegel PI IRTG, Biochemistry, University of Alberta

Welcome, Introductory remarks

08:30-09:15 Dr. Bruce Morgan Molecular Cell Biology, TU Kaiserslautern

NOVEL APPROACHES TO IDENTIFY GLUTATHIONE TRANSPORTERS IN THE SECRETORY PATHWAY

09:15-10:00 Dr. Katrin Philippar Plant Biochemistry and Physiology, LMU Munich

FUNCTION OF PLASTID FATTY ACID EXPORT (FAX) PROTEINS IN DEVELOPMENT AND CELLULAR LIPID DISTRIBUTION

10:00-10:30 Coffee break

Session 2: Membrane Proteins and Immune Function (Chair: Marie-Christine Klein) Li Ka Shing Building, Oborowsky Degner Seminar Hall

10:30-11:00 Chitirala Praneeth Physiology, Saarland University (J. Rettig group)

DESIGNING AN ORGANELLE SPECIFIC CALCIUM AND PH SENSOR TO MEASURE CALCIUM AND PH CONCENTRATIONS INSIDE LYTIC GRANULES OF MURINE CYTOTOXIC T CELLS

11:00-11:30 Swai Mon Khaing Biochemistry, University of Alberta (N. Touret group)

MECHANISM OF CONTROL OF CD36 NANOCLUSTERING BY F-ACTIN AND LIPID NANODOMAINS

11:30 – 13:00 Lunch (sandwich lunch) and poster viewing, KATZ building Atrium

13:00 – 17:30 Trip to West Edmonton Mall by public transit

or hike to river valley, Legislature, via High Level Bridge (Depart from KATZ Atrium for both trips)

17:30 – 19:00 Dinner at KATZ Atrium (Bridges catering)


Day Two - Wednesday, September 7, 2016

07:30 – 08:30 Breakfast/Coffee, Li Ka Shing Building lobby Session 3: Structure/Function Studies of Membrane Proteins (Chair: Sara Schwarz), Li Ka Shing Building, Oborowsky Degner Seminar Hall

08:30-09:00 Dr. Rashmi Panigrahi Biochemistry, University of Alberta (J. Lemieux group)

MECHANISTIC INSIGHT INTO INTRAMEMBRANE ENZYME DIACYLGLYCEROL TRANSFERASE 1

09:00-09:30 Laura Hoffmann Pharmacology and Toxicology (V.Flockerzi group)

IMPACT OF CONSERVED AMINO ACID RESIDUES ON CHANNEL ACTIVITY OF DISTANTLY RELATED TRPs

10:00-10:30 Dr. Debajyoti Dutta Biochemistry, University of Alberta (L. Fliegel group)

CHARACTERIZATION OF TM-11 HELIX OF SpNHE1: THE Na+ /H+ EXCHANGER OF SCHIZOSACCHAROMYCES POMBE

10:00 – 11:30 Coffee / Supervisory Committee Meetings

Office J. Casey

KATZ 4-020E

Office N. Touret

KATZ 4-020H Lunch room KATZ 4-002

Meeting room KATZ 4-125

10:00-10:20 A. Blum M. Schmitt J. Rettig J. Casey

A. Erdogan V. Flockerzi J. Riemer J. Weiner

M.-C. Klein R. Zimmermann B. Niemeyer X.-Z. Chen

D. Hickl T. Möhlmann S. Keller J. Lemieux

10:20-10:40 H. Sarder M. Schmitt E. Neuhaus E. Cordat

S. Marz E. Friauf V. Flockerzi T. Alexander

A. Melnyk R. Zimmermann J. Herrmann X.-Z. Chen

B. Danielczak S. Keller T. Möhlmann H. Young

10:40-11:00 L. Ohler T. Möhlmann E. Neuhaus J. Lemieux

L. Hofmann V. Flockerzi E. Friauf X.-Z. Chen

A.-M. Miederer B. Niemeyer R. Zimmermann L. Fliegel

A Grethen S. Keller J. Engel M. Overduin

11:00-11:20 S. Schwartz M. Schmitt J. Riemer J. Lemieux

P. Chitirala J. Rettig V. Flockerzi J. Casey

R. Stutz R. Zimmermann B. Niemeyer E. Cordat

K. Patzke E. Neuhaus T. Möhlmann

11:30 – 12:30 Lunch (pizza lunch) and poster viewing KATZ Atrium

12:30 – 17:00 Trip to Elk Island National Park or free time

(Depart from KATZ Atrium for Elk Island trip)

18:00 – 20:30 BBQ Dinner in Emily Murhpy Park, Picnic Site #1, with musical performance by Impact Drum and Bugle Corps


Day Three - Thursday, September 8, 2016

08:00 – 09:00 Breakfast/Coffee, Li Ka Shing Building lobby

Session 4: Cell Biology of Membrane Proteins (Chair: Hasib Sarder) Li Ka Shing Building, Oborowsky Degner Seminar Hall

09:00-09:30 Armin Melnyk Medical Biochemistry and Molecular Biology, Saarland University (R. Zimmermann group)

SEALING THE HUMAN SEC61 TRANSLOCATION CHANNEL - LUMINAL CHAPERONE BIP AND THE IMPACT OF THE CO-CHAPERONE SPECIFICITY

09:30-10:00 Rawad Lashhab Physiology, University of Alberta (E. Cordat group)

ROLE OF kAE1/CLAUDIN-4 INTERACTION IN MAINTAINING ACID/BASE AND ELECTROLYTE HOMEOSTASIS

10:00-10:30 Andrea Blum Cell & Molcular Biology, Saarland University (M. Schmitt group)

FUNCTIONAL ANALYSIS OF KDEL RECEPTORS IN YEAST AND MAMMALIAN CELLS

10:30 – 11:00 Coffee break

Session 5: Diseased Membrane Proteins (Chair: Darpan Malhotra) Li Ka Shing Building building, Oborowsky Degner Seminar Hall

11:00-11:30 Dr. Alka Kumari Biochemistry, University of Alberta (group J. Casey)

A PERSONALIZED MEDICINE APPROACH TO CORNEAL DYSTROPHY: ASSESSMENT OF ALL REPORTED MISSENSE SLC4A11 MUTANTS AS CANDIDATES FOR FOLDING CORRECTION

11:30-12:00 Shahid Ullah Physiology, University of Alberta (group E.Cordat)

SLC26A7 DOES NOT COMPENSATE THE LOSS OF KAE1 IN DISTAL RENAL TUBULAR ACIDOSIS DUE TO ITS PH DEPENDENCY

12:00 – 13:30 Lunch and poster viewing, KATZ Atrium


Session 6: Calcium Homeostasis (Chair: Joe Primeau), Li Ka Shing Building building, Oborowsky Degner Seminar Hall

13:30-14:00 Megan Beggs Pediatrics, University of Alberta (group T. Alexander)

NOVEL CALCIUM TRANSPORT PATHWAYS MEDIATE INTESTINAL CALCIUM ABSORPTION PRE-WEANING

14:00-14:30 Sabrina Marz Animal Physiology, TU Kaiserslautern (group E. Friauf)

CALCIUM DEPENDENT SECRETION ACTIVATOR 1 MOST LIKELY INTERACTS WITH GLYCINE TRANSPORTER 2

14:30 – 15:00 Coffee break

15:00-15:30 Wang Zheng Physiology, University of Alberta (group X.-Z. Chen)

EVOLUTIONARILY CONSERVED INTRACELLULAR GATE OF TRANSIENT RECEPTOR POTENTIAL CHANNELS

15:30-16:00 Anna-Maria Miederer Biophysics, Saarland University (group B. Niemeyer)

PROTEOMIC SCREEN FOR NOVEL STIM2 INTERACTION PARTNERS

16:00 – 17:00 Trainee Meetings / Principal Investigator Discussions

(rooms: Oborowsky Hall for PI meeting KATZ 4003, KATZ 4-125, KATZ 4020 for trainee meetings)

17:00-17:15 Concluding remarks

Li Ka Shing Building building, Oborowsky Degner Seminar Hall

EndofMeeting

19:00-21:00 Mixer at O’Byrne’s Irish Pub, 10616 Whyte Ave

Friday, September 9, 2016

10:00 - 12:00 Industrial visit for IRTG trainees to biotech company Phytola with presentations by Drs. Randall Weselake, Ela Mietkiewska and Saleh Shah

(meet at 9:45 am in the lobby of Lister Centre and walk together to Phytola)

5th IRTG Meeting Guidance Committee Meetings page 13

GUIDANCE COMMITTEES

5th IRTG Meeting Guidance Committee Meetings page 14

Guidance Committee Meetings

Wednesday, September 7, 2016

PhD student Main Supervisor Co-Supervisor (D) Co-Supervisor (C) 1. Blum, Andrea M. Schmitt J. Rettig J. Casey 2. Chitirala, Praneeth J. Rettig V. Flockerzi J. Casey 3. Danielczak, Bartholomäus S. Keller T. Möhlmann H. Young 4. Erdogan, Alican J. Riemer V. Flockerzi J. Weiner 5. Finger, Yannik J. Riemer - - 6. Grethen, Anne S. Keller J. Engel M. Overduin 7. Hickl, Daniel T. Möhlmann S. Keller J. Lemieux 8. Hofmann, Laura V. Flockerzi E. Friauf X.-Z. Chen 9. Klein, Marie-Christine R. Zimmermann B. Niemeyer X.-Z. Chen 10. Marz, Sabrina E. Friauf V. Flockerzi T. Alexander 11. Melnyk, Armin R. Zimmermann J. Herrmann X.-Z. Chen 12. Miederer, Anna-Maria B. Niemeyer R. Zimmermann L. Fliegel 13. Ohler, Lisa T. Möhlmann E. Neuhaus J. Lemieux 14. Patzke, Kathrin E. Neuhaus T. Möhlmann 15. Sarder, Hasib M. Schmitt E. Neuhaus E. Cordat 16. Schwartz, Sara M. Schmitt J. Riemer J. Lemieux 17. Stutz, Regine R. Zimmermann B. Niemeyer E. Cordat Schedule

Office J. Casey

KATZ 4-020E

Office N. Touret

KATZ 4-020H Lunch room KATZ 4-002

Meeting room KATZ 4-125

10:00-10:20 A. Blum M. Schmitt J. Rettig J. Casey

A. Erdogan V. Flockerzi J. Riemer J. Weiner

M.-C. Klein R. Zimmermann B. Niemeyer X.-Z. Chen

D. Hickl T. Möhlmann S. Keller J. Lemieux

10:20-10:40 H. Sarder M. Schmitt E. Neuhaus E. Cordat

S. Marz E. Friauf V. Flockerzi T. Alexander

A. Melnyk R. Zimmermann J. Herrmann X.-Z. Chen

B. Danielczak S. Keller T. Möhlmann H. Young

10:40-11:00 L. Ohler T. Möhlmann E. Neuhaus J. Lemieux

L. Hofmann V. Flockerzi E. Friauf X.-Z. Chen

A.-M. Miederer B. Niemeyer R. Zimmermann L. Fliegel

A Grethen S. Keller J. Engel M. Overduin

11:00-11:20 S. Schwartz M. Schmitt J. Riemer J. Lemieux

P. Chitirala J. Rettig V. Flockerzi J. Casey

R. Stutz R. Zimmermann B. Niemeyer E. Cordat

K. Patzke E. Neuhaus T. Möhlmann

5th IRTG Meeting Speaker Abstracts page 15

SPEAKER ABSTRACTS IN CHRONOLOGICAL ORDER


SESSION 1 (Chair: Gareth Armanious) Tuesday, 6 September 2016 8:30 – 9:15 NOVEL APPROACHES TO IDENTIFY GLUTATHIONE TRANSPORTERS IN THE SECRETORY PATHWAY

Bruce Morgan, Cellular Biochemistry, University of Kaiserslautern, Kaiserslautern, Germany.

Glutathione is a ubiquitous tripeptide found in all eukaryotes and many prokaryotes. Glutathione is present at

high concentrations (1-10 mM) in cells and tissues and plays important roles in diverse cellular processes including iron homeostasis and the detoxification of toxic electrophiles, heavy metals and certain reactive oxygen species. In yeast and mammalian cells glutathione is synthesized exclusively in the cytosol yet is found in virtually all cellular organelles and compartments thereby implying that there must be glutathione transport across intracellular membranes. Surprisingly, the identity of the glutathione transporters in most intracellular membranes remains unknown. This is particularly true of the secretory pathway, despite extensive efforts to identify transporters for example in the endoplasmic reticulum (ER). We are applying a range of techniques to investigate glutathione homeostasis in the ER, in particular focusing on the extent of communication between the ER and cytosol glutathione pools and the identification of candidate glutathione transporters.

In a first approach we have developed novel genetically-encoded reporters of the glutathione redox potential, which we have targeted to the ER and cytosol in yeast. We have performed real-time dynamic measurements of the response of ER and cytosolic glutathione pools to appropriate chemical and genetic manipulations. Our preliminary data suggests that the both the steady state glutathione redox potential in the ER, and the dynamic response to perturbation, closely mimics that of the cytosol suggesting that there is rapid and dynamic movement of glutathione across the ER membrane. Furthermore, by differential targeting of Gsh1 and Gsh2, the proteins responsible for glutathione synthesis, we observe that glutathione synthesis in the ER is sufficient to maintain cellular viability. We plan to cross our strains with the yeast gene deletion library to screen for candidate glutathione transporters.

Supported by EMBO, DKFZ, BioComp and TU Nachwuchsring. 9:15 – 10:00 FUNCTION OF PLASTID FATTY ACID EXPORT (FAX) PROTEINS IN DEVELOPMENT AND CELLULAR LIPID DISTRIBUTION

Katrin Philippar. DFG-Heisenberg group "Plastid fatty acid & iron transport", Department Biology I - Botany, Biocenter LMU Munich, Großhaderner-Str. 2-4, D-82152 Planegg-Martinsried, Germany.

Fatty acids (FAs) are building blocks for the majority of cellular lipids, which are essential throughout life of all

organisms. Besides their role as constituents of biological membranes, plant acyl-lipids are used for diverse functions at different destinations and tissues. Thus, membrane transport and distribution of FAs and lipids is crucial for plant growth and development. Further, plant-derived lipid compounds are of biotechnological importance, e.g. for production of biodiesel or nutrient improvement. Since plant de novo FA synthesis essentially takes place in plastids, export to the endoplasmatic reticulum (ER) for acyl editing and lipid assembly is necessary. Although it is generally agreed that free FAs are shuttled across plastid envelope membranes, the mode of export - simple diffusion or protein-mediated - still is a matter of debate. The identification of FAX1, a novel protein for FA-export across the inner envelope of chloroplasts, gave an answer to this question. FA-transport function of FAX1 as demonstrated in yeast cells is crucial for plant biomass production, male fertility and synthesis of FA-derived compounds such as lipids, ketone waxes, or pollen cell wall material. ER-derived lipids decrease when FAX1 is missing, but levels of plastid-produced species increase. FAX1 over-expressing lines show the opposite, including a pronounced increase of TAG oils in flowers and leaves. In the model plant Arabidopsis, 7 proteins belong to the FAX family and since besides FAX1 also FAX2 and FAX3 are predicted to be plastid targeted, tissue-specific expression and/or complementary functions are likely. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems with potential impact on FA/lipid metabolism and/or disease in vertebrates. Supported by DFG.


SESSION 2: Membrane Proteins and Immune Function (Chair: Marie-Christine Klein)10:30 – 11:00 DESIGNING AN ORGANELLE SPECIFIC CALCIUM AND PH SENSOR TO MEASURE CALCIUM AND PH CONCENTRATIONS INSIDE LYTIC GRANULES OF MURINE CYTOTOXIC T CELLS

Praneeth Chitirala, Dr. Jens Rettig lab, Institute of Physiology and Center for Integrative Physiology and Molecular Medicine (CIPMM), Saarland University, Homburg/Saar, Germany.

Cytotoxic T lymphocytes (CTLs) play an important role in our body's immune system. Their main effector

function is to recognize and destroy viral-infected and tumorigenic target cells. They contain cytotoxic proteins such as granzymes and perforin in specialized secretory granules termed cytotoxic granules (CGs). CG contents are released upon target cell recognition at the highly dynamic CTL:target cell interface called the immune synapse. Ca2+ signaling inside the CTL is essential for its activation, effector function, tolerance of self-antigens and homeostasis. Despite it being such an important parameter, the Ca2+ concentration inside CGs is unknown. We aim to estimate the Ca2+

concentration inside CGs and examine how this concentration regulates CG function. To this end, we have generated organelle-specific Ca2+and pH sensors, which we attempt to use as a tool to calculate the absolute calcium concentration and pH of CGs. We selected ratiometric, FRET based Troponin C Ca2+ sensors (Twitch calcium sensors), which have a Kd ranging from 150 nM to 9.5 µM (Griesbeck et al., 2014). The Ca2+ sensors were fused to the C-terminus of the CG membrane protein Synaptobrevin2 (Syb2) targeting the sensor into the lumen of the CG. The Ca2+ concentration inside the CG was measured by generating in vivo calibration curves at pH 7.3 using Ionomycin. However, since the pH of CGs is acidic, knowledge of the absolute pH inside CGs is required for the calibration of the Ca2+ sensor. Therefore, the ratiometric pH sensor clopHensorN(Q69M) was generated as a fusion protein to the C-terminus of granzymeB (GraB-clopHensorN(Q69M)). The correct localization was verified by co-staining with a granzymeB antibody and by co-transfection with either Syb2-Twitch or granzymeB-mTFP. Using GraB-clopHensorN(Q69M), the pH in CGs was found to be 5.9 ± 0.2. RT-PCR in CTLs showed that the V0 domain subunits a1, a2 and a3 are expressed. Next, we aim to investigate which a-isoform of the V-ATPase is functional for CGs by down-regulating the expression of the three subunit isoforms using RNAi and then measuring CG pH using GraB-clopHensorN(Q69M). Furthermore, we would like to calibrate the Ca2+ sensor at pH 6 to obtain the absolute Ca2+ concentration inside CGs. We also aim to stimulate CTLs to induce CG fusion and then measure the Ca2+ concentration in CGs during maturation. Supported by DFG (IRTG 1830) 11:00 – 11:30 MECHANISM OF CONTORL OF CD36 NANOCLUSTERING BY F-ACTIN AND LIPID NANODOMAINS

Swai Khaing, and Nicolas Touret. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7.

CD36, a multi-ligand plasma membrane receptor, has been implicated in immunity, metabolism and angiogenesis. We have recently demonstrated that CD36 nanoclustering at the plasma membrane is key to the initiation of CD36 signaling. In endothelial cells (ECs), the binding of thrombospondin-1 (TSP-1, an endogenous extracellular matrix anti-angiogenic factor) to CD36 nanoclusters activates an associated Src family kinase, Fyn, leading to ECs apoptosis, hence, inhibiting angiogenesis. CD36-Fyn association at the plasma membrane is primarily over region rich in filamentous actin (F-actin) and its depolymerization results in inhibition of signalling. We are interested in elucidating the mechanisms and functions of CD36-Fyn enrichment on the actin cytoskeleton during TSP-1induced signaling in ECs.

We hypothesize that lipid nanodomains play a role in bringing together CD36-Fyn to F-actin regions through adaptor molecules, and that it enables the formation of signaling platform necessary for signal transduction. Using microscopy methods on HeLa cells co-transfected with Fyn and various fluorescent lipid biosensors and stained for F-actin (Phalloidin-AF647), we determined that Fyn is enriched on F-actin area at sites of phosphatidylinositol 4,5-bisphosphate enrichment (PIP2). During TSP-1 stimulation on Human Microvascular Endothelial Cells (HMEC), the CD36-Fyn-F-actin enrichment shift to domains containing PI(3,4,5)P3, suggesting a role for the phosphoinositide 3-kinase in signaling. The role of this kinase will be further investigated using inhibitor or RNA depletion methods targeting PI3-Kinase subunits. Furthermore, to characterize the adaptor molecules involved in connecting F-actin to lipid nanodomains and/or CD36 nanoclusters, we are conducting fractionation and immunoprecipitation approaches followed


by mass spectrometry (MS) analysis. This method relies on the separation of soluble cytosolic and membrane components from those associated with F-actin or CD36, and identification of proteins by proteomics. The screen will be further narrow down on proteins known to contain lipid binding domains and/or known to have lipid interacting partners. Altogether, our investigation will enable the characterization of a cellular components regulating the formation and activity of plasma membrane receptor nanoclusters.

Supported by IRTG and CIHR. SESSION 3: Structure/Function Studies of Membrane Proteins (Chair: Sara Schwarz) Wednesday, 7 September 2016 08:30 – 09:00 MECHANISTIC INSIGHT INTO INTRAMEMBRANE ENZYME DIACYLGLYCEROL TRANSFERASE1

Rashmi Panigrahi1, Kristian Mark P. Caldo2, Wei Shen3, Jeella Acedo4, Yang Xu1, Tsutomu Matsui5 Andrew Song1, Linda Hanely-Bowdoin3, John C. Vederas4, Randall J. Weselake2 and M. Joanne Lemieux1

1Department of Biochemistry, University of Alberta, Canada T6G 2H7 2Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 3Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA 27695 4Department of Chemistry, University of Alberta, Canada T6G 2G2 5Stanford Synchrotron Radiation Lightsource, California 94025

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent formation of triacylglycerol (TAG)

and the activity of the enzyme may have a substantial effect on the flow of carbon into the seed oil of oleaginous plants. DGAT1 is a unique and essential membrane –bound enzyme that shares no homology with other family members involved in TAG production. Recombinant DGAT1 from oilseed rape (Brassica napus) (BnaDGAT1) was used for the study. It was purified in active form, reconstituted in phospholipids which increase its activity. The enzyme exhibited a hyperbolic response in activity to increasing bulk concentration of sn-1, 2-dioleoylglycerol. The enzyme demonstrated allostery exhibiting positive cooperativity. Previous research by our group has shown that an allosteric exosite for acyl-CoA is present in the hydrophilic N-terminal region of the enzyme. An N-terminal truncation of the enzyme, consisting of residues 1-113, was mainly random coil and could interact with oleoyl-CoA displaying positive cooperativity. In silico and in vitro studies demonstrated the truncated construct 81-113 was the most structured region of the N terminus possessing the necessary attributes to interact with acyl-CoA. Further activators were identified for BnaDGAT1 effective at micromolar concentration. Interestingly, it was identified that phosphorylation decreased BnDGAT1 activity. Our findings pave way to plausible development of strategies for increasing TAG content in plant biomass.

Supported by MITACS, IRTG

09:00 – 09:30 IMPACT OF CONSERVED AMINO ACID RESIDUES ON CHANNEL ACTIVITY OF DISTANTLY RELATED TRPs

Laura Hofmann, Wang Zheng, Xing-Zhen Chen and Veit Flockerzi. Department of Experimental and Clinical Pharmacology, Saarland University, 66421 Homburg; Department of Physiology, 7-29A Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada

The < 4 Å structure of the rat TRPV1 channel published in 2013 by Liao et al. was only the starting point to

solve an additional number of high-resolution TRP structures, the structures for TRPA1 (Paulsen et al., 2015), TRPV2 (Zubcevic et al., 2016) and TRPV6 (Saotome et al., 2016). From these data it is clear that four TRP proteins co-assemble to functional channels. At the same time they give detailed insights into intra- and intermolecular interactions of the functional channel complex. On basis of these structures which resolve interacting amino acids on a single amino acid level it is possible to narrow down putative inter- and intramolecular interaction sites. By site directed mutagenesis and expression of mutated cDNAs followed by functional characterization of the channels, these structural information can be directly correlated with channel function in vivo. Furthermore open state structures of TRPV1 induced by potent agonists are available allowing a deeper understanding of the mechanism of channel gating.


Our aim is to identify conserved amino acids within the TRP channel superfamily contributing to common mechanisms in TRP channel activity. Based on sequence alignments we examined conserved amino acid residues in the N-terminal pre-S1 helix, the S6 and C-terminal TRP domains of TRPC4, TRPC1, TRPM4, TRPV6 and other TRP members. Within these domains we focused on conserved tryptophan (W) residues which can be found in the pre-S1 domain as well as in the TRP-like domain. The corresponding and additional mutations have been inserted into the mTRPC4α, mTRPM4 and hTRPV6 cDNAs, the cDNAs were subcloned into the mammalian bicistronic TRP-IRES-GFP expression vector to yield independent TRP and GFP transcripts. Ca Imaging and Patch Clamp experiments were carried out to characterize mutant and wild-type channel function. Interestingly the replacement of the invariant tryptophan residues by alanine within the TRP domains of mTRPC4α, mTRPM4 and hTRPV6 cDNA, are gain-of-function mutations. One TRPC member, TRPC1, apparently does not form functional channels by itself. With the constant aim to bring also homotetrameric TRPC1 channels to life we are currently introducing the various “gain-of-function” mutations including the replacement of tryptophan residues by alanine into the mTRPC1 cDNA. In general the pursued approach supports the idea derived from the structure of TRPV1, that the TRP domain is a lever to affect the channel pore.

Supported by DFG (IRTG 1830) 10:00 –10:30 CHARACTERIZATION OF TM-11 HELIX OF SpNHE1: THE Na+/H+ EXCHANGER OF SCHIZOSACCHAROMYCES POMBE

Debajyoti Dutta and Larry Fliegel Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

With the emerging problems in the changing global environment, soil salinity poses an even more serious

challenge in agriculture. The Na+/H+ exchanger (NHE) plays a critical role in controlling the resistance to salinity in plant cells. The fission yeast S. pombe is used as a model organism to study salt tolerance. S. pombe has a limited number of Na+/H+ exchangers and its NHE1 protein has close sequence homology (31%) with the major plant Na+/H+ exchanger SOS1. SpNhe1 deletion results in a salt sensitive phenotype. SpNhe1 has 12 transmembrane (TM 1-12) segments and two transmembrane segments of SpNhe1, TM-4 and TM-11, are predicted to be critical in Na+ transport. We chose to explore the role of TM-11 by alanine scanning site - specific mutagenesis. SpNhe1 mutants were expressed and characterized in a S. pombe strain with the endogenous SpNHE1 knocked out. Growth in sodium and lithium containing medium was used to characterize SpNhe1 activity. Wild type SpNhe1 protein restored growth to the knockout strain in liquid media. Cells were able to grow well in solution containing 500 mM NaCl. In contrast the knock out strain showed no growth in 500 mM NaCl and compromised growth in 200 mM NaCl. When amino acids K360 and E361 were mutated to Ala, there was severe impairment of growth in high concentrations of NaCl, almost comparable to the knock out strain. Mutation of amino acid H367A also caused growth to be largely compromised in 500 mM NaCl. Several other mutants had more minor effects on the ability to tolerate higher concentrations of NaCl. On solid media, the pattern of effects of the mutations on the ability to confer salt tolerance was largely the same as in liquid media. Mutant proteins with the K360A and E361A were unable to convey NaCl tolerance. Additionally, the H367A, K383A and L386A mutant proteins were largely ineffective in conferring NaCl tolerance. More minor, but consistent effects were shown by the F364A, I371A, G372A, Y377A and F380A. Initial studies also suggest amino acids L381, L384 are critical for Li+ tolerance. Overall, results show that TM XI is a critical transmembrane segment modulating SpNHE1 cation transport. Supported by NSERC and the International Research Training Group.


SESSION 4: Cell Biology of Membrane Proteins (Chair: Hasib Sarder) Thursday, 8 September 2016 09:00 – 09:30 SEALING THE HUMAN SEC61 TRANSLOCATION CHANNEL - LUMINAL CHAPERONE BIP AND THE IMPACT OF THE CO-CHAPERONE SPECIFICITY

Armin Melnyk1, Stefan Schorr1, Nico Schäuble1, Martin Jung1 & Richard Zimmermann1

¹Department Of Medical Biochemistry And Molecular Biology, Saarland University, Building 44 Homburg 66421, Germany.

Beside its relevance for protein synthesis and folding the mammalian endoplasmic reticulum (ER) constitutes

also a main storage compartment for free calcium ions (Ca2+). During translocation of newly synthesized proteins into the ER lumen by the membrane resident Sec61 complex calcium ions leak from the ER into the cytosol due to the ion concentration gradient between ER and cytosol. In previous work, calmodulin was shown to bind to the cytosol located IQ motif of Sec61α in a calcium dependent manner, thus leading to channel closure after calcium has started to leak out. Furthermore, the luminal Hsp70 chaperone BiP, an essential component of the translocation machinery, has been shown to counteract the loss of Ca²+ during translocation by binding to the ER luminal loop7 of the Sec61 channel and closing of the latter. To investigate and characterize this interaction of BiP and Sec61α we performed binding studies such as surface plasmon resonance (SPR) spectroscopy, peptide arrays, pulldown- and photo-cross-linking experiments. Based on the findings that Kar2p, the yeast orthologue of BiP as well as a BiP mutant which does not bind the J-domain of Hsp40 co-chaperones are both unable to seal the channel, we focused on the interactome of the luminal chaperone network, to gain knowledge about the mechanism of recruiting BiP to the Sec61 channel. In combination with live-cell Ca2+ imaging we identified two Hsp40 co-chaperones, ERj3 and ERj6 whose depletion lead to an increased cytosolic calcium influx which is even more pronounced after simultaneously depletion of BiP, which leads us to the model that these two ERj-proteins are co-chaperones of BiP in limiting ER Ca²+ leakage at the level of the Sec61 complex.

Supported by DFG 09:30 – 10:00 ROLE OF kAE1/CLAUDIN-4 INTERACTION IN MAINTAINING ACID/BASE AND ELECTROLYTE HOMEOSTASIS

Rawad Lashhab, Alina Rumley, Ensaf Almomani, Emmanuelle Cordat

Department of Physiology. Membrane Protein Diseases Research Group. University of Alberta, Edmonton, AB, Canada. Bicarbonate transporters play an essential role in maintaining a neutral plasma pH. One of these transporters, the

kidney Anion exchanger 1 (kAE1), is located at the basolateral membrane of Type A-Intercalated Cells (A-IC) in the collecting duct of the kidney, and functions as Cl-/HCO3

- exchanger. A yeast two-hybrid assay showed that kAE1 interacts with Claudin-4 (Cldn-4). Cldn-4 is a membrane protein located in the tight junctions of A-IC. It acts as a paracellular Cl- pore by facilitating Cl- reabsorption in the collecting duct. We hypothesized that kAE1/Cldn-4 interaction regulates pH and electrolyte homeostasis. In polarized renal epithelial cells expressing kAE1-wild type (kAE1-WT), immunofluorescence and Proximity Ligation Assays showed a co-localization between kAE1 and Cldn-4 at the basolateral membrane. Immunoprecipitations confirmed the interaction. Although qRT-PCR results showed a slight increase in Cldn-4 mRNA, immunoblots showed no effect on Cldn-4 total protein expression upon kAE1 expression. Ussing Chamber experiments revealed a decreased trans-epithelial electrical resistance (TEER) and an increased paracellular Na+, Cl- & HCO3

- permeability upon kAE1 expression. Our data support that kAE1 alters tight junction properties independent of changes in intracellular pH. Our results confirm the interaction between kAE1 and Cldn-4 and support a role for this interaction in electrolyte homeostasis, acid/base balance and possibly in hypertension, a condition that affects a third of North American adults. Supported by CIHR, the Canadian Foundation for Innovation, the Kidney Foundation of Canada, the NSERC CREATE Program & IRTG.


10:00 – 10:30 FUNCTIONAL ANALYSIS OF KDEL RECEPTORS IN YEAST AND MAMMALIAN CELLS

Andrea Blum and Manfred Schmitt, Molecular & Cell Biology, Department of Biosciences, Saarland University, D-66123 Saarbrücken, Germany.

A/B toxins such as cholera toxin, Pseudomonas exotoxin and killer toxin K28 contain a KDEL-like motif at one of their subunits which ensures retrograde toxin transport through the secretory pathway of a target cell. As key step in host cell invasion, each toxin binds to plasma membrane (PM) receptors that are utilized for cell entry. We recently identified Erd2p, the yeast KDEL receptor (KDELR), as PM receptor of the viral K28 toxin carrying a C-terminal HDEL motif at its cell binding β-subunit. Consistent with its function at the cell surface, Erd2p was demonstrated to colocalize in the PM and to internalize H/KDEL-cargo such as K28 toxin, GFPHDEL and Kar2p. While PM localization of Erd2p was demonstrated by live cell imaging and cell surface biotinylation, we are currently extending these studies by immunogold labeling and EM imaging. In an approach to identify components involved in antero- and retrograde KDELR transport and endocytic internalization, we aim to establish an APEX screen and protein proximity labeling in yeast. Since it is unknown how cells ensure that a major KDELR fraction can be retained in the ER and Golgi while a minor fraction resides in the PM, we analyzed the lysine cluster near the C-terminus of Erd2p for its potential function as KKXX-like retention motif. Our studies on KDELR dynamics in mammalian cells revealed that KDEL-cargo binding induces dose-dependent receptor cluster formation and subsequent KDELR internalization from the PM which is counteracted by microtubule-assisted anterograde receptor transport to preferred PM docking sites. Since KDELRs have recently been shown to function in intra-Golgi/ER signaling and maintenance of Golgi homeostasis, we propose a similar signaling function of KDELRs after cargo binding at the cell surface. To address these novel functions in more detail, we are currently performing a CRISPR/Cas9-mediated KDELR1 knock-out in HeLa cells for further analysis.

Supported by Saarland University & DFG (IRTG 1830) SESSION 5: Diseased Membrane Proteins (Chair: Darpan Malhotra) Thursday, 8 September 2016 11:00 – 11:30 A PERSONALIZED MEDICINE APPROACH TO CORNEAL DYSTROPHY: ASSESSMENT OF ALL REPORTED MISSENSE SLC4A11 MUTANTS AS CANDIDATES FOR FOLDING CORRECTION

Kumari Alka and Joseph R. Casey. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.

Mutations of SLC4A11 cause genetic endothelial corneal dystrophies: Fuchs endothelial corneal dystrophy,

congenital hereditary endothelial corneal dystrophy type 2 (CHED2) and Harboyan syndrome. The molecular defect associated with these mutations include mis-folding of the protein, leading to its retention in endoplasmic reticulum (ER), mis-trafficking to the apical side of endothelium and a loss of SLC4A11 water flux function. Current treatments for these diseases, including corneal transplant, are inadequate. This is the first attempt to characterize the molecular phenotype of all reported disease-causing mutations of SLC4A11 protein with an overall aim of identifying ER-retained missense mutants that can be trafficked to the cell surface. This will establish the SLC4A11 genotypes of individuals who might benefit from folding correction therapy, setting the stage for personalized medicine. This study measured relative cell surface abundance of 58 SLC4A11 missense mutants using bioluminescence resonance energy transfer (BRET) based assay. 35 out of 58 missense mutants were ER-retained when expressed in HEK293 cells. Interestingly, G709E, G583D, R282P, W240S, R209W, A269V, T271M, T379P, L843P and S213P SLC4A11 mutants exhibited increased processing to the plasma membrane when grown at 30 °C. As a proof-of-concept, the drug glafenine was shown to rescue some of the ER-retained mutants to the cell surface. Immunolocalization with e-cadherin in MDCK cells established basolateral location of SLC4A11 in polarized epithelia. Mis-trafficking of mutant SLC4A11 to the apical membrane or loss of water flux function was investigated as a possible cause of the disease for some of the mutants. Further, the mutants were mapped on the homology model of SLC4A11 based on its processing to the cell membrane. In conclusion, SLC4A11 mutants were categorized as 1. Candidates for folding correction therapy on the basis of their relative abundance at the cell surface and 2. Mutants with no processing defect were further sub-categorized as apically-mistargated or catalytically inactive.

Supported by IRTG and CIHR.


11:30 – 12:00 SLC26A7 DOES NOT COMPENSATE THE LOSS OF KAE1 IN DISTAL RENAL TUBULAR ACIDOSIS DUE TO ITS PH DEPENDENCY

AKM Shahid Ullah1, Carly Rumley1, Valentina Peleh2, Mattia Berrini1, Rawad Lashhab1, R. Todd Alexander1, Johannes Herrmann2, Emmanuelle Cordat1 1Department of Physiology, University of Alberta, Edmonton, AB, Canada; 2Kaiserslautern University, Kaiserslautern, Germany.

SLC26A7 is a basolateral glycoprotein expressed in acid-secreting intercalated cells of the collecting duct, which also contain the kidney chloride/bicarbonate exchanger 1 (kAE1). SLC26A7 has been reported to function either as a chloride/bicarbonate exchanger or a chloride channel. As it is expressed in the same cells as kAE1, we asked why a second anion exchanger is unable to compensate for the loss of kAE1 in patients with distal renal tubular acidosis (dRTA). We found that SLC26A7 abundance increases with extracellular osmolarity and extracellular pH of the growth medium. When co-expressed with kAE1 WT or the dRTA mutant, R901X SLC26A7 chloride/bicarbonate exchange rate is decreased compared to when expressed individually. These results support that acidic extracellular pH destabilizes SLC26A7 protein and provides a plausible explanation for the absence of compensatory bicarbonate reabsorption via SLC26A7 in patients with dRTA due to a mutation in the SLC4A1 gene encoding for kAE1. Supported by NSERC CREATE Program, International Research training Group (IRTG) Canadian Institute of Health Research (CIHR) The Kidney Foundation of Canada Women and Children’s Health Research Institute (WCHRI) Canada Foundation of Innovation SESSION 6: Calcium Homeostasis (Chair: Joe Primeau) 13:30 – 14:00 NOVEL CALCIUM TRANSPORT PATHWAYS MEDIATE INTESTINAL CALCIUM ABSORPTION PRE-WEANING

Megan R. Beggs, Justin J. Lee, R. Todd Alexander Department of Physiology, University of Alberta, Edmonton, Alberta, Canada

Calcium is essential to vital physiological functions including bone mineralization where the rate of deposition peaks in infancy and mineral content peaks by early adulthood. Maintenance of a positive calcium balance is thus crucial during development. Calcium homeostasis is mediated by interactions between the intestine, kidneys, and bones. Intestinal absorption occurs by either an active transcellular or passive paracellular pathway. Currently, the duodenum is thought to be the site of largely transcellular absorption whereas the jejunum and ileum are proposed to mediate exclusively paracellular absorption. We set out to describe calcium absorption pathways pre and post weaning. We hypothesized a transition from predominantly paracellular to transcellular intestinal absorption at weaning (three weeks of age in mice). Wildtype FVB/N mice (n=12) at 7 ages from 1 day to 6 months old were examined. Levels of Trpv6 and CalbD9k mRNA, mediators of transcellular calcium transport, show a six-fold increase between two weeks and one month of age in the duodenum with a corresponding increase in CalbindinD9k protein. In the jejunum and ileum, mediators of transcellular transport – Trpv6, Cav1.3, and CalbD9k – are highly expressed prior to weaning, suggesting novel pathways of calcium absorption during development. Abundance of Cldn-2 and -15 mRNA, mediators of paracellular absorption, peak at 7 days in the duodenum. In the ileum, Cldn-2 mRNA peaks at 14 days with a 10-fold decrease by 1 month, where abundance of Cldn-15 increases 3-fold during this time to peak after weaning. These observations suggest that weaning marks a significant shift in mechanisms mediating calcium transport from the duodenum, jejunum, and ileum. Functional studies using Ussing chambers are required to confirm these expression results. Future studies to delineate the mechanisms underlying the observed changes are required in order to understand how a positive calcium balance is maintained during development.

This research has been funded by NSERC and the generous support of the Stollery Children’s Hospital Foundation through the Women and Children’s Health Research Institute


14:00 – 14:30 CALCIUM-DEPENDENT SECRETION ACTIVATOR 1 MOST LIKELY INTERACTS WITH GLYCINE TRANSPORTER 2

Sabrina Marz1, Mattson Jones1, Claudia Fecher-Trost2, Martin Jung3, and Eckhard Friauf1. 1Animal Physiology Group, Department of Biology, University of Kaiserslautern, D-67663 Kaiserslautern, Germany. 2Institute for Experimental and Clinical Pharmacology and Toxicology, Saarland University, D-66424 Homburg, Germany. 3Medical Biochemistry and Molecular Biology, Saarland University, D-66424 Homburg, Germany.

Glycine is an important inhibitory neurotransmitter in the central nervous system. The neuronal glycine transporter 2 (GlyT2) is localized in presynaptic terminals and mediates glycine uptake from the synaptic cleft, contributing to glycine homeostasis. GlyT2 has received growing attention as potential target for treatment of pain and hyperekplexia. Since protein-protein interactions can regulate protein function, it is mandatory to investigate the interactome of GlyT2. The aim of this study is the establishment of a sophisticated proteomic approach to identify and validate GlyT2 interactors. To do so, co-immunoprecipitations (co-IPs) of GlyT2 and GST-Pull-Down assays of GlyT2 C- and N-termini (CT, NT) with subsequent mass spectrometry were performed in triplicate. 427 proteins were identified as potential interactors. GlyT2 co-IPs of GlyT2+/+ animals revealed 265 exclusive proteins, of which 45 were identified in triplicate. Among these, the calcium-dependent secretion activator 1 (CAPS1) was identified with nine exclusive unique peptides. CAPS1 is essential for trafficking of vesicles, exocytosis of neurotransmitters, and modulates uptake of monoamines. Co-IPs with overexpressed and endogenous protein followed by immunoblotting verified the binding of CAPS1 to GlyT2. Additionally, GlyT2 efficiently bound to CAPS1 in reverse co-IPs. The binding of CAPS1 to GlyT2 is independent of its localization in lipid rafts, where GlyT2 is known to be most active. A peptide spot array was performed, in which GlyT2-CT, -NT and parts of the CAPS1 sequence in 15 amino acids long peptides were spotted onto a membrane and incubated with brain lysate followed by an antibody labelling. The results propose binding of CAPS1 to GlyT2-CT and binding of GlyT2 mainly to Munc homology domain of CAPS1. Biotinylation assays indicate a lower surface expression of GlyT2 when co-expressed with CAPS1, which propose a reduced trafficking of GlyT2. Taken together, our data suggest that CAPS1 likely interacts with GlyT2 and potentially regulates GlyT2 activity. Supported by DFG. 15:00 – 15:30 EVOLUTIONARILY CONSERVED INTRACELLULAR GATE OF TRANSIENT RECEPTOR POTENTIAL CHANNELS

Wang Zheng1, Ruiqi Cai1, Laura Hofmann2, Veit Flockerzi2, and Xing-Zhen Chen1. 1Department of Physiology, University of Alberta, Edmonton, AB, Canada T6G 2H7; 2Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, 66421, Homburg, Germany

Members of the transient receptor potential (TRP) channel superfamily detect a wide range of stimuli from mechanical force to noxious temperature and initiate electrical signals or Ca2+ signaling pathway by opening and closing pore gates. The location of the gate in TRP channels remain unresolved even recently resolved structures of several members including TRPA1, TRPV1, 2, and 6 point to hydrophobic residues in the intracellular part of transmembrane segment 6 (S6). Here, we investigate the gate residue within the sequence “NMLIALM”, a highly conserved motif within S6 segment across all TRP channels. By systematically mutating individual hydrophobic residue to hydrophilic residue asparagine, we find that A558N mutant gained the most significant increased channel activity in TRPP3 in the absence of activator when expressed in Xenopus oocytes, suggesting that A558 residue form the most restricted area along the permeation pore to function as a gate in the closed state. In support of gate function of A558 residue, we further find that TRPP3 channel activity is strongly correlated with hydrophilicity of residues in 558 position when different amino acids were introduced. With similar strategy, we also identified A616 in TRPV6 as the gate residue, which is different with that identified in structures of TRPV1, 2 or 6. In comparison with TRPP3 and TRPV6, we identified the residue I617 but not A618 as the gate in TRPC4. Interestingly, the similar position was shown to be the gate in the structure of TRPV1. Like in TRPC4, we further identified the residue V976 right before A977 to be the gate residue in TRPM8. Taken together, our data show that TRP channels share a similar hydrophobic gate residue, either alanine or isoleucine (valine in TRPM8) within the conserved motif NMLIALM in S6 segment. Supported by NSERC and KFoC. 15:30 – 16:00


PROTEOMIC SCREEN FOR NOVEL STIM2 INTERACTION PARTNERS

Anna-Maria Miederer, Larry Fliegel, Barbara A. Niemeyer.

Molecular Biophysics, Saarland University, 66421 Homburg, Germany.

Store-operated calcium entry (SOCE) is a ubiquitous process of critical relevance in immune cells among

others. In T cells this entry is mainly mediated by STIM1 mediated activation of Orai1 but prominent neuronal expression of the homolog STIM2 shows that STIM2 is the main activator for neuronal SOCE and plays an important role for Ca2+ homeostasis in spine maturation. Much less is known about function and regulation of STIM2 compared to STIM1. We discovered previously a novel inhibitory splice variant of STIM2, named STIM2.1. We choose STIM2.1 and STIM2.2 (previously STIM2) to find common and individual interaction partners for these two known splice variants of STIM2. The strategy involves the cloning and expression of a tagged STIM2 splice specific construct that contain the coding region for ascorbate peroxidase (APEX2). The thereby acquired enzymatic activity allows for localized generation of biotin-phenol radicals that immediately interact with proteins in the vicinity (~20 nm). The biotinylated proteins were detected using different methods like immunocytochemistry and Westernblot to proof the functionality of the approach. The final identification of interaction partners was done using mass spectrometry. Therefore cells were cultivated in SILAC (stable isotope labeling with amino acids in cell culture) media to identify unspecific binding to the beads or background during the mass spectrometry analysis. For the detection of the interaction partners the biotinylated proteins were enriched using streptavidin magnetic beads and subjected to mass spectrometry analysis.

Supported by IRTG1830.

5th IRTG Meeting List of poster presentations page 25

POSTER PRESENTATIONS


List of posters

1 Alshumaimeri, Nada IDENTIFICATION OF PROTEIN-PROTEIN INTERACTORS OF THE CORNEAL DYSTROPHY-CAUSING PROTEIN, SLC4A11

2 Armanious, Gareth FUNCTIONAL UNDERPININGS OF HEREDITARY DCM: FROM CARDIAC CALCIUM HANDLING PROTEINS TO SARCOMERIC SCAFFOLDING PROTEINS

3 Arutyunova, Elena RHOMBOID-MEDIATED INTRAMEMBRANE PROTEOLYSIS OF PINK1 IN THE MITOCHONDRION

4 Baronas, Victoria A. Kv1.2 CHANNELS AT THE INTERFACE OF REDOX AND ELECTRICAL EXCITABILITY

5 Cai, Ruiqi INTRA-MOLECULAR N- AND C- INTERACTION IN TRANSIENT RECEPTOR POTENTIAL POLYCYSTIN-3 (TRPP3) AND CANONICAL-4 (TRPC4) CHANNELS

6 Danielczak, Bartholomäus

POLYMER-MEDIATED MEMBRANE SOLUBILIZATION MONITORED BY MULTI-PARAMETRIC SURFACE PLASMON RESONANCE

7 Erdogan, Alican REDOX PROCESSES IN IMS PROTEIN IMPORT AND COMPLEX I ASSEMBLY

8 Finger, Yannik HUMAN ADENYLATE KINASE HSAK2 IS A SUBSTRATE OF MIA40

9 Grethen, Anne COMPARISON OF MEMBRANE-SOLUBILIZING EFFICIENCIES OF AMPHIPHILIC POLYMERS

10 Hu, Qiaolin INTERACTION BETWEEN THE N- AND C- TERMINI OF THE TRANSIENT RECEPTOR POTENTIAL VANILLOID TYPE 6 (TRPV6) CHANNEL

11 Kaur, Gurnit ELIMINATION OF ARSENIC SPECIES BY SINGLE NUCLEOTIDE POLYMORPHIC VARIANTS OF THE HUMAN MULTIDRUG RESISTANCE PROTEIN 2 (MRP2/ABCC2)

12 Klein, Marie-Christine FUNCTIONAL CHARACTERIZATION OF A COMMON VARIABLE IMMUNE DISEASE-RELATED SEC61A1 MUTATION IN HUMANS

13 Kumari, Alka & Badior, K

SLC4A11 THREE-DIMENSIONAL MODEL EXPLAINS STRUCTURAL BASIS FOR ENDOTHELIAL CORNEAL DYSTROPHY-CAUSING MUTATIONS


14 Kurata, Harley T. SUBUNIT SPECIFICITY AND STOICHIOMETRY OF KCNQ CHANNEL OPENERS

15 Marensi, Vanessa GLUTATHIONE TRANSFERASE P1 (GSTP1) IS MODIFIED BY PALMITATE

16 Ohler, Lisa THE PROTEIN FAMILY OF EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS – CURRENT STATE OF KNOWLEDGE AND FUTURE PERSPECTIVES

17 Overduin, Michael STRUCTURE AND LIPID BINDING FUNCTION OF PLPA AT THE BACTERIAL OUTER MEMBRANE BY SOLUTION STATE NMR

18 Patzke, Kathrin CHLOROPLASTIDIC SUGAR METABOLISM AND IT´S INFLUENCE ON CELLULAR SIGNALING

19 Primeau, Joseph O. CRYSTALS, CLEAVAGE, AND CROSS-LINKS: INVESTIGATING THE SERCA ACCESSORY SITE

20 Panigrahi, Rashmi MECHANISTIC INSIGHT INTO INTRAMEMBRANE ENZYME DIACYLGLYCEROL TRANSFERASE

21 Rehman, Saba MUTATIONS IN CLAUDIN-14 ASSOCIATED WITH HYPERCALCIURIA

22 Sarder, Hasib A. M. DISSECTING INTRACELLULAR TRAFFICKING AND MIS-TRAFFICKING OF HUMAN KIDNEY AE1 IN YEAST AND MAMMALIAN CELLS

23 Schwartz, Sara STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE YEAST VIRAL A/B TOXIN K28

24 Stutz, Regine MECHANISMS OF PROTEIN TRANSPORT INTO THE HUMAN ENDOPLASMIC RETICULUM

25 Wang, Caroline K. STATE- AND USE-DEPENDENT BINDING OF KCNQ CHANNEL OPENERS

26 Wong, Ka Yee STRUCTURAL INVESTIGATION OF THE INTRACELLULAR LOOP OF THE HUMAN SODIUM/HYDROGEN EXCHANGER ISOFORM 1 USING NUCLEAR MAGNECTIC RESONANCE SPECTROSCOPY


5th IRTG Meeting Poster abstracts page 29

POSTER ABSTRACTS IN ALPHABETICAL ORDER


Alshumaimeri, Nada IDENTIFICATION OF PROTEIN-PROTEIN INTERACTORS OF THE CORNEAL DYSTROPHY-CAUSING PROTEIN, SLC4A11

Nada Alshumaimeri, and Joseph Casey. Department of Biochemistry, University of Alberta, Edmonton.

SLC4A11 is a membrane transport protein found at the basolateral surface of corneal endothelial cells.

Mutations of SLC4A11 cause Endothelial Corneal Dystrophies (ECD), specifically Fuchs ECD and Congenital Hereditary Endothelial Dystrophy (CHED), both marked by endothelial cell loss and corneal stromal edema. We hypothesize that protein interactors of SLC4A11 influence the function and localization of SLC4A11. Identification of SLC4A11-protein interactors will lead us to understand the full physiological function of SLC4A11 and role in endothelial corneal dystrophies. The Membrane Yeast two Hybrid (MYTH) technique was used to identify SLC4A11-protein interactors. A cDNA library was constructed from bovine cornea mRNA and cloned into the prey vector (pPR3-N). Yeast were transformed with human SLC4A11 cDNA cloned in bait vector (pCMBV). The ability of cotransformed yeast to grow on auxotrophic selective plates indicated colonies where SLC4A11 (Bait) interacted with prey protein. We screened 60 x 106 total transformants. 431 clones grew as interactors and 273 clones survived after confirmatory screening. Clones were sequenced indicating six unique SLC4A11-membarne protein candidate interactors: Ovarian Carcinoma Immunoreactive Antigen Domain containing protein 1 (OCIAD1), Transmembrane 254 (TMEM 254), Leptin receptor overlapping transcript like 1 (LEPROT1), ORM1 like protein 2 (ORMDL2), CMP sialic acid transporter and Transmembrane 128 (TMEM 128). TM254 protein has no known function and shares sequence similarity with Glycophorin A, a chaperone protein for Band 3 protein (AE1/SLC4A1) we hypothesize that TM254 acts to chaperone the folding and trafficking of SLC4A11 from the site of biosynthesis (endoplasmic reticulum) to the plasma membrane. OCIAD1 is involved in cell-matrix interaction that explains the loss of endothelial cells in ECDs when SLC4A11 is mutated.

Supported by CIHR. Armanious, Gareth FUNCTIONAL UNDERPININGS OF HEREDITARY DCM: FROM CARDIAC CALCIUM HANDLING PROTEINS TO SARCOMERIC SCAFFOLDING PROTEINS

Gareth Armanious, Laine Lysyk, Jessi Bak, and Howard Young. Department of Biochemistry, University of Alberta.

SERCA achieves the majority of the calcium removal from the cytosol of cardiomyocytes by transporting

calcium from the cytosol into the sarcoplasmic reticulum during diastole. 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 SR lumen. This determines the rate of diastole 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 PLB have been identified in patients with heart failure, and are being discovered at an 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. Interestingly, the 4-year-old patient also has a mutation in myosin binding protein C3 (MYBPC3) listed as “likely benign” that may contribute to the surprisingly young age of diagnosis. The effects that these variants of PLN have on the kinetics of SERCA, PKA, and PP1 is currently under investigation. P21T PLN showed increased helical content by CD compared to WT PLN, while A15T mutation resulted in no change in helical content. The inhibitory effects of P21T phospholamban were more potent than those of WT PLN, while A15T PLN further decreased the apparent calcium affinity of SERCA compared to WT PLN. When these variant of phospholamban were phosphorylated however, their inhibition on SERCA was relieved to a level similar to that of phosphorylated WT PLN. A15T mutation of phospholamban severely decelerates phosphorylation of the regulatory protein, as do other clinical mutations in close proximity to serine 16. MD simulation of MYBP shows decreased stability in the structural domain of the mutation site, and increased domain dynamics. Supported by CIHR, HSF, and NSERC CREATE.


Arutyunova, Elena RHOMBOID-MEDIATED INTRAMEMBRANE PROTEOLYSIS OF PINK1 IN THE MITOCHONDRION

Elena Arutyunova, Rashmi Panigrahi, Stephania Irwin, Andrew Song, Laine Lysyk, Nicolas Touret, and M. Joanne Lemieux Department of Biochemistry, University of Alberta, Edmonton, Canada, T6E2H7 International Research Training Group Annual Meeting. September 6-8, 2016. Edmonton Alberta

PINK1, a PTEN-kinase, is a regulatory protein that gauges mitochondrial health. The processing of PINK1

plays a key role in its signaling capacity. In healthy mitochondria, PINK1 is proteolysed by the inner mitochondrial membrane protease PARL (Presenilin Associated Rhomboid Like), which belongs to the rhomboid family of serine intramembrane proteases. Damaged mitochondrion accumulates PINK1 leading to recruitment and phosphorylation of ubiquitin, which triggers the reaction cascade, resulting in mitophagy – the selective degradation of damaged mitochondria. Aberrant accumulation of PINK1 results in an imbalance in mitophagy with healthy mitochondrion being flagged for destruction, leading to overall mitochondrial fragmentation and cell death. Inherited mutations in PINK1 are associated with an autosomal recessive PARK6 form of Parkinson’s disease. The PARK6 Parkinson’s disease presentation is idiopathic with the sporadic forms, linking the process of mitophagy to Parkinson’s disease. We are investigating the molecular basis for the accumulation of mitochondrial PINK1 in Parkinson’s disease. We hypothesize that accumulation of PINK1 could be due to its import defects into the inner mitochondrial membrane or defects in its clearance from the inner mitochondrial membrane where it encounters the PARL protease. Focusing of the transmembrane region of PINK1, we examine whether mutations in this region influences its cleavage by PARL using recombinantly expressed human PINK1 and PARL protease. By examining the kinetic parameters of truncated versions of recombinant human PARL, we provide insight into its regulation. We also examine using 3DSIM superresolution microscopy whether variants found in the transmembrane region of PINK1 influences its import from the outer to the inner mitochondrial membranes. Molecular dynamics simulations reveal the structural defects associated with PINK1 variants and their insertion into lipid bilayers. This work reveals the molecular etiology with PINK1 variants associated with Parkinson’s disease. Support: NSERC-CREATE, CIHR, AIHS, CFI, Parkinson’s Society Baronas, Victoria Kv1.2 CHANNELS AT THE INTERFACE OF REDOX AND ELECTRICAL EXCITABILITY

Victoria A. Baronas, Yury Y. Vilin, Runying Y. Yang, Harley T. Kurata.

Voltage gated potassium channels (Kv) are the largest and most diverse family of ion channels. They are ubiquitously located within the central and peripheral nervous system where they are essential regulators of action potential threshold and morphology. Within this diverse Kv channel family, Kv1.2 channels have a unique phenotype that sets them aside from all other Kv channels. They are subject to a regulatory mechanism that allows channels to progressively increase activity during trains of repetitive stimuli. We term this phenotype ‘use-dependent activation’. This is an unusual and unique phenotype for a potassium channel, which may act as an intrinsic timing mechanism to silence excessively firing neurons. We hypothesize that this regulator is extrinsic to the Kv1.2 channel, and demonstrate that the regulator affects the channel in a state-dependent fashion. Surprisingly, the extent of use-dependent activation is highly sensitive to redox state. When cells expressing Kv1.2 are exposed to reducing conditions, they become extremely susceptible to use-dependent activation such that a train of depolarizations will elicit a 100-fold plus increase in current. We show that this effect is specifically due to the extracellular redox environment, and that the response is not mediated by modification of cysteines intrinsic to the Kv1.2 channel, but rather an extrinsic redox-sensitive regulator. Altogether, we demonstrate a novel mechanism of regulation of a potassium channel, which allows Kv1.2 to act as a transducer of the extracellular redox environment into electrical activity. Supported by CIHR, NSERC, AIHS, CFI and HFSP.


Cai, Ruiqi INTRA-MOLECULAR N- AND C- INTERACTION IN TRANSIENT RECEPTOR POTENTIAL POLYCYSTIN-3 (TRPP3) AND CANONICAL-4 (TRPC4) CHANNELS

Ruiqi Cai1, Wang Zheng1, Qiaolin Hu1, Laura Hofmann2, Veit Flockerzi2 and Xing-Zhen Chen1. 1Membrane Protein Disease Research Group, Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Canada T6G 2H7; 2Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, 66421, Homburg, Germany.

The transient receptor potential (TRP) superfamily of cation channels are activated by stimuli like light,

temperature, mechanical force, acid and chemicals, and are divided into seven sub-families. TRP polycystin-3 (TRPP3) is activated by calcium and acid, and has been reported to involve in sour tasting and hedgehog signaling. TRP canonical-4 (TRPC4) was found to play important roles in the phosphoinositide signaling. With mutagenesis, two-electrode voltage clamp and Xenopus oocytes expression, we identified the N-terminal W81 and C-terminal K568 of human TRPP3 to be essential for TRPP3 calcium-induced activation, which are highly conserved across TRP channels and different species. Similarly, the N-terminal W314 and C-terminal R639 of mouse TRPC4 G504S are critical for its large basal current. Recently resolved structures of TRPA1, -V1, -V2 and -V6 all indicate a co-localization between the N- and C-termini but whether they do interact with each other and whether the interaction is functionally important have remained unclear. We hypothesize that the N- and C-termini of a TRP channel interact through an aromatic residue and a cationic residue and that this interaction is functionally essential. We first found that TRPP3 channel activity is substantially reduced by co-expression of an N-terminal peptide I40-L95 containing W81; it thus acted as a blocking peptide (BP). With the whole- or sliced-oocyte immunofluorescence, BP was found to co-localize with full-length (FL) TRPP3, whereas BP with W81A mutation wasn’t. Co-immunoprecipitation (co-IP) experiments support that BP is in the same complex with FL-TRPP3, and that the W81A mutation in BP or the K568A mutation in FL-TRPP3 disrupts the complex. Consistently, TRPC4 N-terminal peptide E280-K329 containing W314 inhibits the channel activity of the TRPC4 G504S mutant, which was trapped in an activated state. As assessed by co-IP, peptide E280-K329 interacted with FL-TRPC4 G504S and this interaction was significantly reduced when R639A mutation was introduced into TRPC4 G504S. Taken together, our data strongly support that the N- and C-termini interaction is functionally critical and mediated through an N-terminal aromatic residue and a C-terminal cationic residue in both TRPP3 and TRPC4.

Supported by NSERC Discovery Grant (to XZC) and NSERC CREATE IRTG Studentship (to RC and QH). Danielczak,Bartholomäus POLYMER-MEDIATED MEMBRANE SOLUBILIZATION MONITORED BY MULTI-PARAMETRIC SURFACE PLASMON RESONANCE

Bartholomäus Danielczak and Sandro Keller. Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany.

The isolation and the purification of integral membrane proteins (MPs) are crucial steps to render these

important but challenging research subjects amenable to in vitro investigations. For decades, MP extraction has been accomplished by the use of detergents to incorporate MPs into micelles. However, detergent micelles can be harsh, that is, they provide only limited stability and insufficient membrane-mimicking properties.

Recently, a promising detergent-free approach has emerged that is based on the use of styrene/maleic acid (SMA) copolymers. SMA solubilizes biological and model membranes into nanodiscs by forming patches of lipid bilayers bounded by an SMA belt. The thermodynamics of solubilization by SMA has recently been studied using liposomes of different lipid compositions.1 However, liposomes in suspension tend to fuse and aggregate rapidly, hampering the detection of SMA-mediated solubilization kinetics. By contrast, supported lipid bilayers, once deposited on hydrophilic solid supports, are mechanically and thermodynamically stable and, thus, facilitate the measurement of polymer-mediated solubilization kinetics. In this study, we aim to measure the solubilization kinetics and thermodynamics of supported lipid bilayers by copolymers such as SMA and the related copolymer diisobutylene/maleic acid (DIBMA) with the aid of multi-parametric surface plasmon resonance (SPR) spectroscopy. Our results challenge a three-step kinetic model for SMA-mediated solubilization recently proposed by Scheidelaar et al, according to which SMA is supposed to initially adhere superficially to the lipid bilayers before inserting into the hydrophobic core and


finally assembling into nanodiscs.2

1 Cuevas Arenas et al, Nanoscale, 2016, DOI: 10.1039/C6NR02089E. 2 Scheidelaar et al, Biophys. J., 2015, 108(2):279–90. Supported by University of Kaiserslautern & DFG (IRTG 1830) Erdogan, Alican REDOX PROCESSES IN IMS PROTEIN IMPORT AND COMPLEX I ASSEMBLY

Alican Erdogan and Jan Riemer. Institute of Biochemistry, University of Cologne, Cologne, Germany.

Complex I of the respiratory chain of mitochondria is crucial for cellular energy production. It is also a major

source of reactive oxygen species, and dysfunctions of the complex have been implicated in the pathogenesis of a variety of neurodegenerative disorders. Dysfunctions often occur as a result of an impaired assembly, but so far only little is known about the biogenesis and maintenance of Complex I in mammalian cells. Seven of its subunits are encoded in the mitochondrial genome, while the remaining 38 subunits have to be imported from the cytosol. Subunits are sequentially assembled to give rise to the holoenzyme supported by assembly factors. We aim to characterize the function of proteins in the assembly/maintenance of Complex I:

The four uncharacterized nuclear-encoded Complex I subunits NDUFS5, NDUFB7, NDUFB10 and NDUFA8 lack typical mitochondrial import signals but instead contain conserved cysteine residues. The proteins are likely imported and trapped in the intermembrane space in a redox-dependent manner (twin-CxC9 proteins). This process of oxidative folding is facilitated by the oxidoreductase Mia40 and is coupled to the activity of the respiratory chain. It is unknown how this redox pathway affects the assembly and maintenance of Complex I and whether the coupling to the respiratory chain provides a feedback control for respiratory chain assembly. We will therefore study the import and the function of these proteins as well as the role of Mia40 and AIF in Complex I biogenesis and maintenance on the molecular level. Here, we will present our findings on the import and the function of these proteins as well as the role of Mia40/AIF in Complex I biogenesis and maintenance on the molecular level.

Supported by DFG. Finger, Yannik HUMAN ADENYLATE KINASE HSAK2 IS A SUBSTRATE OF MIA40

Yannik Finger

In a proteomic approach we identified novel substrates of Mia40 including the human adenylate kinase HsAK2. Adenylate kinases are a group of phosphotransferases with high importance for cellular energy balance and signalling, controlling the cellular levels of adenine nucleotides. The human isoform HsAK2 is found in mitochondria, located in the intermembrane space. The mature HsAK2 has a length of 239 amino acids and the crystal structure shows a disulfide bond between C42 and C92. In the intermembrane space, HsAK2 exchanges phosphate groups between ATP and AMP, forming two molecules of ADP and vice versa. This constant phosphoryl-transfer keeps the ATP level up, contributes to AMP signalling and is part of a shuttle system to move ATP from the ATP/ADP-translocase and ADP back to the matrix. HsAK2 appears to have a very low KM towards AMP (<10 µM), rendering it the main cellular AMP sink.

The interaction of HsAK2 with Mia40 and the role of Mia40 in HsAK2 import and maturation is not understood yet. We propose that Mia40 uses its redox-active CPC motif to import and oxidize HsAK2. We found that a redox-inactive variant of Mia40 was unable to bind HsAK2 and depletion of Mia40 results in absence of HsAK2. Moreover, we identified a potential Mia40 recognition motif in HsAK2, confirmed the existence of one disulfide bond in HsAK2 and that mutation of specific cysteines in HsAK2 (C42 and C92) results in absence of HsAK2 from mitochondria. Using an AK activity assay we showed that formation of the disulfide bond is only important for mitochondrial import, but not for HsAK2 activity.

Supported by DFG (IRTG 1830)


Grethen, Anne COMPARISON OF MEMBRANE-SOLUBILIZING EFFICIENCIES OF AMPHIPHILIC POLYMERS

Anne Grethen, Rodrigo Cuevas Arenas, and Sandro Keller. Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany.

In order to study and characterize membrane proteins in vitro, they need to be extracted from their native lipid

environment while maintaining their integrity and activity. Conventional membrane-mimetic systems, such as detergent micelles, often poorly resemble the native membrane environment and thus can lead to fast denaturation, misfolding, or activity loss of target membrane proteins. Styrene/maleic acid (SMA) copolymers are able to directly self-insert into artificial or native lipid membranes and solubilize membrane proteins embedded in lipid patches to form nanodiscs called SMA/lipid particles (SMALPs), which are stabilized by an SMA belt. The availability of straightforward extraction protocols in the absence of conventional detergents and the high thermal and temporal stability of membrane proteins incorporated into SMALPs underline the high potential of this new membrane-mimetic system.

SMA copolymers exist in different forms with various styrene/maleic acid molar ratios, which affect their amphiphilic properties. Since the insertion of SMA into the lipid bilayer is thought to be mainly driven by the hydrophobic effect, it is to be expected that different SMA copolymers show different solubilization behavior. Therefore, we systematically characterized and compared solubilization efficiencies of SMA(2:1) and SMA(3:1), which are the two most popular types of SMA used for solubilizing membrane proteins. The solubilization of artificial lipid vesicles (liposomes) of different phospholipid composition was monitored by 31P nuclear magnetic resonance spectroscopy (31P-NMR) dynamic light scattering (DLS), and multidetection size exclusion chromatography (SEC). Thermodynamic parameters of solubilization were determined from 31P-NMR data using a three-stage model that describes the different lipid/polymer assemblies as pseudophases. These measurements were complemented by intensity size distributions, total scattering intensities, and polydispersity values obtained from DLS and by SEC profiles simultaneously monitored with the aid of absorbance, refractometry, viscometry, and light scattering.

Supported by DFG (IRTG 1830)

Hu, Qiaolin INTERACTION BETWEEN THE N- AND C- TERMINI OF THE TRANSIENT RECEPTOR POTENTIAL VANILLOID TYPE 6 (TRPV6) CHANNEL

Qiaolin H1, Wang Zheng1, Ruiqi Cai1, Laura Hofmann2, Veit Flockerzi2, and Xing-Zhen Chen1. 1Membrane Protein Disease Research Group, Dept of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7; 2Dept of Experimental and Clinical Pharmacology, Faculty of Medicine, Saarland University, 66421 Homburg, Germany

Transient receptor potential (TRP) channels belongs to a large family of polymodal nonselective cation channels involved in sensing different stimuli such as mechanical force, light, chemicals, acid, and temperature. It has so far remained unknown as to whether these different stimuli activate TRP channels with a shared mechanism. TRPV6 and TRPV5 are the only Ca2+ selective members of the TRP superfamily whereas most other TRP channels are Ca2+ permeable non-selective cation channels. TRPV6 is involved in calcium absorption in the intestine, and has been implicated in cancer, sperm maturation, and male fertility. Here, with mutagenesis, two-electrode voltage clamp and Xenopus oocyte expression, we first found that TRPV6-mediated Ca2+ current is increased more than 10 folds when highly conserved aromatic residue W361 in the N-terminus or W633 in the C-terminus is mutated to alanine, suggesting that W361 and W633 are essential to maintain TRPV6 in its closed state. Since recently resolved structure of TRPA1, TRPV1, -2 and -6 all indicated physical proximity between the N- and C-termini, we hypothesize that W361 and W633 mediate TRPV6 N-to-C-termini interaction to stabilize its closed conformation. In order to determine the physical interaction between the human TRPV6 N- and C-termini, we co-expressed full length (FL) and a N-terminal peptide (NP, X319-X368) in Xenopus oocytes. With co-immunoprecipitation, we indeed found that FL TRPV6 and NP are in the same complex. More importantly, the interaction between FL TRPV6 and NP was disrupted either with W361A mutation in NP or W633A mutation in FL TRPV6. In summary, our data strongly suggested that W361 and W633 together mediate interaction between the N- and C-termini of TRPV6, which is functionally critical. Further investigations are required to elucidate how the interaction regulates channel function or gating.

Supported by NSERC Discovery Grant (to XZC) and NSERC IRTG Studentship (to QH, RC)


Kaur, Gurnit ELIMINATION OF ARSENIC SPECIES BY SINGLE NUCLEOTIDE POLYMORPHIC VARIANTS OF THE HUMAN MULTIDRUG RESISTANCE PROTEIN 2 (MRP2/ABCC2)

Gurnit Kaur1, and Elaine M. Leslie1,2. 1Department of Laboratory Medicine and Pathology and 2Department of Physiology, University of Alberta, Edmonton, AB, Canada T6G2H7.

Arsenic and selenium are toxic compounds, however in vivo exposures to arsenite and selenite result in mutual

detoxification. The molecular basis of this can be explained by the biliary excretion of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]- by the ATP-binding cassette (ABC) transporter, multidrug resistance protein 2 (MRP2/ABCC2). The ABCC2 gene is highly variable; >150 single nucleotide polymorphisms (SNPs) have been identified. Several SNPs have been shown to alter the toxicokinetics of important therapeutic agents. The objective of this study was to determine whether ABCC2 SNPs that result in the amino acid changes, R412G, V417I, S789F, R1150H, R1181L, N1244K, P1291L, V1188E, A1450T, T1477M, C1515Y and C1515Y/V1188E, displayed altered [(GS)2AsSe]- transport activity in comparison to wild-type (WT) MRP2. ABCC2 SNPs were generated using site-directed mutagenesis and expressed in HEK293T cells. Plasma membrane-enriched vesicles were isolated and relative MRP2 levels were determined by western blotting. Transport activities of WT and variant MRP2 were compared using [(GS)2AsSe]-. All mutants were detected in whole cell lysates except for T1477M. S789F and A1450T were not detected in plasma membrane enriched vesicles. R412G and R1150H displayed lower [(GS)2AsSe]- transport activity compared to WT. The differences in cellular localization of S789F, A1450T and T1477M suggest that these amino acids may contribute to correct folding and trafficking of MRP2. Arsenic exposed individuals with ABCC2 SNPs that display reduced transport activity and mislocalization may not benefit from selenium supplementation.

Supported by CIHR, AIHS and ACF. Kim, Robin Y. USING VOLTAGE CLAMP FLUOROMETRY TO UNDERSTAND KCNQ CHANNEL PHARMACOLOGY Robin Y. Kim1, Stephan A. Pless2, Harley T. Kurata3 1Pharmacology Anesthesiology and Therapeutics, University of British Columbia, Vancouver, BC, Canada,2Center for Biopharmaceuticals, University of Copenhagen, Copenhagen, Denmark, 3Pharmacology, University of Alberta, Edmonton, AB, Canada.

KCNQ2 and KCNQ3 subunits form heteromeric “M-channels” that are widely expressed in the brain and

play a critical role in controlling neuronal excitability. While loss of function mutations in these channels result in neonatal epilepsy, promoting their opening through the use of the novel anti-convulsant retigabine (RTG) can provide therapy to epileptic patients. In this study, we set out to explore the effects of RTG on the voltage sensing mechanism of homomeric KCNQ3[A315T] channels using voltage clamp fluorometry (VCF). Introducing a cysteine residue into the extracellular region of the voltage sensing domain of the channel (VSD) enables us to measure fluorescence to track conformational changes in this region critical for channel function. The voltage dependence of the fluorescence is similar to channel gating, suggesting that movements of the VSD is tightly coupled to the opening and closing of the channel pore (PD, pore domain). In the presence of RTG, the fluorescence-voltage relationship is shifted to hyperpolarized potentials, indicating that RTG acts to stabilize the activated VSD. Furthermore, this effect depends residue TRP265, previously identified to be an essential component of the RTG binding site. To probe whether RTG can activate the VSD independently from the PD, we used CiVSP (voltage sensitive phosphatase) to deplete membrane PIP2 and uncouple the two domains. In the absence of PIP2, RTG loses its ability to stabilize the active VSD. In addition, mutating a cluster of potential PIP2 interaction residues in the C-terminus reduces RTG’s stabilizing effects on the VSD. Further examination of the fluorescence reports reveal both PIP2 and RTG can alter the magnitude of the fluorescence change induced by channel activation, suggesting the presence of multiple VSD conformations. Taken together, our data suggest that RTG stabilizes a unique PIP2 dependent channel open state, and that conformational flexibility of activated KCNQ channels may contribute to their sensitivity to channel openers. This work is supported by CIHR.


Klein, Marie-Christine FUNCTIONAL CHARACTERIZATION OF A COMMON VARIABLE IMMUNE DISEASE-RELATED SEC61A1 MUTATION IN HUMANS

Marie-Christine Klein1, Desirée Schubert2, Bodo Grimbacher2, Adolfo Cavalié3 & Richard Zimmermann1. ¹Department Of Medical Biochemistry And Molecular Biology, Saarland University, Homburg 66421, Germany. ²Center for Chronic Immunodeficiency, University Medical Center Freiburg and University of Freiburg, Freiburg 79108, Germany. 3Department Of Experimental And Clinical Pharmacology, Saarland University, Homburg 66421, Germany.

The endoplasmic reticulum (ER) of human cells is a major storage compartment for calcium ions (Ca²⁺) and a

main site for protein synthesis. The translocation and membrane integration of newly synthesized proteins into the ER lumen and ER membrane, respectively, by the membrane resident Sec61 complex represents a crucial point in intracellular calcium homeostasis: Ca²⁺ leaks from the ER into the cytosol due to the steep ion concentration gradient between these two compartments. That's why the Sec61 channel needs to be tightly controlled by 'gate keepers' like Calmodulin on the cytosolic and BiP on the ER lumenal side of the ER membrane. Their mechanism of limiting the calcium leakage from the ER has been investigated by our lab employing live cell Ca²⁺ imaging of HeLa cells using the ratiometric Ca²⁺ indicator FURA-2 in combination with siRNA mediated cellular depletion of proteins of interest or their inhibition of small molecules.

Recently, a SEC61A1 mutation which may lead to a Sec61 channel defect attracted our attention. This mutation is associated with the common variable immunodeficiency (CVID) which is the most abundant symptomatic primary immunodeficiency. Patients display a very heterogenous clinical phenotype which ranges from recurrent infections of the upper respiratory tract, an increased risk of developing autoimmune diseases to selective cancers. A hallmark of the phenotype is the complete absence of plasma cells from the peripheral blood. According to our model, the underlying defect in B cell differentiation into plasma cells may result from an altered calcium homeostasis in affected cells. This would again emphasize the importance of preserving the Sec61 ion permeability barrier and its impact on the calcium homeostasis even with a pathophysiological background. To test this hypothesis live cell Ca²⁺ imaging experiments are pursued in a transient HeLa model cell system as well as further functional characterization. Supported by Saarland University, IRTG.


Kumari, Alka & Badior, Katherine E. SLC4A11 THREE-DIMENSIONAL MODEL EXPLAINS STRUCTURAL BASIS FOR ENDOTHELIAL CORNEAL DYSTROPHY-CAUSING MUTATIONS

Alka Kumari, Katherine E. Badior and Joseph R. Casey. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.

Purpose: SLC4A11 is an integral membrane protein abundant in corneal endothelium. Point mutations of SLC4A11 protein cause genetic endothelial corneal dystrophies manifesting very early in life (congenital hereditary endothelial corneal dystrophy (CHED) and Harboyan syndrome (HS)) or in the fifth to sixth decade of life (Fuchs endothelial corneal dystrophy (FECD)). The molecular defect associated with these mutations include mis-folding of the protein, leading to its retention in endoplasmic reticulum (ER) and a loss of SLC4A11 water flux function. We aim to elucidate the effect of disease-causing SLC4A11 mutations on protein structure. This is a first attempt to classify SLC4A11 mutations into structural sub-categories, to provide a molecular explanation for SLC4A11- associated disease. Methods: A 3D homology model of the human SLC4A11 protein membrane domain was created on the basis of the crystal structure of human SLC4A1 structure at 3.5 Å. The homology modelling was performed, using the SWISS-MODEL server. To test the validity of the SLC4A11 homology model, mutations were made at positions corresponding to the protein’s predicted catalytic site. Functional activity of these mutants was assessed by whole cell swelling assay. Results: Amongst 29 disease-causing mutations of SLC4A11 membrane domain, 17 altered helix packing and two were in the dimeric interface. The other mutations mapped to the SLC4A11 transport catalytic site. Consistent with the homology model, protein-packing mutants were associated with protein mis-folding and ER retention. Catalytic site mutants affected SLC4A11 water flux activity. Conclusions: Missense mutants of SLC4A11 were categorized as disrupting 1. Helix packing, 2. Catalytic activity, or 3. Dimerization. The 3D SLC4A11 model provides the ability to predict pathogenicity of variants of this protein identified in the future. Supported by IRTG and CIHR. Kurata, Harley SUBUNIT SPECIFICITY AND STOICHIOMETRY OF KCNQ CHANNEL OPENERS

Alice W. Wang, Michael C. Yau, Runying Yang, Harley T. Kurata

Retigabine is the first approved anti-epileptic drug that acts via activation of voltage-gated potassium channels,

targeting KCNQ channels that underlie the neuronal M-current. Retigabine exhibits little specificity between KCNQ2-5, which all contain a Trpresidue in the pore domain that is essential for retigabine actions. The retigabine analog ICA-069673 (‘ICA73’) exhibits much stronger effects than retigabine on KCNQ2 channels, including a large hyperpolarizing shift of the voltage-dependence of activation, and roughly two-fold enhancement of peak current. In addition, ICA73 exhibits strong subtype specificity for KCNQ2 over KCNQ3, and appears to have a unique mechanism because pore mutations that abolish retigabine action (KCNQ2 Trp236Phe) do not affect ICA73 sensitivity. Based on ICA73 sensitivity of chimeric constructs of the transmembrane segments of KCNQ2 and KCNQ3, this drug appears to interact with the KCNQ2 voltage sensor (S1-S4) rather than the pore region targeted by retigabine. KCNQ2 point mutants in the voltage sensor were generated based on KCNQ2/KCNQ3 sequence differences, and screened for ICA73 sensitivity. These experiments reveal that KCNQ2 residues Phe168 and Ala181 in the S3 segment are essential determinants of ICA73 subtype specificity. Mutations at either position in KCNQ2 abolish the ICA73-mediated gating shift, while retaining retigabine sensitivity. Interestingly, A181P mutant channels show little ICA73-mediated gating shift, but retain current potentiation by the drug. These findings demonstrate that retigabine and ICA73 act via distinct mechanisms, and provide the first insights into channel residues that underlie subtype specificity of KCNQ channel openers. Exploiting these specific determinants of ICA73-mediated channel potentiation, we have also generated tetrameric channel constructs with varying numbers of ICA73-sensitive subunits, revealing the stoichiometric requirement for ICA73 effects. Supported by HFSP (Human Frontiers in Science Program) and CIHR.


Marensi, Vanessa GLUTATHIONE TRANSFERASE P1 (GSTP1) IS MODIFIED BY PALMITATE

Marensi V1, Yap M2, Ji Y3, Lin C3, Berthiaume LG2, Leslie EM1.

Departments of Physiology1 and Cell Biology2, University of Alberta, Edmonton, Alberta, Canada; Centre for Biomedical Mass Spectrometry, Department of Biochemistry, Boston University School of Medicine3, Massachusetts, Boston, USA.

Glutathione transferase P1 (GSTP1) protects cells from carcinogens by catalyzing their conjugation with the tripeptide glutathione (γ-Glu-Cys-Gly). It is also involved in cell signalling, proliferation and apoptosis. Overexpression of GSTP1 in tumours and single nucleotide polymorphic variants are associated with anti-cancer drug resistance and poor prognosis. In contrast, inactivation of GSTP1 due to epigenetic gene silencing increases susceptibility to certain cancer types, with prostate cancer as the best studied example. GSTP1 is classically described as a cytosolic enzyme; however, we have reported that it is strongly associated with the plasma membrane, and the strength is comparable to the integral membrane protein Na+/K+ATPase. We hypothesize that the addition of a hydrophobic component is required to allow its strong interaction with membranes. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. We found that GSTP1 is modified by palmitate. However, Cys-less mutants expressed in MCF7 cells surprisingly retained palmitoylation. In addition, treatment of palmitoylated GSTP1 with NaOH, which cleaves ester bonds, did not remove palmitate. These data suggested that GSTP1 is modified by palmitate on at least one non-Cys residue. We also demonstrated that GSTP1 can be non-enzymatically palmitoylated. Peptide sequencing, by ESI-MS/MS, of the palmitoylated GSTP1 revealed that Cys48 and Cys102 undergo S-palmitoylation and Lys103 undergoes N-palmitoylation. N-palmitoylation of an internal Lys residue is a rare observation and provides an explanation for the resistance of GSTP1 palmitoylation to NaOH treatment. In conclusion, we have identified palmitoylation as a novel post-translational modification of GSTP1. This research lays the foundation for understanding the fundamental biology of GSTP1 and how palmitoylation may influence its structure, function and membrane association.

Supported by CIHR, ACF and AIHS.

Ohler, Lisa THE PROTEIN FAMILY OF EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS – CURRENT STATE OF KNOWLEDGE AND FUTURE PERSPECTIVES

Christopher Girke1, Manuel Daumann1, Lisa Ohler1, Daniel Hickl1, Joe Casey2, M. Joanne Lemieux3 and Torsten Möhlmann1. 1Department of Plant Physiology, University of Kaiserslautern, Germany 2Dept. of Physiology and Biochemistry, University of Alberta, Canada 3Dept. of Biochemistry, University of Alberta, Canada

Nucleoside transporters play an important role in many organisms as they transport hydrophilic nucleosides across membranes. In human, these transporters can either act as equilibrative (ENT) or concentrative (CNT) nucleoside transporters and are possible targets for the treatment of cancers and viral diseases. In plants, nucleoside transporters from four species are characterized: AtENT1, 3, 4, 6 and 7 (Arabidopsis), OsENT2 (rice), HvENT1 (barley) and StENT1 and 3 (potato). Plant ENTs are structurally related to the human ENTs, but act – except for AtENT7 – as substrate-proton-symporters. As integral membrane proteins they consist of 11 predicted transmembrane domains with a cytosolic loop between domain 6 and 7, a cytosolic N- and extracellular C-terminus. All plant ENTs studied so far have a broad substrate specificity and transport purine nucleosides (adenosine and guanosine) and pyrimidine nucleosides (cytidine and uridine) with apparent KM values in a range from 3-94 µM. Nucleoside transport is inhibited by deoxynucleosides and nucleobases, but in contrast to hENTs not by dilazep and nitrobenzylmercaptopurine ribonucleoside (NBMPR). Arabidopsis ent3 knock out mutants are highly resistant towards the toxic substrate analogs 5-fluorouridine and 2-chloroadenosine. Therefore, AtENT3 seems to be the major pyrimidine importer in Arabidopsis seedlings. AtENT1 is located in the vacuolar membrane and exports nucleosides derived from RNA degradation. No structure of any ENT has been solved so far. However, topological and mutational analysis of mammalian and plant members as well as purification approaches of mammalian, yeast and plant members combined with functional studies have been performed. AtENT7 was purified as recombinant protein with retained function in binding pyrimidine and


purine nucleosides and nucleobases with high affinities. Supported by DFG (IRTG 1830) Overduin, Michael STRUCTURE AND LIPID BINDING FUNCTION OF PLPA AT THE BACTERIAL OUTER MEMBRANE BY SOLUTION STATE NMR

Faye C. Morris1, Timothy J. Knowles2, Riyaz Maderbocus1,2, Mark Jeeves2, Jennifer Kirwan3, Eva Heinz4, Timothy J. Wells1, Douglas F. Browning1, Yanina R. Sevastsyanovich1, Denisse L. Leyton4, Amanda E. Rossiter1, Vassiliy N. Bavro1, Pooja Sridhar2, Douglas G. Ward2, Neil J. Shimwell2, Ashley Martin2, Jason W. Sahl5, Catherine A. Wardius1, Daniel Walker6, Trevor Lithgow4, Mark R. Viant3, David A. Rasko5, Adam F. Cunningham1, Ian R. Henderson1, Michael Overduin2 1Institute of Microbiology and Infection, 2School of Cancer Studies and 3School of Biosciences, University of Birmingham, Edgbaston, UK. 4Department of Microbiology, Monash University, Clayton, Australia. 5Institute of Genome Sciences, University of Maryland, Baltimore, USA. 6Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

The Gram-negative outer membrane is an asymmetric lipid bilayer composed of lipopolysaccharide and

phospholipid decorated with integral outer membrane proteins and peripheral lipoproteins. The lipopolysaccharide and protein components are synthesised in the cytoplasm and trafficked to the outer membrane by known, dedicated systems. How phospholipid is trafficked to the outer membrane (OM) has remained unclear. PlpA is a conserved outer membrane lipoprotein with an unresolved structure and function. Here we present its solution structure, both free and inserted into outer-membrane mimicking micelles. This reveals that PlpA is composed of two BON domains which form an interconnected opposing pair. This is an unprecedented finding with functional repercussions. The second BON domain interfaces phosphatidylglycerol (PG)-based membrane surfaces. Loss of PlpA resulted in decreased levels of phosphatidylglycerol in the outer membrane and impaired its barrier function. The Mla retrograde phospholipid transport system is linked to PlpA through suppressor mutations, suggesting PlpA forms a crucial component of the machinery that binds and traffics phospholipids to the outer membrane. The discovery of the fold and function of PlpA provides a new target for developing therapies that disrupt the integrity of the bacterial outer membrane.

Supported by BBSRC and CAIP funding. Patzke, Kathrin CHLOROPLASTIDIC SUGAR METABOLISM AND IT´S INFLUENCE ON CELLULAR SIGNALING

Kathrin Patzke, Patrick Klemens, Pratiwi Prananingrum and Ekkehard Neuhaus. Department of Plant Physiology, University of Technology, Kaiserslautern, Germany

Chloroplasts are the most noticeable components of plant cells and represent, next to the vacuole, the biggest

cellular compartment. During photosynthesis, chloroplasts produce high amounts of carbohydrates acting as the main energy source in plants. These carbohydrates are stored as transitory starch in the chloroplast itself in order to be remobilized at night, or are directly transported to the cytosol to serve as precursors for sucrose synthesis. Sucrose is exclusively synthesized in the cytosol, but accumulates during cold in the plastidic stroma to protect the chloroplasts i.a. against ROS (reactive oxygen species). Accordingly, there should be transporters localized in the chloroplast envelope. The COR413IM proteins (cold regulated inner membrane proteins) as well as the VGT3 (vacuolar glucose transporter 3), both localized in the chloroplast envelope (i.a. shown by GFP-fusion-analysis) represent suitable candidates for the sugar transport across the plastidic membrane. Furthermore, stromal sugars do not only act as protectants but are also involved in cellular signaling processes. The chloroplast-located Invertase (InvE) (Vargas et al. 2008), catalyzing the hydrolysis of sucrose to glucose and fructose, seems to take part in such signaling processes. A point-mutation in this protein (C294Y) causes higher stability and an increased catalytic activity. Moreover, exogenously applied sugars, especially sucrose, lead to yellow cotyledons in the early developmental stage. This gain-of-function mutant is so called sicy-192 (sugar induced cotyledon yellow-192). The phenotype and the fact that sicy-192 plants show an altered expression of photosynthesis-associated nuclear genes (PhANGs) in the presence of exogenously supplied sucrose, causes the consideration of inter-


organellar communication by retrograde signaling with InvE as a necessary participant (Tamoi et al. 2010). Preliminary results display a significant increase of anthocyanins, plant stress indicators, after exposure to 4°C for 17days in all mutant plants. Within our work, we are trying to gain more insights into the sugar mediated retrograde signaling processes from chloroplast to nucleus, focussing on the putative sugar carriers COR413IM and VGT3, and the plastidic Invertase InvE. This project is supported by DFG (T-SFB 175, The Green Hub) Primeau, Joseph O. CRYSTALS, CLEAVAGE, AND CROSS-LINKS: INVESTIGATING THE SERCA ACCESSORY SITE

Joseph O. Primeau1 and Howard S. Young1

1University of Alberta, Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7.

Dilated cardiomyopathy (DCM) is a fatal form of heart disease characterized by dilation in one or both ventricles, and systolic dysfunction. ~30% of DCM cases are of a familial or hereditary origin, suggesting a strong genetic component to the disease. At the molecular level, extensive dysregulation of calcium metabolism in the heart has been linked to heart failure. Calcium transients responsible for contraction and relaxation of heart-muscle cells are achieved by calcium release from calcium stores, and subsequent refilling by calcium handling proteins, such as the Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), within the cell. SERCA is regulated by a number of small inhibitory proteins, in particular a calcium transport regulatory protein that has been linked to heart failure – phospholamban (PLN). PLN regulates SERCA by decreasing cardiac contractility and relaxation rate, causing decreased stroke volume and heart rate. PLN inhibition of SERCA can be reversed through adrenaline stimulation and structural modification of PLN by Protein Kinase A (PKA), increasing cardiac output and heart rate.

PLN self-assembles into homopentamers that have been proposed to serve as an inactive storage form of PLN monomers that occupy the inhibitory groove of SERCA. This imparts an additional level of sophistication to SERCA regulation by having PLN in a state of dynamic equilibrium in the SR membrane. However, two-dimensional co-crystals of SERCA and PLN suggest that the PLN pentamer directly interacts with SERCA around trans-membrane (TM) segment 3 distinct from the inhibitory groove. This indicates that the PLN pentamer may play a larger role in SERCA regulation than just serving as pool of inactive inhibitory monomers. The PLN pentamer may sense subtle changes to the TM domain of SERCA and release/sequester inhibitory monomers during Ca2+transport, quickly regulating SERCA's activity in response to stimuli.

Here, we report evidence that the PLN pentamer does interact with SERCA in regions outside of the inhibitory groove. Using a combination of chemical cross-linking and CNBr cleavage, I have observed the interaction of the PLN pentamer with the TM domain of SERCA at areas consistent with TM segments 1 or 3. This molecular interaction is also being further investigated using electron cryo-microscopy (Cryo-EM) of two-dimensional crystals of SERCA and PLN. Utilizing a Falcon II direct electron detector, we can investigate the fine structural details of SERCA and the PLN pentamer at a much higher resolution than before. Structural information gleaned from Cryo-EM will provide a greater understanding to the complex interaction between SERCA and PLN and how it affects regulation of Ca2+transport in the heart.


Panigrahi, Rashmi MECHANISTIC INSIGHT INTO INTRAMEMBRANE ENZYME DIACYLGLYCEROL TRANSFERASE

Rashmi Panigrahi1, Kristian Mark P. Caldo2, Wei Shen3, Jeella Acedo4, Yang Xu1, Tsutomu Matsui5 Linda Hanely-Bowdoin3, John C. Vederas4, Randall J. Weselake2 and M. Joanne Lemieux1 1Department of Biochemistry, University of Alberta, Canada T6G 2H7 2Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P 3Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA 27695 4Department of Chemistry, University of Alberta, Canada T6G 2G2 5Stanford Synchrotron Radiation Lightsource, California 94025

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent formation of triacylglycerol (TAG)

and the activity of the enzyme may have a substantial effect on the flow of carbon into the seed oil of oleaginous plants. DGAT1 is a unique and essential membrane –bound enzyme that shares no homology with other family members involved in TAG production. Recombinant DGAT1 from oilseed rape (Brassica napus) (BnaDGAT1) was used for the study. It was purified in active form, reconstituted in phospholipids which increase its activity. The enzyme exhibited a hyperbolic response in activity to increasing bulk concentration of sn-1, 2-dioleoylglycerol. The enzyme demonstrated allostery exhibiting positive cooperativity. Previous research by our group has shown that an allosteric exosite for acyl-CoA is present in the hydrophilic N-terminal region of the enzyme. An N-terminal truncation of the enzyme, consisting of residues 1-113, was mainly random coil and could interact with oleoyl-CoA displaying positive cooperativity. In silico and in vitro studies demonstrated the truncated construct 81-113 was the most structured region of the N terminus possessing the necessary attributes to interact with acyl-CoA. Further activators were identified for BnaDGAT1 effective at micromolar concentration. Interestingly, it was identified that phosphorylation decreased BnDGAT1 activity. Our findings pave way to plausible development of strategies for increasing TAG content in plant biomass. Supported by MITACS and IRTG. Rehman, Saba MUTATIONS IN CLAUDIN-14 ASSOCIATED WITH HYPERCALCIURIA

Saba Rehman, Dr. Todd Alexander, Department of Physiology, University of Alberta, Edmonton, Canada

Idiopathic hypercalciuria (IH) is the most common metabolic abnormality in children causing increased risk of kidney stones. IH is a polygenic trait with increased incidence in first-degree relatives. IH is the excretion of too much calcium in the urine when plasma calcium levels are normal. Children with IH fail to reabsorb calcium from the proximal part of the nephron and some have increased circulating 1,25 dihydroxyvitamin D levels. Proximal calcium reabsorption occurs through tight junction proteins including claudin-2 and -12. Moreover increased claudin-14 in the thick ascending limb (TAL) prevents proximal calcium reabsorption. We hypothesized therefore that loss of function mutations in claudin-2 or -12, or gain of function mutations in claudin-14 would cause kidney stone formation. Moreover, a loss of function mutation in CYP24A1 would cause hypercalciuria. To test these hypothesizes we collected DNA from a cohort of children (N = 30) with Idiopathic hypercalciuria and are sequencing the candidate genes, claudin-2, -12 , -14 and CYP24A1 for mutations that could cause this disease. Sequence analysis of CYP24A1, which encodes 25-hydroxyvitamin D 24-hydroxylase, revealed 1 coding, and 6 non-coding variations in exons 3, 6 and 8 respectively. Sequence analysis of claudins-2 and -12 did not revealed variations in claudin-2 and a single variation in claudin-12.Sequence analysis of claudin-14 revealed 6 non-coding mutations in an intronic region of DNA. Of 6 variations discovered, we observed that 16/30 children were heterozygous for the single nucleotide polymorphism (SNP) Rs 128494. This frequency is higher than expected when compared to 1868 control subjects from1000 genome project, p=0.019725 by chi-squared. In future we will clone this intronic claudin-14 SNP into a reporter vector to determine if it has an effect on claudin-14 expression.

Supported by CIHR


Sarder, Hasib A. M. DISSECTING INTRACELLULAR 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 Cl- ions, thereby ensuring acid excretion in the urine [1]. Various genetically inherited mutations in the kAE1 encoding gene have been reported to negatively affect HCO3

-/Cl- exchange and to ultimately result in clinical disorders known as distal renal tubular acidosis (dRTA). Until now, autosomal dominant (AD) and recessive (AR) mutations have been identified that are either linked to false kAE1 localization or mistrafficking [2]. Since the underlying molecular mechanisms for proper kAE1 targeting are still poorly understood, we are using yeast as simple eukaryotic model organism to dissect the intracellular targeting and trafficking of wild-type kAE1 and its mutant variants. Since yeast Bor1p is proposed to be the homologue of mammalian kAE1 [3], we are currently focusing on three major aspects: (i) Expression of codon-optimized wild-type kAE1 and its clinically relevant mutant variants in a yeast Δbor1 knock-out mutant for functional complementation; (ii) Analysis of intracellular kAE1 trafficking by life cell imaging (CLSM) and determination of kAE1 plasma membrane localization through mRAS recruitment and cell surface biotinylation; (iii) Genetic screen of selected yeast knock-out and overexpressing mutants to identify cellular components involved in proper kAE1 trafficking and/or capable to restore kAE1 mistrafficking by redirecting mutant kAE1 back to the plasma membrane. The results obtained from the yeast screen will be translated into the situation of mammalian cells to get a deeper mechanistic understanding of kAE1 mis-targeting/trafficking in dRTA associated clinical disorders.

Supported by DAAD-GSSP & DFG (IRTG 1830) Schwartz, Sara STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE YEAST VIRAL A/B TOXIN K28

Sara Schwartz, Molecular & Cell Biology, Saarland University, Saarbrücken, Germany (supervised by Manfred Schmitt, Joanne Lemieux and Barbara Niemeyer)

Controlled disulfide bond reduction is a key step in the intracellular trafficking of A/B toxins such as

cholera toxin, Pseudomonas exotoxin and the yeast viral K28 toxin. To dissect this process at the molecular level, we use K28 as model which consists of two subunits (α and β) connected by a single inter-chain disulfide. During intoxication by receptor endocytosis and retrograde transport through the secretory pathway, K28 enters the cytosol from the ER. After reductive cleavage of the α/β disulfide in the cytosol, cytotoxic α is released and kills by blocking DNA synthesis and arresting cells at the G1/S boundary. While K28 secreting cells likewise internalize their own toxin, the retrotranslocated α/β heterodimer is rapidly degraded after complexation with the cytosolic preprotoxin precursor; to achieve this self-protection, S-S bond reduction must be tightly controlled. In the present project we investigate the properties of cysteine residues and thiol redox control in K28 and try to solve its 3D crystal structure. Mature K28 possesses five cysteines from which C333 in β was demonstrated to form the single inter-chain disulfide with C56 in α. In addition, we found that K28 is irreversibly inactivated at pH higher than 6 through the formation of S-S-linked higher order oligomers. Since such pH conditions also occur in vivo during toxin passage through the Golgi (pH 6) and the ER (pH 7.2), we propose an essential role of soluble ER residents (in particular PDI) to tightly control conformational rearrangements in K28 and prevent the undesired release of cytotoxic α.


Stutz, Regine MECHANISMS OF PROTEIN TRANSPORT INTO THE HUMAN ENDOPLASMIC RETICULUM

Regine Stutz, Department Of Medical Biochemistry and Molecular Biology, Saarland University, Germany

The translocation into the human endoplasmic reticulum (ER) is a decisive step in the biogenesis of many

secretory and membrane proteins. This is mediated by a complex machinery of membrane embedded and associated proteins, termed translocon. The protein transport across the ER membrane can occur either co- or post-translationally through the highly conserved heterotrimeric Sec61-complex. The translocon-associated protein (TRAP) complex is an integral component of the translocon and comprises four transmembrane subunits. It is proposed to influence the gating of the polypeptide-conducting Sec61 channel. Moreover, the TRAP complex may play a role in the topogenesis of polytopic membrane proteins, which could also be due to a chaperone like function in the interaction with the nascent polypeptides being transported. Recently, mutations in the human TRAP subunits were observed to result in congenital disorders of glycosylation (CDG). That leads to the suggestion that the TRAP complex may be required for increasing the efficiency of co-translational N-glycosylation by the oligosaccharyltransferase (OST) complex. 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. The overall aim of the project is to gain better insight into the substrate specific functions of components of the ER translocation machinery. Our unbiased proteomic approach after siRNA mediated knockdown of TRAP genes showed that the TRAP complex is required for efficient translocation of various proteins. These candidate TRAP-substrate proteins are currently evaluated one-by-one in cellular protein transport assays following siRNA mediated knockdown. With these experiments we hope to be able to define the substrate characteristics of the TRAP complex. Supported by IRTG1830 Wang, Caroline K. STATE- AND USE-DEPENDENT BINDING OF KCNQ CHANNEL OPENERS

Caroline K. Wang, Alice W. Wang, Harley T. Kurata

Retigabine, a KCNQ2-5 channel opener, is the first and only anticonvulsant approved for human use that acts

via opening of voltage-gated potassium channels. An important unexplored feature of retigabine and its derivatives is their state- and use-dependent properties. As described for voltage-gated sodium channe

l blockers, drugs that exhibit use-dependence may have a stronger drug effect with more frequent channel stimulation, making them especially useful for selectively targeting hyperactive cells that trigger seizures in epilepsy. We aimed to generate a detailed understanding of the mechanism of action of KCNQ channel openers. We tested the state- and use-dependence of interaction of retigabine and a ‘second-generation’ KCNQ opener (ICA069673) with WT KCNQ2 and KCNQ2[A181P] channels. We assessed drug binding to pre-open states by applying drugs at different holding potentials, and subjected cells to frequent repetitive stimulations to assess use-dependent drug interactions. WT KCNQ2 channels showed marked state- and use- dependent binding to ICA73, with the drug preferentially binding to the open channel state and having a greater effect with more frequent pulses. Unlike WTQ2, A181P appears to lack state-dependent binding to ICA73, and does not show use-dependent activation. Retigabine binds to both the open and closed WT KCNQ2 channel states, although it may have a higher affinity for the open state, allowing it to retain some use-dependent activation. These findings highlight an important property of KCNQ channel openers and will facilitate the development of more efficacious drugs. Supported by CIHR.


Wong, Ka Yee STRUCTURAL INVESTIGATION OF THE INTRACELLULAR LOOP OF THE HUMAN SODIUM/HYDROGEN EXCHANGER ISOFORM 1 USING NUCLEAR MAGNECTIC RESONANCE SPECTROSCOPY

Ka Yee Wong, Ryan Mckay, Larry Fliegel. Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7.

The mammalian sodium/hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein

that regulates intracellular pH in many types of cells. The NHE1 is an integral membrane protein that exchanges intracellular H+ for extracellular Na+. It is involved in intracellular pH regulation in the heart, and has been implicated in ischemic heart disease and cardiac hypertrophy. There is currently no high resolution structure of the entire NHE1 protein, however we have previously used nuclear magnetic resonance spectroscopy to determine the structures of individual transmembrane helices of NHE1 in membrane mimetics. Detailed structural information would be useful for understanding the mechanism of NHE1 and for the development of inhibitors.

To gain further structural insight into the protein, including helix-helix interactions and to build up a more complete structure of NHE1, we have produced a peptide containing the intracellular loop of NHE1 protein between transmembrane segments X and XI. One amino acid of this intracellular loop (R440) has been previously shown to be important in pH sensing by the NHE1 protein. Circular Dichroism Spectroscopy has been performed. The spectra obtained have indicated that the peptide’s secondary structure is mainly alpha helical and somewhat beta sheet. A 1H-TOCSY and 1H- NOESY were taken on an Inova 800 mHz spectrometer for the purpose of structural analysis. A preliminary structure has been deduced by the program, Cyana (ver2). However, further refinement and optimization are required. Supported by: IRTG Canadian Institutes of Health Research (CIHR) Natural Sciences and Engineering Research Council of Canada (NSERC)

5th IRTG Meeting Participant email list page 45

CONTACTS participant email list


FirstName Surname Affiliation emailDr. Todd Alexander DivisionofNephrology,Departmentof

Pediatrics,[email protected]

Nada Alshumaimeri Biochemistry,UniversityofAlberta(groupofJoeCasey)

[email protected]

Gareth Armanious Biochemistry,UniversityofAlberta(groupofHowardYoung)

[email protected]

Elena Arutyunova Biochemistry,UniversityofAlberta(groupofJoanneLemieux)

[email protected]

Katie Badior Biochemistry,UniversityofAlberta(groupofJoeCasey)

[email protected]

Victoria Baronas Pharmacology,UniversityofAlberta(groupofHarleyKurata)

[email protected]

Megan Beggs Pediatrics,UniversityofAlberta(groupofToddAlexander)

[email protected]

Andrea Blum Cell&MolecularBiology,SaarlandUniversity(groupofManfredSchmitt)

[email protected]

Ruiqi Cai Physiology,UniversityofAlberta(groupofXing-ZhenChen)

[email protected]

Dr. Joe Casey Director,IRTGinMembraneBiology,Biochemistry,UniversityofAlberta

[email protected]

Dr. Xing-Zhen Chen IRTGPI,Physiology,UniversityofAlberta [email protected] NaEun Chin Pharmacology,UniversityofAlberta(groupof

HarleyKurata)[email protected]

Praneeth Chitirala Physiology,SaarlandUniversity(groupofJensRettig)

[email protected]

Dr. Emmanuelle Cordat IRTGPI,Physiology,UniversityofAlberta [email protected] Bartholomäus Danielczak MolecularBiophysics,TUKaiserslautern

(groupofSandroKeller)[email protected]

Dr. Joachim Deitmer GeneralZoology,TUKaiserslautern [email protected]. Debajyoti Dutta Biochemistry,UniversityofAlberta(groupof

LarryFliegel)[email protected]

Alican Erdogan CellBiology,UniversityofCologne(groupofJanRiemer)

[email protected]

Mansoore Esmaili Biochemistry,UniversityofAlberta(groipofMichaelOverduin)

[email protected]

Yannik Finger CellBiology,UniversityofCologne(groupofJanRiemer)

[email protected]

M'Lynn Fisher Biochemistry,UniversityofAlberta(groupofHowardYoung)

[email protected]

Dr. Larry Fliegel IRTGPI,Biochemistry,UniversityofAlberta [email protected]. Eckhard Friauf AnimalPhysiology,TUKaiserslautern [email protected]


FirstName Surname Affiliation email Anne Grethen MolecularBiophysics,TUKaiserslautern

(groupofSandroKeller)[email protected]

Dr. Johannes Herrmann CellBiology,TUKaiserslautern [email protected]

Laura Hofmann PharmacologyandToxicology(groupofVeitFlockerzi)

[email protected]

Qiaolin(Lin) Hu Physiology,UniversityofAlberta(groupofXing-ZhenChen)

[email protected]

Gurnit Kaur Physiology,UniversityofAlberta(groupofElaineLeslie)

[email protected]

Dr. Sandro Keller MolecularBiophysics,TUKaiserslautern [email protected] SwaiMon Khaing Biochemistry,UniversityofAlberta(groupof

NicolasTouret)[email protected]

Robin Kim Pharmacology,UniversityofAlberta(groupofHarleyKurata)

[email protected]

Marie-Christine

Klein MedicalBiochemistryandMolecularBiology,SaarlandUniversity(groupofRichardZimmermann)

[email protected]

Dr. Alka Kumari Biochemistry,UniversityofAlberta(groupofJoeCasey)

[email protected]

Dr. Harley Kurata Pharmacology,UniversityofAlberta [email protected] Rawad Lashhab Physiology,UniversityofAlberta(groupof

EmmanuelleCordat)[email protected]

Dr. Joanne Lemieux IRTGPI,Biochemistry,UniversityofAlberta [email protected]. Elaine Leslie Physiology,UniversityofAlberta [email protected] Laine Lysyk Biochemistry,UniversityofAlberta(groupof

JoanneLemieux)[email protected]

Darpan Malhotra Biochemistry,UniversityofAlberta(groupofJoeCasey)

[email protected]

Vanessa Marensi Physiology,UniversityofAlberta(groupofElaineLeslie)

[email protected]

Sabrina Marz AnimalPhysiology,TUKaiserslautern(groupofEckhardFriauf)

[email protected]

Armin Melnyk MedicalBiochemistryandMolecularBiology,SaarlandUniversity(groupofRichardZimmermann)

[email protected]

Anna-Maria Miederer Biophysics,SaarlandUniversity(groupofBarbaraNiemeyer)

[email protected]

Dr. Torsten Möhlmann PlantPhysiology,TUKaiserslautern [email protected]. Bruce Morgan MolecularCellBiology,TUKaiserslautern [email protected]. Ekkehard Neuhaus PlantPhysiology,TUKaiserslautern [email protected]. Barbara Niemeyer Biophysics,SaarlandUniversity [email protected]


FirstName Surname Affiliation email Lisa Ohler PlantPhysiology,TUKaiserslautern(groupof

TorstenMöhlmann)[email protected]

Dr. Michael Overduin Biochemistry,UniversityofAlberta [email protected]. Rashmi Panigrani Biochemistry,UniversityofAlberta(groupof

JoanneLemieux)[email protected]

Kathrin Patzke PlantPhysiology,TUKaiserslautern(groupofEkkehardNeuhaus)

[email protected]

Dr. Katrin Philippar PlantBiochemistryandPhysiology,LMUMunich

[email protected]

Joe Primeau Biochemistry,UniversityofAlberta(groupofHowardYoung)

[email protected]

Saba Rehman Pediatrics,UniversityofAlberta(groupofToddAlexander)

[email protected]

Dr. Jens Rettig Physiology,SaarlandUniversity [email protected]. Jan Riemer CellBiology,UniversityofCologne [email protected] Hasib Sarder Cell&MolcularBiology,SaarlandUniversity

(groupofManfredSchmitt)[email protected]

Sara Schwartz Cell&MolcularBiology,SaarlandUniversity(groupofManfredSchmitt)

[email protected]

Cameron Smithers Biochemistry,UniversityofAlberta(groipofMichaelOverduin)

[email protected]

Regine Stutz MedicalBiochemistryandMolecularBiology,SaarlandUniversity(groupofRichardZimmermann)

[email protected]

Dr. Nicolas Touret PIIRTG,Biochemistry,UniversityofAlberta [email protected] Nicholas Twells Pharmacology,UniversityofAlberta(groupof

HarleyKurata)[email protected]

Shahid Ullah Physiology,UniversityofAlberta(groupofEmmanuelleCordat)

[email protected]

Caroline Wang Pharmacology,UniversityofAlberta(groupofHarleyKurata)

[email protected]

Dr. Joel Weiner Biochemistry,UniversityofAlberta [email protected] KaYee Wong Biochemistry,UniversityofAlberta(groupof

LarryFliegel)[email protected]

Runying Yang Pharmacology,UniversityofAlberta(groupofHarleyKurata)

[email protected]

Dr. Howard Young Biochemistry,UniversityofAlberta [email protected] Wang Zheng Physiology,UniversityofAlberta(groupof

Xing-ZhenChen)[email protected]

Dr. Richard Zimmermann MedicalBiochemistryandMolecularBiology,SaarlandUniversity

[email protected]

5th IRTG Meeting Appendix page 49

APPENDIX Getting Around in Edmonton


HOTEL

Varscona Hotel

The hotel Varscona is approx. 20 min walking distance to the meeting venue, Li Ka Shing Building (see the map in this booklet). There is also a shuttle service to the university operated by Diamond Limo until 9 am from Varscona hotel. The service is on first come, first served basis, book in advance at 780-465-4002 (ask for Diamond). Other option would be a taxi, which the hotel can book for the guests. Contact person for the IRTG group at Varscona hotel is Ankush Chodvadiya.

Lister Centre

The Lister Centre features 20 hotel-style guestrooms offering a choice of one queen bed, one queen bed with a double sofa bed, or two double beds. All guestrooms feature windows that open. Each room features a private bathroom, clock radios, cable TV, telephones with free local calls, hair dryers, irons and ironing boards, duvets and extra pillows, flat screen TV's, Keurig Coffee Machines and mini fridges. Services: coin-operated laundry, vending and ice machines. Complimentary services: High-speed internet access (requires ethernet cable), Free local calls with long distance access (requires a calling card that contacts a toll free number) Check-in is after 4:00 pm and check-out is by 11:00 am. Contact person for our group is: Sheri Penner Guest Services Coordinator, Reservations 1-044 Lister Centre University of Alberta Edmonton, Alberta T6G 2H6 P:780.492.9300


WHERE TO EAT

Restaurants near Lister Centre, University:

Earls Kitchen and Bar 8629 112 St NW, Edmonton, AB T6G 1K8 Phone: (780) 439-4848 Hours: 11AM–12AM Menu: earls.cahttps://earls.ca/locations/campus/menu/kitchen https://earls.ca/locations/campus/menu/kitchen Sherlock Holmes Pub 8519 112th Street Northwest, Edmonton, AB T6G 2T9 Phone: (780) 431-0091 Hours: 11AM–12AM Menu: http://www.thesherlockspubs.com/ Sugarbowl Eatery & bar serving many craft beers, breakfast-to-dinner bistro meals & weekend brunch. 10922 88th Avenue, Edmonton, AB T6G 0Z1 Hours: 8AM–11:30PM Phone: (780) 433-8369 Menu: thesugarbowl.org http://thesugarbowl.org/wine-cocktails Also see page 5 of the program

Restaurants near Varscona Hotel, Whyte Avenue:

https://www.zomato.com/edmonton/university-old-strathcona-restaurantshttps://www.zomato.com/edmonton/university-old-strathcona-restaurants Also see page 5 of the program


FITNESS

Butterdome, Van Vliet Centre and Wilson Climbing Centre on Campus On the North campus are many fitness facilities, including a gym and a swimming pool. Day passes can be purchased at the information counter for $9 (gym and pool) and $4 (pool). Day users are issued temporary VVC membership card by Facility Services. Photo ID required, e.g. Driver’s license, passport or other government-issued picture identification. Hours: Monday to Friday: 0600 - 2200 Saturday to Sunday: 0900 - 1900 For more information see: https://www.ualberta.ca/physical-education-recreation/facilities/north-campus/access-membership-and-services https://www.ualberta.ca/physical-education-recreation/facilities/north-campus/wilson-climbing-centre

Rent a bike U of Alberta sustainability club provides a service for bicycle rentals: Phone: 780-492-1648 Email: [email protected] Hours: Wednesday/Friday 1:00 p.m. - 5:00 p.m. Address: SAB 1-13 South Academic Building (SAB) University of Alberta Edmonton, Alberta T6G 2G7 http://su.ualberta.ca/services/sustainsu/projects/bikelibrary/http://su.ualberta.ca/services/sustainsu/projects/bikelibrary/ http://su.ualberta.ca/services/sustainsu/projects/bikelibrary/

PUBLIC TRANSPORT For public transport schedules see Edmonton Transport System (ETS) website: http://www.edmonton.ca/edmonton-transit-system-ets.aspx ETS trip planer: http://etstripplanner.edmonton.ca/PlanYourTrip.aspx

5th IRTG Meeting Notes page 53

NOTES




Documents

5th Joint Symposium University of Alberta Edmonton, Alberta, … · 2016. 8. 29. · 5th Joint Symposium University of Alberta Edmonton, Alberta, Canada September 6th to 8th, 2016