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2nd International Conference
“
Peroxisome Formation,
Function and Metabolism
20 – 22 June 2016
| EMBL Hamburg
”
| Germany
Sessions
• Peroxisomes in human health and disease
• Computational analyses of peroxisomes
• Peroxisome biogenesis / de novo formation and fission
© EMBL 2016
• Plant, fungal and protist peroxisomes
• Structural biology of peroxisomes
• Peroxisomes as biological systems
www.itn-perfume.eu
www.embl-hamburg.de
Table of contents
Welcome……………………………………………………………………………….. . 2
Programme
Monday, June 20……………………………………………………………… . 3
Tuesday, June 21…………………………………………………….. ............ 4
Wednesday, June 22……………………………………………………….… . 6
Logistic Information
The DESY Campus…………………………………………………………… 7
Poster session information……………………………………………….... ... 8
Information for speakers………………………………………………… ....... 8
Internet, electricity, currency…………………………………………… ........ 9
Useful telephone numbers……………………………………………………. 9
Printing and photography……………………………………………………. . 9
Catering………………………………………………………………………. ... 9
Hamburg public transport……………………………………………….. ..... 10
Sight-seeing information about Hamburg…………………………………… .... 13
Speakers’ abstracts…………………………………………………………….. ..... 17
Poster abstracts…………………………………………………………….. ........... 48
List of conference participants………………….………………………….......... 60
1
WELCOME
Dear conference participant,
We very warmly welcome you to the 2nd International Conference “Peroxisome Formation,
Function and Metabolism” and to the city of Hamburg.
The conference is jointly organized by two partners of the European Research & Training
Network PERFUME (www.itn-perfume.eu/): the University of Hamburg and the European
Molecular Biology Laboratory (EMBL), in collaboration with the PERFUME network
coordinator from the University of Groningen, The Netherlands.
The conference venue will be on campus of the German Research Center DESY, which
hosts a leading synchrotron radiation facility (Petra III) and the European X-ray Free Electron
Laser, which will start operation next year 2017. In life sciences, these facilities are mainly
used for getting highest quality X-ray diffraction data, to determine structures of biological
macromolecules at atomic resolution. The EMBL Unit on this site operates three synchrotron
radiation beamlines at the Petra III ring for applications in life sciences.
We are extremely pleased to host a number of leading scientists from the field of
peroxisomal research, both at the senior and junior level. Some additional presentations will
be given by members of the PERFUME network. We hope that, apart from the scheduled
oral and poster presentations, there will be ample of opportunities for interactions and
discussions.
Please make of use of all opportunities offered, to visit our research facilities on DESY
campus or explore the many different flavors of the city of Hamburg. Our staff will be more
than happy to advise you on potential idea.
Last but not least, we would like to acknowledge the support of the EU Marie Curie ITN grant
PERFUME (Grant Agreement Number 316723). We are very grateful for the generous
financial support from our sponsors Beckman Coulter, Nikon, Jena Bioscience, Seqlab/
Microsynth and Carl Roth.
With our warmest regards,
Sigrun Reumann, University of Hamburg and University of Stavanger (Norway)
Matthias Wilmanns, European Molecular Biology Laboratory, Hamburg Unit
Ida van der Klei, University of Groningen, The Netherlands
Sponsors:
2
PROGRAMME
Monday, June 20
12:30
13:30
Registration
13:30
13:45
Matthias Wilmanns
(EMBL Hamburg, Germany)
Welcome
Session 1:
Peroxisomes in human health and disease
(chair: Michael Schrader, Exeter, UK)
13:45
14:15
Ronald Wanders
(Amsterdam,
The Netherlands)
Metabolic functions of peroxisomes and
their interaction with other organelles in
health and disease
14:15
14:45
Hans Waterham
(Amsterdam,
The Netherlands)
Human peroxisomal disorders: novel
phenotypes, novel genotypes
14:45
15:00
Kim Falkenberg
(Amsterdam,
The Netherlands)
ACBD5 deficiency is a novel peroxisomal
disorder
15:00
15:15
Katharina Herzog
(Amsterdam,
The Netherlands)
Lipidomic analysis of fibroblasts from
Peroxisomal Disorder patients identifies
disease-specific phospholipid ratios
15:15
15:30
Afsoon Sadeghi Azadi
(Exeter, UK)
Elucidating novel signalling pathways in
peroxisome proliferation in mammals
15:30
16:00
Coffee/tea
Session 2:
Computational analyses of peroxisomes
(chair: Barbara Bakker, Groningen, The Netherlands)
16:00
16:30
Vitor Martins Dos Santos
(Berlin, Germany)
Semantic technologies for data integration
and network modeling
16:30
16:45
Robert Smith
(Berlin, Germany)
Providing a framework for in silico analysis
of large-scale dynamic metabolism
16:45
17:00
Nicola Bordin
(Sevilla, Spain)
Functional and structural characterization
of peroxisomal proteins through integrative
computational biology
17:00
17:15
Agnieszka Wegrzyn
(Groningen,
The Netherlands)
Genome-scale constraint-based model of
Refsum’s disease at work – predictions
and validation
17:15
17:45
Ralf Erdmann
(Bochum, Germany)
The PerTrans Network
18:00
19:30
Dinner
19:30
Open
end
Networking and socializing
with beer and wine
3
Tuesday, June 21
Session 3:
Peroxisome biogenesis / de novo formation and fission
(chair: Matthias Wilmanns, Hamburg, Germany)
99:00
99:30
Suresh Subramani
(San Diego, USA)
Functional characterization of peroxin
domains required for peroxisome
biogenesis
9:30
9:45
Julia Ast
(Marburg, Germany)
Pex5-dependent peroxisomal import in
Ustilago maydis
9:45
10:00
Justyna Wroblewska
(Groningen,
The Netherlands)
A variety of proteins localize to S.
cerevisiae pre-peroxisomal membrane
vesicles in the absence of Pex3
10:00
10:15
Anirban Chakraborty
(Bochum, Germany)
Involvement of PMP Pex34 in de novo
synthesis of peroxisomes
10:15
10:45
Coffee/tea
10:45
11:15
Ida van der Klei
(Groningen,
The Netherlands)
Peroxisomal membrane biogenesis
11:15
11:30
Katharina Haupenthal
(Lübeck, Germany)
Pex19 regulates Pex3 integration into the
endoplasmic reticulum membrane
11:30
11:45
Daniel Luis Cruz Zaragosa
(Bochum, Germany)
Analysis of pex3 mutant reveals early
stages of the de novo formation of
peroxisomes in Saccharomyces cerevisiae
12:00
14:00
Lunch and
Poster session
Session 4:
Plant, fungal and protist peroxisomes
(chair: Sigrun Reumann, Hamburg/Stavanger)
14:00
14:30
Jianping Hu
(East Lansing, USA)
A role for the ubiquitin-proteasome system
in regulating plant peroxisome protein
import
14:30
15:00
Imogen Sparkes
(Exeter, UK)
Peroxisome dynamics in plants
15:00
15:15
Louisa Sandalio
(Granada, Spain)
Peroxisomal dynamics regulate rapid cell
responses to environmental stresses
through a reactive oxygen speciesmediated pathway: role of peroxin PEX11a
15:15
15:30
Delphine Crappe
(Stavanger, Norway)
Pathogen defense by plant peroxisomes:
Protein targeting and functional analyses
of peroxisome-targeted NHL proteins
15:30
16:00
Coffee/tea
4
Session 4:
Plant, fungal and protist peroxisomes (cont.)
16:00
16:30
Paul Michels
(Edinburgh, UK)
The occurrence and functions of
peroxisomes in trypanosomatids and other
protists
16:30
16:45
Eglys Gonzalez-Marcano
(Merida, Venezuela)
Possible function of the pyruvate
phosphate dikinase (PPDK) located at the
glycosomal membrane in Trypanosoma
cruzi
16:45
17:00
Ritika Singh
(Groningen,
The Netherlands)
Identification of novel stress related
proteins in yeast peroxisomes
17:00
17:15
Piotr Lisik
(Stavanger, Norway)
Identification and characterization of a
novel survival protein, SurE-like
phosphatase/
nucleotidase, in Arabidopsis peroxisomes
17:15
17:30
José M. Palma
(Granada, Spain)
Peroxisomes from pepper fruits: New
perspectives on the organelle’s
metabolism
17:30
19:00
DESY tour
19:30
open
end
BBQ Dinner
5
Wednesday, June 22
Session 5:
Structural Biology of peroxisomes
(chair: Damien Devos, Sevilla, Spain)
9:00
9:30
Hiroaki Kato
(Kyoto, Japan)
Recognition mechanism of the second
peroxisomal targeting signal, PTS2, by its
receptor complex Pex7p and Pex21p
9:30
10:00
Matthias Wilmanns
(Hamburg, Germany)
Peroxisomes as tools in structural biology
10:00
10:15
Evdokia-Anastasia
Giannopoulou
(Hamburg, Germany)
Structural analysis of proteins involved in
peroxisome biogenesis
10:15
10:30
Coffee/tea
Session 6:
Peroxisomes as biological systems
(chair: Ida van der Klei, Groningen, The Netherlands)
10:30
11:00
Einat Zalckvar
(Rehovot, Israel)
Systematic characterization of proteome
localization dynamics during growth in
oleic acid reveals a novel peroxisome
targeting receptor
11:00
11:30
Marc Fransen
(Leuven, Belgium)
Imaging peroxin-protein interactions in
mammalian cells: challenges and
opportunities
11:30
12:00
Ida van der Klei
(Groningen,
The Netherlands)
Closure
12:00
12:30
Lunch
6
Logistic Information
The DESY campus
The main entrance to the DESY site is located at the following address:
Notkestrasse 85, 22607 Hamburg. The conference will take place in the CFEL building
(building 99, circled). The seminar room can be found on the ground floor.
For the participants staying on campus, there is a cafeteria in the canteen building (9) where
breakfast is available from 7 a.m., Monday to Friday. A bistro can be found in the adjacent
building (9a, marked on the map below) where dinner is served from 17:00 to 23:00, Monday
to Friday.
Map of DESY Campus and CFEL Building
7
Poster session information




The exact number of your poster has been shared via email and will be available on
the notice board.
You could already set up your board when you arrive and keep it for the duration of the
conference.
Size of the poster board: A0, 120 x 180 cm. These panels have a portrait orientation.
You can mount your poster with the pins we supply on-site.
Information for speakers
All talks will be held in the seminar room in the CFEL building (99). Please get in touch with
a member of the EMBL IT group in the room in advance of the time of your talk in
preparation. We would prefer to run your presentation on our machines due to
incompatibilities that might appear if running from your own workstations.
If your presentation contains movies, please save the movie files and the presentation in
one folder (with an indication of order). The file name should be labeled with your name and
the date that the file was last modified or created.
WE ASK YOU TO PLEASE MAKE SURE TO KEEP WITHIN YOUR PRESENTATION TIME LIMIT.
8
Internet, electricity and currency
Wi-fi is available over the ‘DESY-guest’ network. No username and password are required.
Currency:
1 euro = approximately USD 1.11 (indicative rate 17 June 2016)
1 euro = approximately GBP 0,793 (indicative rate 17 June 2016)
1 euro = approximately JPY 115,7 (indicative rate 17 June 2016)
1 euro = approximately NOK 9.42 (indicative rate 17 June 2016)
Electricity: 220 volts, 50 Hz European plugs with two circular metal pins are used
Local time: GMT + 1 hour
Useful telephone numbers
Below are the most important emergency service phone numbers for Hamburg.
Police 110
Ambulance 112
Fire Department 112
Printing and photography
During the conference, an EMBL staff member will be taking photographs. If you would not
like to appear in these, please inform the photographer or a member of the Administration
Team.
If you need to print your boarding pass or train ticket, please contact the EMBL admin staff.
Catering
All meals and coffee breaks are included in the registration fee.
Coffee breaks and lunch will take place in the foyer of the CFEL building.
Dinner:
- Dinner on Monday will be served in CFEL
- Dinner on Tuesday will be in the DESY Bistro
There will be beverages served in the CFEL building during the poster sessions.
9
Hamburg Public Transport
Train tickets can be bought from the DB ticket machines at every station and cost 3.20 € for a single
ticket. A one way journey within the Greater Hamburg area (includes DESY, airport and main train
station) can be purchased by selecting the Großbereich tariff. Tip: Change the language on the ticket
machine by selecting the corresponding flag on the main screen.
A day ticket (Tageskarte) for use on buses and trains can be purchased for 7.20 €.
Three-day public transport tickets are available by buying a Hamburg Card available online, at the
airport or from main Hamburg train station. A three-day ticket costs 21.90€ and offers discounts at a
variety of restaurants, cruises and attractions within Hamburg.
www.hamburg-travel.com/search-booking/hamburg-card/hamburg-card-savings/
Taxi
Local taxis can be ordered from the following companies:
Taxi Hamburg: +49 (0) 40 666 666
Hansa Taxi:
+49 (0) 40 211 211
Car
There are designated parking spaces along the road leading to the CFEL building on campus. Unless
the area is reserved, these can be used by participants and speakers who are travelling by car.
Train
If you plan to travel by train from or to somewhere outside of Hamburg, please check the German Rail
website for details on times and prices:
http://www.bahn.de/i/view/USA/en/index.shtml
Travel to and from Hamburg International Airport
Hamburg airport is directly connected to the city centre with the S1 train line. The S1 train leaves from
the lower ground floor of the airport and travels via the main station (Hamburg Hbf) to Altona,
Othmarschen and Blankenese. For those taking the train from the airport to DESY, please take either
the S1 or S11 and change to the number 1 bus at Othmarschen station.
10
Train network in Hamburg
.
11
Bus
Public buses leave from outside the main entrance to the DESY campus regularly. To head towards
the centre or east of Hamburg, take the bus in the direction of Altona. By getting off at the stop
‘Othmarschen’, you can change to the local train service which will take you directly to the airport or
main train station.
Public bus timetable (Sundays)
Zum Hünengrab (DESY)
Othmarschen
dep. 9:24 Every 20 12:24 Every 10 20:04 20:12 Every 10 22:12
arr. 9:31 minutes 12:31 minutes 20:11 20:19 minutes 22:19
Othmarschen
Zum Hünengrab (DESY)
dep. 8:24 Every 20 11:04 11:21 Every 20 13:01 Every 10 20:01
arr. 8:31 minutes 11:11 11:28 minutes 13:08 minutes 20:08
Public bus timetable (Monday - Friday)
Zum Hünengrab (DESY)
dep. 9:02 Every 10 18:42
18:54 Every 10 20:04
Othmarschen
arr.
9:10 minutes 18:50
19:01 minutes 20:11
Othmarschen
dep.
13:00
Every 10
17:40
17:51
Every 10
20:21
Zum Hünengrab (DESY)
arr.
13:07
minutes
17:47
17:58
minutes
20:28
Please make sure that you are at the bus stop 5 minutes before the departure time.
12
Sight-seeing information for Hamburg
Alster
The beautiful lake which you will see in the
heart of Hamburg is the Alster. The
Binnenalster, or Inner Alster, is one of
two artificial lakes within the city limits
of Hamburg, which are formed by the
river Alster. The other lake is the Aussenalster, Outer Alster, which used to be outside
of the city walls many years ago. In the
summer, locals come to the Alster to relax on
the banks or to do water sports such as
sailing, rowing and kayaking. In the winter if
the temperature falls well under 0 °C and the
ice is thick enough, it also offers wonderful
skating opportunities.
Shopping
The main shopping areas in the city centre are the Europa Passage, Jungfernstieg, Neuer
Wall, Grosse Bleichen and Gänsemarkt. Please note that shops close at 20:00 on working
days. Shops do not open on Sundays.
Eating out in Hamburg
Recommended restaurants in Hamburg:

Jochen Albrecht Brewery - Adolphsbrücke 7, 20457 Hamburg, Tel: 040 - 36 77 40

Restaurant Raven – Mittelweg 161, 20148 Hamburg, Tel: 04041424550

Gröninger
Privatbrauerei
Willy-Brandt-Str.
47,
20457
Hamburg,
Tel: 040 570 105-100

Blockbräu* – Landungsbruecken, 20359 Hamburg, Tel: 040 4450 500 114
*This restaurant offers excellent beer, a wonderful view of the harbour and is 10 min from the
Portuguese quarter ad 20 min on foot from the Reeperbahn.
13
Social activities
There are plenty of things to do in Hamburg. Please contact the admin team for assitance in
planning any free time you may have prior to or following the worksho
Harbour Boat Trip
Discover the Elbe with a harbour boat trip.
The boats begin their one-hour tour every
day at 12:00, 15:30 and 16:30 (German
commentary).
There is a daily tour at 12:00 in English.
The tickets cost 17.00 € and are available
at the office of “Elbe Erlebnistoerns”,
Bridge 2 in Landungsbrücken.
Tel: + 49 (0) 4021 4627
Nearest station: Landungsbrücken (S1,
S2, S3). Please make sure that you are at
the harbour 20-30 minutes beforthe
departure time.
The ‘Miniature Wonderland’
Another exciting and popular attraction is the “Miniature Wonderland” in the harbour area of
Hamburg. It is the largest model railway in the world and includes replicas of Hamburg, the
Harz mountain range, Austria, the USA, Scandinavia, Switzerland and even boasts a
miniature airport with aircraft.
Ticket price: 12.00€
Opening hours:
Sun.08.09: 8:30 - 20:00
Mon. 09.09, Wed. 11.09 and Thurs. 12.09: 9:30 18:00, Tue. 10.09: 9:30 - 21:00
Address: Kehrwieder 2-4, Block D 20457 Hamburg
– Speicherstadt
Nearest station: Baumwall (U3) or Messberg (U1)
14
Reeperbahn
Do not miss out on a visit to the heart of Hamburg's infamous nightlife – the Reeperbahn.
Visited and loved by The Beatles and many other celebrities, the Reeperbahn has a lot to
offer - pubs, bars, discos, live bands, music clubs, erotic shows and other “sinful” nightspots.
You can get there with the S1 train by getting off at the “Reeperbahn” station.
Speicherstadt-Tour
The Speicherstadt (lit. city of warehouses,
meaning warehouse district) in Hamburg,
Germany is the largest timber-pile founded
warehouse district in the world. It is located
in the port of Hamburg—within the HafenCity
quarter—and was built from 1883 to 1927.
The district was built as a free zone to
transfer goods without paying customs. As of
2009 the district and the surrounding area is
under redevelopment.
Please contact the admin team for more
information.
Hafencity Tour including Historic Speicherstadt
Booking in advance is required. Please contact the admin team if you are interested in
taking the tour.
With the development of HafenCity on the
Elbe waterfront, Hamburg is setting new
standards, in Europe. By 2025 a city brimming
with life will have taken shape on a site
covering 157 hectares. In contrast to other city
areas dominated purely by offices and retail, it
will blend maritime atmosphere with work,
living, the arts, leisure, tourism and shopping.
With its many shops, cafés and restaurants,
the western section is already an urban space
with nearly 2,000 residents and attracts a
growing number of visitors. Nowadays some
9,000 people are at work in HafenCity,
employed by more than 450 companies,
including 40 larger businesses with space requirements exceeding 1,000 sqm for up to 1,400
employees.
(Source: http://www.alstertouristik.de/English/classics/alster-round-trip.html)
15
HafenCity is a quarter in the district of Hamburg-Mitte in Hamburg, Germany. It is located on
the Elbe river island that was formerly called Kehrwieder and Wandrahm. HafenCity
Hamburg is a project of city-planning where the old port warehouses of Hamburg are being
replaced with offices, hotels, shops, official buildings, and residential areas. The project is the
largest rebuilding project in Europe in scope of landmass (approximately 2,2 km²).
Enjoy your stay in Hamburg!
16
SPEAKERS’ ABSTRACTS
Session 1, Monday 13:45-14:15:
Functions of peroxisomes in human metabolism and interplay with
other subcelluar organelles
Ronald J.A. Wanders
University of Amsterdam, Academic Medical Center, Departments Clinical Chemistry and Pediatrics,
Emma Children’s Hospital, Laboratory Genetic Metabolic Diseases, Meibergdreef 9, 1105 AZ,
Amsterdam, The Netherlands
The essential role of peroxisomes in cellular metabolism is emphasized by the existence of a
number of different genetically determined diseases in which one of these peroxisomal
functions is impaired. The four major functions of peroxisomes, at least in humans, from the
perspective of human diseases include: (1.) fatty acid (FA) beta-oxidation; (2.) ether
phospholipid (EPL) synthesis; (3.) fatty acid alpha-oxidation, and (4.) glyoxylate
detoxification. Peroxisomes can only fulfill their role in cellular metabolism in close
collaboration with other organelles. This is true for fatty acid oxidation and basically all other
functions of peroxisomes. Indeed, whereas mitochondria can catalyze the oxidation of fatty
acids down to CO2 and H2O thanks to the presence of the citric acid (Krebs) cycle and
respiratory chain in mitochondria, peroxisomes can only chain shorten fatty acids, followed
by the export of the products of peroxisomal beta-oxidation (acetyl-CoA, propionyl-CoA, and
various medium-chain acyl-CoAs) to mitochondria either in the form of the corresponding
carnitine ester or in their free acid form. With respect to the second major function of
peroxisomes, i.e. ether phospholipid synthesis, peroxisomes only catalyze the first initial
steps of EPL biosynthesis leading to the synthesis of alkyldihydroxyacetone phosphate
and/or alkyl-glycerone phosphate followed by transfer of these metabolites to the
endoplasmic reticulum (ER) for all subsequent reactions. Furthermore, all enzymatic steps
involved in FA alpha-oxidation do take place in peroxisomes but, again, alpha-oxidation is
fully dependent upon the interaction with other organelles, notably mitochondria for provision
of 2-oxyglutarate, reoxidation of NADH, removal of end products of alpha-oxidation, et
cetera. Finally, also with respect to glyoxylate detoxification, peroxisomes are heavily
dependent upon the interaction with mitochondria again since glycine, the final end product
of glyoxylate detoxification in peroxisomes, is degraded to CO2 and H2O in mitochondria.
17
Session 1, Monday 14:15-14:45:
Human peroxisomal disorders: novel genotypes, novel phenotypes
Hans R. Waterham
Laboratory Genetic Metabolic Diseases, Academic Medical Center at University of Amsterdam, The
Netherlands
Peroxisomes are dynamic organelles, which, in humans, play an essential role in a variety of
cellular catabolic and anabolic metabolic pathways, including fatty acid alpha- and betaoxidation, and plasmalogen and bile acid synthesis. Defects in genes encoding peroxisomal
proteins can result in a large variety of peroxisomal disorders affecting either specific
metabolic pathways, i.e. the single peroxisomal enzyme deficiencies, or causing a
generalized defect in function, assembly and maintenance of peroxisomes, i.e. peroxisome
biogenesis disorders. Clinically, patients with peroxisomal disorders may present with
variable severity ranging from early lethality to subtle neurosensory aberrations.
I will briefly review clinical, biochemical and genetic aspects of the different human
peroxisomal disorders known to date, with emphasis on some recently discovered defects.
18
Session 1, Monday 14:45-15:00:
ACBD5 deficiency is a novel peroxisomal disorder
Sacha Ferdinandusse1, Kim D. Falkenberg 1, Janet Koster1, Petra A. Mooyer1, Richard
Jones2, Carlo W.T. van Roermund1, Michael Schrader3, Amy Pizzino4, Ronald J.A.
Wanders1, Adeline Vandever4, Hans R. Waterham1
1
Laboratory Genetic Metabolic Diseases, Department of Clinical Chemistry, Academic Medical Center,
2
3
Amsterdam, the Netherlands Kennedy Krieger Institute, Baltimore, Maryland, USA Department of
Biosciences, College of Life and Environmental Sciences, University of Exeter Exeter, UK.
4
Department of Neurology, Children's National Health System, 111 Michigan Avenue, Northwest,
Washington, DC 20010, USA
In a patient presenting with progressive motor skill, neurological and visual impairments, we
detected abnormalities in peroxisomal metabolism. More precisely, the patient showed an
impaired beta-oxidation of very-long-chain fatty acids (VLCFAs) leading to their
accumulation, whereas other peroxisomal parameters were normal. Although the
biochemical profile was characteristic for a defect of peroxisomal VLCFA oxidation, we could
not identify a mutation in any of the known proteins involved in this process.
Unexpectedly, we identified a homozygous deletion in the ACBD5 gene of the patient,
resulting in the complete absence of ACBD5 proteins. ACBD5 is a peroxisomal membrane
protein with a cytosolic acyl-CoA binding domain. The exact function of ACBD5 has not been
established, but it had been suggested to be involved in peroxisome degradation or in
intracellular transport of acyl-CoA esters.
We could confirm that the identified ACBD5 mutation is the cause of the patient’s disease by
performing complementation assays in the patient’s cells. Additionally – by generating an
ACBD5 knock out cell line using CRISPR-Cas9 genome editing – we verified that an ACBD5
deficiency indeed causes accumulation of VLCFAs. Taken together, our studies indicate that
ACBD5 plays a crucial role in the peroxisomal processing of VLCFAs, extending our
understanding of peroxisomal metabolism. Furthermore, we discovered that ACBD5
deficiency is a novel peroxisomal single enzyme deficiency.
19
Session 1, Monday 15:00-15:15:
Lipidomic analysis of fibroblasts from Peroxisomal Disorder
patients identifies disease-specific phospholipid ratios
Katharina Herzog1, Mia L. Pras-Raves1,2 , Martin A. T. Vervaart1, Angela C. M. Luyf1,2 ,
Antoine H. C. van Kampen1,2,3 , Ronald J. A. Wanders1, Frederic M. Vaz1#, Hans R.
Waterham1#
1
2
Laboratory Genetic Metabolic Diseases, and Bioinformatics Laboratory, Department of Clinical
Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam,
3
Amsterdam 1105 AZ, The Netherlands. Biosystems Data Analysis, Swammerdam Institute for Life
Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.;
#
shared last authors
Peroxisomes are subcellular organelles involved in various metabolic processes, including
fatty acid and phospholipid homeostasis. The peroxisomal disorders (PD) represent a group
of genetically heterogeneous metabolic diseases caused by the dysfunction of peroxisomes.
Accordingly, cells from PD patients are expected to have an altered composition of fatty
acids and phospholipids.
Using an LC/MS-based lipidomics approach, we show that the phospholipid composition is
characteristically altered in cultured primary skin fibroblasts from PD patients when
compared to healthy controls. We observed a marked overall increase of phospholipid
species containing very long chain fatty acids, and a decrease of phospholipid species with
shorter fatty acid species in PD patient fibroblasts.
Based on our data, we present a set of specific phospholipid ratios for fibroblasts that clearly
discriminate between PD patients, and those from healthy controls. Our findings will aid in
the diagnosis and prognosis of PD patients, including an increasing number of mild patients
in whom hardly any abnormalities are observed in biochemical parameters commonly used
for diagnosis.
20
Session 1, Monday 15:15-15:30:
Elucidating novel signalling pathways in peroxisome proliferation in
mammals
Afsoon Sadeghi Azadi & Michael Schrader
Biosciences, University of Exeter, UK
The ability of peroxisomes (PO) to respond dynamically to environmental changes and
stresses, for example by increasing their number and/or enzymatic capacity, is essential for
cellular and organismal function and health (1-3). Despite the importance of PO proliferation,
the signalling pathways regulating this process are poorly understood, in particular in
humans. In order to identify novel signalling pathways and associated factors involved in PO
proliferation, we have developed a cell-based PO proliferation assay to investigate different
stimuli and their capacity to induce PO proliferation. Using qPCR, differential expression
patterns for human peroxins were revealed. Combining this assay with pharmacological and
transcriptomic analyses resulted in the identification of novel regulatory signalling pathways.
We applied bioinformatics approaches to screen promoter regions of PO genes for regulatory
elements and performed network analyses. A map of regulatory motif sites across the human
PO genes has been developed. Our analysis revealed differences in transcription factor
binding sites between metabolic and biogenetic PO genes suggesting differential regulation.
For the first time, we demonstrate a link between TGFβ signalling and Pex11-mediated PO
proliferation. Our findings suggest an involvement of PO function in cell growth,
differentiation and inflammatory processes.
(1) Schrader et al. 2016, BBA 1863: 971–983
(2) Ebberink et al. 2012, J Med Genet 49:307-13
(3) Delmaghani et al. 2015, Cell 163: 894–906
21
Session 2, Monday 16:00-16:30:
Semantic web technologies applied to integration of biological data
JC. J. van Dam1, J.J. Koehorst 1, E Saccenti1, , P.J Schaap1,
V. A. P. Martins dos Santos1,2 , M Suarez-Diez1
1
Laboratory of Systems and Synthetic Biology, Wageningen University,
LifeGlimmer GmbH, Berlin, Germany. [email protected]
The Netherlands
2
1. Introduction
Systems Biology applies various computational strategies to integrate heterogeneous data to
model and to discover properties of biological systems. Semantic web technologies have a
tremendous potential for the integration of heterogeneous data sets.
The RDF data model is a mature W3C standard designed for the integrated representation
of heterogeneous information from disparate sources and it is proving effective for creating
and sharing biological data. RDF is not a data format, but a data model for describing
resources in the form of self-descriptive subject, predicate and object triples that can be
linked in an RDF-graph. Integration of heterogeneous data from different sources in a single
graph relies on using retrievable controlled vocabularies, which is essential to access and
analyse integrated data Once data sources are converted into the semantic Web,
SPARQLcan be used to integratively query them no matter their origin. Therefore, an
increasing number of widely used biological resources are becoming available in the RDF
data model.
Genome annotation is the first step in understanding bacterial species and paves the way to
the development of metabolic models and engineering strategies. Currently, de novo
genome annotation is performed through semi-automated work-flows integrating
complementary methods relying on database search. The queried databases are constantly
updated, therefore, for each annotation, provenance has to be stored. Provenance includes
likelihood values that can be re-evaluated when integrating different results for specific
requirements.
2. Results
We developed a Semantic Annotation Platform for Prokaryotes (SAPP) that includes the full
chain of provenance for every step of the annotation work-flow. Data is stored in RDF which
allows sharing across different applications and integration of new data.
RDF resources can be readily queried with SPARQL. However, constructing these queries
requires knowledge of the underlying structure of the integrated database(s). This structure
can be complex, since RDF allows the integration of a heterogeneous data sets that can be
added at any time, which in turn expands the underlying structure. Therefore we have
developed RDF2GRAPH a tool that automatically recovers the structure of an RDF resource,
such as the one created through SAPP. The recovered structure can be used to efficiently
generate SPARQL queries.
3. Discussion
This strategy has been successfully applied, among other for the identification of differences
between syntrophic and non-syntrophic butyrate and propionate degraders and to generate
hypothesis, hereby significantly reducing the experimental workload.
The presented examples show the potential of semantic web technologies in their application
to the field of systems biology. The developed tools will be helpful for improving the usability
of semantic web technologies, which is required for data integration in (computational)
biology, systems biology and the emerging field of semantic systems biology.
22
Session 2, Monday 16:30-16:45:
Providing a framework for in silico analysis of large-scale dynamic
metabolism
R. P. van Rosmalen1, A. B. Wegrzyn2, B. Bakker2, C. Fleck1, V. A. P. Martins dos Santos1,3,
R. W. Smith1,3
1
Laboratory of Systems & Synthetic Biology, Wageningen UR, The Netherlands
Systems Biology Centre for Energy Metabolism and Ageing, University Medical Centre Groningen,
3
Groningen, The Netherlands LifeGlimmer GmbH, Berlin, Germany [email protected];
[email protected]
2
Within the PerFuMe (PERoxisome Formation, Function and Metabolism) network, data has
been collected at several biological levels from the genome-scale to detailed kinetics of fattyacid oxidation pathways in peroxisomes, mitochondria and endoplasmic reticulum. As part of
the Systems Biology approach employed by PerFuMe, individual mathematical models have
been constructed to describe each of these data levels and aid the generation of
experimental hypotheses. However, a unified model describing the effects of
compartmentalisation of fatty-acid oxidation on large-scale metabolism is yet to be achieved.
This is, in part, due to the lack of a computational framework whereby the mathematical
description of large-scale metabolism and signaling pathways can be made equivalent. In
this talk I shall introduce a new pipeline that converts genome-scale reaction maps into
parameterized mathematical models that allow for the simulation of dynamic behaviours
within metabolism. We will highlight the generality of these steps by analyzing model
organisms (such as E. coli and S. cerevisiae) and the pathway that regulates central carbon
metabolism in L. lactis. Furthermore, model reduction techniques can be included such that
single pathways, including their feedback with global metabolic effects, can be simulated. By
adding this tool to the repertoire of computational techniques currently used to analyze
metabolism, we hope that the peroxisome community will one day be able to understand the
effects of altered peroxisomal metabolism on global metabolic responses.
23
Session 2, Monday 16:45-17:00:
Functional and structural characterization of peroxisomal proteins
through integrative computational biology
Nicola Bordin
Centro Andaluz de Biologia del Desarollo, Universidad Pablo de Olavide, Sevilla
During last decade, the gap between sequence determination and functional annotation has
increased dramatically, representing an incomplete understanding of the data we have
generated. Automatic annotation pipelines ease the burden of manual annotation, but are
limited in scope and coverage. Computational tools for proteome annotation are intrinsically
conservative in assigning a definitive function (76% of proteins in UniProt/TrEMBL are
annotated as “unknown” or “uncharacterized”) and tend to focus on specific aspects of the
protein such as functional domains, signal peptide prediction, or the presence of
transmembrane helices, among others. Compartmentalizing the annotation gives a very
specific characterization of an aspect of the protein, at the cost of losing the general overview
of the protein's function and role in its environment. Integrating results from several
databases and tools allows us to simultaneously question several related aspects of a
protein's function. We have created a computational pipeline that combines the advantages
of manual curation with the speed and power of bioinformatics. The pipeline is modular,
open, can be installed at your location or run on our server. The pipeline allows the
characterization of whole proteomes as well as single proteins. In collaboration with various
PerFuMe consortium members, we are applying the developed pipeline to subsets of
proteins of interest, determining Pex homologs in various model organisms, characterizing
their function and structure and how they interact within the cellular environment.
24
Session 2, Monday 17:00-17:15:
Genome-scale constraint-based model of Refsum’s disease at work
– predictions and validation
Agnieszka Wegrzyn1, Katharina Herzog2, Hans Waterham2 and Barbara Bakker1
1
Systems Biology Centre for Energy Metabolism and Ageing, University Medical Centre Groningen,
2
University of Groningen, Groningen, The Netherlands Department of Clinical Chemistry and
Pediatrics, Laboratory Genetic Metabolic Disease, Academic Medical Center, University of
Amsterdam, The Netherlands [email protected]
The majority of the fatty acids (FAs) present in the human body are oxidized via the
mitochondrial β-oxidation pathway. Metabolism of very-long chain FAs, phytanic acid and
dicarboxylic acids is, however dependent on peroxisomal α- and β-oxidation, and supported
by ω-oxidation in the endoplasmic reticulum. Peroxisomal FAO plays a crucial role as a
detoxifying pathway which is stressed by the existence of several inborn peroxisomal
deficiencies, such as Refsum’s disease. It is an autosomal-recessive disease characterised
with neurological degeneration. Patients accumulate phytanic acid in cells and plasma due to
a deficiency of phytanoyl-CoA hydroxylase (PHYH). Recently a FA ω-oxidation pathway, has
been proposed as a potential route to eliminate the phytanic acid in Refsum’s patients.
Regretfully, our knowledge about the effect of phytanic acid on the cell metabolism and its
regulation is scarce. In this project, a systems biology approach is applied to study the
systems response to defects in peroxisomal FAO, in particular PHYH deficiency.
Our human genome-scale metabolic model is based on the newest version of Recon 2.
Artificial-centring hit-and-run (ACHR) sampling of the solution space of our manually curated
model was applied to determine the flux distribution and the robustness of the network upon
a single-gene deletion.
To provide a physiological context for model input and output, experiments on fibroblasts
from Refsum’s patients were performed. Briefly, control and patients fibroblasts were grown
in Ham’s F-10 medium with or without a phytanic acid. Cells were collected after 72 hours for
lipidomics, RNA-seq and proteomics. Additionally cells and media were collected at 0, 24, 48
and 72 hours for protein determination as well as metabolomics analysis.
We present the curated and extended fatty-acid oxidation module and show initial predictions
of the effect of Refsum’s disease on the metabolic network. Furthermore, we show the
preliminary results from our in vitro studies comparing various omics readouts with the model
predictions.
We expect that our analysis of metabolic changes upon single enzyme deficiencies in
peroxisomal FAO pathways will contribute to the development of new strategies for treatment
or diagnosis of a broad spectrum of diseases.
25
Session 2, Monday 17:15-17:45:
Protein Import into Peroxisomes
Ralf Erdmann
Ruhr-University Bochum, Medical Faculty, System Biochemistry, D-44780 Bochum, Germany
Peroxisomal matrix proteins contain specific peroxisomal targeting signals (PTS1 or PTS2)
that are post-translationally recognized and bound in the cytosol by the peroxisomal import
receptors, which direct the receptor-cargo complex to the peroxisomal membrane. The
cargo-loaded receptors insert into the peroxisomal membrane and assemble with other
membrane proteins to form the translocon, which as a transient pore allows the translocation
of the folded proteins across the membrane. In the following, import receptors are
ubiquitinated and released from the membrane in an ATP-dependent manner.
The research group PerTrans of the Deutsche Forschungsgemeinschaft brought together
nine scientists, which in a collaborative approach focus on the
• Structure and associated dynamics of the peroxisomal translocon for PTS1-proteins
• Analysis of membrane insertion and pore formation of the import receptors
• Elucidation of the mechanism of the translocation of folded protein across the
peroxisomal membrane
• Regulation of the function of the translocon
• Characterization of the translocon for PTS2-proteins
Here I will report on the dissection of steps in peroxisomal protein import with emphasis on
alternative protein import pathways into peroxisomes. The existence of two distinct PTS1receptors, in addition to two distinct PTS2-dependent import routes, contributes to the
adaptive metabolic capacity of peroxisomes in response to environmental changes.
26
Session 3, Tuesday 9:00-9:30:
Functional characterization of peroxin domains required for
peroxisome biogenesis
Gaurav Agrawal, Helen H. Shang and Suresh Subramani*
Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500
Gilman Drive, UC San Diego, La Jolla, CA 92093-0322, USA.
The peroxins Pex19 and Pex3 play an indispensable role in peroxisomal membrane protein
(PMP) biogenesis, peroxisome division and inheritance. We have mapped, using deletions,
the functional regions of the Pex19 protein that are required for peroxisome biogenesis.
Surprisingly, import-competent peroxisomes were still formed when Pex19 domains
previously believed to be required for biogenesis were deleted. In addition, a delay of 14-24 h
in peroxisome biogenesis was observed in these mutants. Deletions in the N-terminal Pex3binding site disrupted Pex19’s direct interactions with Pex3, but preserved interactions with
an mPTS-binding region of a PMP. In contrast, deletion of the C-terminal mPTS-binding
domain of Pex19 disrupted the interaction between Pex19 and some PMPs, while leaving
Pex19-Pex3 interactions intact. However, Pex11 and Pex17 retained their interaction with
both N and C-terminal deletions. Co-expression of both N and C-terminal constructs
improved growth observed with the single deletions. In conclusion, physical segregation of
the Pex3 and PMP-binding domains of Pex19 has provided novel insights into the modular
architecture of Pex19. We define the minimum region of Pex19 required for peroxisome
biogenesis.
27
Session 3, Tuesday 9:30-9:45:
Pex5-dependent peroxisomal import in Ustilago maydis
Julia Ast1, Domenica Martorana2, Johannes Freitag3, Michael Bölker1
1
Philipps Universität Marburg, Department of Biology, Karl-von-Frisch-Str. 8, D-35032 Marburg;
2
[email protected]
Georg-August Universität Göttingen, Department of Molecular
3
Microbiology & Genetics, Grisebachstr. 8, D-37077 Göttingen Department of Molecular and Cell
Biology, University of California at Berkeley, 475D Li Ka Shing Center #3370, Berkeley, CA 947203370
Peroxisomes are ubiquitous organelles that perform important metabolic reactions such as
the β-oxidation pathway for degradation of fatty acids. Peroxisomal proteins are translated in
the cytosol and are imported as fully folded, co-factor bound proteins and even as oligomers.
Proteins destined for the peroxisomal matrix harbor a short conserved C-terminal targeting
signal (PTS1), which is imported by the conserved cytosolic receptor protein Pex5, or an Nterminal targeting signal (PTS2), which is recognized by the receptor protein Pex7.
In the plant pathogenic fungus Ustilago maydis the vast majority of peroxisomal proteins
contain PTS1 and only a few ones carry PTS2. Remarkably, U. maydis encodes two Pex5
receptors which we named Pex5a and Pex5b. Despite both proteins show a high extent of
sequence similarity, single deletion mutants of pex5a or pex5b cannot be complemented by
expression of the other pex5 gene. Additionally, while deletion of pex5a abolished growth on
oleic acid and had nearly no effect on pathogenic development, Pex5b was found to be
important not only for growth on fatty acids, but also for filament formation and virulence.
Unlike to yeast but similar to mammals and plants the fungus U. maydis lacks a separate
Pex20 protein. Instead, the longer one of the two Pex5 proteins acts as a co-receptor of
Pex7. Pex5b of U. maydis is not only essential for PTS2 protein import but also for the import
of PTS1 proteins: We could show that Pex5a alone is not sufficient and only functions as a
PTS1 import receptor in the presence of Pex5b.
Transcription of pex5a and pex5b is differentially regulated during the life cycle of U. maydis
and both Pex5a and Pex5b show different target specificity suggesting modulation of the
peroxisomal proteome and metabolism during infection of its host plant and in adaption to
changing environmental conditions.
28
Session 3, Tuesday 9:45-10:00:
A variety of proteins localizes to S. cerevisiae pre-peroxisomal
membrane vesicles in the absence of Pex3
Justyna P. Wróblewska1, Kévin Knoops1, Ida J. van der Klei1, Einat Zalckvar2, Maya
Schuldiner2
1
Molecular Cell Biology, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
Molecular Genetics, Weizmann Institute of Science, Rehovot, 7610001, Israel
2
Peroxisomes are important organelles in which eukaryotic cells compartmentalize a variety of
specific metabolic processes. There is an ongoing debate on the molecular mechanisms of
peroxisome proliferation. In wild-type yeast cells peroxisomes multiply mainly by fission of
pre-existing ones, while in cells temporarily devoid of peroxisomes, they can form de novo.
So far, only a few proteins involved in this process are known.
pex3 yeast cells had been considered to lack any peroxisomal membrane structures until
pre-peroxisomal vesicles (PPVs) were identified in H. polymorpha cells upon blocking
autophagy. The peroxisomal membrane remnants in pex3 atg1 cells contain a subset of
peroxisomal membrane proteins (Pex13, Pex14 and Pex8) and are the target of Pex3 during
de novo peroxisome formation.
We have recently confirmed that PPVs are present in S. cerevisiae pex3 cells as well. We
aim to understand how those structures are formed in pex3 atg1 cells. Using a combination
of genetic, biochemical and microscopy approaches, we analyzed the protein composition of
these structures and searched for genes that are required for their formation.
We have performed localization studies of a set of 19 peroxins in the pex3 atg1 mutant. This
analysis indicated that in addition to Pex8, Pex13 and Pex14 several other peroxins
accumulate at pre-peroxisomal vesicles in pex3 atg1 cells. The other peroxins were either
mislocalized to the cytosol or mitochondria (Pex11). None of the PMPs tested accumulated
at the ER in S. cerevisiae pex3 cells. We have also identified many other interesting
candidate proteins that are targeted to pre-peroxisomal membranes in the absence of Pex3
and may be implicated in the formation of PPVs. The most promising candidates are being
analysed in order to uncover their function in relation with PPVs/peroxisome biogenesis.
29
Session 3, Tuesday 10:00-10:15:
Involvement of PMP Pex34 in de novo synthesis of peroxisomes
Anirban Chakraborty
Ruhr University Bochum, Bochum, Germany
Pex34 is an integral peroxisomal membrane protein, which is known to play a role in
peroxisome fission. It interacts with members of the Pex11 family of proteins to maintain this
role(Tower et al, 2011). Yeast two-hybrid analysis showed that it interacts with other PMPs
besides Pex11 family members. Also, it interacts with itself to form a homo dimer. Moreover,
all of these interactions are somehow dependent on Pex3 and Pex19. When either Pex3 or
Pex19 is deleted from the genome, all the interactions are missing. These results stress the
dependency of Pex34 on Pex19 and Pex3 and lay the fact that Pex34 is indeed involved in
peroxisome fission but might also be involved in de novo biogenesis of peroxisomes.
Δpex3Δpex34 cells were transformed with galactose-inducible Pex3p-GFP and time-lapse
fluorescence microscopy was done to see if Pex34 could restore the formation of
peroxisomes in this double deletion strain. After 2 hours, the Pex3p-GFP showed
mislocalised cytosolic pattern and single large punctae, which colocalised with the
peroxisomal marker DsRed SKL. This is different from what is seen in the the wild-type and
supports a function of Pex34p in de novo biogenesis of peroxisomes.
30
Session 3, Tuesday 10:45-11:15:
Peroxisomal membrane biogenesis in yeast
Yuan Wei, Arman Akşit, Arjen M. Krikken, Rinse de Boer, Anita Kram and
Ida J. van der Klei
Molecular Cell Biology, University of Groningen, PO Box 11103, 9300 CC Groningen, The
Netherlands, E-mail: [email protected]
Both vesicular and non-vesicular pathways have been proposed to be responsible for the
transport of lipids to peroxisomes. In yeast peroxisomes are fully devoid of lipid biosynthetic
enzymes. Therefore these organisms are ideal models to study how lipids are transported to
the peroxisomal membrane from their site of synthesis.
Non-vesicular transport of lipids occurs at membrane contact sites (MCS), regions of close
apposition between two membranes. We studied the occurrence and functions of MCSs
between peroxisomes and other cellular compartments using the yeast Hansenula
polymorpha as a model organism.
Electron microscopy analysis indicated that at peroxisome repressing growth conditions
(glucose) the single peroxisome that is present per cell is solely associated with the
endoplasmic reticulum (ER), whereas at conditions that induce peroxisome proliferation
(methanol) MCSs with vacuoles and mitochondria were present as well.
Localisation studies revealed that Pex23 is an ER protein that localizes to ER-peroxisome
MCSs. The absence of Pex23 in combination with the vCLAMP proteins Ypt7, Vam7 or
Vps39 resulted in a peroxisome deficient phenotype, whereas pex23, ypt7, vam7 and vps39
single deletion strains showed minor defects in the formation and abundance of
peroxisomes. Microscopy analysis of the double deletion strains showed that these cells
contain still small peroxisomes that appeared to be unable to expand, explaining why the
bulk of the matrix proteins is mislocalized to the cytosol. This phenotype could be largely
suppressed upon introduction of an artificial ER-peroxisome anchor protein.
Summarizing, our studies suggest that peroxisomes receive membrane lipids from both the
ER and vacuoles via MCSs.
31
Session 3, Tuesday 11:15-11:30:
Pex19 regulates Pex3 integration into the endoplasmic reticulum
membrane
Katharina Haupenthal, Enno Hartmann and Kai-Uwe Kalies
Institute of Biology, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
According to current models, peroxisomes can arise by growth and division of existing
peroxisomes or de novo via the endoplasmic reticulum (ER). In the latter model, it is
assumed that at least a subset of peroxisomal membrane proteins (PMPs) integrate into the
ER membrane before they bud into preperoxisomal vesicles. We investigate the biogenesis
of the single-spanning membrane protein Pex3 that exposes its short N-terminus to the
peroxisomal matrix. In earlier studies we could show that human PEX3 integrates in vitro into
ER-derived microsomes. Unexpectedly, PEX3 is efficiently transported even under
posttranslational conditions. Now we find that this posttranslational ER-integration of PEX3
can be blocked by recombinant PEX19 in a dose-dependent manner. This transport inhibition
is not unique for the mammalian system but can be observed also in the yeast system.
Moreover, farnesylation of PEX19 is not essential for this inhibition in vitro. Employing protein
chimeras of human PEX3 and yeast Pex22p we find that the transport inhibition relies on
interactions between PEX19 and the C-terminal part of PEX3. Using pull-down experiments
we confirm a direct interaction between PEX3 and PEX19. Surprisingly, even under
conditions where cotranslational and posttranslational protein translocation can occur in
parallel the PEX3 integration into microsomes is efficiently blocked by PEX19. This suggests
either that PEX19 also inhibits the cotranslational membrane integration of PEX3 or that
PEX3 integrates mainly posttranslationally under the conditions used. All together, we
speculate that the cellular PEX19 level regulates PEX3 integration into the ER. This could
answer the question how PEX3 decides between the two putative co-existing biogenesis
pathways – direct transport to existing peroxisomes or integration into the ER membrane.
32
Session 3, Tuesday 11:30-11:45:
Analysis of pex3 mutant reveals early stages of the de novo
formation of peroxisomes in Saccharomyces cerevisiae
Luis Daniel Cruz-Zaragoza, Ralf Erdmann
System Biochemistry, Ruhr-Universität Bochum, Germany
Peroxisomes are cellular organelles with a crucial role in the eukaryotic cell’s metabolism
and preservation. To maintain organellar function, the import of peroxisomal matrix and
membrane proteins has to be ensured. Extensive studies on the import mechanism of
luminal proteins led to a well-accepted model of peroxisomal matrix protein import. However,
different models on the targeting and insertion of peroxisomal membrane proteins (PMPs)
are under debate. Even if it seems to be clear that the ER contributes to the biogenesis of
peroxisomes, it remains unsolved how much the ER is involved in this process.
Pex14p play an essential role in peroxisome biogenesis. It has been shown that preperoxisomal structures which appear enriched in Pex13p and Pex14p are present in
Hansenula polymorpha pex3Δatg1Δ mutant strain (Knoop et al., 2014).
In this work, we demonstrate that in Saccharomyces cerevisiae pex3Δ mutant, Pex14-TProtA
is associated to membranes that are independent of the ER and mitochondria. We show that
these structures are not sensitive to degradation by autophagy, which is different from H.
polymorpha. However, the data underline the crucial role of Pex3p in this process also for S.
cerevisiae.
In addition, the composition of the Pex14-TProtA purified complex from solubilized
membranes was determined. We show that members of the docking complex (Pex14p,
Pex13p, and Pex17p), matrix protein import receptors (Pex5p, Pex7p, and Pex18p), and the
Pex4p-Pex22p pair are components of the complex. However, the RING-finger complex and
the AAA-complex were not detected.
These results suggest that a group of PMPs can be inserted into putative preperoxisomal
membranes even in absence of Pex3p. Further analysis of other proteins of these
assemblies shall give additional hints of the origin and nature of these pre-peroxisomal
structures.
References
Knoops K., Manivannan S., Cepińska M.N., Krikken A.M., Kram A. M., Veenhuis M., and van der Klei
I. J. Preperoxisomal vesicles can form in the absence of Pex3. JCB, 204, 5: 659-668.
33
Session 4, Tuesday 14:00-14:30:
A role for the ubiquitin-proteasome system in regulating plant
peroxisome protein import
Jianping Hu
Michigan State University, USA
Peroxisomes play pivotal roles in a suite of metabolic processes and often act coordinately
with other subcellular organelles, such as chloroplasts and mitochondria. Peroxisomes
import their matrix proteins from the cytosol by a number of peroxisome membraneassociated proteins called peroxins or PEX proteins. To understand how the functions of the
PEX proteins are regulated, we identified an Arabidopsis RING-type E3 ubiquitin ligase as a
novel peroxisome membrane protein, which interacts with and destabilizes several peroxins
that act in the early steps of peroxisome matrix protein import. Our data demonstrate that the
same E3 ubiquitin ligase can be shared by two metabolically associated organelles to
degrade components of distinct import machineries. We argue that degradation of organelle
biogenesis factors by the ubiquitin-proteasome system may constitute an important
regulatory mechanism utilized by eukaryotic cells to coordinate the biogenesis of functionally
linked organelles.
34
Session 4, Tuesday 14:30-15:00:
Plant peroxisome dynamics
Imogen Sparkes
Biosciences, University of Exeter, Stocker Rd, Exeter, EX4 4QD
Plant peroxisomes are extremely dynamic organelles. In epidermal cells they display
seemingly erratic movement characteristics; stop-go, change direction and display a range of
speeds over a relatively short time frame. During this process they appear to ‘sit’ next to
chloroplasts for prolonged periods of time. This association has been proposed to be a
requirement for shuttling of intermediates during photorespiration. The erratic nature of
peroxisome movement is shared with other organelle classes, all of which appear to respond
to external factors including pathogens.
Organelle movement in higher plants is mainly governed by actomyosin dependent
processes. Arabidopsis encodes for 17 myosins six of which appear to control global
organelle movement. Our understanding of the motor components that specifically drive
peroxisome movement is therefore still fairly rudimentary. More recently, it has emerged that
organelles appear to physically interact and ‘attach’ to other organelles. Using optical
tweezers we have probed whether peroxisomes physically interact with chloroplasts. Here, I
will present our recent published findings on the interaction and highlight that peroxisomes
are also attached to other unknown structures within the cell (Gao et al. 2016 Plant Physiol.
170:263-272).
35
Session 4, Tuesday 15:00-15:15:
Peroxisomal dynamics regulate rapid cell responses to
environmental stresses through a reactive oxygen speciesmediated pathway: role of peroxin PEX11a
María Rodríguez-Serrano1, María C. Romero-Puertas1, María Sanz-Fernández1, M. Cristina
López1, Ana M. Laureano-Martín2, Cecilia Gotor2, Jianping Hu3, Luisa M. Sandalio1
1
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del
2
Zaidín-CSIC, 18008 Granada, Spain, Institute of Plant Biochemistry and Photosynthesis, CSIC and
3
the University of Seville, 41092 Spain . Michigan State University-Department of Energy Plant
Research Laboratory and Department of Plant Biology, Michigan State, East Lansing MI, 48824, USA
Peroxisomes are highly dynamic and metabolically active organelles that play an important
role in cellular functions, including the reactive oxygen species (ROS) metabolism [1,2].
Peroxisomal morphological dynamics involving proliferation, movement and production of
dynamic extensions called peroxules have been associated with ROS in plant cells.
However, the consequences and the regulation of these changes are still unknown. In this
study, we show that treatment of Arabidopsis leaves with the heavy metal cadmium (Cd)
produces time course-dependent changes in peroxisomal dynamics, starting with peroxule
formation, followed by peroxisome proliferation and finally returning to their normal
morphology and number of peroxisomes. Peroxisomes abundance was regulated by
autophagy processess. The velocity of peroxisomes was also modified by halting the
peroxisomes while at the same time producing peroxules and more rapid peroxisomes after
24 h of treatment. We found that changes in the speed and peroxules formation were
regulated by NADPH oxidase (C and F)-related ROS production during Cd treatment, while
peroxisomal sources of ROS were not apparently involved. Peroxule formation was not
specific to Cd but a general response to other stimuli such as arsenic (As) and jasmonic acid
(JA) and was regulated by peroxin 11a (PEX11a), because Arabidopsis pex11a RNAi lines
were unable to produce peroxules under stress conditions. Concerning the role of peroxules,
the pex11a line showed higher lipid peroxidation content and lower GST, CAT2, CSD3 and
RRTF1 expression after a short period of Cd treatment as compared to WT plants,
suggesting that these extensions are involved in regulating ROS accumulation and ROSdependent gene expression. These results indicate that peroxule formation and PEX11a may
govern stress perception and the fast cell responses to environmental cues.
References[1] Hu, J., Baker, A., Bartel, B, Linka, N., Mullen, R.T., Reumann, S., and Zolmanh B. K.
(2012) Plant peroxisomes: Biogenesis and function. The Plant Cell 24(6): 2279–2303.2] Sandalio,
L.M., Romero-Puertas, M.C. (2015). Peroxisomes sense and respond to environmental cues by
regulating ROS and RNS signalling networks. Annals of Botany 116 (4): 475–485.
This work was financially supported by ERDF co-financed grant BIO2012-36742 and BIO2015-67657P from MICINN and the Junta de Andalucía (BIO-337).
36
Session 4, Tuesday 15:15-15:30:
Pathogen defense by plant peroxisomes: Protein targeting and
functional analyses of peroxisome-targeted NHL proteins
Delphine Crappe1, Kirsti Sørhagen1, Gopal Chowdhary1, Amr Kataya1 and
Sigrun Reumann1,2
1
Center for Organelle Research, University of Stavanger, Stavanger, Norway
Plant Biochemistry and Infection Biology, Biocentre Klein Flottbek, University of Hamburg,
Hamburg, Germany
2
Around 14% of crops produced worldwide are lost by plant diseases. Therefore, efforts have
been focused on understanding natural plant defense mechanisms in order to boost the
innate immune system of crop plants and reduce losses to increase agricultural productivity.
In plants NHL (NDR1/HIN1-like) genes play crucial roles in pathogen induced plant
responses to biotic stress. Based on sequence similarity, 45 NDR1/HIN1-like proteins (NHLs)
have been identified in Arabidopsis. None of them has been experimentally associated with
peroxisomes. Our new PTS1 protein algorithms, however, predicted that three family
members (NHL4, NHL6 and NHL25) are PTS1 proteins. Moreover, microarray-based
expression analyses indicate pronounced pathogen responsiveness for AtNHL25 and
especially AtNHL6. Our co-localization studies indeed demonstrated that AtNHL4 and
AtNHL25 are peroxisomal but, surprisingly, the proteins do not seem to be directed to
peroxisomes by the standard PTS1 pathway for soluble matrix proteins. The three NHL
proteins are also observed in smaller vesicle-like non-peroxisomal structures that often are
found attached to mature peroxisomes that were also observed for AtNHL6. In addition,
AtNHL4 is detected in the endoplasmic reticulum suggesting that the protein transits through
the ER and leaves the compartment in some type of ER-derived vesicles that further move to
and merge with in order to reach their final destination. Consistent with ER targeting, all three
NHLs contain a predicted transmembrane domain. We confirmed membrane localization of
AtNHL4 experimentally. To gain deeper insights into the targeting mechanism, several
deletion constructs of AtNHL4 have been generated. Deletion of the PTS1 strongly reduced
peroxisome targeting. To unravel the identity of the vesicular structures, the effect of COPII
inhibitors is presently investigated, and several PEX markers are generated.
Functional analyses have been initiated to characterize AtNHL6 and AtNHL25. Semiquantitative and real-time PCR indicate that AtNHL6 is strongly induced in response to
flagellin22. Single and double knock-out mutants of NHL6 and NHL25 have been generated.
Metabolomics analyses of the double mutant (nhl6-1xnhl25-1) are being performed to
investigate their role in response to biotic stress.
37
Session 4, Tuesday 16:00-16:30:
Occurrence and functions of peroxisomes in trypanosomatids and
other protists
Paul Michelsa, Toni Gabaldónb, Michael Gingerc a
b
School of Biological Sciences, University of Edinburgh, UK The Barcelona Institute of Science and
c
Technology, Spain Department of Biological Sciences, University of Huddersfield, UK
Peroxisomes can be detected in representatives of all eukaryotic superphyla. The similar
morphology and mode of biogenesis of the organelles indicate a monophyletic origin within
the Last Eukaryotic Common Ancestor (LECA). Despite a common origin and shared
morphological features, peroxisomes from different organisms show a remarkable diversity of
enzyme content. Moreover, the metabolic processes present in the organelles can vary
importantly dependent on nutritional or developmental conditions. Peroxisomes probably
originated from the endoplasmic reticulum (ER); the common involvement of all peroxisomes
in lipid metabolism, notably H2O2-dependent fatty-acid oxidation suggests that sequestering a
H2O2-producing FAD-dependent acyl-CoA oxidase from the ER in a newly formed organelle,
limiting the rate of oxidative damage within the ER, has been the evolutionary driver for the
peroxisome’s origin. Subsequent evolution of peroxisomes in different lineages involved
multiple acquisitions of metabolic processes – often involving retargeting enzymes from other
cell compartments – and losses. Available information about peroxisomes in protists, albeit
scarce, and new bioinformatics data indicate a striking diversity among free-living and
parasitic protists from different phylogenetic supergroups. Peroxisomes in only some protists
show major involvement in H2O2-dependent metabolism, as in peroxisomes of mammalian,
plant and fungal cells. Within the excavate supergroup of eukaryotes, the presence of
glycolytic and gluconeogenic enzymes inside peroxisomes is characteristic for kinetoplastids
(parasites such as Trypanosoma and Leishmania and the free-living Bodo) and diplonemids
(free-living protists abundantly present in oceans), where the organelles are hence called
glycosomes. The role of glycosomes in trypanosomatid metabolism, and the changes of the
organelles’ enzyme content during the parasites’ life cycle, have been studied in great detail.
Regarding other excavate protists, bioinformatics analysis suggests the presence of more
‘classical’ peroxisomes in the free-living Naegleria, whereas the parasites Giardia and
Trichomonas have lost peroxisomes altogether. Among alveolates (including parasites such
as Plasmodium, Toxoplasma, Cryptosporidium) and amoebozoans (free-living Dictyostelium,
parasitic Acanthamoeba and Entamoeba) patterns of peroxisome loss are more complicated.
Often, a link is apparent between the niches occupied by the protists, nutrient availability,
and the absence of the organelles or their presence with a specific enzymatic content.
38
Session 4, Tuesday 16:30-16:45:
Possible function of the pyruvate phosphate dikinase (PPDK)
located at the glycosomal membrane in Trypanosoma cruzi
González-Marcano Ea*, Cáceres Aa, Quiñones Wa, Concepción JLa
a
Laboratorio de Enzimología de Parásitos. Facultad de Ciencias. Universidad de Los Andes. Mérida,
*
Venezuela [email protected]
In Latin America, the morbidity and mortality associated to American Trypanosomiasis
(Chagas disease), caused by the parasitic protist Trypanosoma cruzi are much higher than
those associated with malaria, schistosomiasis or leishmaniasis. Chagas disease is
distributed in America from the south of the U.S.A. to the south of Argentina. At present,
about 6 to 7 million people worldwide are estimated to be infected. Globally, the calculated
annual burden for health-care costs is $ 627.46 million, not including the expenses in
spraying insecticide to control the vectors transmitting the parasite. The economic burden of
Chagas disease is similar to or exceeds those of other prominent globally occurring
diseases, signifying an economic argument for more attention and efforts towards control of
this disease. Rational design of new drugs is in progress. The more developed agents are
the triazole derivatives, which inhibit the synthesis of ergosterol. Glycolytic enzymes have
also been studied as possible chemotherapeutic targets due to their particular characteristic
of compartmentalization within glycosomes. The enzyme Pyruvate Phosphate Dikinase
(PPDK), absent in mammals, has been found in T. cruzi’s glycosomes, where it is both
associated to the membrane (but in an inactive form) and in the matrix. PPDK is until now the
only enzyme known to hydrolyze the high-energy compound PPi in the glycosomes. Indeed,
its importance in the maintenance of the energy and redox balance inside the organelle has
been recently confirmed. However, the specific topology of PPDK at the membrane (facing
the matrix or cytosol) and its possible function are not clear yet. Protease protection assays
of integral glycosomes suggested that the enzyme is located at the outer face of the
glycosomal membrane. Probably it is attached to a membrane protein, as it sequence does
not show any membrane integral domain. Additionally, experiments were performed to
determine if the function of the membrane-associated PPDK could be related with the
parasite’s metabolism. To that end, glucose consumption was measured using partially
permeabilized parasites with their glycosomes still intact. When such cells were treated with
monoclonal anti-PPDK antibodies, glucose consumption was arrested. This result may
indicate that the enzyme could be related to the intake of PEP into the glycosome for further
catabolism by PEPCK and/or the active, matrix-located PPDK. Obviously, more specific
assays should be made in order to unambiguously establish the function of the membranelinked PPDK.
Keywords: Trypanosoma cruzi, Glycosome, metabolism, Pyruvate Phosphate Dikinase (PPDK).
39
Session 4, Tuesday 16:45-17:00:
Identification of novel stress related peroxisomal proteins
in Hansenula polymorpha
Ritika Singh1, Harshitha S. Kumar1, Thomas Lingner2, Sigrun Reumann3 and
Ida J. van der Klei1
1
Molecular Cell Biology, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
2
3
[email protected] Abteilung Bioinformatik (IMG), Goldschmidtstr. 1, 37077 Göttingen Plant
Biochemistry and Infection Biology, Department Head, Biozentrum Klein Flottbek, Ohnhorststr. 18,
22609 Hamburg, Germany
Peroxisomes are ubiquitous cell organelles that play a central role in cellular lipid
metabolism. Peroxisomes also possess enzymes to maintain redox balance and counteract
oxidative stress. Recent studies resulted in the identification of several novel and often
unexpected peroxisome functions, including non-metabolic roles. These novel functions are
often related to stress and stress adaptations. The present study aims to extend the atlas of
peroxisome functions by the identification of novel stress-related, yeast peroxisomal proteins.
Through the use of novel peroxisome targeting signal (PTS) prediction tools as well as
proteomics of organelles isolated from stressed yeast cells, we have identified two novel
putative peroxisomal proteins in Hansenula polymorpha named HpPrx1 and HpAhp1.
Sequence analysis suggests that both proteins belong to the superfamily of peroxiredoxins.
Peroxiredoxins are ubiquitous thiol-specific proteins that have multiple functions in stress
protection, including protection against oxidative stress.
We show that N-terminally GFP-tagged HpPrx1 and HpAhp1 proteins localize to
peroxisomes when grown on glucose, whereas HpPrx1 (but not HpAhp1) was also
peroxisomal in methanol-grown cells. In parallel, deletion strains were constructed. These
mutants display hypersensitivity to various stress conditions, with cells lacking HpAhp1 being
more sensitive than those lacking HpPrx1. Furthermore, the double mutant showed further
increased susceptibility to stress conditions. Taken together, our preliminary data suggests
that both proteins participate in combating oxidative stress in H. polymorpha.
40
Session 4, Tuesday 17:00-17:15:
Identification and Characterization of a Novel Survival Protein,
SurE-like Phosphatase/Nucleotidase, in Arabidopsis Peroxisomes
Piotr Lisik1, Gopal Chowdhary1, Amr Kataya1, and Sigrun Reumann1,2
1
Centre for Organelle Research, University of Stavanger N-4021 Stavanger, Norway
Biozentrum Klein Flottbek, University of Hamburg, D-22609 Hamburg, Germany
2
Bacterial stationary-phase survival protein (SurE) is a well-studied enzyme that exhibits
phosphatase activity as a nucleotidase or polyphosphate phosphohydrolase with varying
substrate specificity in different organisms. Interestingly, the enzyme plays a significant role
in response to abiotic stresses such as nutrient deprivation. SurE orthologs have also been
found in archaea and eukaryotes but none of them has been reported to be associated with
peroxisomes. Of two SurE-like phosphatase/nucleotidase (AtSurE1) in Arabidopsis one is a
newly predicted plant peroxisomal PTS1 protein that might contribute in the plant response to
abiotic stress. In vivo subcellular localization studies in our laboratory showed that AtSurE1 is
weakly targeted to peroxisomes in tobacco protoplasts and possesses a non-canonical PTS1
domain terminating with SSL>. To investigate the physiological function of AtSurE1, His 6tagged recombinant protein was produced in E. coli and purified to high homogeneity.
Enzymatic analyses confirmed that AtSurE1 is a divalent-metal-ion-dependent phosphatase
showing highest activity in the presence of cobalt, magnesium and manganese ions.
Screening for natural substrates revealed that AtSurE1 exhibits highest activity towards
3’AMP, dAMP and 5’AMP, which suggests that AtSurE1 plays a significant role in purine
metabolism. Microarray database analysis and experimental data confirmed also a role of
AtSurE1 in the response to environmental stresses in Arabidopsis thaliana. According to
qPCR results AtSurE1 gene is highly overexpressed in response to heat and salt stress, and
down-regulated in response to cold stress. AtSurE1 knock-out mutants were isolated and
subjected to metabolome analyses in collaboration with Metabolic Discoveries (N. Schauer)
but did not reveal any phenotype up to now. For further functional AtSurE1 characterizations,
structural analyses are performed in collaboration with Natasha Giannopoulou and Matthias
Wilmann’s group.
41
Session 4, Tuesday 17:15-17:30:
Peroxisomes from pepper fruits: new perspectives on the organelle
metabolism
Marta Rodríguez-Ruiz1, Paz Álvarez de Morales1, Sigrun Reumann1, Francisco J Corpas1,
José M. Palma1
1
Dpt. Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, E2
18008 Granada, Spain; Pflanzenbiochemie und Infektionsbiologie, Biozentrum Klein Flottbek, D22609 Hamburg, Germany
Pepper (Capsicum annuum L.) is an annual plant species whose fruits undergo important
metabolic changes through development and ripening. These changes reported include
emission of volatile organic compounds associated with respiration, destruction of
chlorophylls and synthesis of new pigments (red/yellow carotenoids plus xanthophylls and
anthocyans) responsible for the color shift, protein degradation/synthesis, and shifts in total
soluble reducing equivalents [1]. At the subcellular level, chloroplasts turn into chromoplasts
during this developmental stage with major metabolic and structural alterations, whereas
mitochondria and peroxisomes do not show any apparent morphological modifications [1].
Accordingly, the study of pepper fruit peroxisomes was initiated, and the full characterization
of these organelles was firstly reported [2]. Contrary to the dual metabolism (glyoxysome-like
type versus leaf peroxisome type), it was found that peroxisomes from pepper fruits contain
at the same time the enzymes from both the photorespiratory and the glyoxylate cycle
pathways [2]. Regarding the metabolism of reactive oxygen species (ROS), two superoxide
dismutase (SOD) isozymes were detected by enzymatic, immunological and proteomic
approaches: one Mn-SOD and one Fe-SOD [1,2]. Mn-SODs are commonly present in plant
peroxisomes, but the presence of an Fe-SOD was only reported before in carnation petals
[3]. On the other hand, catalase was found to be involved in the ripening process, where
post-translational modification and regulation through nitration events seems to occur [4]. By
in silico analysis of the peroxisomal proteome from pepper fruits, four new potential proteins
bearing PTS1 have been found by bio-computing comparisons with orthologs from
Arabidopsis databases. Thus, a putative terpene synthase 14, an annexin 3, a cytochrome
P450 and an oxide-reductase protein have been postulated as novel peroxisomal
constituents. Overall, our results provide new perspectives on the metabolism of plant
peroxisomes, especially in fruits where information on the role of these organelles during
fundamental processes such as ripening have not been established yet.
[1] Palma JM et al. (2015) Ann. Bot. 116: 627-636.
[2] Mateos RM et al. (2003) J. Plant. Physiol. 160: 1507-1516.
[3] Droillard MJ, Paulin A (1990) Plant Physiol 94: 1187-1192.
[4] Chaki M et al. (2015) Ann. Bot. 116: 637-647.
[Supported by Grant AGL2015-65104-P from MINECO, Spain]
42
Session 5, Wednesday 9:00-9:30:
Recognition mechanism of the second peroxisomal targeting
signal, PTS2 by its receptor complex Pex7p and Pex21p
Hiroaki Kato
Graduate School of Pharmaceutical Sciences, Kyoto University, JAPAN
There are two types of targeting signals PTS1 and PTS2 for the import of peroxisomal matrix
proteins and recognized by Pex5p and Pex7p, respectively. While Pex5p alone is sufficient
to recognize PTS1, Pex7p requires a co-receptor to stabilize its interaction with PTS2. In
Saccharomyces cerevisiae, Pex18p and Pex21p serve as the co-receptor with redundant
function whereas the longer variant of Pex5p (Pex5pL) also acts as the co-receptor in
mammals and in Arabidopsis thaliana. Several crystal structures of Pex5p with or without
PTS1 had been reported, while no crystal structure of Pex7p had been determined. To
elucidate the molecular mechanism of PTS2 recognition by Pex7p and its co-receptor, we
solved the crystal structure of PTS2 of the peroxisomal 3-ketoacyl-CoA thiolase (Fox3p)
complexed with Pex7p and Pex21p C-terminal domain from S. serevisiae. The crystal
structure unveiled the detailed mechanism of the PTS2 recognition (1). A nonapeptide motif
of PTS2 R-[L/V/I/Q]-xx-[L/V/I/H]-[L/S/G/A]-x-[H/Q]-[L/A] (x is any residue) adopted an
amphipathic α-helix fold. The hydrophobic amino-acid side chains of PTS2 lined up at one
side and interacted directly with both Pex7p and Pex21p whereas the conserved arginine
and histidine of PTS2 (Arg4 and His11 in S. cerevisiae Fox3p) interacted only with Pex7p.
Pex7p adopted a 7-bladed β-propeller fold and its PTS2 binding surface shaped an acidic
groove containing Asp61, Glu106 and Glu222 in the middle to accommodate the basic
arginine and histidine side chains of PTS2. The hydrophobic side chains of PTS2 lay on the
hydrophobic edge along the side of the acidic groove. These features fixed the orientation of
PTS2 properly. In addition, the extended Pex21p C-terminal domain bound to the side of
Pex7p, and the helical region of Pex21p formed a lid to cover the remaining hydrophobic
surface of PTS2. This tripartite hydrophobic interaction clearly explained how the co-receptor
stabilizes the PTS2 binding of Pex7p. Thus, the recognition mechanism of PTS2 is much
different from that of the other helical N-terminal targeting signals, such as the signal peptide
for ER and mitochondrial targeting signal, MTS.
(1) Pan, D. et al., Nat. Struct. Mol. Biol., 20, 987—993 (2013)
43
Session 5, Wednesday 9:30-10:00:
Peroxisomes as tools in structural biology
Matthias Wilmanns
EMBL Hamburg, Germany
Peroxisomal matrix protein translocons are unique by means of their ability to import large,
oligomeric and functional proteins assemblies. However, the overall architecture of
peroxisomal translocons remains unknown to date, despite various genetic, biochemical and
electrophysiological studies. Detailed structural knowledge on such translocons would open
novel routes for mechanistic studies, which are otherwise limited in terms of design
opportunities. The main underlying challenge is that peroxisomal translocons are
nonpermanent, making it difficult to capture them as an intact molecular machinery system.
We show previous and present work mostly focusing on the initial steps of the translocation
process, cargo recognition and docking, and present mechanistic insights emerging from our
structure-based work. We wrap up in presenting ideas how to advance our knowledge
towards capturing the overall architecture of an entire translocon.
44
Session 5, Wednesday 10:00-10:15:
Structural analysis of proteins involved in peroxisome biogenesis
Evdokia-Anastasia Giannopoulou, Matthias Wilmanns
EMBL Hamburg, Germany
Peroxins are proteins essential for the biogenesis of peroxisomes. Two peroxins, Pex3 and
Pex19 are involved in the insertion of various Peroxisomal Membrane Proteins (PMPs) in the
peroxisomal membrane and are thus crucial for its formation. Pex3 is a PMP that acts as a
docking receptor for Pex19. Pex19, in turn, functions as a chaperone and import receptor for
newly synthesized PMPs. Pex19 binds and stabilizes the PMPs and guides them to Pex3,
where they are inserted into the peroxisomal membrane by an as-of-yet unknown
mechanism. This project aims to elucidate the PMP import mechanism by structural
characterization of the Pex3-Pex19-PMP ternary complex using high- (X-ray Crystallography)
and low- resolution (Small Angle X-ray Scattering) structural determination techniques, in
addition to complementary biophysical methods.
Keywords: peroxisome biogenesis, Pex3, Pex19, X-ray Crystallography
45
Session 6, Wednesday 10:30-11:00:
Systematic characterization of proteome localization dynamics
during growth in oleic acid reveals a novel peroxisome targeting
receptor
Eden Yifrach1, Silvia G. Chuartzman1, Noa Dahan1, Shiran Maskit1, Lior Zada1, Uri Weill1, Ido
Yofe1, Maya Schuldiner*1 and Einat Zalckvar*1
1
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel
* Co-corresponding
To optimally perform the diversity of metabolic functions that occur within peroxisomes, cells
must dynamically regulate peroxisome size, number and content in response to cell state
and the environment. Except for transcriptional regulation little is known about the
mechanisms used to perform this complicated fete. We used complementary high content
screens to follow changes in most of the yeast, S. cerevisiae, proteins during growth in
oleate. We found extensive changes in cellular architecture and identified several proteins
co-localizing with peroxisomes that have not previously been considered peroxisomal
proteins. One of the newly identified peroxisomal proteins, Ymr018w, is a protein with an
unknown function that is similar to the yeast peroxisomal targeting receptor Pex5 as well as
to the human Pex5 protein. We demonstrate that Ymr018w is a novel peroxisomal targeting
receptor that targets a subset of matrix proteins to peroxisomes. We have therefore renamed
Ymr018w, Pex9, and suggest that Pex9 is a condition-specific targeting receptor that enables
the dynamic rewiring of peroxisomes in response to metabolic needs.
46
Session 6, Wednesday 11:00-11:30:
Imaging peroxin-protein interactions
challenges and opportunities
in
mammalian
cells:
Marc Fransen
KU Leuven – University of Leuven, Department of Cellular and Molecular Medicine, Laboratory of Lipid
Biochemistry and Protein Interactions, B-3000 Leuven, Belgium, [email protected]
Many biological processes are mediated through specific protein association and dissociation
events and, to gain more insight into these processes, it is essential to have access to a
variety of cell-based and biochemical approaches for assessing protein-protein interactions.
Among those, the yeast two-hybrid system and co(immuno)precipitation assays are most
popular, most likely due their simplicity and ease of implementation. Some other, more
recently developed methodologies, include isothermal titration calorimetry, fluorescence/bioluminescence resonance energy transfer techniques, and MAPPIT, a cytokine
receptor-based two-hybrid method in mammalian cells [1]. However, a major drawback of at
least some of the latter approaches is that they require high tech equipment and technical
expertise.
To gain a better insight into the complex network of peroxin-protein interactions in living
mammalian cells, we initially employed a commercially available cell-based system claimed
to be suitable for monitoring reversible protein-protein interactions between GFP- and RFPtagged proteins in real time (http://www.chromotek.com/products/f2hr-assay/f2hr-kit-basic/).
Unfortunately, as this assay does not appear to be generally applicable for imaging
interactions between proteins containing a peroxisomal targeting signal, we designed a
simple and powerful alternative approach to visualize protein-protein interactions in all types
of living cells. In this talk, I will first briefly outline this approach. Next, to demonstrate the
strengths (and potential weaknesses) of this method, I will present the interaction datasets
we currently have for human PEX5, PEX14, and PEX19. Where relevant, I will compare the
results with those previously obtained in the yeast and bacterial two-hybrid systems [2]. In
addition, I will discuss how some of these findings challenge our current understanding of
how these proteins may function in a complex. Finally, I will demonstrate that our in cellulo
assay is suitable to monitor changes in protein-protein interactions under conditions affecting
peroxisome biology.
References
[1] Stynen B et al. (2012) Microbiol Mol Biol 76, 331-382.
[2] Fransen M et al. (2002) Mol Cell Proteomics 1, 243-252.
This work was supported by grants from the KU Leuven (OT/14/100) and the Fonds voor
Wetenschappelijk Onderzoek-Vlaanderen (Onderzoeksproject G095315N).
47
POSTER ABSTRACTS
Understanding the physiopathogenesis of peroxisomal leukodystrophies by generating novel microglial cell models with
CRISPR/Cas9-mediated genome editing.
RAAS Q. 1,2, GONDCAILLE C. 1,2, SAIH F.-E. 1,2,3, TROMPIER D. 1,2, ANDREOLETTI P. 1,2,
VEJUX A. 1,2, NURY T. 1,2, LIZARD G1, 2, CHERKAOUI-MALKI M. 1, 2& SAVARY S. 1,2
1
2
Univ. Bourgogne-Franche Comté, 21000 Dijon, France; Laboratoire de Biochimie du Peroxysome,
Inflammation et Métabolisme des Lipides (BioPeroxIL EA 7270), Faculté des Sciences Gabriel, Dijon,
3
F21000, France; Laboratoir de Biochimie et Neurosciences, Faculté des Sciences et Techniques,
Université Hassan I, BP 577 26 000 Settat, Morocco.
Peroxisomal leukodystrophies are severe neurodegenerative disorders without any
satisfactory therapy. Among these peroxisomal disorders, X-ALD is caused by mutations in
the ABCD1 gene while ACOX1 deficiency is associated with deletion or mutations in the
ACOX1 gene. ABCD1 encodes for a peroxisomal ABC transporter involved in the transport,
through peroxisomal membrane, of the very-long-chain fatty acids, which then are processed
by ACOX1 as the first rate-limiting enzyme of the peroxisomal β-oxidation system. The
understanding progress in the physiopathogenesis of these leukodystrophies suffers from the
lack of appropriate cell and animal models. Since peroxisomal defect in microglia seems to
be a key element of this physiopathogenesis, we proposed to generate microglial cell lines
unable to transport and/or β-oxidize very-long-chain fatty acids into the peroxisome, i.e; BV-2
microglial cell lines deficient in ABCD1, ABCD2 or ACOX-1. To that end, we used the
CRISPR/Cas9 genome editing technology since it permits a complete and stable elimination
of gene function by homologous recombination. Preliminary results indicate that the strategy
succeeded to knock out the genes of interest. Monoallelic or biallelic mutated cell clones
were selected by flow cytometry and antibiotic treatment and will be further characterized to
evaluate the consequences of peroxisomal defects on redox and inflammatory status,
phagocytosis and antigen presentation.
48
Unveiling the role of contact
and the plasma membrane.
sites
between
peroxisomes
Noa Dahan, Nadav Shai, Silvia Chuartzman, Maya Schuldiner and Einat Zlackvar1
1
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
In order to optimize their multiple cellular functions, peroxisomes must collaborate and
communicate with surrounding organelles. A common way of communication between
organelles is through membrane contact sites where membranes of two organelles create
close-range interactions facilitating exchange of small molecules and intracellular signaling.
The extent of contact sites formed by peroxisomes as well as how peroxisomes rely on
contact sites for their various cellular activities are not yet fully characterized.
A split fluorescence reporter was developed in our lab to detect peroxisomal contact sites in
yeast by which one part of a fluorophore is fused to the outer membrane of peroxisomes
while the other is fused to the outer membrane of other organelles. An interesting proximity
was detected between peroxisomes and the plasma membrane (PM-PEX). PM-PEX
proximity region is detectable in 40% of the cells in a typical position adjacent to the budneck. To uncover the molecular players that are involved in the formation and regulation of
PM-PEX interaction we are currently using an automated high content screen to detect the
effect of different yeast mutants on the fluorescence reporter. Finally we aim to characterize
the physiological role of this novel interaction between the plasma membrane and
peroxisomes.
49
Trypanosoma
cruzi
contains
two
glycosomally
located
galactokinases; Molecular and biochemical characterization
Lobo-Rojas Aa, González-Marcano Ea, Valera-Vera Ea, Acosta Ha, Quiñones W a, Burchmore
Rb, Concepción J.L.a, Cáceres Aa*.
a
Laboratorio de Enzimología de Parásitos. Facultad de Ciencias. Universidad de Los Andes. Mérida,
Venezuela.
b
Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences,
University of Glasgow. Glasgow, Scotland.
[email protected]
Trypanosoma cruzi, the causative agent of Chagas’ disease, is a protist which undergoes
multiple morphological and metabolic changes during its complex life cycle involving two
hosts, a vertebrate and an insect. This transition is accompanied by changes in the
carbohydrate composition of the macromolecules on the cell surface. The cell is coated with
a dense glycocalyx composed mainly of glycoconjugates, some of which play an essential
role in parasite survival, infectivity and virulence. These glycoconjugates are made up of
monosaccharides such as D-galactose (D-Gal: D-galactopyranose and D-galactofuranose)
among others. In all trypanosomatids, D-galactose is a particularly abundant constituent of
the glycoclayx. Two different putative galactokinase genes were found in the genome
database of T. cruzi. Both genes, TcGALK-1 and TcGALK-2, were cloned and expressed in
Escherichia coli. The calculated molecular masses of the encoded proteins were 51.9 kDa
(TcGALK-1) and 51.3 kDa (TcGALK-2). The values determined for the affinity (Km) of the
recombinant proteins were for galactose 0.108 mM (TcGALK-1) and 0.091 mM (TcGALK-2)
and for ATP 0.36 mM (TcGALK-1) and 0.1 mM (TcGALK-2); substrate inhibition by ATP (Ki
0.414 mM) was only detected for TcGALK-2. Gel-filtration chromatography assays showed
that both the natural TcGALKs, in a purified glycosome preparation, and recombinant
TcGALK-1 are monomeric. Additionally, in agreement with the possession of a type-1
peroxisome-targeting signal (PTS-1) by both TcGALKs, they are indeed located in the
glycosomes. Both genes are expressed in the epimastigote (insect) and trypomastigote
(mammalian) stages of the parasite. In accordance with the presence of a functional
galactokinase, epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium
supplemented with 22 mM of galactose, suggesting that this hexose could be used in the
synthesis of UDP-galactose and also as a possible carbon and energy source.
Keywords: Galactokinase; Trypanosoma cruzi; Glycosome; GHMP superfamily; Kinetic analysis;
Galactose metabolism; Nucleotide sugars; UDP-galactose.
50
Structural characterization of yeast PTS1 cargo protein Pcs60
N. Hanna*, A.E. Giannopoulou*, D. Passon, M. Wilmanns
EMBL Hamburg –Notkestrasse 85, 22607 Hamburg
Peroxisomal matrix proteins can be imported into the peroxisomal lumen using peroxisomal
targeting signal 1 (PTS1) or peroxisomal targeting signal 2 (PTS2) import pathways. The
PTS1 pathway is the most common means to transport folded cargo protein in peroxisomes,
utilizing peroxin Pex5. Pex5 is a soluble receptor cycling between a free cytoplasmic state,
where it can recognise and bind Peroxisomal matrix proteins and a membrane bound state,
forming the transient PTS1 pore. The interaction between Pex5 and the cargo proteins
occurs via the TPR domains (tetratricopeptide repeats) of Pex5 and a tripeptide signal
sequence on the C-terminus of the cargo protein. Pcs60 is a cargo oxalyl-CoA synthetase
containing the tripeptide SKL, which results in high affinity binding with Pex5. In this study,
Pcs60 was crystallised and its structure was determined by means of X-ray Crystallography
in a resolution of 2.4 Å. Further characterisation of Pcs60 and its complex with Pex5 was
performed by biophysical methods, Small Angle X-ray Scattering (SAXS) and ongoing
crystallography experiments.
Keywords: Peroxisomes, Pcs60, Pex5, PTS1 pathway, peroxisomal matrix protein, Structural Biology
51
Peroxisomal dysfunction is associated with up-regulation of
apoptotic cell death via miR-223 induction in knee osteoarthritis
patients with type 2 diabetes mellitus
Dongkyun Kima#, Jinsoo Songa#, Chiyeon Ahna, Yeonho Kanga, Churl-Hong Chunb,
Eun-Jung Jina*
a
Department of Biological Sciences, College of Natural Sciences, Wonkwang University, Iksan,
b
Chunbuk, 570-749, Korea Departments of Orthopedic Surgery, Wonkwang University School of
Medicine, Iksan, Chunbuk, 570-749, Korea.
Recent increasing evidences showing the interconnection between mitochondria and
peroxisome in performing metabolic functions imply that peroxisome dysfunction could lead
to a wide variety of human diseases including cancer and osteoarthritis (OA) as mitochondria
dysfunction. Even though higher incident and development of OA in diabetes mellitus (DM)
patients, not much of evidential mechanism study in this inter-regulation between OA and OA
with DM in a new view of peroxisome. In this study, we analyzed the alteration of
peroxisomal gene expression that could responsible for pathological difference between OA
chondrocytes and OA/DM chondrocytes. To discriminate responsible genes in the OA/DM
pathogenesis, the expressions of three hundred sixty-two genes reported to differentially
related to peroxisome were analyzed with OA chondrocytes in OA cartilage and OA/DM
chondrocytes in the cartilage of OA with DM patient. Among them, PEX-16, a component of
peroxisome, was significantly down-regulated in OA/DM chondrocytes and this downregulation of PEX-16 was increased the miR-223 induction. Knock-down studies using PEX16 null cell line and PEX-16 specific siRNA showed the significant increase in apoptotic cell
death. Moreover, over-expression of miR-223 stimulates apoptotic cell death in human
articular chondrocytes and induced severe cartilage destruction in db/db mice. In conclusion,
our study showed the differential peroxisomal gene expression profiles for OA/DM
chondrocytes from OA chondrocytes and suggest the possibility that peroxisomal dysfunction
in OA/DM could responsible for early incident and development of OA in DM patients.
Keywords: Peroxisome dysfunction, osteoarthritis, diabetes mellitus, PEX-16, miR-223
52
Microalgae 2021: Molecular design of improved production
microalgae to accelerate the establishment of an algae-based
bio-economy in Norway
Dmitry Kechasov1, Manish Budathoki1, Imke Büsing2, Amit Sharma3, Thomas Lingner4,
Svein Dahle5, Dominic Nanton6, Jodi Maple Grødem1 and Sigrun Reumann1,2
1
2
Center for Organelle Research (CORE), University of Stavanger, 4021, Norway Biocenter Klein
3
Flottbek, Plant Biochemistry and Infection Biology, University of Hamburg, 22609, Germany
Department of Biology, Norwegian University of Science and Technology, N-7491 Trondheim,
4
Norway. Institute for Microbiology and Genetics, Dept. of Bioinformatics, D-37077 Goettingen,
5
6
Germany MicroA AS, Oljeveien 4, 4056 Tananger, Norway EWOS Innovation AS, Gtnr 78 Bnr 3,
4335 Dirdal, Norway
Fish industry is the second largest industry in Norway. One of the major limiting factors of
aquaculture are long-chain polyunsaturated fatty acids (LC-3/-6 FAs, e.g. EPA,
DHA, ARA), which have clinically proven, positive effect on cardiovascular and mental health
of humans. These essential fatty acids cannot be produced de novo by animals. Their main,
but non-sustainable source for fish farming is marine fish oil. However, the primary producers
of LC-PUFAs are microalgae. Molecular R&D of microalgae, however, is still in its infancy. To
understand and optimize lipid metabolism and productivity, we analyzed the predicted
proteome of peroxisomes from representative microalgae such as Nannochloropsis and their
important role in metabolism and particularly in the biosynthesis and degradation (ßoxidation) of LC-PUFAs. Molecular genetic methods to validate subcellular targeting of
predicted peroxisomal proteins of Nannochloropsis are presently established. We also work
on the genetic optimisation of Nannochloropsis to increase the omega-3 fatty acid production
and thereby increase the LC-PUFA content in natural microalgae. This will be achieved by
the development of a toolbox for recombinant protein production (the GMO approach) and for
technology-assisted breeding of microalgae (the non-GMO approach using random
mutagenesis and FACS methodology). Our research will help to significantly reduce the
production costs and in the same time promote commercial exploitation of microalgal biocompounds in Norway, from high-value products in the short-run and to low-value mass
products such as 4th generation biofuels in the long-run.
53
Peroxisome Targeting and Functional Analyses of Prenylated
Small GTPases (AtIANs) Involved in Pathogen Defence
Saugat Pokhrel1, 2, Christian Falter2, Ulrike Peters2, Sigrun Reumann1, 2
1
2
Centre for Organelle Research, University of Stavanger N-4021 Stavanger, Norway
Biocenter Klein Flottbek, University of Hamburg, D-22609 Hamburg, Germany
Vertebrate GIMAP proteins (GTPases of the immune associated nucleotide binding protein)
and plant IAN proteins (Immune Associated Nucleotide Binding) are small GTPases with
important functions in innate immunity. Although first discovered in plants, most of the
information available till date originates from vertebrates. Sequence analysis supports
closest similarity of GIMAP/IANs with the TRAFAC class of small GTPases. Structural
analysis also revealed dynamin-like structural features. In vertebrates GIMAP homologs are
located in the ER, Golgi and mitochondria and play important roles in the regulation of T-cell
selection and apoptosis. In Arabidopsis 13 IAN/GIMAP genes have been identified by
computational methods. Recently, AtIAN12 has been the first GIMAP/IAN homolog to be
localized to peroxisomes (Crappe, Kataya and Reumann, unpublished data) although it does
not contain any PTS1/2. The targeting to peroxisomes may occur by a novel mechanism
through protein prenylation. Here, we investigated subcellular targeting of a closely related
homolog, namely AtIAN13, which is also predicted to be prenylated and might follow the
same targeting pathway. The gene was successfully cloned by RT-PCR. Localization studies
of full-length AtIAN13 fused N-terminally with EYFP revealed that the protein is targeted to
vesicle-like structures of approx. 1 µm in diameter in tobacco protoplasts. Specific membrane
staining was clearly visible and contrasted matrix targeting. In onion epidermal cells, EYFPAtIAN13 was primarily cytosolic but also weakly localized to unknown structures of different
morphology. Interestingly, a cell death-like phenotype was observed, as cytoplasmic
streaming and organelle movement was arrested in the transformed cells but not in the
neighboring untransformed cells. Evan’s blue cell death assay is currently applied.
Localization studies of the reporter protein extended C-terminally by the C–terminal 10 amino
acid of IAN13 (including the CaaX prenylation motif) showed membrane targeting in
unknown spherical vesicle-like structures in onion epidermal cells and larger spherical
unknown structures in tobacco protoplasts, all of which resemble lipid bodies. Bodipy and
Nile red staining are presently applied to characterize the vesicles. Site-directed mutagenesis
of the predicted prenylation motif (C-to-A in the CaaX motif) combined with comparative
subcellular localization studies showed completely cytosolic targeting in different expression
systems. These results provide strong evidence that AtIAN13 indeed is prenylated and that
this post-translational modification determines protein anchoring in membranes and
subcellular targeting of AtIAN13 to its final destination. In addition, one IAN homolog
(AtIAN1) was predicted and validated to carry a non-canonical PTS1 (VKL>). Attempts are
still ongoing to clone the full-length CDS of this gene.
54
Allosteric Modulation of Peroxisomal Membrane Protein
Recognition by Farnesylation of the Peroxisomal Import
Receptor PEX19
Leonidas Emmanouilidis, Ulrike Schütz, Konstantinos Tripsianes, Tobias Madl,
Juliane Radke, Robert Rucktäschel, Matthias Wilmanns, Wolfgang Schliebs, Ralf Erdmann,
Michael Sattler
The peroxin PEX19 is a soluble protein which functions as chaperone and as import receptor
for peroxisomal membrane proteins (PMPs). The interaction between PEX19 and its cargo
takes place at the conserved, farnesylated C-Terminus of PEX19. NMR analyses of the
farnesylated C-terminal domain (CTD) of PEX19 with cargo peptides demonstrated that the
farnesyl moiety is buried in an internal hydrophobic cavity within the CTD. Farnesylation
induces substantial conformational changes that reshape the PEX19 surface to form two
hydrophobic pockets that recognize conserved aromatic/aliphatic side chains in PMP ligands.
Mutations of PEX19 residues involved in farnesyl or PMP recognition were not able to
completely complement pex19 phenotype in human Zellweger patient fibroblasts. Our
results demonstrate an allosteric mechanism for the regulation of protein function by
farnesylation.
55
Peroxisomal dysfunction is associated with up-regulation of
apoptotic cell death via miR-223 induction in knee osteoarthritis
patients with type 2 diabetes mellitus
Dongkyun Kima#, Jinsoo Songa#, Chiyeon Ahna, Yeonho Kanga, Churl-Hong Chunb,
Kyung Songc, Eun-Jung Jina*
a
Department of Biological Sciences, College of Natural Sciences, Wonkwang University, Iksan,
b
Chunbuk, 570-749, Korea; Departments of Orthopedic Surgery, Wonkwang University School of
c
Medicine, Iksan, Chunbuk, 570-749, Korea; Department of Pharmacy, College of Pharmacy,
Wonkwang University, Iksan, Chunbuk, 570-749, Korea
Recent increasing evidences showing the interconnection between mitochondria and
peroxisome in performing metabolic functions imply that peroxisome dysfunction could lead
to a wide variety of human diseases including cancer and osteoarthritis (OA) as mitochondria
dysfunction. Even though higher incident and development of OA in diabetes mellitus (DM)
patients, not much of evidential mechanism study in this inter-regulation between OA and OA
with DM in a new view of peroxisome. In this study, we analyzed the alteration of
peroxisomal gene expression that could responsible for pathological difference between OA
chondrocytes and OA/DM chondrocytes. To discriminate responsible genes in the OA/DM
pathogenesis, the expressions of three hundred sixty-two genes reported to differentially
related to peroxisome were analyzed with OA chondrocytes in OA cartilage and OA/DM
chondrocytes in the cartilage of OA with DM patient. Among them, PEX-16, a component of
peroxisome, was significantly down-regulated in OA/DM chondrocytes and this downregulation of PEX-16 was increased the miR-223 induction. Knock-down studies using PEX16 null cell line and PEX-16 specific siRNA showed the significant increase in apoptotic cell
death. Moreover, over-expression of miR-223 stimulates apoptotic cell death in human
articular chondrocytes and induced severe cartilage destruction in db/db mice. In conclusion,
our study showed the differential peroxisomal gene expression profiles for OA/DM
chondrocytes from OA chondrocytes and suggest the possibility that peroxisomal dysfunction
in OA/DM could responsible for early incident and development of OA in DM patients.
Keywords: Peroxisome dysfunction, osteoarthritis, diabetes mellitus, PEX-16, miR-223
56
In the yeast Hansenula polymorpha the peroxisomal enzyme
aspartate aminotransferase-2 (AAT2) is required for ethanol growth,
but not for aspartate biosynthesis
Ann S. Thomas, Chris P. Williams and Ida J. van der Klei
Attempts to study if an aspartate /malate shuttle is operative in peroxisomes revealed the
presence of the enzyme Aspartate aminotransferase-2 (Aat2) in peroxisomes of oleic acid
grown Saccharomyces cerevisiae1. Sequence alignment of various AAT2 proteins showed
that while most other organisms contain either a PTS1 or PTS2 signal to target the protein to
peroxisomes, there is no recognizable PTS1 or PTS2 in Hansenula polymorpha AAT2.
Surprisingly, preliminary data on Hp AAT2 suggest that this protein still localizes to
peroxisomes. We furthermore showed that H. polymorpha cells lacking AAT2 (aat2) were
unable to grown on ethanol, but did not require aspartate. This contrasts the phenotype of S.
cerevisiae aat2 cells, which are auxotrophic for aspartate.
The localisation of HpAat2 to peroxisomes and its function in ethanol-growth leads us to
speculate that it is probably involved in the glyoxylate cycle, a pathway that is localized to
peroxisomes in non-conventional yeasts and plant. Here, it possibly functions in the
continuous oxidation of NADH.
It appears that the enzymatic reactions required for C-2 metabolism proceed in opposite
directions in H. polymorpha and S. cerevisiae. This may be because unlike most other
yeasts, S. cerevisiae has the ability to produce ethanol, and is therefore less suited to
execute efficient C2 metabolism.
1
Verleur N, Elgersma Y, Van Roermund CW, Tabak HF, Wanders RJ. (1997) Cytosolic aspartate
aminotransferase encoded by the AAT2 gene is targeted to the peroxisomes in oleate-grown
Saccharomyces cerevisiae. Eur J Biochem. 247:972-80.
57
Identification of novel proteins involved in peroxisome-vacuole
membrane contact sites in Saccharomyces cerevisiae
Huala Wu, Arjen M. Krikken, and Ida J. van der Klei
Molecular Cell Biology, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
[email protected]
Peroxisomes play a vital role in a variety of specific metabolic processes. Since yeast
peroxisomes lack their own lipid biosynthesis machinery, they have to receive membrane
lipids from other cellular organelles. These lipids can be transported through vesicle
dependent or non-vesicular transport pathways.
A non-vesicular mode of lipid transport occurs at membrane contact sites (MCSs), regions of
close apposition between two membranes, separated by approx.. 10-30nm. Extensive
research is carried out to provide insights on the function and mechanisms of MCSs existing
between various organelles. So far, many studies have reported the existence of MCSs
between peroxisomes and ER, mitochondria, chloroplasts, lipid droplets and lysosomes.
We recently obtained evidence that yeast peroxisomes may form contacts with the vacuole.
This study aims to identify Saccharomyces cerevisiae proteins which could be involved in
this peroxisome-vacuole MCS. We first focus on the possible role of Vps39, a main
component of HOPS (homotypic fusion and protein sorting) and vCLAMP (Vacuole and
mitochondria path).
58
Investigating the peroxisomal membrane associated degradation
(PMAD) pathway in yeast
Xin Chen1, Srishti Devarajan1, Natasha Danda1, Ida J. van der Klei1, Chris Williams1
1
Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of
Groningen, the Netherlands
In yeast cells, peroxisomes play an indispensable role in oxidation reactions. In Hansenula
polymorpha (Hp), peroxisomes are involved in the oxidation of methanol, together with the
degradation of toxic hydrogen peroxide.
The import of peroxisomal membrane proteins (PMP) is well documented in previous studies,
while little is known about how PMPs are removed from the membrane and degraded. So far
only one bona fide example is known: redundant Pex3 in Hp cells is degraded by the
ubiquitin proteasome system (UPS) (Williams & van der Klei, BBRC 2013). However, Pex3p
and Pex16p in mammalian cells display increased half-lives in cells treated with the
proteasome inhibitor MG132 (Huybrechts et al, Traffic 2009), suggesting that UPS mediated
PMP degradation is a common occurrence.
Here, we have studied degradation of the PMP Pex13, a member in docking complex. Pex13
displays a rapid turnover in cells treated with the protein synthesis inhibitor cycloheximide,
while the levels of Pex11p, another PMP, remained steady under these conditions. The
levels of Pex13 were increased in cells lacking the E2 enzyme Pex4, the E3 ligases
Pex2,Pex10 or Pex12, as well as in cells expressing the ubiquitin mutant K48R, indicating a
role for ubiquitination in Pex13 levels. The activity of the Pex13 promoter in these mutants
was not up-regulated compared to wild type cells, indicating that the increased level of Pex13
in these strains was caused by inhibited degradation.
Furthermore, fluorescent microscopy indicated that Pex13-GFP displays a punctate signal
that co-localized with Pex14-mCherry, suggesting that Pex13 accumulates on the
peroxisomal membrane when its degradation is inhibited. In addition, we observed polyubiquitinated forms of Pex13, which suggests that Pex13 ubiquitination targets the protein for
degradation by the proteasome. Finally, Pex13 degradation is inhibited in cells deleted for
PEX5 or PEX8, which could suggest that Pex13 ubiquitination and degradation are linked to
the import of matrix proteins.
In summary, Pex13 is involved in import process of peroxisomes and ubiquitinated Pex13
suggests it can be degraded by the proteasome. There would be growth defect and
peroxisome remnants if the degradation of Pex13 is affected in ubiquitination defect mutants.
59
List of participants
Ast, Julia
Azimian, Amir
North Khorasan University of Medical Sciences
North Khorasan University of Medical
Sciences
[email protected]
[email protected]
Philipps University of Marburg
[email protected]
North Khorasan University of Medical
Sciences
[email protected]
Bakker, Barbara
Bordin, Nicola
University Medical Centre Groningen
Universidad Pablo de Olavide
[email protected]
[email protected]
Brauns, Ann-Kristin
Chakraborty, Anirban
UKE Hamburg
Ruhr University Bochum
[email protected]
[email protected]
Chen, Xin
Cherkaoui Malki, Mustapha
University of Groningen
University of Bourgogne
[email protected]
[email protected]
Chowdhary, Gopal
Corpas, Francisco
University of Stavanger
Spanish National Research Council
[email protected]
[email protected]
60
Costello, Joseph
Crappe, Delphine
University of Exeter
University of Stavanger
[email protected]
[email protected]
Cruz Zaragoza, Luis Daniel
Dahan, Noa
Ruhr-Universität Bochum
The Weizmann Institute of Science
[email protected]
[email protected]
Devos, Damien
Dos Santos, Vitor
Universidad Pablo de Olavide
Life Glimmer GmbH Berlin
[email protected]
[email protected]
Erdmann, Ralf
Falkenberg, Kim
Ruhr-University Bochum
Academic Medical Centre Amsterdam
[email protected]
[email protected]
Falter, Christian
Fransen, Marc
University of Hamburg
KU Leuven
[email protected]
[email protected]
61
Giannopoulou, Evdokia
Gonzalez, Eglys
EMBL Hamburg
Universidad de Los Andes
[email protected]
[email protected]
Hanna, Nabil
Haupenthal, Katharina
EMBL Hamburg
University of Luebeck
[email protected]
[email protected]
Herzog, Katharina
Hu, Jianping
Academic Medical Center Amsterdam
Michigan State University
[email protected]
[email protected]
Jin, Eun-Jung
Kato, Hiroaki
Wonkwang University
Kyoto University
[email protected]
[email protected]
Kechasov, Dmitry
Lisik, Piotr
University of Stavanger
University of Stavanger
[email protected]
[email protected]
62
Michels, Paul
Nguyen, Thu
University of Edinburgh
University of Hamburg
[email protected]
[email protected]
Palma, Jose Manuel
Pokhrel, Saugat
Estacion experimental del Zaidin CSIC
University of Stavanger,
University of Hamburg
[email protected]
[email protected]
Radke, Juliane Lara
Reumann, Sigrun
Ruhr-University Bochum
University of Hamburg and Stavanger
[email protected]
[email protected]
Sadeghi Azadi, Afsoon
Sandalio, Luisa Maria
University of Exeter
E stacion experimental del Zaidin CSIC
[email protected]
[email protected]
Schauer, Nicolas
Schrader, Michael
Metabolomic Discoveries GmbH
University of Exeter
[email protected]
[email protected]
63
Singh, Ritika
Smith, Robert
University Of Groningen
LifeGlimmer GmbH
[email protected]
[email protected]
Song, Kyung
Sparkes, Imogen
Wonkwang University
University of Exeter
[email protected]
[email protected]
Thomas, Ann
Subramani, Suresh
University of Groningen
University of California
[email protected]
[email protected]
van der Klei, Ida
Veenhuis, Marten
University of Groningen
University of Groningen
[email protected]
[email protected]
Wanders, Ronald
Waterham, Hans
Academic Medical Centre Amsterdam
Academic Medical Centre Amsterdam
[email protected]
[email protected]
64
Wegrzyn, Agnieszka
Wilmanns, Matthias
University Medical Centre Groningen
EMBL Hamburg
[email protected]
[email protected]
Wroblewska, Justyna
Wu, Huala
University of Groningen
University of Groningen
[email protected]
[email protected]
Zalckvar, Einat
Weizmann Institute of Science
[email protected]
65
Notes:
66
Notes:
67
Notes:
68