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Dwarfs and Giants: Mechanism of ABCB regulation by immunophilins

English title Dwarfs and Giants: Mechanism of ABCB regulation by immunophilins
Applicant Geisler Markus
Number 125001
Funding scheme Project funding
Research institution Unité de biologie végétale Département de Biologie Université de Fribourg
Institution of higher education University of Zurich - ZH
Main discipline Cellular Biology, Cytology
Start/End 01.07.2009 - 31.12.2012
Approved amount 331'000.00
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Keywords (9)

auxin; P-glycoprotein; ABCB; protein-protein interaction; FKBP; PPIase; auxin transport; immunophilins; calmodulin

Lay Summary (German)

Lead
Lay summary
Lokale Gradienten des Pflanzenhormons Auxin bestimmen weitgehend die Physiologie und die Entwicklung der Pflanzen. Diese werden auf zellulärer Ebene durch die Aktivität von Exportern kontrolliert, die entweder zur Familie der PIN-Proteine oder der ABCBs/PGPs gehören. Letztere werden durch Protein-Protein Interaktion mit dem Immunophilin TWISTED DWARF1 (TWD1) reguliert.Im Gegensatz zur Physiologie sind die molekularen Mechanismen dieser Interaktion weitgehend unverstanden. In diesem Projekt möchten wir daher gerne, den Mechanismus der ABCB Aktivierung durch Interaktion mit dem TWD1 Protein untersuchen. In einem ersten Schritt, werden wir versuchen Bindungsoberflächen in beiden Molekülen durch bioinformatische Analysen, biochemische Methoden und Strukturanalysen zu vorauszusagen und zu verifizieren. Darüber hinaus möchten wir das Konzept bestätigen, dass in Analogie zu tierischen Systemen, TWD1 ABCB1 durch intramolekulare Konformationsänderungen in der C-terminalen Nukleotidbindungsdomäne aktiviert. Für das nahe verwandte FKBP38 konnte nämlich gezeigt werden, dass eine versteckte PPIase-Aktivität durch das Calcium-bindende Molekül Calmodulin reaktiviert werden kann.Diese Calmodulin-abhängige PPIase-Aktivität sollte den TWD1-ABCB1 Komplex stabilisieren. Mithilfe von NMR-Technologie möchten wir darüber hinaus demonstrieren, dass diese induzierte PPiase-Aktivität zu intramolekularen Konformationsänderungen in ABCB1 führt. Diese wiederum sollen durch Mutagenese in vitro und final in der Pflanze verifiziert werden.Die Ergebnisse sollten unseren Erkenntnisstand hinsichtlich der ABCB-Transporter Regulation erweitern, könnten aber auch von agronomischem und klinischem Interesse sein.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Arabidopsis TWISTED DWARF1 Functionally Interacts with Auxin Exporter ABCB1 on the Root Plasma Membrane.
Wang Bangjun, Bailly Aurélien, Zwiewka Marta, Henrichs Sina, Azzarello Elisa, Mancuso Stefano, Maeshima Masayoshi, Friml Jirí, Schulz Alexander, Geisler Markus (2013), Arabidopsis TWISTED DWARF1 Functionally Interacts with Auxin Exporter ABCB1 on the Root Plasma Membrane., in The Plant cell, in press(in press), 1-13.
A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants.
Barbez Elke, Kubeš Martin, Rolčík Jakub, Béziat Chloé, Pěnčík Aleš, Wang Bangjun, Rosquete Michel Ruiz, Zhu Jinsheng, Dobrev Petre I, Lee Yuree, Zažímalovà Eva, Petrášek Jan, Geisler Markus, Friml Jiří, Kleine-Vehn Jürgen (2012), A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants., in Nature comm., 485(7396), 119-22.
Arabidopsis ABCB21 is a facultative auxin importer/exporter regulated by cytoplasmic auxin concentration.
Kamimoto Yoshihisa, Terasaka Kazuyoshi, Hamamoto Masafumi, Takanashi Kojiro, Fukuda Shoju, Shitan Nobukazu, Sugiyama Akifumi, Suzuki Hideyuki, Shibata Daisuke, Wang Bangjun, Pollmann Stephan, Geisler Markus, Yazaki Kazufumi (2012), Arabidopsis ABCB21 is a facultative auxin importer/exporter regulated by cytoplasmic auxin concentration., in Plant & cell physiology, 53(12), 2090-100.
ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis.
Ding Zhaojun, Wang Bangjun, Moreno Ignacio, Dupláková Nikoleta, Simon Sibu, Carraro Nicola, Reemmer Jesica, Pěnčík Aleš, Chen Xu, Tejos Ricardo, Skůpa Petr, Pollmann Stephan, Mravec Jozef, Petrášek Jan, Zažímalová Eva, Honys David, Rolčík Jakub, Murphy Angus, Orellana Ariel, Geisler Markus, Friml Jiří (2012), ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis., in Nature communications, 3, 941-941.
NMR assignments of the FKBP-type PPIase domain of FKBP42 from Arabidopsis thaliana.
Burgardt Noelia Inés, Linnert Miriam, Weiwad Matthias, Geisler Markus, Lücke Christian (2012), NMR assignments of the FKBP-type PPIase domain of FKBP42 from Arabidopsis thaliana., in Biomolecular NMR assignments, 6(2), 185-8.
Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation.
Henrichs Sina, Wang Bangjun, Fukao Yoichiro, Zhu Jinsheng, Charrier Laurence, Bailly Aurélien, Oehring Sophie C, Linnert Miriam, Weiwad Matthias, Endler Anne, Nanni Paolo, Pollmann Stephan, Mancuso Stefano, Schulz Alexander, Geisler Markus (2012), Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation., in The EMBO journal, 31(13), 2965-80.
The AGC kinase, PINOID, blocks interactive ABCB/PIN auxin transport.
Wang Bangjun, Henrichs Sina, Geisler Markus (2012), The AGC kinase, PINOID, blocks interactive ABCB/PIN auxin transport., in Plant signaling & behavior, 7(12), 1515-1518.
Flavonols accumulate asymmetrically and affect auxin transport in Arabidopsis.
Kuhn Benjamin M, Geisler Markus, Bigler Laurent, Ringli Christoph (2011), Flavonols accumulate asymmetrically and affect auxin transport in Arabidopsis., in Plant physiology, 156(2), 585-95.
Plant Lessons: Exploring ABCB Functionality Through Structural Modeling.
Bailly Aurélien, Yang Haibing, Martinoia Enrico, Geisler Markus, Murphy Angus S (2011), Plant Lessons: Exploring ABCB Functionality Through Structural Modeling., in Frontiers in plant science, 2, 108-108.
Arabidopsis PCR2 is a zinc exporter involved in both zinc extrusion and long-distance zinc transport.
Song Won-Yong, Choi Kwan Sam, Kim Do Young, Geisler Markus, Park Jiyoung, Vincenzetti Vincent, Schellenberg Maja, Kim Sun Ha, Lim Yong Pyo, Noh Eun Woon, Lee Youngsook, Martinoia Enrico (2010), Arabidopsis PCR2 is a zinc exporter involved in both zinc extrusion and long-distance zinc transport., in The Plant cell, 22(7), 2237-52.
Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid.
Ruzicka Kamil, Strader Lucia C, Bailly Aurélien, Yang Haibing, Blakeslee Joshua, Langowski Lukasz, Nejedlá Eliska, Fujita Hironori, Itoh Hironori, Syono Kunihiko, Hejátko Jan, Gray William M, Martinoia Enrico, Geisler Markus, Bartel Bonnie, Murphy Angus S, Friml Jirí (2010), Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid., in Proceedings of the National Academy of Sciences of the United States of America, 107(23), 10749-53.
Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters.
Song Won-Yong, Park Jiyoung, Mendoza-Cózatl David G, Suter-Grotemeyer Marianne, Shim Donghwan, Hörtensteiner Stefan, Geisler Markus, Weder Barbara, Rea Philip A, Rentsch Doris, Schroeder Julian I, Lee Youngsook, Martinoia Enrico (2010), Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters., in Proceedings of the National Academy of Sciences of the United States of America, 107(49), 21187-92.
Identification of an ABCB/P-glycoprotein-specific inhibitor of auxin transport by chemical genomics.
Kim Jun-Young, Henrichs Sina, Bailly Aurélien, Vincenzetti Vincent, Sovero Valpuri, Mancuso Stefano, Pollmann Stephan, Kim Daehwang, Geisler Markus, Nam Hong-Gil (2010), Identification of an ABCB/P-glycoprotein-specific inhibitor of auxin transport by chemical genomics., in The Journal of biological chemistry, 285(30), 23309-17.
The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning.
de Carbonnel Matthieu, Davis Phillip, Roelfsema M Rob G, Inoue Shin-Ichiro, Schepens Isabelle, Lariguet Patricia, Geisler Markus, Shimazaki Ken-Ichiro, Hangarter Roger, Fankhauser Christian (2010), The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 protein is a phototropin signaling element that regulates leaf flattening and leaf positioning., in Plant physiology, 152(3), 1391-405.

Collaboration

Group / person Country
Types of collaboration
Prof. A. Murphy/Purdue University United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Prof. S. Mancuso/University of Firenze Italy (Europe)
- Publication
- Research Infrastructure
Dr. Y. Fukao, NAIST Japan (Asia)
- Publication
- Research Infrastructure
Prof. K. Yazaki/University of Kyoto Japan (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. S. Pollmann/University of Madrid Spain (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. C. Fankhauser/University of Lausanne Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
AUXIN2012 Poster REGULATION OF ABCB AND PIN-MEDIATED AUXIN TRANSPORT BY PINOID 03.12.2012 Big Island, Hawaii, USA, United States of America Geisler Markus;
8th Tri-National Arabidopsis meeting Poster TWISTED DWARF1 regulates auxin transport by interfering with the actin cytoskeleton 12.09.2012 Lausanne, Switzerland, Austria Geisler Markus;
ISLSWG Satellite Symposium „Plant Biology in Space“ of the EPSO Plant Biology Congress 2013 Talk given at a conference Control of root gravitropism by auxin-mediated cell elongation 30.07.2012 Freiburg, Germany, Germany Geisler Markus;
ASPB meeting “Plant Biology 2009” Poster unknown 18.07.2012 Honolulu, Hawaii, USA, United States of America Geisler Markus;
International Arabidopsis meeting (ICAR2012) Poster PINOID REGULATES ABCB1/PGP1-CATALYZED AUXIN TRANSPORT BY LINKER PHOSPHORYLATION 03.07.2012 Vienna, Austria, Austria Geisler Markus;
20th International Plant Growth Substances (IPGSA) Conference Talk given at a conference unknown 28.06.2012 Tarragona, Spain, Spain Geisler Markus;
Regulation of Plant growth Talk given at a conference unknown 12.04.2012 Potsdam, Germany, Germany Geisler Markus;


Knowledge transfer events



Self-organised

Title Date Place
Fleischfressende Pflanzen 09.01.2013 Rietberg Montessori Schule Zürich, Switzerland

Communication with the public

Communication Title Media Place Year
New media (web, blogs, podcasts, news feeds etc.) ABCB-Transporter: Lassen Wurzeln nach unten wachsen und stärken Krebszellen Kommunikation und Medien (press release) Western Switzerland 2012

Awards

Title Year
Cozzarelli Prize (Class VI) from the National Academy of Sciences (USA) 2011

Associated projects

Number Title Start Funding scheme
111912 Regulation of AtPGP1-mediated auxin transport by phosphorylation 01.07.2006 Project funding
197563 Mechanistic and evolutionary insight into chaperon-mediated ABC transporter homeostasis in plants 01.01.2021 Project funding

Abstract

Local gradients of the signaling molecule auxin (IAA) are the primary determinants for many aspects of plant physiology and development. In agreement with the chemiosmotic theory, auxin efflux catalyzed by members of PIN-FORMED (PIN) and ABCB/P-GLYCOPROTEIN (PGP) families is the rate-limiting step and requires therefore strict regulation.ABCB-mediated export has been shown to be modulated by PINs and the FKBP (FK506-binding protein)-like TWISTED DWARF1 (TWD1)/FKBP42 by means of protein-protein interaction. TWD1 positively regulates the activity of ABCB1/PGP1 and close ABCB19/PGP19/MDR1 which is documented by a close overlap between twd1 and abcb1 abcb19 phenotypes. Interestingly, TWD1-ABCB1 interaction is disrupted by auxin transport inhibitors, like NPA or flavonols, and enhanced by IAA itself, resulting in blocked or stimulated auxin transport in planta, respectively. As a consequence, twd1 plants are NPA-insensitive suggesting that TWD1 functions as a flux sensor of ABCB-mediated auxin transport.However, in contrast to its physiological relevance, the underlying mechanisms are entirely unclear. The N-terminal PPIase domain, functioning as cis-trans peptidylprolyl isomerase in canonical FKBPs, binds the C-terminal nucleotide binding fold (NBD2) of ABCBs. Initially it was therefore thought that TWD1 interaction would block IAA or ATP access. In light of recent findings and the unequal ABCB1/TWD1 stoichiometry a transient mode of action was predicted. In this project we intend to precisely map TWD1-ABCB interfaces and to elucidate the mechanism of ABCB activation by TWD1 docking.Previous unsuccessful mapping trials suggest that the TWD1-ABCB1 interaction employs 3D binding surfaces in both molecules. Therefore, we aim to precisely determine essential surface residues by use of combinations of bioinformatical (in silico docking), biochemical (ABCB1 peptide scans) and structural tools (co-crystallization of purified ABCB1-NBD2 and TWD1 proteins). Moreover we aim to demonstrate intramolecular, conformational changes in the NBD2 of ABCBs that are proposed to be catalyzed by a TWD1 intrinsic chaperone or a hidden PPIase activity, that would account for altered substrate affinities and specificities. In agreement, a stabilizing, chaperone activity of TWD1 was detected in vitro but all attempts to detect a PPIase activity for TWD1 have been unsuccessful. This is in analogy to closely related human FKBP38, that was recently shown to own a PPIase activity upon Ca2+/calmodulin-binding, forming the basis for target protein binding. Ca2+/calmodulin binding, established also for TWD1, leads apparently to a conformational change in the docking PPIase domain that is only able to bind FK506 upon activation.In a first step we will prove a hidden PPIase activity of the TWD1 FKBD that is activated by Ca2+/calmodulin. Consequently, we aim to show that activation stabilizes NBD2-TWD1 complex formation. Second, in order to demonstrate intramolecular NBD2 shifts, caused by TWD1 docking, as the primary cause of altered ABCB activity, we will employ NMR spectroscopy, quenching of intrinsic tryptophane fluorescence and NEM labeling of single-cysteine NBD2. Mechanistic implications from the structure analysis will be verified by site-directed mutagenesis of key residues analyzed in vitro and in yeast using BRET and auxin transport assays. Finally this novel mechanism will be validated by functional complementation of abcb mutants with mutated, genomic ABCB-NBD2 constructs, followed by a detailed physiological analysis.The outcome should provide a deep insight into the regulation of auxin transport via plant ABCBs and the establishment of local auxin gradients controlling virtually all steps of plant development. Transfer of this knowledge might later on open new strategies for the directed genetic or chemical manipulation of plant size or stem diameter, being critical for agricultural productivity. Finally, this novel mechanism controlling ABCB activity and substrate specificity by immunophilins might as well be of clinical relevance, as multidrug resistance attributed mainly to ABCBs are the major cause for chemotherapy failure.
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