Biochemical characterization of the self-splicing GIN1 protein in arbuscular mycorrhizal fungi [Elektronische Ressource] / vorgelegt von Esther Serrano Padial

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• Biologie

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• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2005
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 13 Mo

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Biochemical characterization of the self-splicing
GIN1 protein in arbuscular mycorrhizal fungi







Dissertation



der Fakultät für Biologie
der Eberhard Karls Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften






vorgelegt von
Esther Serrano Padial
aus Elda, Spanien
2005





























Tag der mündlichen Prüfung 24.06.2005
Dekan Prof. Dr. Friedrich Schöffl
1. Berichterstatter PD Natalia Requena
2. Prof. Dr. Rüdiger Hampp


































To my family Acknowledgments

My special acknowledgement to PD. Dr. Natalia Requena, for giving me the chance
to work in this interesting subject, her constant patience and supervision of my work.

I would like to thank the group of Prof. Dr. R. Hampp for nice working atmosphere
and the good friends I left there: Alonso, Mónica, Anita, Martin, Silvia, Marion and in
special M. Ecke, for being able to solve any problem.

My special regards to Dr. Christian Ottmann and Prof. Dr. C. Oecking and her group.
It was really nice to work with you, thanks for being always willing to help and solving
so many proteomics and other problems.

My special acknowledgement to Thomas Härtner and Prof. Dr. Wohlleben. Without
your advice, patience and good humor I could have not make it with the lipid
extraction and characterization. Danke viel mals! Es war wirklich schön mit dir
zusammen zum arbeiten.

Specially thanks to G. Mander and Prof. Dr. Reinhardt Fischer and his group, for
scientific discussion and that hard week of work.

Thanks to Prof. Dr. R. Süssmuth and his group, Kathrin, Falco, Diane, Timo and
Simone, and in special Dr. M. Bertazzo, the chemistry connection, for solving
analytical problems and HPLC-MS analyses.

Although we never met, my special regards to Prof. Dr. Sonia Paris. Thanks for your
fast email advice. Your protocols, experience and professional discussion brought
light to my GTP binding experiments.

Thanks to Dr. T. Haug and all the people in the Isotopenlabour, for being always so
nice to me and helping in the set up of the experiments related to radioactivity.

My acknowledgement to Prof. Dr. M. Bölker for providing the Umcdc42 and
Umcdc42(Q-L) constructs and P. A. Beachy for the DmHh-C construct and advice.

I would like to thank many groups and people in the Botanical Institute for their
contribution to this work in many aspects: Anne, Luz Irina, F. de Courcy, Dr. M. de Simone, Dr. K. Schumacher, Dr. M. Tarkka, PD. Dr. U. Nehls, Dr. Swechheimer, PD.
Dr. D. Begeroff, Prof. Dr. Zeits, Prof. Dr. Kottke, Prof. Dr. Nürnberger, Prof. Dr. Harter.

To Dr. Nina Grunze, for personal and professional advice and critical correction of
the thesis. For being a great person, friend and researcher.

To Dr. Mark Trautwein, for hearing and solving my “never ending little lab problems”,
thanks for sharing your “common sense”, reviewing my work and unconditional help
and friendship.

To Dr. Claudia Lemmel, for supporting me being a great friend and researcher,
opening your house and your heart to me. I am sure we will be for long good friends.

Gracias al Prof. Dr. Juan Niclós y Prof. Dr. Pepa González, por introducirme en el
campo de la investigación y apoyarme en mi decisión de venir a Alemania.

A mi gran amigo Alonso Leyva por todas las risas, lágrimas y sudores que
compartimos estos años.

A Patricia, por las miles de horas y risas al teléfono; a André, por tu guitarra y Ney; a
Carla, Cleo, Cintia y Guillerme, por los buenos churrascos; a Malú, por tu gran
amistad; a Claudinha, por tu gran corazón; a Dino, Elisette, Renata, Alexandre,
Simone y Reiane, por su alegría.

A Daniela, Xavi, Mario, Andrea, Alejandro y Justine, por los buenos momentos.

A mi madre y hermano, a pesar de no comprender mis inquietudes, siempre he
contado con vuestro apoyo.

A mi padre, por las cientos de llamadas telefónicas y cartas mandadas todos estos
años. No sabes lo que me han ayudado.

Mi agradecimiento especial a Marcelo Bertazzo, por tu incondicional amor y apoyo,
tu generosidad, valía profesional y humana. No cambies jamás. Index
________________________________________________________________________
1 Sumary 1

2 Introduction 3
2.1 Mycorhiza 3
2.1.1 Definition and importance 3
2.1.2 Establishment of the symbiosis 4
2.2 Signaling events in the symbiosis 5
2.2.1 The response of the plant 9
2.3 The Glomus mosseae GIN1 gene (GmGIN1) 12
2.4 The GmGIN1-N terminus orthologues: GTPase proteins 14
2.4.1 GTPases: conserved structure for a variety of functions 14
2.4.2 Lipid modification and targeting of proteins, particularly GTPases 19
2.4.3 GIMAP family of GTP binding proteins 22
2.5 The GmGIN1-C terminus orthologues: Hedgehog (Hh) proteins 25
2.5.1 Protein splicing: Inteins and Hedgehog proteins 25
2.5.2. Lipid modification of Hedgehog proteins and localization to rafts 28
2.5.3 The Hog domain is found in other proteins 31
2.6 Aim of this work 33

3 Materials and methods 34
3.1 Equipment
3.2 Standard buffers and solutions 34
3.3 Culture media 36
3.4 Protein SDS-PAGE reagents 39
3.4.1 In gel trypsin digest 42
3.5 Western blot detection buffers and solutions 42
3.5.1 Primary antibodies 43
3.5.2 Secondary
3.6 Ni-NTA purification buffers 43
3.6.1 Native purification buffers
3.6.2 Denaturing buffers 44
3.7 Organisms and culture conditions 45
3.7.1. Escherichia coli 45
3.7.1.1 Strains 45 Index
________________________________________________________________________
3.7.1.2 Standard culture conditions 46
3.7.1.3 Standard protein induction conditions 46
3.7.2 Mycorrhizal fungi 46
3.7.2.1 Strains
3.7.2.2 Standard culture conditions 46
3.8 Molecular biological methods 47
3.8.1 Plasmids
3.8.2 Oligonucleotides 48
3.8.3 Plasmid DNA extraction from E. coli 49
3.8.4 Restriction enzyme analysis of plasmid DNA preparations 49
3.8.5 PCR 49
3.8.6 Synthesis of DIG-labelled probes by PCR 50
3.8.7 Ligation of PCR products 50
3.8.8 Cloning for protein expresion 50
3.8.9 E. coli chemically competent cells 51
3.8.10 E. coli electro-competent cells 52
3.8.11. E. coli transformation
3.8.12 Northern and Southern Blot 52
3.8.13 Bacterial colony hybridization 53
3.9 Biochemical methods 54
3.9.1 Protein purification
3.9.1.1 Native of His-tagged proteins from E. coli 54
3.9.1.2 Denaturing purification of His-tagged proteins from E. coli 55
3.9.1.3 Ion Exchange purification of GmGIN-C terminus 55
3.9.2 Protein precipitation 56
3.9.3 Splicing reaction 56
3.9.4 Production of antibodies directed against the N- and C-terminus
of GmGIN1 56
3.9.5 SDS-Polyacrylamide gel electrophoresis of proteins 58
3.9.5.1 Tris-Glycine gels 58
3.9.5.2 Schägger-Jagov gels
3.9.5.3 Casting and running of gels 59
3.9.6 Western blot transfer
3.9.7 detection 60 Index
________________________________________________________________________
3.9.8 Stripping and reprobing 61
3.9.9 In-gel trypsin digest 61
3.9.10 Determination of protein concentration by Bradford reagent 62
3.9.11 Protein extraction of mycorrhizal samples 63
3.9.12 Lipid extraction 63
3.9.12.1 Folch method (Folch et al., 1957) 63
3.9.12.2 Lipid extraction according to Beilby et al. (1980) 64
3.9.12.3 Lipid extraction according to Nichols et al. (1965) 64
3.9.12.4 Lipid extraction according Blight and Dyer (1959) 65
3.9.12.5 Extraction of unsaponified fraction (Entenman, 1957) 65
3.9.13 Lipid column chromatography 66
3.9.14 Thin layer chromatography (TLC) 66
3.9.15 Lipid acetylation 67
3.9.16 Sterol content quantification
3.9.17 Gas liquid chromatography - mass spectrometry (GC-MS) 67
3.9.18 GTPase activity 68
3.9.18.1 GTP overlay assays
3.9.18.2 GTP binding filtration assays 69
3.9.18.3 Dissociation assay using competing nucleotides 69
3.9.18.4 Enzymatic activity of GmGIN1-N 70

4 R