Protein import into the inner envelope membrane of chloroplasts [Elektronische Ressource] / vorgelegt von Ewa Firlej-Kwoka

Ewa Firley-Kwoka

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 17 Mo

Extrait

Protein Import into the Inner Envelope
Membrane of Chloroplasts




Dissertation
Zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München








Vorgelegt von
Ewa Firlej-Kwoka


München, 26. Juni 2008



























Gutachter:
1. Prof. Dr. Jürgen Soll
2. Prof. Dr. Jörg Nickelsen

Tag der mündlichen Prüfung: 26.06.08



Contents
Contents



Abbreviations................................................................................................... 1
Summary .......................................................................................................... 3
Zusammenfassung........................................................................................... 4
1. Introduction ................................................................................................. 6
1.1 Protein import into chloroplasts .................................................................................. 6
1.2 Transit peptides............................................................................................................ 7
1.3 Import regulation at the level of the Toc complex and in the intermembrane space .. 9
1.4 Import regulation at the level of the Tic complex ..................................................... 10
1.5 Soluble stromal factors, protein processing and folding ........................................... 11
2. Aim of this work ........................................................................................ 13
2.1. Facts about the proteins chosen as import substrates ............................................... 14
3. Materials..................................................................................................... 16
3.1 Chemicals and membranes ........................................................................................ 16
3.2 Kits ............................................................................................................................ 16
3.3 Molecular weight and size markers........................................................................... 16
3.4 Enzymes .................................................................................................................... 17
3.5 Oligonucleotides........................................................................................................ 17
3.5.1 For cloning of HP17 into pSP65 vector ............................................................. 17
3.5.2 For cloning of HP29b into pSP65 vector ........................................................... 17
3.5.3 For cloning of HP28 into pSP65 vector ............................................................. 17
3.5.4 For cloning of HP36 into pSP65 vector ............................................................. 17
3.5.5 For cloning of IEP37 into pSP65 vector............................................................. 18
3.5.6 For cloning of HP17 from pSP65 into pET21d vector....................................... 18
3.5.7 For cloning of mOE33 from pOE33/pET21c into pET21d vector..................... 18
3.6 Vectors....................................................................................................................... 18
3.7 Clones ........................................................................................................................ 19
3.7.1 HP17, HP28, HP29b, HP34, PIC1, PPT and XPT ............................................. 19
3.7.2 HP36 and IEP37 ................................................................................................. 19
3.7.3 pSSU................................................................................................................... 19
i Contents
3.7.4 Tic32................................................................................................................... 19
3.7.5 tpSSU-110N-mSSU............................................................................................ 20
3.7.6 pOE33 and mOE33............................................................................................. 20
3.8 Bacterial strains ......................................................................................................... 20
3.9 Growth media ............................................................................................................ 20
3.10 Radioisotopes .......................................................................................................... 21
3.11 Plant material........................................................................................................... 21
4. Methods ...................................................................................................... 22
4.1 General molecular biology methods.......................................................................... 22
4.1.1 Standard methods ............................................................................................... 22
4.1.2 Plasmid DNA isolation....................................................................................... 22
4.1.3 Polymerase chain reaction (PCR)....................................................................... 22
4.1.4 Cloning techniques ............................................................................................. 23
4.1.5 In vitro transcription and translation .................................................................. 23
4.2 Isolation of intact chloroplasts from pea ................................................................... 24
4.3 Treatment of chloroplasts and translation product before import ............................. 24
4.3.1 ATP depletion from chloroplasts and in vitro translation product ..................... 24
4.3.2 Protease pre-treatment of isolated intact chloroplasts ........................................ 25
4.3.3 Chloroplasts pre-treatment with Ophiobolin A and ionophore A23187 ............ 25
4.4 Import experiments and chloroplasts post-treatment................................................. 25
4.4.1 Import of radioactively labelled proteins into intact chloroplasts ...................... 25
4.4.2 Chloroplasts post-treatment with thermolysin.................................................... 26
4.4.3 ATP concentration scale..................................................................................... 26
4.4.4 Pulse-chase import experiment........................................................................... 26
4.4.5 Competition for import with mOE33 and pOE33 .............................................. 27
4.5 Suborganellar localization of imported proteins ....................................................... 27
4.5.1 Fractionation of chloroplasts into soluble and membrane fractions after import27
4.5.2 Extraction of proteins with 6 M urea.................................................................. 27
4.6 Stromal processing assay........................................................................................... 27
4.7 Overexpression and purification of pOE33 and mOE33........................................... 28
4.8 Methods for separation and identification of proteins............................................... 28
4.8.1 SDS-Polyacrylamide-Gel-Electrophoresis (SDS-PAGE) .................................. 28
4.8.2 Detection of proteins .......................................................................................... 28
4.8.3 General methods of protein biochemistry .......................................................... 29
ii Contents
5. Results......................................................................................................... 30
5.1 Protein import into chloroplasts and post-treatment with thermolysin ..................... 31
5.2 Stromal processing assay........................................................................................... 34
5.3 Pulse-chase import experiments ................................................................................ 36
5.4 Energy requirement for import into chloroplasts ...................................................... 39
5.5 Chloroplast fractionation into membrane and soluble fractions after import............ 41
5.6 Protein extraction with 6M urea ................................................................................ 44
5.7 Competition for import with mOE33 and pOE33 ..................................................... 46
5.8 Chloroplast pre-treatment with thermolysin.............................................................. 51
5.9 Chloroplasts pre-treatment with Ophiobolin A and the ionophore A23187 ............. 54
6. Discussion................................................................................................... 59
References ...................................................................................................... 67
Acknowledgements........................................................................................ 75
Curriculum vitae ...............................