Members of the Preprotein and Amino Acid Transporter Family Constitute Components of Novel Protein Import Pathways into Chloroplasts [Elektronische Ressource] / Claudia Roßig. Betreuer: Christiane Reinbothe

Claudia Berger

Informations

• Publié par: universitat_bayreuth
• Publié le: 01 janvier 2011
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 14 Mo

Extrait

Members of the Preprotein and Amino Acid Transpr oFratemily
Constitute Components of Novel Protein
Import Pathways into Chloroplasts




DISSERTATION



zur Erlangung des Grades
- Doktor der Naturwissenschaften -
der Fakultät für Biologie, Chemie und Geowissenscfhtean
der Universität Bayreuth





vorgelegt von
Claudia Roßig



Bayreuth, im Mai 2011





Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenschaften
der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften (Dr. rer. nat.).





Die vorliegende Arbeit wurde unter der Betreuung von Frau PD Dr. Christiane Reinbothe
und Herrn Prof. Dr. Steffen Reinbothe in der Zeit vom Dezember 2006 bis November 2009
im Laboratoire Plastes et Différenciation Cellulaire (UJF/CNRS/FRE3017) der Université
Joseph Fourier 1 in Grenoble (Frankreich) und vom Januar 2010 bis Mai 2011 am Lehrstuhl
für Pflanzenphysiologie der Universität Bayreuth angefertigt.





Das Promotionsgesuch wurde eingereicht am: 04.05.2011

Das Rigorosum fand statt am: 04.10.2011



Der Prüfungsausschuss bestand aus:

PD Dr. Christiane Reinbothe (Erstgutachterin)
Prof. Dr. Angelika Mustroph (Zweitgutachterin)
Prof. Dr. Stephan Clemens (Vorsitz)
Prof. Dr. Konrad Dettner
Prof. Dr. Benedikt Westermann



Folgende Publikationen sind u. a. aus der vorliegenden Arbeit entstanden:

SAMOL I, BUHR F, SPRINGER A, POLLMANN S, LAHROUSSI A, ROSSIG C, VON WETTSTEIN D,
REINBOTHE C & REINBOTHE S (2011a) Implication of the oep16-1 mutation in a flu-
independent, singlet oxygen-regulated cell death pathway in Arabidopsis thaliana. Plant
Cell Physiol 52, 84-95.

SAMOL I, ROSSIG C, BUHR F, SPRINGER A, POLLMANN S, LAHROUSSI A, VON WETTSTEIN D,
REINBOTHE C & REINBOTHE S (2011b) The outer choroplast envelope protein OEP16-1
for plastid import of NADPH:protochlorophyllide oxidoreductase A in Arabidopsis
thaliana. Plant Cell Physiol 52, 96-111.

BARTSCH S, SAKSOU J, YANG M, GRAY J, ROSSIG C, REINBOTHE C & REINBOTHE S – A
protochlorophyllide (Pchlide) a oxygenase involved in plastid import of NADPH:Pchlide
oxidoreductase (POR) A is essential for plant viability. Submitted for publication in
Plant Physiology.



































For my parents



















CONTENTS

CONTENTS

ABBREVIATIONS ............................................................................................................................. V

SUMMARY/ ZUSAMMENFASSUNG................................................................................................ 1
SUMMARY........................................................................................................................................... 2
ZUSAMMENFASSUNG...................................................................................................................... 4

INTRODUCTION............................................................................................................................... 7
1.1 The Evolutionary Origin of Chloroplasts................................................................................ 8
1.2 The Photoprotective Role of PORA during Plant Greening ................................................... 9
1.3 The Role of Plastid Envelope Membranes in Protein Import................................................ 11
1.4 Canonical Protein Import Pathways into Chloroplasts.......................................................... 12
1.4.1 The TIC/TOC Pathway – Protein Translocation into the Stroma................................... 13
1.4.2 Substrate-Specificity and Regulation of the TIC/TOC Pathway .................................... 14
1.5 Diversity of Novel Protein Import Pathways ........................................................................ 16
1.5.1 Substrate-specific Import of PORA involving the PTC Complex.................................. 17
1.5.2 Import of Transit Sequence-less Proteins ....................................................................... 19
1.6 The Preprotein and Amino Acid Transporter (PRAT) Family.............................................. 20
1.7 Aim of this Work................................................................................................................... 22

RESULTS ........................................................................................................................................... 24
2.1 Isolation of Components of the ceQORH-specific Translocon Complex ............................. 25
2.1.1 Production of Import Intermediates................................................................................ 25
2.1.2 Purification and Identification of Envelope Proteins Involved in ceQORH-Import....... 28
2.2 Expression and Purification of HP20-(His) and HP30-(His) – Production and 6 6
Characterization of Antibodies.............................................................................................. 30
2.2.1 Expression and Purification of HP20-(His) and HP30-(His) ....................................... 30 6 6
2.2.2 Antibody Characterization.............................................................................................. 33
2.3 Molecular-biological Characterization of A. thaliana Knock-out Lines............................... 37
2.4 Expression and Localization of HP20 and HP30 .................................................................. 42
2.4.1 Database Analysis and Prediction of the Subcellular Localization of HP20 and HP30 . 42
2.4.2 Subcellular Localization of HP20 and HP30.................................................................. 43
2.4.3 Biochemical Localization of HP20/QTC24 and its Characterization as Envelope
Membrane Protein .......................................................................................................... 49
2.4.4 Topology of HP20 and HP30 as Integral Membrane Proteins........................................ 51
2.5 Functional Analysis of HP20 and HP30 as Components of Protein Import Pathways ......... 52
2.5.1 Role of HP20/QTC24 during ceQORH-Import into Plastids ......................................... 52
2.5.2 In planta Targeting of ceQORH and TIC32 in Athp20 Mutants .................................... 57
2.6 Phenotypic Characterization of the Athp20 and Athp30 Mutants.......................................... 60
2.6.1 Plant Growth under Standard Light Conditions.............................................................. 61
2.6.2 Greening of Etiolated Seedlings under Low Light Conditions....................................... 63
2.6.3 Greening of Etiolated Seedlings under Light Stress Conditions..................................... 68

I
CONTENTS

2.6.4 Analysis of Protein Expression during Senescence........................................................ 70
2.7 Post-transcriptional Silencing of HP20 and HP30 in A. thaliana......................................... 75
2.7.1 Created RNA Silencing Constructs ................................................................................ 75
2.7.2 Preliminary Phenotypic Characterization of RNAi Plants.............................................. 76
2.8 Analysis of the Role of OEP16-1.......................................................................................... 78
2.8.1 Characterization of the Mutants Atoep16-1;6 and Atoep16-1;7 ..................................... 80
2.8.2 Analysis of a Complemented Atoep16-1;6 Line............................................................. 85

DISCUSSION..................................................................................................................................... 92
3.1 The Physiological Role of HP20/HP22 and HP30/HP30-2 in the Chloroplast Envelopes ... 94
3.1.1 HP20/QTC24 mediates the Import of the Transit Peptide-less Precursor Protein .............
ceQORH ......................................................................................................................... 94
3.1.2 Localization of HP20 and HP30 and their Topology in Envelope Membranes.............. 96
3.1.3 Athp20 and Athp30 Plants are Not Defective in the Plastid Import of Standard Precursor
Proteins and Amino Acids .............................................................................................. 98
3.1.4 Analysis of the Phenotype of Athp20 and Athp30 Plants cultivated under Standard
Growth Conditions.......................................................................................................... 99
3.1.5 The Accumulation of Plastid-encoded Proteins is delayed during the De-etiolat