Phosphorylation of chloroplast preproteins and the characterisation of nucleoside diphosphate kinase 2 [Elektronische Ressource] / vorgelegt von Rita Sharma

Ruben Martinez Buey

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Phosphorylation of chloroplast preproteins
and
the characterisation of nucleoside diphosphate
kinase 2






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



vorgelegt von
Rita Sharma


München
2006




















Gutachter:
1. Prof. Dr. Jürgen Soll
2. Prof. Dr. Hans-Ulrich Koop
Tag der mündlichen Prüfung: 22.08.2006









Dedicated to my spiritual
guru.












Learning
is finding out
what you already know.
Doing is demonstrating that
you know it.
Teaching is reminding others
that they know just as well as you.
We are all learners,
doers, teachers.









...........................................................................................................................Table of contents

Table of contents


Abbreviations..............................................................................................................................iv

1. Abstract...…………….....…………………………………………………………………… ..……1

2. Zusammenfassung...........………………………………………………………………………….2

3. Introduction…………………………………………………………………………………………..3

4. Materials…………………………………………………………………………………………….10

4.1 Chemicals…..……………………………………………………………………………………..10
4.2 Enzymes and Kits ………………………………………………………………………………..10
4.3 Molecular weight and size markers…………………………………………………………….10
4.4 DNA primers………………………………………………………………………………………10
4.5 Bacterial strains……
4.6 Plant material………………………………………………………………………………...…...11
4.7 Vectors …………………………………………………… ..…………………….………………11
4.8 Membranes and Filters…………………………………………………………………………..11
4.9 Antisera……………………………………….……………………… ..……………………….. 11
4.10 Films and imaging plates………………………………………………………………………11
4.11 Columns and column materials……………………………………………………………….11
4.12 Equipment for Microinjection…………………….…………………………………………….12
4.13 Bioimaging……………………………………………………………………………………….12

5. Methods…………………………………………………………………………… ………………13
5.1 Molecular biological methods…….…………………………………………… ………………13
5.1.1 General molecular biological methods… …………..…..………………… ……………….13
5.1.2 Plasmid DNA isolation ……………………………………………………… ……………….13
5.1.3 Cloning techniques…………………………………………………………… … …………..13
5.1.3.1 Polymerase chain reaction (PCR) ……………….……………..………… … ………….13
5.1.3.2 General cloning methods…………………………………………………… ……………..13
5.1.3.3 Cloning Strategy……………………………………………………………… …………… 13
5.1.4 RT- and RACE-PCR…………………….…………………………………………… …….19
5.1.5 Isolation and purification of chloroplast DNA………………………………………. ……...19
5.2 Biochemical methods……………………………………… . …………………………….. ….20
5.2.1 General biochemical methods……….…………………………………………………….. ..20
5.2.2 Overexpression of recombinant proteins and preparation of inclusion bodies………….20
5.2.3 Purification of proteins by affinity chromatography…. …….……….………………………21
...........................................................................................................................Table of contents
5.2.4 In vitro transcription and translation….………………………………………………….……21
5.2.5 Phosphorylation assay and Phospho-amino acid analysis…………………….…….…….21
5.2.6 Immuno-precipitation under denaturating conditions after phosphorylation
of proteins…………………………………………………………………....….………….21
5.3 Cell biological methods…………………………………………………….……………….……22
5.3.1 Isolation of intact chloroplasts from pea………………………………………………………22
5.3.2 ATP depletion from isolated chloroplasts and in vitro translation product………………..22
5.3.3 Import of radioactive-labeled proteins into intact chloroplasts……………………………..22
5.3.4 Protease treatment of intact chloroplasts after import………………………………….…...22
5.3.5 Chloroplast fractionation into soluble and insoluble fractions………………………………23
5.3.6 Chloroplast fractionation into suborganellar compartments……………..…………………23
5.3.7 Isolation of chloroplasts from Arabidopsis thaliana leaves…………………………...….…23
5.3.8 Isolation of Arabidopsis thaliana protoplasts…………………………....23
5.3.9 Isolation of protoplasts from Nicotiana tabacum leaves………………………………….…24
5.3.10 PEG Transformation of Nicotiana tabacum protoplasts…………………………………...24
5.3.11 Isolation and PEG Transformation of Arabidopsis thaliana protoplasts…………………24
5.3.12 Isolation of pea protoplasts…………………………………………………………………...24
5.3.13 PEG Transformation of pea protoplasts……………...……………………………………..25
5.3.14 Isolation of cytoplasm from pea and Arabidopsis thaliana protoplasts………………..…25
5.3.15 Preparation of Arabidopsis Leaf Extract…………………………………………………….25
5.3.16 Embedding of protoplasts in alginic acid……………………………………………………25
5.3.17 Embedding of protoplasts in agarose…………………………………………………….…25
5.3.18 Attachment of protoplasts using Poly-L-Lysine…………………………………………….26
5.3.19 Staining of PEG transformed protoplasts with DAPI………………………………………26
5.3.20 In vivo labelling technique……………………………………………………………….……26
5.3.20.1 Isolation and PEG transformation of tobacco protoplasts for in vivo labelling………..26
5.3.20.2 Radioactive labelling of protoplasts……………………………………………………….27
5.3.20.3 Protoplast homogenisation…………………………………………………………………27
5.3.20.4 Separation of soluble and pellet fractions from labelled protoplasts………….………28
5.3.20.5 Fractionation of chloroplasts isolated from labelled protoplasts….……………………28
5.3.20.6 Immunoprecepitation of proteins from labelled protoplasts.……………………………28
5.3.21 Isolation of nucleoids from isolated pea chloroplasts…………………………………..…29

6 Results………………………………………………………………………………………………..30
6.1 Study of the role of phosphorylation in the transit peptide of preproteins…………………..30
6.1.1 Determination of the sites of phosphorylation in the transit peptides of preproteins…….30
6.1.1.1 Determination of the site of phosphorylation in the transit peptide of APC1…………...30
6.1.1.2 Determination of the site of phosphorylation in the transit peptide of HCF136………...32
6.1.1.3 Determination of the site of phosphorylation in the transit peptide of CAO…………….33
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6.1.1.4 Determination of the site of phosphorylation in the transit peptide of LHCP…………...34
6.1.2 Attempts to study the effect of phosphorylation on the import of preproteins…………….36
6.1.2.1 Microinjection of mRNA/DNA into isolated protoplasts…………………………………...37
6.1.2.1.1 Construction of GFP-fusion constructs.…………………………………………………..37
6.1.2.1.2 The microinjection technique and the prerequisites for it………………………………38
6.1.2.1.3 In vivo labelling of protoplasts and analysis of expressed proteins…………………...44
6.2 The characterisation of the two forms of psNDPK2 and the study of the localisation of
atNDPK2………………….………………………………. …………………………………48
6.2.1 Detailed study of the two proteins encoded by the psNDPK2 gene……………………….48
6.2.1.1 Primary structure of psNDPK2………………………………………………………………48
6.2.1.2 Import of psNDPK2 into isolated chloroplasts……………………………………………..49
6.2.1.3 Import characteristics of the two forms of psNDPK2……………………………………...50
6.2.1.4 Suborganellar localisation of the two forms of psNDPK2………………………………...52
6.2.1.5 Localisation of the two forms of psNDPK2 in vivo………………………………………...56
6.2.1.6 The in vivo expression levels of the two forms of psNDPK2……………………………..57
6.2.1.7 Intra-organellar localisation of psNDPK2 in chloroplasts…………………………………58
6.2.1.8 Investigation into the proposed DNA binding sites in psNDPK2………………………...62
6.2.2 Localisation of atNDPK2 within the plant cell……………….…………………………….….65
6.2.2.1 Phylogenetic analysis of plant NDPK proteins…………….……………………………….65
6.2.2.2 Targeting of atNDPK2 into isolated chloroplasts………….……………..………………...66
6.2.2.3 Transient expression of atNDPK2 into protoplasts………. ……………………….………67
6.2.2.4 Distribution of atNDPK isoforms in the cell……………….………………………………..68
6.2.2.5 atNDPK2 is not re-translocated into the cytosol after import into chloroplasts………..70
6.2.2.6 A single mRNA is observed for atNDPK2 in Arabidopsis thaliana……………………...71

7. Discussion…………………………………………………………………………………………...73

8. References…………………………………………………………………………………………..83

9. Acknowledgements…………………………………………………………………………………91

10. Curriculum vitae..………………………………………………………………………………….92

11. Ehrenwoertliche Versicherung..…………………………………………………………………93


................................................................................................................................Abbreviations
Abbreviations
Amino acid aa
Bovine serum albumin BSA
Chlorophyll A Oxygenase CAO
Carboxyl terminus C-terminus
Cysteine Cys
4’,6-diamidino-2-phenylindol DAPI
Dithiothreitol DTT
Ethylenediaminetetraacetic acid EDTA
ferredoxin-NAD(P)+ oxidored