Characterisation of mechanisms and components of protein phosphorylation in photosynthetic membranes of Synechocystis sp. PCC 6803 [Elektronische Ressource] / vorgelegt von Irina Piven

Irina Piven

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Characterisation of mechanisms and components of
protein phosphorylation in photosynthetic membranes of
Synechocystis sp. PCC 6803




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






vorgelegt von
Irina Piven
aus Moskau, Russland


2006























1. Gutachter: PD. Dr. Anna Sokolenko
2. Gutachter: Prof. Dr. Reinhold G. Herrmann




Tag der mündlichen Prüfung: 15.12.2006 Content I
CONTENT

Abbreviations………………………………………………………………………….… VI

1. Introduction……………………………………………………………………... 1
1.1 Phosphorylation in higher plants……………………………………………..….. 1
1.2 Protein phosphorylation in various photosynthetic organisms…………..…….. 4
1.3 Energy distribution between photosystems I and II through
state transitions mechanisms……………………………………………….……... 6
1.3.1. State transitions in chloroplasts of higher plants……….……………………. 6
1.3.2 State transitions in cyanobacterial cells……..………………………………... 7
1.4 Phosphorylation in cyanobacterial cells…………………………………………. 8
1.5 Characterisation of protein kinases and phosphatases……………………..…. 10
1.6 Structure, degradation and role of PBSs in cyanobacteria …………………... 12
1.7 Possible roles of peptidyl-prolyl cis/trans isomerases………………………….. 16

2. Materials and Methods…………………………………………..………….. 19
2.1 Materials………………………………………………………………………….. 19

2.1.1 Chemicals and enzymes…………………………………………….............. 19
2.1.2 Molecular weight markers…………………………………………………… 19
2.1.3 Bacterial strains……………………………………………………………... 19
2.1.4 Vectors……………………………………………………………………..... 20
2.1.5 Oligonucleotides……………………………………………………………... 20
2.1.6 Media for bacterial growth………………………………………………….. 21
2.1.7 General buffers and solutions………………………………………………... 22
2.1.8 Antibodies………………………………………………………………….... 23
2.1.9 Transfer membranes……………………………………………………….... 23

2.2 Methods…………………………………………………………………………... 24
2.2.1 Standard methods………………………………………………………….... 24
2.2.2 Sequence analysis………………………………………………………..….. 24
2.2.3 Growth conditions for Synechocystis…………………………………….…. 24
2.2.4 Construction of Synechocystis mutants…………………………………..…. 25
2.2.4.1 Transformation of Synechocystis………………………………….. 25 Content II
2.2.4.2 Conjugal transfer of plasmid into cyanobacterial cells…………… 26
2.2.5 DNA analysis…………………………………………………………….…. 26
2.2.5.1 DNA isolation from Synechocystis………………………………... 26
2.2.5.2 Isolation of plasmid DNA from E. coli by
,,boiling” lysate method……………………………………..…….. 27
2.2.6 Site-directed mutagenesis in vitro…………………………………….…….. 27
2.2.7 Protein overexpression and generation of antisera……………………….…. 28
2.2.7.1 Protein overexpression in E. coli cell lysates…………………..…. 28
2.2.7.2 Preparation of the probes for rabbit immunization………………... 28
2.2.7.3 Injection of rabbits and antiserum preparation………………….… 28
2.2.8 Pigment analysis of Synechocystis cells…………………………………….. 29
2.2.8.1 Determination of chlorophyll a concentrations………………….... 29
2.2.8.2 Determination of carotenoid concentrations………………………. 29
2.2.8.3 Determination of phycocyanin concentrations……………………. 29
2.2.9 Protein analysis…………………………………………………………….... 30
2.2.9.1 Protein gel electrophoresis……………………………………….... 30
2.2.9.1.1 SDS-denaturing PAA gels……………………………..... 30
2.2.9.1.2 Urea-containing SDS-denaturing PAA gel................….... 30
2.2.9.1.3 Laemmli gel system (Laemmli, 1970)…………………... 30
2.2.9.2 Staining of PAA gels
2.2.9.2.1 Coomassie Brilliant Blue staining of PAA gels…………. 31
2.2.9.2.2 Imidazol staining………………………………………… 31
2.2.9.2.3 Silver staining of PAA gels……………………………... 32
2.2.9.2.4 Luminescent staining……………………………………. 32
2.2.9.3 Analysis of proteins on the membranes…………………………… 33
2.2.9.3.1 Transfer of SDS-denatured proteins onto
nitrocellulose and PVDF membranes…………………... 33
2.2.9.3.2 Membrane staining with Ponceau S…………………….. 33
2.2.9.4 Immunological detection of proteins……………………………… 33
2.2.9.4.1 Western analysis using horseradish
peroxidase-conjugated antibodies………………………. 33
2.2.9.4.2 Western analysis with phospho-specific antisera……….. 34
2.2.10 Isolation and fractionation of thylakoid membranes………………………. 35
2.2.10.1 Isolation of total cellular and membrane proteins
from Synechocystis……………………………………………..... 35
2.2.10.2 Isolation of lumenal proteins…………………………………..… 36
2.2.10.3 Isolation of peripheral membrane proteins……………………..... 36
2.2.10.4 Preparation of phycobilisomes…………………………………... 36
2.2.10.5 Isolation of photosynthetic complexes from
Synechocystis cells by sucrose gradient………………………….. 37
2.2.11 Phosphorylation of cyanobacterial proteins in vivo……………………...... 37 Content III
2.2.12 Dephosphorylation of proteins by alkaline phosphatase in vitro………….. 38
2.2.13 Chlorophyll fluorescence measurements using a PAM fluorometer…….… 38
2.2.14 Low temperature (77K) fluorescence analysis………………………….…. 38

3. Results……………………………………………………………………………..…. 39

3.1 Protein phosphorylation in Synechocystis sp. PCC 6803
wild-type……………………………………………………………………….…. 39
3.1.1 Detection of protein phosphorylation in Synechocystis thylakoids…………. 39
3.1.2 Phosphorylation of PBS antenna proteins in the wild-type
Synechocystis sp. PCC 6803……………………………………..………….. 41
3.1.3 Phosphorylation of thylakoid proteins in PBS-deficient mutants…………... 43
3.1.4 Dephosphorylation of PBS linker proteins in vitro…………………………. 45
3.1.5 Dephosphorylation of PBS linkers is involved in protease recognition…….. 46
3.1.6 Dephosphorylation occurs only in partially disassembled
phycobilisomes……………………………………………………………... 47
3.1.7 Level of phosphorylation of linker proteins during state transition……….… 48
3.1.8 Dephosphorylation of PBS linker proteins is enhanced under high
light or under nitrogen limiting conditions………………………………….. 50
3.1.9 Characterisation of thylakoid protein phosphorylation in Synechocystis
kinase and phosphatase mutants…………………………….……………… 51
3.1.9.1 Dephosphorylation of linker proteins in the mutants
deficient in serine/threonine phosphatases……………………...… 51
3.1.9.2 Phosphorylation state of linker proteins in the
kinase mutants……………………………………………………... 54

3.2 Functional analysis of the cTLP40 PPIase…………………………..….…..….. 57
3.2.1 Analysis of the cTLP40 protein sequence…………………………………... 57
3.2.2 Localisation of the cTLP40 protein within the cyanobacterial cell………..… 59
3.2.2.1 Overexpression of the cTLP40 protein in E. coli lysates………….. 59
3.2.2.2 Intracellular localisation of the cTLP40 protein………………..…. 60
3.2.2.3 Association of the cTLP40 with thylakoid membranes………….... 62
3.2.2.4 Localisation of the cTLP40 within photosynthetic
membranes……………………………………………………….... 63
3.2.3 Functional analysis of the cTLP40 by interposon mutagenesis
in cyanobacterial cells in vivo………………………………………………. 64
3.2.3.1 Construction of the ∆sll0408 knock-out and complementation
strains…………………………………………………………….... 64 Content IV
3.2.3.2 Physiological characterisation of the ∆ctlp40 mutant under
standard and stress conditions of growth………………………….. 66
3.2.3.2.1 Phenotypical analysis of the ∆ctlp40 mutant……………. 66
3.2.3.2.2 Pigment analysis under standard and high light
conditions……………………………………………..…. 70
3.2.4 Chlorophyll fluorescence analysis of photosynthetic complexes
in the ∆ctlp40 mutant with the PAM device……………………………….. 74
3.2.5 Light-dependent photoinhibition in wild-type and ∆ctlp40 strain…….……. 75
3.2.6 Fluorescence analysis of the ∆ctlp40 mutant strain under
LL and HL conditions by 77K fluorescence spectra……………………….. 77
3.2.7 Analysis of photosynthetic proteins in the ∆ctlp40 mutant
under LL and HL regimes…………………………………………………... 79
3.2.8 Localisation and contents of the cTLP40 under
different stress conditions………………………………………………….... 80
3.2.8.1 Analysis of the steady state levels of the cTLP40 under
various growth conditions……………………………….………… 80
3.2.8.2 Localisation of the cTLP40 in thylakoid membrane under
various light and temperature stress conditions………………........ 81
3.2.9 Protein domain analysis of the cTLP40 in vivo…………………………….. 82
3.2.9.1 Construction of various mutant strains of the sll0408 gene……..... 82
3.2.9.1.1 Construction of mutant strain lacking the putative
phosphatase-binding site of the cTLP40 protein……...… 82
3.2.9.1.2 Construction of N- and C-terminal deletion mutants……. 85
3.2.9.1.3 Point mut