Characterization of the Alb3-Oxa1-YidC protein family in Arabidopsis thaliana [Elektronische Ressource] / vorgelegt von Monique Karl

Monique Karl

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Characterization of the
Alb3/Oxa1/YidC protein family
in Arabidopsis thaliana





Dissertation der Fakultät für Biologie
der
Ludwig-Maximilians-Universität München




vorgelegt von

Monique Karl
aus Salzgitter







München
2009













































Erstgutachter: Prof. Dr. J. Soll
Zweitgutachter: PD Dr. C. Bolle

Tag der mündlichen Prüfung: 22.06.2009
LITTERIS ET FLORIBUS




Abbreviations
Abbreviations

2D two dimensional
AA amino acid
Alb Albino
acc. according (to)
At Arabidopsis thaliana
AGI Arabidopsis Genome Initiative
AOX mitochondrial alternative oxidase
AP alkaline phosphatase
ATPase CF CF -ATP synthase (complex) O 1
-ME -mercaptoethanol
BLAST basic local alignment search tool
BN-PAGE blue-native polyacrylamide gel electrophoresis
bp basepair
CC coiled-coil
cDNA copy-DNA
CF membrane-embedded part of CF CF ATP synthase (complex) O O 1
CF soluble part of CF CF ATP synthase (complex)1 O 1
Chl chlorophyll
Col Columbia
Cox cytochrome c oxidase
cp chloroplastidic
C-(terminus) carboxy terminus
cTP chloroplast transit peptide
Cytb f cytochrome b f (complex) 6 6
Cyt c e c
Da Dalton
DoMa n-dodecyl- -D-maltoside
DTT dithiothreitol
ECL enhanced chemiluminescence
E. coli Escherichia coli
ER endoplasmic reticulum
Fd ferredoxin
FNR ferredoxin-NADP(H) oxidoreductase
GFP green fluorescent protein
HMW high molecular weight
IE inner envelope / inner envelope membrane
IMP inner membrane protein
IMS intermembrane space
k, K kilo, times 1000
LHC(P) light-harvesting chlorophyll binding (protein)
LMW low molecular weight
Met methionine
Mt mitochondria(l)
mTP mitochondrial transit peptide
mRNA messenger-RNA
MS mass spectrometry
N-(terminus) amino terminus
OE outer envelope / outer envelope membrane
I Abbreviations
OE33 oxygen evolving complex protein of 33 kDa
OEC plex (of PSII)
Oxa cytochrome c oxidase assembly
PAGE polyacrylamide gel electrophoresis
PCR polymerase chain reaction
PEG polyethylene glycol
Ps Pisum sativum
PSI, PSII photosystem I, II
RACE rapid amplification of cDNA ends
ret. retention signal
RNAiRNA interference
rpm revolutions per minute
RT room temperature
RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase
SD standard deviation
SDS sodium dodecyl sulphate
Sec secretory
SP signal peptide
SRP recognition particle
SSU ribulose-1,5-bisphosphate carboxylase/oxygenase, small subunit
Tat twin-arginine translocon
TEM transmission electron microscopy
Tic translocon at the inner envelope of chloroplasts
TILLING targeting induced local lesions in genomes
TM transmembrane (domain)
Toc translocon at the outer envelope of chloroplasts
TP transit peptide
TPR tetratricopeptide repeat
TX-100 Triton X-100
UTR untranslated region
v/v volume per volume
WT wild-type
w/v weight per volume
x g times the force of gravity




II Table of contents
Table of contents
Abbreviations ............................................................................................................................. I
Table of contents .....................................................................................................................III
1 Summary ................................................................................................................................ 1
2 Zusammenfassung .... 2
3 Introduction ........................................................................................................................... 3
3.1 Chloroplasts, the power house ......................................................................................... 3
3.2 Molecular organization of the thylakoid membrane ........................................................ 3
3.3 Protein translocation into and across the thylakoid membrane ...................................... 5
3.4 The Alb3/Oxa1/YidC protein family ................................................................................. 7
3.4.1 Oxa1, the mitochondrial homologue .................................................................................................... 8
3.4.2 YidC, the bacterial homologue ........................................................................................................... 10
3.4.3 Slr1471 in Synechocystis .................................................................................................................... 11
3.4.4 Alb3, the homologue in chloroplasts .................................................................................................. 11
3.4.5 Alb3.1 and Alb3.2 in Chlamydomonas ............................................................................................... 13
3.4.6 Further homologues in Arabidopsis thaliana ..................................................................................... 13
4 Materials .............................................................................................................................. 15
4.1 Chemicals ....................................................................................................................... 15
4.2 Detergents ................................................................................................................
4.3 Enzymes ....... 15
4.4 Kits ................................................................................................................................. 15
4.5 Molecular weight markers and DNA standards ............................................................. 15
4.6 Antibodies ....... 16
4.7 Strains, vectors, clones and oligonucleotides ................................................................ 16
4.8 Plant material ................................................................................................................. 16
4.9 Clones used in this work ................................................................................................. 17
5 Methods ................................................................................................................................ 18
5.1 Growth conditions .......................................................................................................... 18
5.1.1 Growth of Arabidopsis thaliana ......................................................................................................... 18
5.1.2 Growth of Escherichia coli ................................................................................................................. 18
5.2 Molecular biological methods ........................................................................................ 18
5.2.1 General molecular biological methods ............................................................................................... 18
5.2.2 Homologous recombination................................................................................................................ 18
5.2.3 Isolation of genomic DNA from Arabidopsis thaliana ...................................................................... 19
5.2.4 Characterization of ALB4 TILLING lines .......................................................................................... 19
5.2.5 Polymerase Chain Reaction (PCR) ..................................................................................................... 19
5.2.6 RNA isolation, cDNA preparation and real-time PCR ....................................................................... 20
5.3 Biochemical methods ...................................................................................................... 21
5.3.1 General biochemical methods ............................................................................................................. 21
5.3.2 SDS-polyacrylamide electrophoresis (SDS-PAGE) ........................................................................... 21
5.3.3 Western blotting and immunodecoration with antibodies .................................................................. 21
5.3.4 Two-dimensional blue native gel electrophoresis / SDS-PAGE and immunoblotting ....................... 21
5.3.5 Immunoblot development ................................................................................................................... 22
5.3.6 Extraction of soluble and total membrane proteins from Arabidopsis thaliana ................................. 23
5.3.7 Protein identification by mass spectrometry (MS) ............................................................................. 23
5.4 Cell biological methods .................................................................................................. 24
5.4.1 Isolatio