Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts:
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| Format: | Thesis Electronic eBook |
| Language: | English |
| Published: |
2012
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| Online Access: | Volltext http://d-nb.info/1027066291/34 https://nbn-resolving.org/urn:nbn:de:bvb:19-149042 Inhaltsverzeichnis |
| Physical Description: | 81 S. graph. Darst. |
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| 100 | 1 | |a Studte, Carsten |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts |c Carsten Studte |
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Record in the Search Index
| _version_ | 1804149981014130688 |
|---|---|
| adam_text | IMAGE 1
TABLE OF CONTENTS
EIDESSTATTLICHE ERKLARUNG I
SUMMARY II
ZUSAMMENFASSUNG III
TABLE O F CONTENTS : V
ABBREVIATIONS VIII
1 INTRODUCTION 1
1.1 GENERAL IMPORT PATHWAY INTO CHLOROPLASTS 1
1.2 THE PRAT PROTEIN FAMILY 4
1.3 POTENTIAL FUNCTION O F PRATL 6
1.4 AIM O F THIS WORK 8
2 MATERIALS 9
2.1 CHEMICALS 9
2.2 ENZYMES 9
2.3 ASSAY KITS AND COLUMN MATERIALS 10
2.4 MOLECULAR WEIGHT MARKERS AND DNA STANDARDS 10
2.5 OLIGONUCLEOTIDES 10
2.6 VECTORS, CLONES AND STRAINS 11
2.7 ANTIBODIES 12
2.8 E.COLI MEDIA AND PLATES 12
2.9 PLANT MATERIAL 13
3 METHODS 13
3.1 MOLECULAR BIOLOGICAL METHODS 13
3.1.1 GENERAL MOLECULAR BIOLOGICAL METHODS 13
3.1.2 POLYMERASE CHAIN REACTION (PCR) 13
3.1.3 IN VITRO TRANSCRIPTION AND TRANSLATION 14
3.1.4 GENOMIC DNA ISOLATION FROM ARABIDOPSIS THALIANA 14
3.1.5 RNA EXTRACTION FROM ARABIDOPSIS THALIANA AND QUANTITATIVE RT-PCR
14
3.1.6 SEQUENCING 15
3.2 BIOCHEMICAL METHODS 15
3.2.1 SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) AND STAINING 15
3.2.2 DETERMINATION O F PROTEIN AND CHLOROPHYLL CONCENTRATION 16
3.2.3 PRECIPITATION O F PROTEINS WITH TRICHLOROACETIC ACID (TCA) 16
3.2.4 IMMUNOBLOTTING AND VISUALIZATION 16
HTTP://D-NB.INFO/1028353278
IMAGE 2
3.2.5 PROTEIN EXPRESSION AND PURIFICATION 17
3.2.6 TWO-DIMENSIONAL BLUE NATIVE (BN) ELECTROPHORESIS 18
3.2.7 PROTEOLYSIS ASSAYS WITH THERMOLYSIN, TRYPSIN AND GLUC 19
3.2.8 PEGYLATION ASSAY 20
3.2.9 AMS ASSAY 20
3.2.10 EXTRACTION O F INNER ENVELOPE 21
3.2.11 LIPOSOME PREPARATION 21
3.2.12 FLOTATION ASSAY 22
3.2.13 THIN-LAYER CHOMOTOGRAPHY 22
3.2.14 SWELLING ASSAY 22
3.2.15 ISOELECTRIC FOCUSING (1EF) 23
3.2.16 PHOSPHORYLATION AND DE-PHOSPHORYLATION ASSAY 24
3.2.17 CROSS-LINKING 25
3.2.18 METABOLITE ANALYSES 25
3.2.19 PROTEIN IDENTIFICATION BY MASS SPECTROMETRY (MS) 26
3.3 CELL BIOLOGICAL METHODS 26
3.3.1 ISOLATION O F INTACT CHLOROPLASTS FROM PISUM SATIVUM 26
3.3.2 PREPARATION O F INNER AND OUTER ENVELOPE VESICLES FROM PISUM
SATIVUM 27
3.3.3 ISOLATION O F STROMA FROM PISUM SATIVUM 27
3.3.4 ISOLATION AND FRACTIONATION O F ARAB IDOPS IS THALIANA
CHLOROPLASTS 27
3.3.5 PROTEIN IMPORT EXPERIMENTS 28
3.4 PLANT GROWTH CONDITIONS AND PHENOTYPING 28
3.5 ELECTROPHYSIOLOGICAL MEASUREMENTS 30
3.6 CRYO-ELECTROTOMOGRAPHY 31
4 RESULTS 32
4.1 PURIFICATION O F PRATL 32
4.2 GENERATING AN ANTIBODY AGAINST PSPRATL 33
4.3 LOCALISATION O F PRATL 34
4.4 TOPOLOGY O F PRATL IN THE INNER ENVELOPE 35
4.4.1 PRATL IS PROTEASE RESISTANT 37
4.4.2 PEGYLATION O F PRATL IDENTIFIES CYSTEINS IN TRANSMEMBRANE HELICES
38
4.4.3 AMS TREATMENT FURTHER SUPPORTS LOCATION O F CYSTEINS IN
TRANSMEMBRANE HELICES .... 39
4.4.4 PRATL CAN BE PHOSPHORYLATED AT THE FIRST SERINE IN THE N-TERMINUS
41
4.5 PRATL FORMS SMALL COMPLEXES 42
VI
IMAGE 3
4.6 FLOATATION O F PRATL PROTEOLIPOSOMES DEMONSTRATES STABLE INSERTION
44
4.7 ELECTROPHYSIOLOGICAL MEASUREMENTS 46
4.8 APRATL.L PRATL.2 REPRESENTS A TRUE LOSS O F FUNCTION LINE IN
ARABIDOSIS THALIANA 48
4.9 PHENOTYPING O F THE ATPRATL DOUBLE MUTANT 50
4.9.1 ROOT GROWTH IN THE PRATL DOUBLE MUTANT IS SLIGHTLY INCREASED 52
4.10 RNA EXPRESSION IS REGULATED WITH THE CIRCADIAN CLOCK 54
4.11 PI DM IS AFFECTED IN PHOTORESPIRATION, THE TCA CYCLE AND THE
OXIDATIVE PENTOSE PHOSPHATE CYCLE (METABOLIC INFLUENCES) 57
5 DISCUSSION 60
5.1 THE MEMBRANE-SPANNING PROTEIN PRATL IN THE IE O F CHLOROPLASTS 60
5.2 POSSIBLE ROLE O F PRATL-PHOSPHORYLATION 63
5.3 RNA EXPRESSION O F PRATL FOLLOWS A CIRCADIAN RHYTHM 64
5.4 PRATL INFLUENCES CERTAIN METABOLIC PATHWAYS 67
6 REFERENCES 70
8 ACKNOWLEDGEMENTS 80
9 CURRICULUM VITAE 81
|
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| author_facet | Studte, Carsten |
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| discipline | Biologie |
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| language | English |
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| physical | 81 S. graph. Darst. |
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| spelling | Studte, Carsten Verfasser aut Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts Carsten Studte 2012 81 S. graph. Darst. txt rdacontent c rdamedia cr rdacarrier München, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content http://edoc.ub.uni-muenchen.de/14904/ Verlag kostenfrei Volltext http://d-nb.info/1027066291/34 Langzeitarchivierung Nationalbibliothek https://nbn-resolving.org/urn:nbn:de:bvb:19-149042 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025666418&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Studte, Carsten Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts |
| subject_GND | (DE-588)4113937-9 |
| title | Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts |
| title_auth | Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts |
| title_exact_search | Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts |
| title_full | Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts Carsten Studte |
| title_fullStr | Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts Carsten Studte |
| title_full_unstemmed | Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts Carsten Studte |
| title_short | Characterization of the membrane protein Prat1 in the inner envelope of chloroplasts |
| title_sort | characterization of the membrane protein prat1 in the inner envelope of chloroplasts |
| topic_facet | Hochschulschrift |
| url | http://edoc.ub.uni-muenchen.de/14904/ http://d-nb.info/1027066291/34 https://nbn-resolving.org/urn:nbn:de:bvb:19-149042 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025666418&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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