Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | German |
| Published: |
2003
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 128 Bl. Ill., graph. Darst. 21 cm |
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MARC
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Record in the Search Index
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| adam_text | Table of Contents 4.
1 Introduction 8
1.1 Signal transaction 8
1.2 Phosphorylation 8
1.3 Phosphatidylinositol (Ptdlns) phosphates in signal transduction 9
1.4 Ptdlns-phosphatases and disease 10
1.4.1 SAC 1 -domain containing family of proteins 13
1.4.2 SAC1 cellular functions 15
1.5 Vesicular trafficking 16
1.5.1 Clathrin coated vesicles (CCV) 17
1.5.2 COPII vesicular coats 17
1.5.3 COPI vesicular coats 18
1.6 The role of phosphoinositides and Ptdlns phosphatases in membrane transport 20
1.7 Rationale 21
2 Materials 22
2.1 Security measures 22
2.2 Chemical reagents, enzymes and kits 22
2.2.1 Primer and oligonucleotides. 24
2.2.2 Plasmids and constructs 25
2.2.3 Usedpeptides 28
2.2.4 Primary antibodies: 28
2.3 Commonly used buffers and solutions: 29
3 Methods 30
3.1 Molecular biology methods 30
3.1.1 Concentration determination of nucleic acids 30
3.1.2 Precipitation of nucleic acids 30
3.1.3 Agarose gel electrophoresis 30
3.1.4 Recovery of DNA from agarose gels 31
3.1.5 DNA enzymatic digestion (restriction enzymes, phosphatase treatment) 31
3.1.6 DNA ligation using RapidLigation Kit 31
3.1.7 Ligation of DNA into TA vector (TOPO-cloning) 32
3.1.8 Transformation of competent cells and bacterial culture 32
3.1.9 Cultivation of bacteria 33
3.1.9.1 Storage of bacteria (glycerol stocks) 33
3.1.10 Plasmid preparation 33
3.1.11 Reverse transcriptase-PCR (RT-PCR) of hSACl open reading frame 34
Table of Contents 5
3.1.12 Site directed mutagenesis 34
3.2 Bioinformatics 34
3.3 Methods of tissue culture 35
3.3.1 Cultivation and passaging of mammalian cells 35
3.3.2 Counting of cells 35
3.3.3 Freezing, long term storage and thawing of cells 36
3.3.4 Transfection of mammalian cells using Fugene6 36
3.3.5 Generation of cell strains stably expressing ectopical proteins 36
3.3.6 FACS analysis for expression of fluorescent proteins 37
3.3.7 FACS analysis for induction of apoptosis 37
3.3.8 Determination of cumulative cell numbers 37
3.3.9 Focus forming assay 38
3.4 Expression and analysis of cellular proteins 38
3.4.1 Preparation of cell lysates 38
3.4.2 Concentration determination of proteins 38
3.4.3 SDS gel electrophoresis (SDS-PAGE) 39
3.4.4 Staining of SDS -polyacrylamide gels 39
3.4.4.1 Coomassie-Stain 39
3.4.4.2 Silver Stain 39
3.4.5 Immunoblot 39
3.4.6 Generation of hSACl-specific antibodies 40
3.4.7 Immunoprecipitation _ 40
3.4.8 Peptide competition experiments 40
3.4.9 Peptide precipitation experiments 41
3.4.10 hSAC 1 binding experiments 41
3.4.11 Immunofluorescent staining 41
3.4.12 DAPI nuclear staining and embedding of glass slides 41
3.5 Expression, purification and analysis of recombinant proteins in S. pombe 42
3.5.1 Transformation of S. pombe L40 strain with pESP expression plasmids 42
3.5.2 Purification of GST-hSACl-variant proteins 43
3.5.3 Lipid binding assay 44
3.5.4 Determination of phosphatase activity of GST- hSACl variants 44
3.5.4.1 Malachite green assay 44
3.5.4.2 Protein phosphatase assay 45
4 Results 46
4.1 Cloning of human SAC1 46
4.2 Expression profile of hSACl 47
4.2.1 hSACl shows ubiquitous gene expression in human tissues on mRNA level 48
Table of Contents 6.
4.2.2 hSACl expression varies on protein level 51
4.3 Analyzing cellular functions of hSACl 54
4.3.1 Ectopicoverexpression of hSACl variants in mammalian cells 55
4.3.2 EffectofhSACl overexpression on cellular proliferation 57
4.3.3 Overexpression of GFP-hSAClwt and GFP-hSACl-C/S in U373MG does not induce
apoptosis 58
4.3.4 Both, hSAClwt and hSACl-CS expression increase colony numbers in U373MG cells _61
4.4 hSACl wildtype complements inositol auxotrophy of ASAC1 yeast cells 63
4.5 Expression and purification of recombinant GST-hSACl variants in S. pombe 64
4.6 The hSACl-C/S mutant protein displays reduced lipid phosphatase activity 65
4.7 Protein phosphatase activity of hSACl variants 66
4.8 Wildtype hSACl forms oligomers 67
4.8.1 Wildtype hSACl forms oligomers with recombinant GST-hSACl in vitro 68
4.8.2 Wildtype hSACl forms oligomers in vivo, whereas hSACl-C/S is defective in
oligomerization 70
4.9 hSAClwt and hSACl-C/S display equivalent phosphoinositide-binding properties 71
4.10 Subcellular localization of hSACl 72
4.10.1 GFP-hSAC 1 wt localizes to the ER/Golgi, but GFP-hSAC 1 -C/S accumulates in the Golgi 73
4.11 hSACl interacts with coatomer complex I (COPI) 76
4.11.1 Co-immunoprecipitates of GFP-hSAClwt 76
4.11.2 Identification via MALDI-TOF-MS 76
4.11.3 hSAC 1 interacts with COPI in vitro and in vivo 77
4.12 The C-terminal hSACl sequence (KEKID) acts as COPI recognition signal 81
4.12.1 GST-hSACl quenches hSACl-COPI interaction 81
4.12.2 A putative COPI-interaction motif, KEKID, is essential for the hSACl/COPI interaction 82
4.12.3 The KEKID-mutant accumulates in the Golgi 84
4.13 The leucine zipper domain is responsible for hSACl oligomerization 85
4.13.1 hSAC 1 mutants display defects in oligomerization 85
4.13.2 The leucine zipper variant GFP-hSACl-LZ displays an ER-restricted localization
phenotype 88
4.13.3 Regulation of hSACl oligomerization 90
4.13.3.1 hSACl oligomerization is decreased by phosphoinositides 90
4.13.3.2 hSAClwt binds only to phosphorylated hSACl peptides encompassing the leucine zipper
region 93
Table of Contents 7
5 Discussion 95
5.1 Characterization of human SAC1 96
5.1.1 hSAC 1 is a phosphoinositide phosphatase 96
5.1.2 hSACl displays protein phosphatase activity 97
5.1.3 Cellular effects of hSACl overexpression 99
5.2 hSACl interacts with the coatomer I (COPI) complex 100
5.2.1 Coatomer binding might regulate ER-localization of hSAC 1 102
5.3 Subcellular localization of hSACl and hSACl mutants 103
5.4 hSAClwt oligomerizes 105
5.5 Phosphoinositides inhibit hSACl oligomerization 107
5.6 Model of dynamic hSACl ER/Golgi distribution 109
6 References 112
7 Summary 122
8 Appendix 124
8.1 Acronyms and Abbreviations 124
8.2 Lebenslauf 125
8.3 Akademische Lehrer 126
8.4 Ehrenwortliche Erklarung 127
8.5 Danksagung 128
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| any_adam_object | 1 |
| author | Rohde, Holger Maria 1974- |
| author_GND | (DE-588)129236497 |
| author_facet | Rohde, Holger Maria 1974- |
| author_role | aut |
| author_sort | Rohde, Holger Maria 1974- |
| author_variant | h m r hm hmr |
| building | Verbundindex |
| bvnumber | BV019603135 |
| ctrlnum | (OCoLC)249639012 (DE-599)BVBBV019603135 |
| discipline | Medizin |
| format | Thesis Book |
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| id | DE-604.BV019603135 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T20:01:06Z |
| institution | BVB |
| language | German |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-012933018 |
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| physical | 128 Bl. Ill., graph. Darst. 21 cm |
| publishDate | 2003 |
| publishDateSearch | 2003 |
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| spelling | Rohde, Holger Maria 1974- Verfasser (DE-588)129236497 aut Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 vorgelegt von Holger Maria Rohde 2003 128 Bl. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Marburg, Univ., Diss., 2003 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012933018&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rohde, Holger Maria 1974- Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 |
| subject_GND | (DE-588)4113937-9 |
| title | Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 |
| title_auth | Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 |
| title_exact_search | Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 |
| title_full | Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 vorgelegt von Holger Maria Rohde |
| title_fullStr | Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 vorgelegt von Holger Maria Rohde |
| title_full_unstemmed | Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 vorgelegt von Holger Maria Rohde |
| title_short | Molekulare Charakterisierung der humanen Phosphatidylinositol-Phosphatase SAC1 |
| title_sort | molekulare charakterisierung der humanen phosphatidylinositol phosphatase sac1 |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012933018&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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