Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis:
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| Format: | Abschlussarbeit Mikrofilm Buch |
| Sprache: | English |
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Köln
2015
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| Beschreibung: | 2 Mikrofiches (104 Seiten) Illustrationen 21 cm |
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| adam_text | Titel: Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis
Autor: Bakka, Kavya
Jahr: 2015
Table of Contents
Abstract.......................................................................................................................................4
Table of Contents......................................................................................................................5
1. Introduction............................................................................................................................g
1.1 Mitochondria.........................................................................................................................8
1.2 The mitochondrial Quality Control (QC).........................................................................9
1.3 The mitochondrial protein biogenesis.............................................................................10
1.4 Proteases in the mitochondrial protein biogenesis........................................................10
1.5 AAA proteases in the mitochondria................................................................................12
1.5.1 The i-AAA protease.........................................................................................13
1.5.2 The m-AAA protease.......................................................................................14
1.5.3 Putative interactors of the yeast m-AAA protease......................................17
1.5.4 Putative interactors of the mammalian m-AAA protease..........................18
1.6 The mitochondrial prohibitin complex...........................................................................18
1.7 Phospholipids in the mitochondrial membranes...........................................................19
1.8 MAIP1 (m-AAA protease Interacting Protein 1)............................................................21
1.9 The Mitochondrial Calcium Uniporter (MCU)..............................................................22
1.9.1 The mitochondrial calcium uniporter regulation........................................24
1.9.2 The Essential MCU REgulator (EMRE)........................................................24
Objectives of the thesis..........................................................................................................27
2. Materials and methods.......................................................................................................28
2.1 Tissue culture methods......................................................................................................28
2.1.1 Cell lines and cell culture medium................................................................28
2.1.2 Transfection of cultured cells.........................................................................28
2.2 Yeast growth and biochemical techniques.....................................................................29
2.2.1 Yeast growth conditions.................................................................................29
2.2.2 Yeast genetic techniques.................................................................................29
2.2.3 Preparation of yeast whole-cell fractions.....................................................29
2.2.4 In organelle labeling and cyclohexamide chase of mitochondrial
translated proteins....................................................................................................30
2.2.5 In vivo labeling of translation products.......................................................30
2.3 Molecular Biology techniques..........................................................................................31
2.3.1 Cloning techniques..........................................................................................31
2.3.2 RNA isolation and quantitative RT-PCR analysis......................................31
2.4 Biochemistry techniques...................................................................................................31
2.4.1 Protein extraction from mammalian cells....................................................................31
2.4.2 Immunoblotting methods...............................................................................32
2.4.3 Isolation of mitochondrial fractions..............................................................32
2.4.4 Immunoprecipitation of the MAIP1 interactors..........................................33
2.4.5 Proteomic analysis of the MAIP1 interactome.............................................33
2.4.6 Solubility analysis and sub-cellular localization.........................................34
2.5 Microscopy techniques - Fluorescent microscopy.........................................................34
2.6 Data analysis and statistics...............................................................................................35
3. Results...................................................................................................................................36
3.1 Mammalian m-AAA protease...........................................................................................36
3.1.1 The m-AAA protease interactor Protein: MAIP1........................................36
3.1.2 Generation of HEK293 MAIPl-knockout (MKO) cells...............................36
3.1.3 Characterization of MKO clones....................................................................38
3.1.4 MKO cells show lower PRELID1 steady state levels..................................41
3.1.5 Generation of MKO cell lines stably expressing MAIP1-MYC and MAIP1-
WT...............................................................................................................................41
3.1.6 Phospholipid analysis of MKO cells.............................................................42
3.1.7 MAIP1 interactome..........................................................................................43
3.1.8 Putative interactors of MAIP1........................................................................45
3.1.9 Novel putative interactors of MAIP1............................................................45
3.1.10 Common putative interactors of AFG3L2, SPG7, PHB2 and MAIP1.....47
3.1.11 Functional analysis of MAIP1......................................................................48
3.1.12 Localization of p-EMRE and m-EMRE.......................................................49
3.1.13 Stability of endogenous EMRE....................................................................52
3.1.14 Proteases influencing EMRE biogenesis and stability..............................53
3.1.15 YME1L degrades p-EMRE............................................................................54
3.1.16 MPPa/p and MAIP1 knockdowns have similar EMRE biogenesis defect
.....................................................................................................................................55
3.1.17 EMRE mRNA levels in the knockdown of mitochondrial proteases.....56
3.1.18 m-EMRE is a substrate of the m-AAA protease.........................................58
3.1.19 MCU over expression stabilizes p-EMRE...................................................60
3.1.20 MCU is essential for processing of p-EMRE to m-EMRE.........................62
3.2 Yeast m-AAA protease......................................................................................................64
3.2.1 Identifying new substrates of m-AAA protease in yeast............................64
3.2.2 Precursor and mature Ilv2 show a tendency to aggregate.........................67
3.2.3 Quality control substrates of m-AAA protease have a predisposition to
aggregate....................................................................................................................67
3.2.4 Cardiolipin content in mitochondrial inner membrane (IM) regulates m-
AAA protease activity..............................................................................................69
4. Discussion.............................................................................................................................71
4.1 Yeast m-AAA protease performs quality control of aggregation prone proteins.....71
4.2 Cardiolipin (CL) content in the mitochondrial IM regulates m-AAA protease
activity........................................................................................................................................72
4.3 Inferences from the MAIP1 interactome.........................................................................73
4.4 MAIP1 prefers binding to SPG7 over AFG3L2..............................................................73
4.5 Common interactors of AFG3L2, SPG7, PHB2 and MAIP1.........................................74
4.6 A novel effect of MAIP1 loss on cellular phosphatide acid (PA) levels....................75
4.7 Role of AFG3L2 and MAIP1 in calcium homeostasis...................................................77
4.8 MCU influences EMRE processing..................................................................................80
4.9 Proposed model for p-EMRE biogenesis........................................................................81
REFERENCES..........................................................................................................................83
List of Tables............................................................................................................................93
List of Figures...........................................................................................................................99
Zusammenfassung................................................................................................................101
Abbreviations.........................................................................................................................102
Acknowledgements..............................................................................................................103
Eidesstattliche Erklärung............................................................107
Lebenslauf.......................................Error! Bookmark not defined.
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| spelling | Bakka, Kavya Verfasser (DE-588)1080158359 aut Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis vorgelegt von Kavya Bakka Köln 2015 2 Mikrofiches (104 Seiten) Illustrationen 21 cm txt rdacontent h rdamedia he rdacarrier 24x Dissertation Universität zu Köln 2015 (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=028993485&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Bakka, Kavya Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis |
| subject_GND | (DE-588)4113937-9 |
| title | Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis |
| title_auth | Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis |
| title_exact_search | Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis |
| title_full | Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis vorgelegt von Kavya Bakka |
| title_fullStr | Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis vorgelegt von Kavya Bakka |
| title_full_unstemmed | Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis vorgelegt von Kavya Bakka |
| title_short | Role of the mitochondrial m-AAA protease in quality control and calcium homeostasis |
| title_sort | role of the mitochondrial m aaa protease in quality control and calcium homeostasis |
| topic_facet | Hochschulschrift |
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