Dynamic regulation of function of the mitochondrial TIM23 preprotein translocase [Elektronische Ressource] / von Dušan Popov-Čeleketić

Popov-Celeketic Dusan

Description

??Dynamic Regulation of Function of the Mitochondrial TIM23 Preprotein Translocase Dissertationzur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität MünchenvonDušan Popov- eleketiausBelgrad, Serbien München 2007Ehrenwörtliche VersicherungDiese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet. München, den 8.11.2005 Tag der mündlichen Prüfung: 29. November 20071. Gutachter: Prof. Dr. Jürgen Soll 2. Gutachter: Prof. Dr. Manfred Schliwa Sondergutachter: Prof. Dr. Dr. Walter Neupert e3se e a ekbgm f g b gbETABLE OF CONTENTS1. INTRODUCTION...................................................................................................................................... 11.1. PROTEIN TRAFFIC IN THE CELL............................................................................................................... 11.1.1. Targeting signals of organelle proteins................................................................................................ 11.1.2. Protein translocases ............................................................................................................................. 21.2. BIOGENESIS OF MITOCHONDRIA ............................................................................................................ 31.2.1. Mitochondrial targeting signals .................................................................................................

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Dynamic Regulation of Function of the
Mitochondrial TIM23 Preprotein Translocase
Dissertation
zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
von
Dušan Popov- eleketi
aus
Belgrad, Serbien
München
2007
??Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.
München, den 8.11.2005
Tag der mündlichen Prüfung: 29. November 2007
1. Gutachter: Prof. Dr. Jürgen Soll
2. Gutachter: Prof. Dr. Manfred Schliwa
Sondergutachter: Prof. Dr. Dr. Walter Neupert e e a e
e3s
kbgm f g b gbTABLE OF CONTENTS
1. INTRODUCTION...................................................................................................................................... 1
1.1. PROTEIN TRAFFIC IN THE CELL............................................................................................................... 1
1.1.1. Targeting signals of organelle proteins................................................................................................ 1
1.1.2. Protein translocases ............................................................................................................................. 2
1.2. BIOGENESIS OF MITOCHONDRIA ............................................................................................................ 3
1.2.1. Mitochondrial targeting signals ........................................................................................................... 4
1.2.2. Translocation, sorting, folding and assembly machineries in mitochondria........................................ 6
1.2.3. The TOM complex .............................................................................................................................. 6
1.2.4. Machineries for sorting -barrel proteins in the outer membrane ....................................................... 9
1.2.5. MIA-ERV disulfide relay system ...................................................................................................... 10
1.2.6. The TIM22 complex.......................................................................................................................... 11
1.2.7. Machineries for protein export .......................................................................................................... 12
1.2.8. The TIM23 translocase...................................................................................................................... 13
1.3. THE OBJECTIVE OF THIS WORK............................................................................................................. 17
2. MATERIAL AND METHODS 19
2.1. MOLECULAR BIOLOGY METHODS 19
2.1.1. Isolation of DNA ............................................................................................................................... 19
2.1.1.1. Isolation of yeast genomic DNA .................................................................................................. 19
2.1.1.2. Isolation of plasmid DNA from Escherichia coli ......................................................................... 19
2.1.2. Amplification of DNA sequences by Polymerase Chain Reaction (PCR)......................................... 20
2.1.3. DNA analysis and purification .......................................................................................................... 21
2.1.3.1. Agarose gel electrophoresis of DNA ............................................................................................ 21
2.1.3.2. Isolation of DNA from agarose gels............................................................................................. 21
2.1.3.3. Measurement of DNA concentration........ 21
2.1.4. Enzymatic manipulation of DNA........... 22
2.1.4.1. Digestion of DNA with restriction endonucleases........................................................................22
2.1.4.2. Ligation of DNA fragments.......................................................................................................... 22
2.1.5. Transformation of electrocompetent E. coli cells.............................................................................. 22
2.1.5.1. Overview of E. coli strains used ................................................................................................... 22
2.1.5.2. Preparation of electrocompetent cells........................................................................................... 22
2.1.5.3. Transformation of E. coli cells by electroporation ....................................................................... 23
2.1.6. Bacterial plasmids used and cloning strategies........ 23
2.1.6.1. Overview of constructs used for transcription/translation ............................................................ 23
2.1.6.2. Cloning strategy for Tim21 construct used in transcription/translation........................................ 24
2.1.6.3. Overview of plasmids used for protein expression in bacteria ..................................................... 24
2.1.6.4. Cloning strategies for plasmids used for protein expression in bacteria....................................... 24
2.1.7. Transformation of S. cerevisiae cells (Lithium-acetate method)....................................................... 25
2.1.8. S. cerevisiae strains used and cloning strategies................................................................................ 26
2.1.8.1. Overview of yeast strains used ..................................................................................................... 26
2.1.8.2. Cloning strategies for generation of yeast strains by homologous recombination........................ 27
2.1.8.3. Cloni for plasmids used for the transformation of yeast........................................... 29
2.2. CELL BIOLOGY METHODS ..................................................................................................................... 32
2.2.1. E. Coli – media and growth ............................................................................................................... 32
2.2.1.1. Media for E. coli........................................................................................................................... 32
2.2.1.2. Cultivation of E. coli .................................................................................................................... 32
2.2.2. S.cerevisiae – media and growth ....................................................................................................... 32
2.2.2.1. Media for S.cerevisiae................... 32
2.2.2.2. Cultivation of S.cerevisiae......................................................................................... 33
2.2.2.3. Growth of yeast strains where mitochondria with the TIM23 complex in different translocation
modes are generated .................................................................................................................................... 34
2.2.3. Determination of the growth characteristics of yeast strains ............................................................. 34
2.2.4. Isolation of yeast mitochondria................ 34
E 2.2.4.1. Large scale isolation of yeast mitochondria.................................................................................. 34
2.2.4.2. Isolation of crude yeast mitochondria (“fast mito prep”)..............................................................35
2.2.5. Preparation of mitoplasts ................................................................................................................... 36
2.2.6. Protease treatment and “clipping assay”............................................................................................ 36
2.2.6.1. Protease treatment of mitochondria .............................................................................................. 36
2.2.6.2. Removal of the N-terminus of Tim23 exposed on the mitochondrial surface (“clipping assay”) 36
2.2.7. Carbonate extraction.......................................................................................................................... 37
2.3. PROTEIN BIOCHEMISTRY METHODS..................................................................................................... 37
2.3.1. Protein analysis.................................................................................................................................. 37
2.3.1.1. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)............................................................... 37
2.3.1.2. Blue-Native gel electrophoresis (BNGE) ..................................................................................... 38
2.3.1.3. CBB staining of SDS-PAGE gels................................................................................................. 39
2.3.1.4. Transfer of proteins onto nitrocellulose/PVDF membrane (Western-Blot).................................. 39
2.3.1.5. Protein quantification by autoradiography.................................................................................... 40
2.3.1.6. Determination of protein concentration........................................................................................ 40
2.3.2. Protein preparation ............................................................................................................................ 40
2.3.2.1. Trichloroacetic acid (TCA) precipitation