Isolation and characterisation of the intermembrane space components of the mitochondrial TIM22 protein import machinery of Neurospora crassa [Elektronische Ressource] / von Andreja Vasiljev

Vasiljev, Andreja

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Isolation and characterisation of the
intermembrane space components of the
mitochondrial TIM22 protein import machinery of
Neurospora crassa








Dissertation
zur Erlangung des doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München






von Andreja Vasiljev
aus Subotica,
Serbien und Montenegro





München, 2004































Mündliche Prüfung am: 15.11.2004
Sondergutachter: Herr Prof. Dr.Dr. Walter Neupert
1. Gutachter: Herr Prof. Dr. Reinhold Herrmann
2. Gutachter: Herr PD Dr. Enrico Schleiff


































To my grandmothers, Jelena and Marija



Table of Contents

1. Introduction ....................................................................................1
1.1. Mitochondrial protein translocation machineries........................................................ 1
1.1.1. Mitochondrial structure and function................................................................. 1
1.1.2. Protein translocation in mitochondria of N. crassa and S. cerevisiae................ 4
1.2.2.1. Targeting of preproteins to mitochondria.......................................................... 4
1.2.2.2. Translocases of the outer mitochondrial membrane ......................................... 6
1.2.2.3. Translocases of the inner me 8
1.2. Zinc fingers............................................................................................................... 13
1.3. Aims of the present study.......................................................................................... 17
2. Material and methods ...................................................................18
2.1. Molecular biology methods....................................................................................... 18
2.1.1. PCR (polymerase chain reaction)..................................................................... 18
2.1.2. DNA purification and analysis......................................................................... 19
2.1.2.1. Analytical and preparative gel electrophoresis ............................................... 19
2.1.2.2. DNA concentration measurement ................................................................... 19
2.1.3. Cloning of DNA fragments.............................................................................. 20
2.1.3.1. Enzymatic manipulation of DNA: restriction and ligation reactions.............. 20
2.1.3.2. Preparation and transformation of E. coli competent cells ............................. 20
2.1.4. E. coli strains used............................................................................................ 21
2.1.5. Small and large scale isolation of plasmid DNA from E. coli ......................... 21
2.1.6. Plasmids and genomic library clones used....................................................... 22
2.1.7. Cloning strategies.............................................................................................23
2.1.8. S. cerevisiae strains used.................................................................................. 26
2.1.9. Preparation of yeast DNA ................................................................................ 26
2.1.10. N. crassa strains used....................................................................................... 27
2.1.11. Screening of N. crassa cosmid libraries........................................................... 27
2.1.12. Southern blot....................................................................................................29
2.1.13. Screening of clones through in situ colony-blotting ........................................ 29
2.2. Cell biology methods................................................................................................29
2.2.1. E. coli: Media and culture 29
2.2.2. N. crassa............................................................................ 30
2.2.2.1. Media and solutions for N. crassa................................................................... 30
2.2.2.2. Growth of N. crassa hyphae 32
2.2.2.3. Transformation of N. crassa 34
2.2.3. Isolation of mitochondria from N. crassa hyphae............................................ 35
2.2.4. Crude isolation of mitochondria from N. crassa (“mini” prep) ....................... 36
2.2.5. S. cerevisiae: Culture and Media...................................................................... 37
2.2.5.1. Media for S.cerevisiae..................................................................................... 37
2.2.5.2. S. cerevisiae growth ........................................................................................ 37
2.2.5.3. Transformation of S. cerevisiae (lithium acetate method) .............................. 37
2.2.6. Dilution assay...................................................................................................38
2.2.7. Isolation of mitochondria from S. cerevisiae ................................................... 38
2.2.8. Isolation of crude mitochondria from S. cerevisiae ......................................... 39
2.3. Biochemical methods................................................................................................39
2.3.1. Electrophoretic methods...................................................................................39
2.3.1.1. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) .................................
2.3.1.2. High Tris-urea SDS-Polyacrylamide gel electrophoresis............................... 40
2.3.1.3. Blue-Native gel electrophoresis (BNGE)........................................................ 40
I
2.3.1.4. 2D Blue-Native gel electrophoresis (BNGE).................................................. 41
2.3.1.5. Coomassie blue staining of SDS gels.............................................................. 42
2.3.1.6. Silver staining of SDS gels.............................................................................. 42
2.3.1.7. Transfer of proteins to nitrocellulose/PVDF membrane (Western-Blot)........ 43
2.3.2. Protein concentration determination................................................................43
2.3.3. Protein quantification by autoradiography and phosphorimaging ................... 44
2.3.4. Synthesis of radioactively labelled proteins in vitro ........................................ 44
2.3.5. Import of preproteins into isolated mitochondria............................................. 45
2.3.6. Generation of mitoplasts (“swelling”)..............................................................46
2.3.7. Trichloroacetic acid (TCA) precipitation of proteins.......................................
2.3.8. Ammonium sulphate precipitation of proteins................................................. 46
2.3.9. Carbonate extraction........................................................................................47
2.3.10. Expression and purification of proteins ...........................................................
2.3.10.1 Purification of recombinant proteins expressed in E. coli ............................. 47
2.3.10.2 Purification of immunoglobulin G (IgG) ....................................................... 48
2.3.10.3 Purification of Tim9·Tim10 complex from N. crassa mitochondria ............. 48
2.3.11. Gel filtration.....................................................................................................49
2.3.12. Digitonin fractionation.....................................................................................50
2.3.13. Thin layer chromatography (TLC) for determination of detergent traces in
protein preparations.......................................................................................... 50
2.3.14. Chemical cross-linking51
2.3.15. Screening of peptide libraries with the purified Tim9·Tim10 complex........... 51
2.3.16. Pull-down assay................................................................................................52
2.3.17. In-gel digestion of proteins for sequencing...................................................... 53
2.4. Immunological methods............................................................................................54
2.4.1. Generation of specific antibodies against N. crassa Tim9 and Tim10 proteins
in rabbits........................................................................................................... 54
2.4.2. Affinity purification of antibodies against Tim9 and Tim10 proteins ............. 55
2.4.3. Immunodecoration...........................................................................................56
2.4.4. Immunoprecipitation and co-immunoprecipitation..........................................
3. Results ........................................................................