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 mitochondrial membrane ......................................... 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: Media and culture............................................................................ 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) ................................. 39 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....................................... 46 2.3.8. Ammonium sulphate precipitation of proteins................................................. 46 2.3.9. Carbonate extraction........................................................................................ 47 2.3.10. Expression and purification of proteins ........................................................... 47 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-linking..................................................................................... 51 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.......................................... 56 3. Results ..........................................................................................58 3.1. Identification of the N. crassa tim9 and tim10 genes................................................ 58 3.1.1. Identification of the N. crassa tim10 gene ....................................................... 58 3.1.2. Identification of the N. crassa tim9 gene ......................................................... 59 3.1.3. Tim9 is an essential protein in N. crassa ........................................................