Biochemical analysis of essential components involved in mitochondrial and cytosolic iron-sulfur protein biogenesis in Saccharomyces cerevisiae [Elektronische Ressource] / vorgelegt von Eugen Urzica

Biochemical analysis of essential components
involved in mitochondrial and cytosolic
iron-sulfur protein biogenesis in
Saccharomyces cerevisiae





Dissertation


zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)





dem Fachbereich Biologie
der Philipps-Universität-Marburg


vorgelegt von


Eugen Urzica

aus Botosani, Rumänien




Marburg/Lahn, April 2007

Vom Fachbereich Biologie der Philipps-Universität Marburg
als Dissertation angenommen am 8.06.2007
Erstgutachter: Prof. Dr. Klaus Lingelbach
Zweitgutachter: Prof. Dr. Roland Lill
Tag der mündlichen Prüfung am 14.06.2007



2
Erklärung

Ich versichere, dass ich die vorliegende Dissertation selbständig verfasst, keine anderen als
die angegebenen Hilfsmittel verwendet und sämtliche Stellen, die im Wortlaut oder dem Sinn nach
anderen Werken entnommen sind, mit Quellenangaben kenntlich gemacht habe. Die Versicherung
schließt Zeichnungen und Skizzen mit ein.
Die Dissertation wurde in der jetzigen oder ähnlichen Form noch bei keiner anderen
Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient. Teile dieser
Arbeit sind bereits in Fachzeitschriften veröffentlicht.
Marburg, 18 April 2007
3 Contents
Contents
Contents..................................................................................................................................... 4
Abbreviations............................................................................................................................ 8
Summary ................................................................................................................................... 9
Zusammenfassung.................................................................................................................. 12
1. Introduction .................................................................................................................... 16
1.1. Chemistry and toxicity of iron ................................................................................. 16
1.2. Iron uptake in S. cerevisiae and its cellular distribution .......................................... 16
1.3. Structure and cellular localization of Fe/S proteins ................................................. 18
1.4. Function of Fe/S proteins ......................................................................................... 19
1.4.1. Electron transfer ............................................................................................... 19
1.4.2. Catalysis ........................................................................................................... 20
1.4.3. Regulatory role of Fe/S clusters....................................................................... 20
1.4.4. DNA-binding proteins...................................................................................... 22
1.5. Biogenesis of Fe/S proteins in Bacteria ................................................................... 22
1.6. Biogenesis of Fe/S proteins in S. cerevisiae ............................................................ 24
1.6.1. Biogenesis of mitochondrial Fe/S proteins ...................................................... 24
1.6.2. Biogenesis of extra-mitochondrial Fe/S proteins............................................. 27
1.7. Hydrogenases 30
1.7.1. NiFe hydrogenases ........................................................................................... 31
1.7.2. Fe-only hydrogenases....................................................................................... 31
1.8. Aim of the present study .......................................................................................... 34
2. Materials and methods................................................................................................... 36
2.1. Bacteria and Yeast strains ........................................................................................ 36
2.1.1. Escherichia coli................................................................................................ 36
2.1.2. Saccharomyces cerevisiae................................................................................ 36
2.2. Growth conditions.................................................................................................... 37
2.2.1. E. coli: Culture and Media ............................................................................... 37
2.2.2. S. cerevisiae: Culture and Media...................................................................... 37
2.3. Oligonucleotides : 39
2.4. Plasmids ................................................................................................................... 41
2.4.1. Escherichia coli plasmids: ............................................................................... 41
4 Contents
2.4.2. Saccharomyces cerevisiae plasmids: ............................................................... 42
2.5. Constructions of Gal-ISD11 strain........................................................................... 44
2.6. Site-Directed Mutagenesis ....................................................................................... 44
2.6.1. GeneEditor™ in vitro Site-Directed Mutagenesis System............................... 44
2.6.2. QuikChange® Site-Directed Mutagenesis Kit................................................. 45
2.6.3. PCR-mediated Site-Directed Mutagenesis....................................................... 45
2.7. Molecular Biological Methods................................................................................. 49
2.7.1. Isolation of Plasmid-DNA from E. coli ........................................................... 49
2.7.2. Preparation of genomic DNA from S. cerevisiae............................................. 49
2.7.3. Purification and Analysis of DNA ................................................................... 50
2.7.4. DNA Agarose Gel Electrophoresis ................................................................. 50
2.7.5. Extraction of DNA from Agarose Gels............................................................ 50
2.7.6. Polymerase Chain Reaction (PCR) .................................................................. 50
2.7.7. Determination of Protein Concentration .......................................................... 51
2.7.8. Digestion of DNA with Restriction Endonucleases......................................... 51
2.7.9. Ligation of DNA Fragments ............................................................................ 52
2.7.10. Preparation and Transformation of Competent E. coli Cells ........................... 52
2.7.11. Transformation of Yeast Cells with Recombinant DNA ................................. 53
2.8. Cell Biological Methods........................................................................................... 54
2.8.1. Isolation of Mitochondria from S. cerevisiae................................................... 54
2.8.2. Crude isolation of Mitochondrial Fractions from S. cerevisiae ....................... 55
2.8.3. Preparation of Yeast Cell Extract (Method of Rödel)...................................... 55
2.8.4. Enzyme Activities of Mitochondrial Proteins.................................................. 56
2.8.5. Enzyme Activities of Cytosolic Proteins ......................................................... 59
552.8.6. Determination of de novo Fe/S cluster biogenesis by Fe radiolabelling ....... 60
2.8.7. Determination of Mitochondrial Iron Content ................................................. 61
2.9. Biochemical Methods............................................................................................... 62
2.9.1. Determination of Protein Concentration .......................................................... 62
2.9.2. TCA (Trichloroacetic Acid) Protein Precipitation........................................... 62
2.9.3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)................................. 63
2.9.4. Coomassie Blue Staining of Proteins............................................................... 64
2.9.5. Transfer of Proteins to Nitrocellulose-Membrane (Western-Blot) .................. 64
2.9.6. Immunostaining................................................................................................ 64
2.9.7. Quantification of Protein Levels after Immunostaining................................... 65
5 Contents
2.9.8. Coupling of Antibodies to Protein-A Sepharose.............................................. 65
2.9.9. Pull-Down Assay.............................................................................................. 65
2.9.10. GFP-Reporter Assay ........................................................................................ 66
2.9.11. Overexpression and Purification of Recombinant His-tagged Proteins........... 66
2.9.12. Electron Paramagnetic Resonance (EPR) Spectroscopy.................................. 67
2.9.13. Determination of Iron Content of Purified Fe/S Proteins ................................ 68
2.9.14. Determination of Sulfide Content of Purified Fe