Fingerprint of the mitochondrial ABC transporter Mdl1p from Saccharomyces cerevisiae [Elektronische Ressource] / von Matthias Hofacker

Matthias Hofacker

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2006
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Fingerprint of the mitochondrial ABC transporter Mdl1p
from Saccharomyces cerevisiae





Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften



vorgelegt beim Fachbereich
Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität Frankfurt



von
Matthias Hofacker
aus Langen/Hessen





Frankfurt am Main, 2006
(D30)vom Fachbereich Biochemie, Chemie und Pharmazie der
Johann Wolfgang Goethe-Universität als Dissertation angenommen.



Dekan: Prof. Dr. Harald Schwalbe

1. Gutachter: Prof. Dr. Robert Tampé
2. Gutachter: Prof. Dr. Bernd Ludwig


Datum der Disputation: Index
Index
1 DEUTSCHE ZUSAMMENFASSUNG...................................................................................................... 1
2 SUMMARY.................................................................................................................................................. 6
3 INTRODUCTION ....................................................................................................................................... 7
3.1 FUNCTION AND ORGANISATION OF ABC PROTEINS............................................................................... 7
3.1.1 Structures of nucleotide binding domains...................................................................................... 9
3.1.2 Structures of full-length ABC transporters .................................................................................. 10
3.1.3 Catalytic cycle and proposed transport mechanism .................................................................... 12
3.2 PEPTIDE AND PROTEIN TRANSPORT OVER MEMBRANES....................................................................... 13
3.2.1 Peptide import in prokaryotes driven by ABC transporters......................................................... 15
3.2.2 The haemolysin system................................................................................................................. 15
3.2.3 The a-factor transporter Ste6p..................................................................................................... 16
3.2.4 The transporter associated with antigen processing like (TAPL) ................................................ 16
3.2.5 r associated with antigen processing (TAP) 16
3.3 RELEVANCE OF MITOCHONDRIAL PROCESSES IN CELLULAR BIOLOGY................................................. 17
3.3.1 The proteolytic system in the inner mitochondrial membrane ..................................................... 18
3.4 MITOCHONDRIAL ABC TRANSPORTERS IN S. CEREVISIAE .................................................................. 19
3.4.1 Mdl1p and its physiological function........................................................................................... 20
3.5 AIMS AND MOTIVATION ..................................................................................................................... 23
4 MATERIAL............................................................................................................................................... 24
4.1 CHEMICALS ........................................................................................................................................ 24
4.2 DETERGENTS....... 26
4.3 CELLS AND MEDIA.............................................................................................................................. 27
4.4 ENZYMES, MARKERS AND KITS.......................................................................................................... 27
4.5 VECTORS............. 27
4.6 ANTIBODIES......... 27
4.7 PEPTIDES............. 28
4.8 EQUIPMENT......... 28
4.9 CENTRIFUGES AND ROTORS................................................................................................................ 29
4.10 SUPPLEMENTARY MATERIAL............................................................................................................... 29
4.11 MEDIA, BUFFERS AND SOLUTIONS...................................................................................................... 30
4.11.1 Cell culture media........................................................................................................................ 30
4.11.2 Solutions for E. coli transformation............................................................................................. 30
4.11.3 Sol S. cerevisiae transformation ................................................................................... 30
4.11.4 Solutions for SDS-PAGE.............................................................................................................. 31
4.11.5 Sol Tricine-PAGE ......................................................................................................... 31
4.11.6 Solutions for Blue-native gel electrophoresis .............................................................................. 31
4.11.7 Sol Coomassie staining................................................................................................. 31
4.11.8 Solutions for Silver staining......................................................................................................... 32
4.11.9 Buffers for electroblotting and immunodetection......................................................................... 32
4.11.10 mitochondria isolation............................................................................................... 32
5 METHODS................................................................................................................................................. 33
5.1 MOLECULAR BIOLOGY ....................................................................................................................... 33
5.1.1 Cell culture................................................................................................................................... 33
5.1.2 Competent E. coli cells................................................................................................................. 33
5.1.3 Transformation of competent E. coli cells ................................................................................... 34
5.1.4 maS. cerevisiae cells ........................................................................................... 34
5.1.5 Cryostocks of yeast cells .............................................................................................................. 34
5.2 BIOCHEMICAL METHODS.................................................................................................................... 35
5.2.1 Standard SDS-PAGE.................................................................................................................... 35
5.2.2 Tricine SDS-PAGE....................................................................................................................... 35
5.2.3 Blue Native-PAGE........ 36
5.2.4 Coomassie and silver staining ..................................................................................................... 36
5.2.5 Transfer of proteins to nitrocellulose membranes ....................................................................... 37
5.2.6 Purification of the Mdl1p-NBD.. 38
5.2.7 Dimerisation Assay ...................................................................................................................... 40
5.2.8 Determination of the Nucleotide Stoichiometry ........................................................................... 40
I Index
5.2.9 Thin layer chromatography (TLC)............................................................................................... 41
5.2.10 Nucleotide binding assays............................................................................................................ 41
5.2.11 Isolation of yeast mitochondria.................................................................................................... 42
5.2.12 Tandem Affinity Purification........................................................................................................ 43
5.2.13 In organello translation ............................................................................................................... 44
5.2.14 Monitoring peptide export from mitochondria............................................................................. 44
5.2.15 Detergent screening ..................................................................................................................... 45
5.2.16 Purification of full-length Mdl1p ................................................................................................. 45
5.2.17 ATP hydrolysis assays.................................................................................................................. 46
5.2.18 Reconstitution of Mdl1p ............................................................................................................... 46
5.2.19 Phospholipid concentration determination.................................................................................. 47
5.2.20 Freeze-fracture electron microscopy of reconstituted Mdl1p ...................................................... 47
5.2.21 Peptide photocross-linking and detection....................................................................................