Conformational dynamics of the mitochondrial TIM23 preprotein translocase [Elektronische Ressource] / vorgelegt von Koyeli Mapa

Koyeli Mapa

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• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Aus dem Adolf Butenandt-Institut für Physiologische Chemie der
Ludwig-Maximilians-Universität, München
Direktor: Prof. Dr.med. Dr. rer. nat. W. Neupert


Conformational Dynamics of the Mitochondrial
TIM23 Preprotein Translocase


Dissertation
zum Erwerb des Doktorgrades der Medizin
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München

vorgelegt von
Koyeli Mapa
aus
Chinsurah, West Bengal, India

München
2009








Mit Genehmigung der Medizinischen Fakultät
der Universität München



1. Berichterstatter: Prof. Dr. Dr. Walter Neupert
2. Berichters Priv. Doz. Dr. Andreas Bender
1. Mitberichterstatter: Prof. Dr. Josef Müller-Höcker
2. Mitberichterstatter: Priv. Doz. Dr. Kai Hell

Dekan: Prof. Dr. Dr. h.c. M. Reiser, FACR,FRCR
Tag der mündlichen Prüfung: 23.07.2009.






















“The dream is not what you see in sleep, dream is the thing
which does not let you sleep”
th - Dr. A P J Abdul Kalam, XI President of India

Contents
1  Introduction ........................................................................................... 1 
1.1  Intracellular protein trafficking ............................................................................ 1 
1.2  Overview on protein translocation into mitochondria .......................................... 1 
1.3  Translocases of the outer mitochondrial membrane ............................................ 4 
1.3.1  Translocase of the outer membrane (TOM Complex) .................................. 4 
1.3.2  The TOB Complex ........................................................................................ 5 
1.4  Protein translocation machinery in the intermembrane space (IMS) of
mitochondria ................................................................................................................... 6 
1.4.1  The Mia40-Erv1 disulfide relay system ........................................................ 6 
1.5  Translocases of inner mitochondrial membrane .................................................. 7 
1.5.1  The TIM22 Complex .................................................................................... 7 
1.5.2  The OXA1 Complex ..................................................................................... 8 
1.5.3  The TIM23 complex ..................................................................................... 9 
1.5.3.1  The membrane embedded part of the TIM23 complex ....................... 11 
1.5.3.2  Import motor part of the TIM23 complex ........................................... 12 
1.6  Mitochondrial Hsp70: A chaperone with two functions .................................... 14 
1.6.1  Mitochondrial Hsp70 in preprotein translocation .......................... 15 
1.6.2  Protein folding by mtHsp70 and its cochaperones in the mitochondrial
matrix ..................................................................................................................... 18 
1.7  Aims of the present study ................................................................................... 19 
2  Materials and Methods ....................................................................... 20 
2.1  Molecular biology methods ................................................................................ 20 
2.1.1  Isolation of DNA ......................................................................................... 20 
2.1.1.1  Isolation of yeast genomic DNA ......................................................... 20 
2.1.1.2  Isolation of plasmid DNA from Escherichia coli ................................ 20 
2.1.2  Amplification of DNA sequences by Polymerase Chain Reaction (PCR) . 21 
2.1.3  DNA analysis and purification .................................................................... 22 
2.1.3.1  Agarose gel electrophoresis of DNA ................................................... 22 
2.1.3.2  Isolation of DNA from agarose gels .................................................... 23 
2.1.3.3  Measurement of DNA concentration ................................................... 23 
2.1.4  Enzymatic manipulation of DNA ............................................................... 23 
2.1.4.1  Digestion of DNA with restriction endonucleases .............................. 23 
2.1.4.2  Ligation of DNA fragments ................................................................. 23 
2.1.5  Transformation of electrocompetent E. coli cells ....................................... 24 
i
2.1.5.1  Overview of E. coli strains used .......................................................... 24 
2.1.5.2  Preparation of electrocompetent cells .................................................. 24 
2.1.5.3  Transformation of E. coli cells by electroporation .............................. 25 
2.1.6  Overview of yeast strains used ................................................................... 25 
2.1.7  Cloning strategies for generation of yeast strains by homologous
recombination ........................................................................................................... 26 
2.1.7.1  Deletions of PAM17 gene and double deletion of PAM17/TIM21 .... 26 
2.1.7.2  Disruption of TIM44 in the haploid strain YPH499............................ 26 
2.1.8  Overview of different plasmids used .......................................................... 27 
2.1.9  Cloning strategies for plasmids used for the transformation of yeast ......... 28 
2.1.9.1  pRS314[His6Pam17] ........................................................................... 28 
2.1.9.2  pVT-102U[Tim21] .............................................................................. 28 
2.1.9.3  pVT-W[Pam17] ................................................................................... 29 
2.1.10  Cloning strategies for plasmids used for recombinant proteins expressions
29 
2.1.10.1  Cloning of mature Tim44 in bacterial expression vector .................... 29 
2.1.10.2  Cloning of N-terminal domain of Tim44 (Tim44-NTD) in bacterial
expression vector .................................................................................................. 30 
2.1.10.3  Cloning of C-terminal domain of Tim44 (Tim44-CTD) in bacterial
expression vector .................................................................................................. 30 
2.1.10.4  Cloning of Nucleotide binding domain of Ssc1(Ssc1-NBD) in bacterial
expression vector .................................................................................................. 30 
2.1.10.5  Cloning of Peptide binding domain of Ssc1(Ssc1-PBD) in bacterial
expression vector .................................................................................................. 31 
2.1.10.6  Cloning of mature Mdj1 in bacterial expression vector ...................... 31 
2.1.10.7  Point mutations of Tim44, Ssc1 and Mdj1 .......................................... 31 
2.1.10.8  List of Primers used for site directed mutagenesis .............................. 32 
2.1.11  Cloning strategies for plasmids used for the transformation of yeast for
checking the in vivo functionality ............................................................................. 33 
2.1.11.1  Cloning of full length Tim44 and domains of Tim44 in pRS314 ....... 33 
2.1.11.2  Making of point mutants of Tim44 in yeast vector ............................. 34 
ii
2.1.11.3  Making of point mutants of Ssc1 in yeast vector ................................ 34 
2.1.12  Checking the in vivo functionality of mutant Tim44 .................................. 35 
2.1.13  Checking the in vivo fy of mutant Ssc1 ..................................... 35 
2.2  Methods in cell biology ...................................................................................... 35 
2.2.1  E. coli – media and growth ......................................................................... 35 
2.2.1.1  Media for E. coli .................................................................................. 35 
2.2.1.2  Cultivation of E. coli ........................................................................... 36 
2.2.2  S. cerevisiae – media and growth ............................................................... 36 
2.2.2.1  Media for S.cerevisiae .........................