Analysis of the role of Mdm38 in respiratory chain biogenesis

Abstract

I herewith declare that this thesis has been written independently and with no other sources and aids than explicitly quoted.Christine Vollmer 11 1.2.2 Mitochondrial ribosomes 13 1.2.3 Mitochondrial protein export machinery 15 1.2.4 Mitochondrial translation 18 1.2.4.1 Regulation of mitochondrial translation 21 1.2.5 Mdm38 24 1.3 Aims of the work 27 2. MATERIAL & METHODS 28 2.1 Material 28 2.1.1 Chemicals, reagents and enzymes 28 2.1.2 Disposables 31 2.1.3 Kits 32 2.1.4 Laboratory equipment 32 2.1.5 Vectors 34 2.1.6 Antibodies 34 2.1.7 Microorganisms 36 2.1.7.1 E. coli strains 36 2.1.7.2 S. cerevisiae strains 36 2.2 Media & growth conditions 37 2.2.1 Media and growth conditions for E. coli 37 2.2.1.1 Media for E. coli 37 2.2.1.2 Cultivation of E. coli 40 2.2.1.3 Growth phase analysis of cultures 41 2.2.1.4 Preparation of permanent (glycerol) cryo--stocks 41 2.2.2 Media and growth conditions for S. cerevisiae 41 2.2.2.1 Media for S. cerevisiae 41 2.2.2.2 Cultivation of S. cerevisiae 42 2.2.2.3 Growth test of S. cerevisiae on agar plates 42 2.2.2.4 Preparation of cryo--stocks 43 2.2.2.5 Isolation of mitochondria from S. cerevisiae 43 2.3 Methods in molecular biology 44 2.3.1 Isolation of DNA 44 2.3.1.1 Isolation of yeast genomic DNA 44 2.3.1.2 Isolation of plasmid DNA from E. coli 45 2.3.1.3 Measurement of DNA concentration 45 2.3.2 Cloning of DNA fragments 45 2.3.2.1 DNA amplification by Polymerase Chain Reaction (PCR) 4.1. Function of Mdm38-Mba1 interaction within the mitochondrial translation machinery 105 4.2 Mdm38: The first putative mitochondrial 14-3-3-like protein in S. cerevisiae 110 4.3 Implications between yeast and human proteins Mdm38 and LETM1 116 4.4 Functional model of Mdm38 119 5. SUMMARY 122 REFERENCES 123 ABBREVATIONS 142 CURRICULUM VITAE 145 LIST OF TABLES 1.1 Overview of the five best--studied mRNA translation activator interactions in the yeast S. cerevisiae 19 2.1 Most often used primary antibodies 35 2.2 Auto--induction medium 38 2.3 Reaction setups for FideliTaq and KOD polymerases 46 2.4 PCR--program for FideliTaq and KOD polymerases 46 2.5 PCR--program for sequencing of DNA 49 2.6 Cloning of Mdm38 and LETM1 constructs 50 2.7 Primer sequences used for generation of truncation constructs 50 2.8 Components for 1.5 x translation buffer 63 3.1Data collection and refinement statistics of the Mdm38 CTD crystal 84 Mitochondrial protein translocasesAs mitochondria are involved in numerous cellular processes (see 1.1.2), a huge number of proteins are required to fulfill all their tasks. Recent studies revealed that the