Structure and function of the mitochondrial TIM23 preprotein translocase [Elektronische Ressource] / von Dejana Mokranjac

Mokranjac Dejana

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Structure and function of the mitochondrial
TIM23 preprotein translocase








Dissertation
zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München











von
Dejana Mokranjac
aus
Belgrad, Serbien und Montenegro






München
2004




































Dissertation eingereicht am: 12. Februar 2004.

1. Gutachter: Prof. Dr. Reinhold Herrmann
2. Gutachter: PD Dr. habil. Angelika Böttger
Sondergutachter: Prof. Dr. Dr. Walter Neupert

Tag der mündlichen Prüfung: 12. Mai 2004.






















Nana Levi i deda Mokiju
koji bi bili ponosni TABLE OF CONTENTS
1. INTRODUCTION............................................................................................................................................. 4
1.1. SORTING OF PROTEINS WITHIN A EUKARYOTIC CELL.................................................................................... 4
1.2. PROTEIN TRANSLOCATION INTO MITOCHONDRIA – AN OVERVIEW............................................................... 4
1.3. MITOCHONDRIAL TARGETING SIGNALS ......................................................................................................... 6
1.4. TRANSLOCASE OF THE OUTER MEMBRANE..................................................................................................... 8
1.5. SORTING OF β-BARREL OUTER RANE PROTEINS................................................................................. 10
1.6. TIM23 TRANSLOCASE.... 11
1.7. TIM22.... 15
1.8. OXA1 TRANSLOCASE...... 18
1.9. AIM OF THE PRESENT STUDY......................................................................................................................... 19
2. MATERIAL AND METHODS...................................................................................................................... 20
2.1. MOLECULAR BIOLOGY METHODS................................................................................................................. 20
2.1.1. Isolation of plasmid DNA from E. coli .............................................................................................. 20
2.1.2. Amplification of DNA fragments by polymerase chain reaction (PCR) ............................................ 20
2.1.3. Enzymatic modifications of DNA....................................................................................................... 21
2.1.3.1. Restriction digestion of DNA ....................................................................................................................... 21
2.1.3.2. Ligation of DNA fragments with T4 ligase .................................................................................................. 22
2.1.4. Purification and analysis of DNA...................................................................................................... 22
2.1.4.1. Gel-electrophoresis of DNA......................................................................................................................... 22
2.1.4.2. Isolation of DNA from agarose gels ............................................................................................................ 22
2.1.4.3. Determination of DNA concentration.......................................................................................................... 23
2.1.5. Transformation of E. coli with plasmid DNA .................................................................................... 23
2.1.6. Plasmids used and cloning strategies................................................................................................ 24
2.1.6.1. Overview of used plasmids .......................................................................................................................... 24
2.1.6.2. Cloning strategies........................................................................................................................................ 24
2.1.7. Screening of N. crassa cosmid library............................................................................................... 29
2.1.7.1. Preparation of a membrane containing pMOcosX #X cosmid library......................................................... 29
2.1.7.2. Preparation of DIG-labeled probes............................................................................................................. 30
2.1.7.3. Hybridization of Southern blots with probes 30
2.2. METHODS OF N. CRASSA GENETICS............................................................................................................... 31
2.2.1. Overview of used N. crassa strains.................................................................................................... 31
2.2.2. Preparation of spheroplasts .............................................................................................................. 31
2.2.3. Transformation of spheroplasts......................................................................................................... 32
2.2.4. Selection of transformants ................................................................................................................. 33
2.2.5. Generation of homokaryotic strains by microconidiation ................................................................. 33
2.2.6. Cultivation of N. crassa..................................................................................................................... 34
352.2.7. Labeling of N. crassa cells with S-sulphate .................................................................................... 35
2.3. METHODS IN S. CEREVISIAE GENETICS 35
2.3.1. Overview of used S. cerevisiae strains............................................................................................... 35
2.3.2. Transformation of S. cerevisiae with recombinant DNA ................................................................... 38
2.3.3. Cultivation of S. cerevisiae................................................................................................................ 38
2.4. METHODS IN PROTEIN BIOCHEMISTRY......................................................................................................... 39
2.4.1. Protein preparation ........................................................................................................................... 39
2.4.1.1. In vitro synthesis of mitochondrial preproteins ........................................................................................... 39
2.4.1.2. Purification of recombinant proteins with MBP-tag from E. coli................................................................ 40
2.4.1.3. Purification of recombinant His-tag from E. coli .................................................................. 41
2.4.2. Preparation of affinity matrices for antibody purifications............................................................... 42
2.4.2.1. Coupling of peptides to the SulphoLink beads............................................................................................. 42
2.4.2.2. Coupling of recombinant proteins to the CNBr-activated Sepharose.......................................................... 42
2.4.3. Analytical protein methods................................................................................................................ 43
2.4.3.1. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ............................................................................... 43
2.4.3.2. Staining SDS-PA gels with Coomassie Brilliant Blue (CBB)....................................................................... 44
2.4.3.3. Silver staining of SDS-PA gels..................................................................................................................... 44
2.4.3.4. Transfer of proteins onto nitrocellulose membranes and Ponceau S staining............................................. 44
2.4.3.5. Protein precipitation with trichloracetic acid (TCA)................................................................................... 45
2.4.3.6. Determination of protein concentration ...................................................................................................... 45
2.4.3.7. Detection and quantification of radiolabeled proteins ................................................................................ 45
2.5. METHODS IN CELL BIOLOGY......................................................................................................................... 46
2.5.1. Methods in N. crassa cell biology 46
2.5.1.1. “Fast mito preps”........................................................................................................................................ 46 2.5.1.2. Isolation of mitochondria ............................................................................................................................ 46
2.5.1.3. “Big p