Role of the AAA protease Yme1 in folding of proteins in the mitochondrial intermembrane space Bernadette Schreiner München 2012 Role of the AAA protease Yme1 in folding of proteins in the mitochondrial intermembrane space Dissertation zur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Bernadette Schreiner aus Limburgerhof München 2012 Eidesstattliche Erklärung Ich versichere hiermit an Eides statt, dass die vorgelegte Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt ist. München, den 31. 01. 2013 Bernadette Schreiner Dissertation eingereicht am: 11. 12. 2012 Tag der mündlichen Prüfung: 29. 01. 2013 Erstgutachter: Prof. Dr. Jürgen Soll Zweitgutachter: Prof. Dr. Ute Vothknecht Sondergutachter: Prof. Dr. Dr. Walter Neupert Für meine Familie INTRODUCTION TABLE OF CONTENTS 1. INTRODUCTION ................................................................................................7 1.1 Cellular protein quality control systems ...............................................................7 1.1.1 Proteins - the worker molecules of the cell ........................................................7 1.1.2 Protein quality control and homeostasis (proteostasis) ......................................8 1.1.3 Failure of protein quality control and homeostasis ............................................9 1.2 Molecular chaperones .............................................................................................9 1.2.1 The Hsp70 system ............................................................................................10 1.2.2 The chaperonins ...............................................................................................10 1.2.3 The Hsp90 system ............................................................................................11 1.2.4 The Hsp100 system ..........................................................................................13 1.3 Proteolytic systems ................................................................................................13 1.3.1 The AAA protein family ..................................................................................13 1.3.1 The AAA protease family ................................................................................14 1.3.2 LON proteases..................................................................................................15 1.3.3 ClpP proteases ..................................................................................................16 1.3.4 FtsH proteases ..................................................................................................17 1.4 Mitochondrial biogenesis ......................................................................................18 1.4.1 Mitochondrial subcompartmentalization..........................................................18 1.4.2 Mitochondrial protein import ...........................................................................18 1.5 Mitochondrial protein quality control.................................................................22 1.5.1 Protein quality control in the mitochondrial outer membrane .........................23 1.5.2 Protein quality control in the mitochondrial matrix .........................................23 1.5.3 Protein quality control in the mitochondrial inner membrane .........................25 1.5.4 Protein quality control in the mitochondrial intermembrane space .................25 1.6 Mitochondrial m-and i-AAA protease .................................................................26 1.7 Aim of the present study .......................................................................................30 2. MATERIALS AND METHODS.......................................................................31 2.1 Molecular biology methods...................................................................................31 2.1.1 Strategies for isolation of DNA........................................................................31 2.1.2 Enzymatic editing of DNA...............................................................................33 2.1.3 DNA purification and analysis .........................................................................33 2.1.4 E. coli strains ....................................................................................................34 2.1.5 Plasmids and cloning strategies........................................................................35 2.2 Yeast genetic methods ...........................................................................................38 2.3 Protein biochemistry methods..............................................................................44 2.3.1 Analytical methods...........................................................................................44 2.3.2 Preparation of proteins .....................................................................................46 2.4 Cell biology methods .............................................................................................49 2.4.1 NaOH cell disruption .......................................................................................49 2.4.2 “Rödel’s” cell disruption ..................................................................................49 2.4.3 “Fast Mitoprep”................................................................................................49 2.4.4 “Big Mitoprep”.................................................................................................50 2.4.5 Generation of mitoplasts ..................................................................................51 2.4.6 Digitonin fractionation of mitochondria...........................................................51 2.4.7 Protease treatment ............................................................................................52 2.4.8 Aggregation assay ............................................................................................52 2.4.9 Ni-NTA agarose pulldown ...............................................................................52 4 INTRODUCTION 2.5 Immunological methods........................................................................................53 2.5.1 Overview of antibodies prepared during this thesis .........................................53 2.5.2 Further antibodies used in this study................................................................53 2.5.3 Generation of specific antisera in rabbits .........................................................54 2.5.4 Detection of proteins on nitrocellulose membranes by immuno-staining........55 2.6 Special Methods .....................................................................................................55 2.6.1 Mass spectrometry of elution fractions of Ni-NTA agarose pulldown ............55 2.6.2 Identification of aggregating proteins by SILAC and mass spectrometry .......56 2.7 Chemicals, consumables and equipment.............................................................57 2.7.1 Chemicals .........................................................................................................57 2.7.2 Consumables ....................................................................................................60 2.7.3 Equipment ........................................................................................................60 3. RESULTS............................................................................................................62 3.1 Generation and characterization of model substrates for .................................62 investigating folding in the mitochondrial intermembrane space...........................62 3.1.1 Generation of an IMS-targeted Cytochrome b2-DHFR model substrate..........62 3.1.2 Optimization of induction of model substrate expression................................64 3.1.3 Expression of model substrate in S. cerevisiae ................................................65 3.1.4 Verification of the steady state levels of endogenous proteins in cells............66 expressing the model substrates ......................................................................66 3.1.5 Subcellular localization of model substrates expressed in vivo .......................67 3.1.6 Submitochondrial localization of the model substrates....................................68 3.2 Investigation of the folding behavior of the model substrates...........................70 3.2.1 In vivo protease resistance in the absence and presence of methotrexate ........70 3.2.2 Requirements for folding of DHFR in the IMS and matrix .............................71 3.3 Identification of potential folding helpers of DHFR in the IMS .......................73 3.3.1 Ni-NTA pulldown and label-free quantification by mass spectrometry ..........73 3.3.2 Confirmation of Ni-NTA pulldown by western blot and immuno-staining.....74 3.4 Generation and characterization of Yme1 deletion strain.................................75 3.4.1 Growth phenotype of Δyme1 strain ..................................................................75 3.4.2 Mitochondrial DNA in Δyme1 strain................................................................76 3.5 Behavior of the model substrate in the absence of Yme1 ..................................76 3.5.1 Expression of model substrates in Δyme1 strain ..............................................76 3.5.2 Effect of Yme1 on the folding of DHFR..........................................................77