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Technische Universität München the Small Heat Shock Proteins and The Technische Universität München Fakultät Chemie Lehrstuhl für Biotechnologie The Small Heat Shock Proteins and the Heat Stress Response of Baker’s Yeast Christopher Gordon Stratil Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. Stephan A. Sieber Prüfer der Dissertation: 1. Univ.-Prof. Dr. Johannes Buchner 2. Univ.-Prof. Dr. Sevil Weinkauf Die Dissertation wurde am 29.12.2015 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 15.02.2016 angenommen. III Contents 1. Summary ........................................................................................................................................ 1 2. Introduction .................................................................................................................................... 2 2.1 Investigations on the small heat shock proteins of baker's yeast ............................................. 2 2.1.1 Protein folding ................................................................................................................... 2 2.1.2 Protein folding within a cell .............................................................................................. 4 2.1.3 Protein folding in disease .................................................................................................. 5 2.1.4 Molecular chaperones and protein folding ........................................................................ 5 2.1.5 Cooperation of the chaperone systems .............................................................................. 6 2.1.6 Small heat shock proteins .................................................................................................. 8 2.1.7 Structure of small heat shock proteins .............................................................................. 9 2.1.8 Approaches to study sHsp structure ................................................................................ 11 2.1.9 The small heat shock protein system of Saccharomyces cerevisiae ............................... 12 2.2. Revisiting the heat stress response of baker's yeast .............................................................. 15 2.2.1 A short history on the heat shock response ..................................................................... 15 2.2.2 Influences of heat on the cell ........................................................................................... 16 2.2.3 Transcriptional heat shock response ................................................................................ 18 2.2.4 Crosstalk of stress responses ........................................................................................... 20 2.3. Objective ............................................................................................................................... 21 3. Materials and methods ................................................................................................................. 22 3.1 General equipment and expendable items .............................................................................. 22 3.2 Chemicals ............................................................................................................................... 24 3.3 Software, databases & web-based tools ................................................................................. 25 3.4 Oligonucleotides and plasmids ............................................................................................... 26 3.5 Bacterial and yeast strains ...................................................................................................... 28 3.6 Media and antibiotics ............................................................................................................. 29 3.7 Buffers .................................................................................................................................... 31 3.8 Molecular biology methods .................................................................................................... 32 3.8.1 Cultivation of E. coli and S. cerevisiae ........................................................................... 32 IV 3.8.2 Yeast transformation protocols........................................................................................ 33 3.8.3 Polymerase chain reaction (PCR) .................................................................................... 34 3.8.4 PCR-based gene deletion ................................................................................................. 35 3.8.5 Cloning of yeast shuttle vectors ...................................................................................... 36 3.9 Protein chemical methods ...................................................................................................... 37 3.9.1 Determination of protein concentration ........................................................................... 37 3.9.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .................................................. 38 3.9.3 Immunoblotting ............................................................................................................... 38 3.10 Synthetic genetic array ......................................................................................................... 39 3.11 Methods for transcriptional studies ...................................................................................... 41 3.12 Microscopy ........................................................................................................................... 44 3.12.1 Fluorescence and bright field microscopy ..................................................................... 44 3.12.2 Soft X-ray tomography .................................................................................................. 44 3.13 Metabolic labeling of yeast cells .......................................................................................... 47 4. Results .......................................................................................................................................... 50 4.1 Small heat shock proteins of baker’s yeast............................................................................. 50 4.1.1 SGAs reveals sHsps involved in protein trafficking ....................................................... 50 4.1.2 The sHsps in models of protein aggregation ................................................................... 57 4.2 Heat stress and the inner cellular morphology ....................................................................... 67 4.2.1 X-ray tomography of unstressed S. cerevisiae ................................................................ 67 4.2.2 X-ray tomography of heat stressed yeast ......................................................................... 69 4.2.3 X-ray tomography of gene deletion mutants ................................................................... 71 4.2.4 Generation of 3D-models of baker’s yeast ...................................................................... 73 4.3 Transcriptional studies on wild type S. cerevisiae under heat shock ..................................... 76 4.3.1 The transcriptional response to mild heat stress .............................................................. 77 4.3.2 The transcriptional response to sub-lethal heat stress ..................................................... 81 4.3.3 Origin of transcriptional waves ....................................................................................... 83 4.3.4 The heat shock response and the molecular chaperone system ....................................... 86 V 4.3.5 Transcriptional regulation of selected pathways ............................................................. 93 4.3.6 Transcriptional regulators of the mild heat shock response .......................................... 100 4.4 Transcriptional heat stress response in the genetic background of TF deletions ................. 104 4.4.1 Rpn4 deletion has broad effects on the transcriptome ................................................... 105 4.4.2 Sko1 deletion has minor effects on the transcriptome ................................................... 110 4.4.3 Msn2/4 deletion has vast influences on the transcriptome ............................................ 114 4.5 Transcriptional heat stress response in hsp12 and sHsp deletion yeast ................................ 117 4.5.1 Hsp26/42 deletion results in vast transcriptional changes ............................................. 118 4.5.2 Hsp12 deletion affects ion homeostasis ........................................................................ 124 4.6 Proteomic investigations on S. cerevisiae under heat shock ................................................ 128 4.6.1 The cellular proteome after 10 min of heat stress ......................................................... 128 4.6.2 The cellular proteome after 30 min of stress ................................................................. 132 5. Discussion .................................................................................................................................. 143 5.1 The small heat shock proteins of baker’s yeast .................................................................... 143 5.2 The transcriptional
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