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Principles of RNA and Protein Quality Control at the Eukaryotic Ribosome

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Principles of RNA and Protein Quality Control at the Eukaryotic Ribosome Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Principles of RNA and Protein Quality Control at the Eukaryotic Ribosome Timm Frank Nikolaus Hassemer aus Mainz 2017 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Franz-Ulrich Hartl betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, den 18.08.2017 ____________________________ Timm Hassemer Dissertation eingereicht am: 18.08.2017 1. Gutachter: Prof. Dr. Franz-Ulrich Hartl 2. Gutachter: PD Dr. Dietmar Martin Mündliche Prüfung am: 22.11.2017 Acknowledgements First of all, I would like to thank Prof. Franz-Ulrich Hartl for the opportunity to conduct my PhD thesis in his research group at the Max Planck Institute of Biochemistry. The work described in this thesis would have not been possible without his invaluable scientific expertise, his experience, and his guidance in the last four years. I am also very grateful to Dr. Sae-Hun Park and Dr. Young-Jun Choe for introducing me to yeast cell biology and countless biochemical methods. The quality of the results obtained during the course of my studies has benefited greatly from their scientific experience and support. Furthermore, I would like to thank all members of the Hartl department for their support and the friendly and open-minded atmosphere in the lab, which always allowed for sophisticated discussions and mutual support. I especially want to thank Emmanuel Burghardt, Albert Ries, Nadine Wischnewski, Anastasia Jungclaus and Romy Lange for their invaluable technical support, and Evelin Frey-Royston and Darija Pompino for their administrative support. I am also very grateful to Dr. Roman Koerner for introducing me to proteomics and for measuring and analyzing countless samples. I want to thank the member of my PhD committee PD Dr. Dietmar Martin for evaluating this thesis. I would like to address special thanks to Dr. Thomas Hauser, Dr. Amit Gupta, Dr. Christian Loew, Dr. Tobias Neudegger, Dr. Robert Wilson, Leonhard Popilka, Dr. David Balchin, Dr. Gabriel Thieulin-Pardo, Martin Mueller and Michael Gropp for their friendship and support in the last four years. Finally, my biggest thanks go to my parents and my sister. Without their unconditional love and support, it would have never been possible for me to achieve this goal. I am deeply grateful that you were always believing in me. Table of contents Table of contents Summary ..................................................................................................................................................... 1 I. Introduction ....................................................................................................................................... 3 I.1 Protein folding and molecular chaperones ............................................................................. 3 I.2 Protein aggregation in vivo ........................................................................................................ 7 I.3 The protein homeostasis network ............................................................................................ 9 I.3.1 Protein synthesis and folding in the cytosol ................................................................ 11 I.3.2 Degradation of proteins .................................................................................................. 13 I.3.3 The cellular stress response ............................................................................................ 16 I.3.4 Endoplasmic reticulum-associated degradation ......................................................... 18 I.3.5 The proteostasis network in disease and aging ........................................................... 20 I.4 The ribosomal quality control pathway ................................................................................ 22 I.4.1 Ribosomal stalling ............................................................................................................ 22 I.4.2 Recognition and dissociation of stalled ribosomes ..................................................... 25 I.4.3 Degradation of aberrant polypeptides .......................................................................... 27 I.4.4 mRNA quality control pathways ................................................................................... 31 I.5 Aim of the study ....................................................................................................................... 34 II. Materials & Methods ..................................................................................................................... 36 II.1 Chemicals ................................................................................................................................... 36 II.2 Enzymes and antibodies .......................................................................................................... 40 II.2.1 Enzymes ............................................................................................................................ 40 II.2.2 Antibodies ......................................................................................................................... 40 II.3 Strains ......................................................................................................................................... 41 II.3.1 Bacterial strains................................................................................................................. 41 II.3.2 Yeast strains ...................................................................................................................... 42 I Table of contents II.4 Plasmids ..................................................................................................................................... 43 II.5 Media and buffers ..................................................................................................................... 45 II.5.1 Media ................................................................................................................................. 45 II.5.2 Buffers ................................................................................................................................ 47 II.6 Materials and instruments....................................................................................................... 54 II.6.1 Kits and consumables ...................................................................................................... 54 II.6.2 Instruments ....................................................................................................................... 54 II.7 Molecular biology ..................................................................................................................... 57 II.7.1 DNA quantification ......................................................................................................... 57 II.7.2 Agarose gel electrophoresis ............................................................................................ 57 II.7.3 DNA sequencing .............................................................................................................. 57 II.7.4 Purification of DNA fragments and plasmid DNA .................................................... 58 II.7.5 Polymerase chain reaction .............................................................................................. 58 II.7.6 Restriction digest and DNA ligation ............................................................................. 58 II.7.7 Sequence- and ligation-independent cloning ............................................................... 59 II.7.8 Site-directed mutagenesis ............................................................................................... 59 II.7.9 Preparation and transformation of chemocompetent E. coli cells ............................. 60 II.7.10 Preparation and transformation of chemocompetent S. cerevisiae cells.................... 60 II.7.11 Construction of mutant S. cerevisiae strains .................................................................. 61 II.7.12 Isolation of genomic DNA from S. cerevisiae ................................................................ 61 II.8 Expression and purification of proteins ................................................................................ 62 II.8.1 Expression of proteins in E. coli ...................................................................................... 62 II.8.2 Purification of proteins expressed in E. coli .................................................................. 63 II.8.3 Expression of proteins in S. cerevisiae ............................................................................ 63 II.9 Protein analytics ........................................................................................................................ 64 II.9.1 Preparation of yeast lysates ............................................................................................ 64 II.9.2 Protein quantification ...................................................................................................... 65 II.9.3 Trichloroacetic acid precipitation .................................................................................. 65 II.9.4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis .................................... 65 II
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