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Role of MICOS for Mitochondrial Morphology and Function

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Role of MICOS for Mitochondrial Morphology and Function Role of MICOS for mitochondrial morphology and function Dissertation for the award of the degree "Doctor rerum naturalium" at the Georg-August-University Göttingen within the doctoral program "Molecular Medicine" of the Georg-August University School of Science (GAUSS) submitted by Tobias Müller born in Alfeld (Leine) Göttingen, 2019 Members of the Thesis Committee Prof. Dr. Peter Rehling University Medical Center Göttingen (Supervisor and First Referee) Institute for Cellular Biochemistry Göttingen, Germany PD Dr. Olaf Jahn Proteomics (Second Referee) Max-Planck Institute for Experimental Medicine Göttingen, Germany Prof. Dr. Michael Meinecke University Medical Center Göttingen Institute for Cellular Biochemistry Göttingen, Germany Additional Members of the Examination Board Prof. Dr. Stefan Jakobs Department of Nanobiophotonics Max-Planck Institute for Biophysical Chemistry Göttingen, Germany Dr. Alexander Stein Membrane Protein Biochemistry Max-Planck Institute for Biophysical Chemistry Göttingen, Germany Prof. Dr. Ralph Kehlenbach University Medical Center Göttingen Institute for Molecular Biology Göttingen, Germany Affidativ Herewith I declare that I prepared my dissertation "Role of MICOS for mitochdon- drial morphology and function" on my own and with no other sources and aids than quoted. Göttingen, March 2019 Part of this thesis will be communicated in the following publication: Callegari S., Müller T., Schulz C., Lenz C., Jans D. C., Wissel M., Opazi F., Rizzoli S. O., Jakobs S., Urlaub H., Rehling P., Deckers M. (2019). A MICOS-TIM22 association promotes mitochondrial carrier import in human mitochondria. Journal of Molecular Biology (submitted) Acknowledgements First of all I would like to thank my supervisor Peter Rehling for the opportunity to work in such an interesting and challenging field, for his relentless support throughout the project and his patience with me. Furthemore Michael Meinecke and Olaf Jahn for being part of my thesis committee and interesting and constructive discussions during committee meetings. In addition Ralph Kehlenbach, Stefan Jakobs and Henning Urlaub for joining my extended examination board. A very special gratitude to Markus Deckers for his never-ending support and cascade of ideas to forward this project. Various scientific and personal discussions together with his patience in frustrating moments were deeply appreciated. A big thanks to my collaborators who provided essential data for this work. Daniel C. Jans and Stefan Jakobs for electron microscopy pictures and Christof Lenz, Thierry Wasselin, Henning Urlaub and Monika Raabe for the Mass-Spectrometry analysis and dealing with the various problems that occurred on the way to the final dataset. Special thanks to all current and past members of the department. Markus and Sylvie for correcting my manuscript, dealing with my language skills and a nice collabora- tion on a wonderful publication. Mirjam for always making me laugh and lighten up many dark days and providing a plethora of evenings with nice food and good com- pany. Ridhima, without whom I wouldn’t have survived many frustrating lab days and was always providing cheer ups, food, tea, manuscript proof reading and great Doctor Who and Merlin moments. My labmates Bettina, Abhishek, Natasha, Lioba and Magnus in addition to various great students for creating a unique and crazy working environment. Special thanks to David, Flo, Matt, Alex, Kathi, Moritz, Sabine, Frank, Cong, Arpita, Martina, Christin, 3x Lisa, Lena, Hussein, Rosi, Petra and so many more for enduring me the last couple of years in the lab. People, who are more family than friends and supported me all my way along, the Dödels inclusive attachments, the Knusperkäfer and last but not least my whole Hammer Style group. I would not have managed without anyone of you! Last and most importantly, my family. They supported me throughout all obstacles without questions and complain and no words can describe my gratefulness. Contents Abstract 1 1. Introduction2 1.1. The eukaryotic cell . .2 1.2. Mitochondria . .3 1.2.1. Structure and dynamics of mitochondria . .4 1.2.2. Mitochondrial oxidative phosphorylation . .5 1.3. Mitochondrial protein import . .7 1.3.1. Translocase of the outer membrane: The TOM complex . .9 1.3.2. The SAM complex . 10 1.3.3. The MIA pathway transport into the intermembrane space . 10 1.3.4. Carrier import via the TIM22 complex . 11 1.3.5. Importing presequence proteins via the TIM23 complex . 11 1.4. Mitochondrial dynamics and inner mitochondrial membrane morphology 12 1.4.1. The ATP synthase stabilizes cristae tips . 12 1.4.2. Regulation of cristae junctions . 13 1.4.3. The Mitochondrial Contact site and cristae Organizing System MICOS . 14 1.4.4. MICOS is connect to various mitochondrial processes . 18 1.5. Aim of this study . 19 2. Materials and Methods 20 2.1. Materials . 20 2.1.1. Chemicals . 20 2.1.2. Solutions . 23 2.1.3. Microorganism and cell lines . 25 2.1.4. Oligonucleotides and Plasmids . 26 2.1.5. Antibodies . 26 2.1.6. Kits . 27 2.1.7. Equipment . 27 2.1.8. Software . 28 2.2. Cell cultivation and isolation of mitochondria . 29 2.2.1. Transformation of HEK-cells . 29 2.2.2. Isolation of human mitochondria . 29 2.2.3. Isolation of yeast mitochondria . 29 2.2.4. Transformation of E. coli ....................... 30 2.2.5. Transformation of Saccharomyces cerevisiae ............ 30 2.2.6. Isolation of Yeast mitochondria . 30 2.3. Molecular biology methods . 31 2.3.1. Isolation of plasmids from E. coli .................. 31 2.3.2. Polymerase chain reaction (PCR) . 31 2.3.3. Agarose gel electrophoresis . 32 2.3.4. Gel purification of DNA . 32 2.3.5. Molecular cloning . 32 2.4. Biochemical methods . 33 2.4.1. Immunofluorescence of U2OS-Cells . 33 2.4.2. Swelling of mitochondria . 33 2.4.3. Affinity purifications . 34 2.4.4. In vitro mitochondrial import . 34 2.4.5. Generating and isolating the TOM-TIM23 supercomplex . 35 2.4.6. In organello biotinylation . 35 2.4.7. In vivo biotinylation of HEK-cells . 36 2.4.8. Measurement of mitochondrial membrane potential . 36 2.4.9. TCA precipitation of Saccharomyces cerevisiae ........... 37 2.4.10. SDS-PAGE . 37 2.4.11. Western blotting and immunodecoration . 38 2.4.12. Coomassie Brilliant Blue staining . 38 2.4.13. Colloidal Coomassie staining . 38 2.4.14. Mass-spectrometry . 38 3. Results 40 3.1. Deletion of MIC10 alters inner mitochondrial membrane morphology . 40 3.1.1. Generation of MIC10 knock-out using the CRISPR/Cas9 system . 40 3.1.2. Ablation of MIC10 affects mitochondrial inner membrane mor- phology . 41 3.1.3. MIC10-/- mitochondria showed altered protein levels for MIC13 . 43 3.1.4. Respiration rate of MIC10-/- cells . 45 3.2. Proximity labelling: A powerful tool to investigate transient interactions 48 3.2.1. Biotin-labelling using an enhanced ascorbate C peroxidase . 49 3.2.2. BioID: a milder form of proximity labelling . 52 3.3. Alteration of the inner mitochondrial morphology affects precursor protein import in S. cerevisiae ............................ 58 3.3.1. Deletion of Mic10 does not effect translocase components . 58 3.3.2. Ablation of Mic10 influences the activity of the TIM23 complex . 59 3.3.3. MICOS does not interact with the carrier translocase in yeast . 65 4. Discussion 68 4.1. MIC10 is involved in cristae junction formation and stabilisation . 68 4.2. Altering the inner membrane morphology does not impact on mitochon- drial function . 69 4.3. Unveiling novel MICOS interaction partners via proximity labelling . 70 4.4. MICOS interacts with the TIM23-complex . 73 4.5. Inner mitochondrial ultrastructure affects import of precursor-containing proteins in yeast . 74 4.6. Carrier import via TIM22 does not depend on proper mitochondrial mor- phology in yeast . 75 5. Conclusion and future perspective 77 A. AppendixI A.1. Abbreviations . .I A.2. List of Figures . III A.3. List of Tables . .IV References . .V Abstract 1 Abstract Mitochondria are highly dynamic organelles with a distinct morphological membrane ultrastructure. The mayor protein complex responsible for the formation and mainte- nance of the inner mitochondrial membrane is the mitochondrial contact site and cristae organizing system (MICOS). This complex consists of 6 constituents in Saccharomyces cerevisiae and 7 in the mammalian system, with Mic60 and Mic10 being the core com- ponents. MICOS has been predominantly investigated in S. cerevisiae and studies in human cells have focussed mainly on MIC60. In this study, a CRISPR/CAS9 mediated MIC10 knock-out cell-line was generated and further characterized. Herby MIC10 could be identified to be essential for forming and maintaining proper mitochondrial morphology in mammalian cells. However, the loss of inner membrane ultrastructure did not have an impact on mitochondrial function and health. To better understand MICOS function in human, two different proximity biotinylation approaches were undertaken and compared to determine novel interaction partners. The first approach involved using an enhanced ascorbate C peroxidase APEX fused to MIC10 and the second approach made use of a promiscuous biotin ligase BioID2. The usage of the BioID2 enzyme proved to be more suitable since it already produced a distinct set of mutual interaction partners together with already known interaction partners. On the other hand, the active labelling reagent using the APEX enzyme proved to be more reactive than anticipated and would need a more thorough control system to identify background labelling. Recent findings reported a connection between MICOS via Mic60 and protein translo- cation through the TOM complex and the MIA-pathway (von der Malsburg et al., 2011). In this study, a spatial connection between MICOS and the TIM23 complex mediated via Mic60 could be found. Performing import studies in two different yeast strains with impaired inner membrane ultrastructure mic10∆ and atp20∆ revealed, that independent from MICOS, proper inner mitochondrial membrane morphology is essential for efficient precursor protein translocation via the TIM23-complex.
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