Reconstitution and molecular characterisation of the mitochondrial membrane contact site
by Barbora Turpin Knotkova née Barbora Knotkova
Date of Examination:2024-08-20
Date of issue:2025-03-14
Advisor:Prof. Dr. Michael Meinecke
Referee:Prof. Dr. Michael Meinecke
Referee:Prof. Dr. Silvio Rizzoli
Persistent Address:
http://dx.doi.org/10.53846/goediss-11141
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Abstract
English
Due to their endosymbiotic origin, mitochondria of eukaryotic cells are enveloped by two membranes. While the outer membrane is rather flat, the inner membrane is intricately folded into subcompartments called cristae. Cristae are essential for efficient cellular respiration and for tight control of apoptotic signalling, but the formation and maintenance of crista architecture is only poorly understood on a molecular level. A known key player in the upkeep of cristae is the MICOS complex, a multi-subunit protein complex situated at crista junctions. Deletions of MICOS subunits lead to aberrant inner mitochondrial membrane architecture, often marked by the detachment of crista membranes from the rest of the inner mitochondrial membrane. However, interpreting this phenotype in vivo is rather difficult as MICOS was shown to have two different molecular functions: firstly, the core subunits are membrane remodelling proteins, and secondly the complex acts as a protein interaction hub with contacts to all other mitochondrial compartments. One of the established contacts is the SAM complex in the outer mitochondrial membrane. The interaction is so stable that MICOS and SAM can be detected in a supercomplex termed the mitochondrial intermembrane space bridging (MIB) complex, which tethers the inner membrane to the outer membrane. There are many indications that the MIB complex is required for the maintenance of proper crista architecture, but direct evidence is missing. Furthermore, how exactly the complex is formed on a molecular level in different eukaryotic species from yeast to humans has not been deciphered yet in detail. In this study I take a bottom-up approach to address these open questions and reconstitute a minimal MIB complex from different species on model membranes in vitro. By incorporating Sam50 of the SAM complex into liposomes and Mic60 of the MICOS complex into lipid nanodiscs and performing co-flotation assays, I show that Mic60 and Sam50 from the budding yeast Saccharomyces cerevisiae interact directly, whereas the human protein variants do not. Mic19, which has previously been suggested to be important for membrane tethering in humans, does not affect tethering of yeast proteins in this model system. Furthermore, using a Mic60 construct from the thermophilic fungus Chaetomium thermophilum, I also demonstrate that the intermembrane space domain of Mic60 is sufficient for an interaction with Sam50 to occur. Overall, I have established a model membrane system which allows the testing of membrane protein interactions and can be used in the future to gain deeper molecular insights into Mic60-Sam50 binding on a structural level. Structural information will pave the way for targeted investigations of the importance of the mitochondrial membrane contact site for crista junction maintenance, and other physiological processes, such as intermembrane molecular transfer.
Keywords: Mitochondria, Membrane Contact sites, MICOS, membrane biochemistry, biochemical reconstitution
