Role of MICOS for mitochondrial morphology and function

Role of MICOS for mitochondrial morphology and function

Tobias Müller

Doctoral thesis

Date of Examination: 2019-07-01

Date of issue: 2019-12-17

Advisor: Rehling, Peter Prof. Dr.

Referee: Jahn, Olaf Dr.

Referee: Meinecke, Michael Prof. Dr.

Persistent Address: http://hdl.handle.net/21.11130/00-1735-0000-0005-12D2-9

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Abstract

English

Mitochondria are highly dynamic organelles with a distinct morphological membrane ultrastructure. The mayor protein complex responsible for the formation and maintenance 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 components. 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 translocation 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 mic10Δ and atp20Δ revealed, that independent from MICOS, proper inner mitochondrial membrane morphology is essential for efficient precursor protein translocation via the TIM23-complex. In contrast to the mammalian system, carrier import via the TIM22 complex was verified to be independent of morphological alterations in yeast, thus further confirming the immense evolutionary divergence between the human and yeast TIM22 carrier translocase.

Keywords: MICOS; Translocase