Structural and functional characterization of TIM22 complex in the inner mitochondrial membarne

by Anusha Valpadashi
Doctoral thesis
Date of Examination:2023-08-18
Date of issue:2024-08-16
Advisor:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Fernández-Busnadiego Rubén
Referee:Dr. Alexander Stein
Referee:Dr. Bohnert Maria
Referee:Prof. Dr. Stefan Jakobs
Persistent Address: http://dx.doi.org/10.53846/goediss-10678

 

 

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Abstract

English

Mitochondria have gained significant attention in disease models due to their crucial role in cellular energy production and metabolic activity. Understanding how mitochondria maintain their function and interact with other cellular components during stress is of great importance. Import translocases play a vital role in importing cytoplasmically synthesized mitochondrial proteins. Advancements in analytical techniques have provided insights into the independent translocases embedded in the mitochondrial membrane and their coordination in ensuring the proper assembly of nuclear-encoded proteins. While most translocases have been evolutionarily conserved with some modifications, the TIM22 complex in the inner mitochondrial membrane has shown diversification from yeast to humans. Recent identification of the peripheral subunits, TIM29 and AGK, in metazoans has shed light on their role in the TIM22 complex. AGK acts as both a complex subunit and a lipid kinase, contributing to the lipid composition of the inner mitochondrial membrane and facilitating carrier protein import through the TIM22 complex. TIM29 is known to stabilize the complex and interact with small TIM proteins, but further knowledge about the structure and function of the independent subunits is limited. To address this, we employed two tagging methods and conducted a large-scale proteomic analysis. By combining chemical crosslinking and mass spectrometric analysis (XL-MS), we identified the regions of interaction between the subunits, providing insights into their spatial orientation and the flexibility of the hexamer complex involved in substrate transport. Building on this understanding, our focus shifted to investigating the significance of the diversification of peripheral subunits. We performed siRNA-mediated knockdown of TIM23, TIM22, and TIM29 proteins and used TMT-based tagging and LC-MS/MS analysis to assess changes in the mitochondrial proteome. Surprisingly, we observed pronounced effects on TOM complex subunits following TIM29 knockdown. The TOM22 and TOM20 proteins showed significant depletion, while TOM40 displayed a lower complex of approximately 130 kDa alongside the final TOM complex. We confirmed that the TOM40 lower complex results from diminished TOM22 protein levels, ruling out the involvement of other complexes such as SAM and MICOS. The presence of a similar phenotype in TOM5 under TIM29 knockdown and defects in TOM40 biogenesis suggests that the observed lower complex represents an intermediate stage of TOM40 assembly after its release from SAM50. Our findings uncover a novel function of TIM29 in regulating TOM40 biogenesis. However, the precise mechanism through which TIM29 influences TOM22 abundance and the importance of the association between TOM40 and TIM29 during biogenesis remains unclear. Further investigation is needed to understand the essential role of TIM29 in the complex mitochondrial proteome of humans. To summarize, this thesis enhances our understanding of the intersubunit interactions within the TIM22 complex and reveals an additional novel function of TIM29 in maintaining the overall import function of mitochondria.
Keywords: Mitochondria; TIM22; TIM29; inner membrane; complex
 
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