Visualizing mitochondrial proteostasis using cryo-electron tomography

Mitochondria are the primary site of energy generation in many organisms, including humans, underscoring their fundamental role in numerous cellular processes. Proper mitochondrial function is safeguarded by an extensive quality control (QC) network that responds to diverse stress conditions. Impairment of this QC system can have severe consequences, as mutations in key components are associated with devastating diseases, often affecting highly energy-dependent tissues such as the brain and muscle. Mitochondrial QC operates on multiple levels and is deeply intertwined with other cellular stress responses, forming a highly complex regulatory system. While significant progress has been made in uncovering individual aspects of mitochondrial QC, the precise orchestration of its components and their spatiotemporal dynamics remain only partially understood. In this thesis, we investigated mechanisms and structural alterations associated with mitochondrial stress to better understand distinct elements of mitochondrial QC. In our first study, we focused on the PINK1-TOM complex, which is stabilized during mitochondrial import arrest and marks damaged mitochondria for autophagic degradation. The impairment of this mechanism is linked to familial forms of Parkinson’s disease. Using a combined approach of in silico structural prediction with AlphaFold and biochemical validation through site-directed mutagenesis, we demonstrated that the import receptors TOM20 and TOM70 are essential for PINK1– TOM complex formation. Moreover, we mapped possible interaction sites between PINK1 and TOM20/TOM70, leading to a model in which PINK1 is shuttled to the TOM complex through either simultaneous or sequential receptor engagement. Our second study investigated the effects of intramitochondrial protein aggregation following inhibition of the mitochondrial chaperone TRAP1, using in situ cryo-electron tomography (cryo-ET). We observed local distortions of cristae architecture near aggregates and localized mitochondrial ribosomes as well as mtHsp60:mtHsp10 chaperone complexes through subtomogram averaging (STA). Under aggregate- inducing conditions, fully assembled mitochondrial ribosomes were drastically V reduced, while mtHsp60:mtHsp10 complexes underwent a dramatic remodeling of their conformational landscape. By combining focused STA classification with single- particle analysis of purified complexes, we identified distinct conformations and proposed a functional folding cycle for mtHsp60:mtHsp10, providing mechanistic insight into its mode of action. In our third study, we analyzed intramitochondrial aggregation induced by importing aggregation-prone proteins into mitochondria. Super-resolution microscopy and cryo-ET showed alterations in cristae morphology and aggregate formation. Functional assays and biochemical experiments revealed sequestration of key factors leading to reduced translation. Importantly, we showed that components of the mitochondrial ribosome rely on mtHsp60:mtHsp10-assisted folding to achieve their functional three- dimensional structure; otherwise, they are degraded by the mitochondrial protease LonP1. This finding highlights an intricate interplay between mitochondrial QC components and complements the observations from our second study. Taken together, our studies provide new insights into mitochondrial QC, with a particular emphasis on the impact of intramitochondrial aggregation. We uncovered the vulnerability of mitochondrial ribosomes to aggregation and highlighted the dynamic interdependence of protein homeostasis factors such as mtHsp60:mtHsp10 and LonP1. These findings expand our understanding of the mechanisms safeguarding mitochondrial function and their relevance to cellular health.