Characterisation of the human TIM22 mitochondrial import translocase
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2020-11-16.
© 2018 Dr. Yilin Kang
Mitochondria are essential cellular organelles for cell viability due to their fundamental role in ATP production, programmed cell death and biosynthetic pathways. Dysfunctional mitochondria are implicated in various pathologies including cancers, cardiovascular diseases and neurodegenerative diseases. Mitochondrial function relies on ~1500 mitochondrial proteins, which are predominately nuclear-encoded and must be imported into mitochondria. Protein trafficking to mitochondria is executed by sophisticated multimeric protein import machines, termed, Translocases. One such machine, the Translocase of the Inner Membrane 22 (TIM22) mediates the import of an important class of hydrophobic proteins, the carrier proteins, which facilitate chemical exchange across the inner membrane, contributing to cellular metabolism and bioenergetics. While TIM22 has been well-characterised using Baker’s yeast, very little is known about the human complex. This study has focussed on disentangling the molecular composition of the human TIM22 complex, and mechanisms underpinning the carrier import pathway. Using immunoprecipitation/mass-spectrometric analysis, we uncovered two novel, metazoan-specific subunits of the human TIM22 complex, termed Tim29 and Acylglycerol kinase (AGK). This is the first report of additional subunits of the human TIM22 complex since the initial reports of the human complex in 1999 (Bauer et al., 1999b). We showed that Tim29 is involved in the assembly of the TIM22 complex and creates contacts with the general entry gate of the outer membrane, the TOM complex, providing a novel mechanism for the translocation and import of hydrophobic proteins. Dissecting the molecular function of AGK revealed a lipid kinase-independent role of this previously described mitochondrial lipid kinase in mediating carrier protein import via the TIM22 complex. Identification of AGK at TIM22 also shed additional insight into the pathomechanisms underpinning Sengers syndrome, a mitochondrial disorder uniquely associated with AGK mutations, providing an unexpected link between mitochondrial protein import and Sengers syndrome. The TIM22 complex is also linked to a neurodegenerative disease, Mohr-Tranebjaerg syndrome (MTS), which is caused by mutations in the TIMM8A gene. To date, the pathomechanism underlying this disease remain unknown. Using CRISPR/Cas9-genome editing and label-free quantitative proteomics, we analysed two distinct cell lines (HEK293 and SH-SY5Y) and revealed a cell-specific function of hTim8a in Complex IV biogenesis in neuronal mitochondria. Our findings indicate that loss of hTim8a leads to mitochondrial dysfunctions which amplifies cytochrome c levels in mitochondria and sensitises cells to death. This research has contributed new insights into understanding of the human TIM22 translocase and carrier protein biogenesis. It has revealed novel features and knowledge on the pathomechanism of two TIM22-associated mitochondrial diseases – Sengers syndrome and Mohr-Tranebjaerg syndrome.
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