The biogenesis of many mitochondrial proteins depends on the import of nucleus-encoded precursors synthesised within the cytosol. These precursors are kept unfolded within the cytosol in order to remain import competent, especially inter membrane space (IMS) proteins. Thus, after importation these proteins are folded into their native state, with IMS protein folding driven by oxidative folding. Correct protein folding is critical to maintain proteostasis within the organelle. Accumulation of misfolded and damaged proteins leads to formation of toxic aggregates which can result in mitochondrial dysfunction. Therefore, the recognition and removal of non-native proteins is essential for mitochondrial proteostasis. To facilitate this, a highly conserved proteolytic network of proteases provides protein quality control in different mitochondrial subcompartments. Within the literature inner membrane (IM) mitochondrial protease yeast mitochondrial escape 1 (Yme1) was demonstrated to play an important role in maintaining proteostasis within the IMS, acting with both chaperone-like and proteolytic activity. However, until recently the function and functional mechanism of Yme1 was not fully understood. In this study, the functional mechanism of yeast Yme1 was investigated, as well as the effect of its deletion at the cellular and organeller level. First, in vitro studies were carried out to study the function and activity of different Yme1 domains. This is done by studying three different Yme1 constructs encoding different Yme1 domains. The result for purification and characterisation of these constructs were described in Chapter 3. Characterisation of construct encoding the ATPase domain showed comparable activity to other AAA proteases reported in the literature. However, purification of other Yme1 constructs was not as successful which limits their characterisation in this study. Next, the effect of Yme1 deletion for cellular growth and mitochondrial function was investigated using in vivo analysis methods. The results were described in Chapter 4. There, cellular growth and chronological life span was observed to be impaired in yme1 deletion strain. We also observed increased sensibility towards oxidative stress during cellular growth. Furthermore, yme1 deletion was shown to negatively impact electron transport chain (ETC) activity, hence mitochondrial function, as indicated by decreased oxygen consumption rate. Interestingly, this study showed cardiolipin content, important for ETC complex organization, would increase in yme1 deletion strain. Finally, in this study mitochondrial function was further characterised using isolated mitochondria. The results for mitochondrial function and individual ETC complexes activity assays were described in Chapter 5. Mitochondrial ATP concentration was shown to be lowered significantly in yme1 deletion mitochondria, indicative of impaired mitochondrial function. Moreover, ETC complex III and IV activity was shown to be decreased, especially when under respiratory conditions. Further analysis suggests this may due to decreased level of their respective subunits. Proteomic study on yme1 deletion mitochondria showed proteins involved in mitochondrial dynamic, protein translocation and cardiolipin biogenesis are upregulated. On the other hand, proteins involved in energy production were shown to be downregulated, for example TCA cycle and pyruvate metabolism. In summary, these results suggest Yme1 plays an important role in maintaining mitochondrial function, in particular regulating the organization and turnover of ETC complex subunits.
|Date of Award||1 Aug 2020|
- The University of Manchester
|Supervisor||Hui Lu (Supervisor) & David Leys (Supervisor)|