Abstract
PINK1 is a mitochondrial serine threonine kinase implicated in the selective removal of damaged mitochondria via the process of mitophagy. Under steady state conditions the cell invests a substantial energy supply into the synthesis of PINK1 and its subsequent partial import into mitochondria, only for the protein to be cleaved and degraded in the cytoplasm. This feedback loop signals to the cell a healthy complement of mitochondria. Conversely, onset of one of a variety of mitochondrial stressors disrupts this equilibrium, inducing rapid accumulation of PINK1 at the outer membrane of mitochondria where its kinase activity is activated and mitochondria begin to undergo clearance through mitophagy. For some years now this model of PINK1 function, notably its constitutive degradation in a healthy environment, has remained unchallenged. Despite this, recent identification of internal mitochondrial proteins thought to be substrates of PINK1 kinase activity suggests the PINK1 import and functional model could extend beyond it simply undergoing degradation at steady state.This thesis aimed to comprehensively analyse PINK1 import behaviour and its regulatory control using a high-resolution MitoLuc assay system for monitoring mitochondrial import in intact mammalian cells. The PINK1 kinase domain was observed to undergo a degree of import into both the intermembrane space and matrix of mitochondria under steady state conditions. Traditional techniques and phosphoproteomic analysis were able to verify this and, in the context of the latter, provide clues to additional substrates that may be under the influence of post-translational PINK1 control. Analysis of a subset of recently documented PINK1 mutations revealed additional mechanistic detail behind regulatory control of PINK1 cleavage by the rhomboid protease PARL by means of secondary structural remodelling of the imported precursor.
Ultimately, the collective dataset presented in this thesis proposes an updated model for PINK1 import whereby, critically, a small proportion of synthesised PINK1 precursor may escape PARL cleavage under steady state. This thesis introduces a mechanism by which this may occur and how this may play a role in mitochondrial function.
Date of Award | 5 Dec 2023 |
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Original language | English |
Awarding Institution |
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Supervisor | Ian R Collinson (Supervisor) & Jon D Lane (Supervisor) |