Characterisation of a mammalian mitochondrial AAA+ protein, SKD3

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Victoria.

Members of the AAA postive (ATPase associated with diverse cellular activities) protein superfamily, as the name indicates, contribute to many different cellular processes. For example they play important roles in membrane fusion, organelle and protein trafficking, DNA replication, protein disaggregation and protein degradation. They are characterised by the presence of an 200-250 amino acid AAA domain containing Walker A and Walker B motifs and other conserved structural elements important for ATP binding and hydrolysis. Proteomic data revealed mitochondria contained a ubiquitously expressed AAA postive protein of unknown function. This protein is annotated as both suppressor of potassium transport defect, protein 3 (SKD3) and caseinlytic protein B (ClpB) however these names do not reflect known physiological functions. The aim of this study was to determine the physiological function of SKD3 in mitochondria. Specifically, experiments focussed on determining the sub-mitochondrial location of human SKD3, its substrates and potential adaptors and the relationship between its domain structure and its function. Data presented in this thesis indicates that SKD3 resides in the intermembrane space of mitochondria where it acts as an assembly chaperone for the biogenesis of protein translocation components known to be critical for the import of outer membrane and intermembrane space proteins. Dissection of the structural domains of SKD3 also indicates that the ankyrin domain of the protein mediates direct substrate engagement while the AAA domain assembles into an active ATPase. These data are in agreement with recent human genetic studies which revealed that inherited mutations in SKD3/CLPB led to 3-methylglutaconic aciduria, progressive brain atrophy, defective intellectual and movement development, neutropenia and cataracts in patients. We speculate that impaired mitochondrial intermembrane space protein biogenesis due to loss VIII of SKD3 function causes gross mitochondria dysfunction and dysregulation of cellular process particulary during developmental stages.